RMC Pharmaceutical Solutions

Assessing rapid viral enumeration/detection systems

2012-01-05T15:20:00.000-08:00

In a previous posting, we alluded to the recent availability of rapid methods for identification of viruses. These technologies, together with rapid methods for enumerating viruses, should greatly expedite the quantification and identification of viruses (and bacteriophage) as compared with the existing cell culture-based approaches.

Rapid enumeration technologies are intended to replace the cell-based infectivity endpoints such as plaque assays or tissue-culture infectious dose assays, which typically require 7-10 days for completion. The use of the rapid methods may be appropriate in cases where it is not necessary to determine the infectious titer of a virus stock. An example of this might be for monitoring the amplification of viruses for preparation of live or subunit vaccines. The particle enumeration technologies include those that specifically measure viral particles and those that measure particles in general. As shown in Table 1, the particle enumeration technologies are not specific to any given virus. These are not generally useful, therefore, for viral identification, although the particle detection method associated with the NanoSight system does allow for sizing of the particles. Viral particle size is a key attribute to be aware of when, for instance, attempting to identify an unknown viral contaminant.

Table 1. Characteristics of rapid viral enumeration/identification technologies

The quantitative polymerase chain reaction (Q-PCR) and universal biosensor (Ibis T5000) technologies represent approaches that are capable of providing information both on the relative quantity of a virus in a sample and its identity. The important difference between the two is that in the former case (Q-PCR), the user is typically evaluating the identity and or quantity of a virus which is reactive with the specific primers and probes used in the assay. From an identification standpoint then, the Q-PCR technique has typically been used to confirm whether an unknown virus is related to the virus for which the assay primers and probes was designed. The degree of relatedness required is determined by the specific primers and probes used in the assay, and may be either to the genus level or the species level. Efforts are being made to incorporate primers for more highly conserved sequences to allow for more broad coverage in Q-PCR assays intended for viral screening. In the case of the universal biosensor (Ibis T5000), an unknown virus in a sample may be simultaneously identified and quantified, as long as the virus is or is closely related to one for which mass spectrometry information is present in the software used for assay analysis. Quantification in either case is in genomic units, and as with the particle enumeration methods, the readout of the quantitative nucleic acid methods does not indicate whether the virus detected is infectious. An additional nucleic acid-based method that may prove useful, in cases where relatively rapid identification of an unknown viral contaminant is needed, is deep (massively parallel) sequencing. This method is more labor intensive (and perhaps costly) then the other quantitative nucleic acid methods described above, but has the advantage that it can provide information regarding the completeness (partial vs. full-length) of the viral genomic sequences detected. This approach has displayed utility in identifying a novel picornavirus in harbor seal samples, porcine circovirus in rotavirus vaccines, and a new parvovirus in bovine serum.

Microarray screening is a technology that may be used to rapidly identify (but not enumerate) an unknown virus in a sample, provided that a probe for the virus is part of the microarray chip. Some microarray chips intended for viral identification also contain probes for conserved viral genomic sequences. In this case, the microarray may identify a novel unknown virus, at least to the genus level. As with the other rapid methods that are based on presence of specific genomic material, the assay cannot discriminate between infectious and non-infectious virus.

See Table 1 for some of the important characteristics and limitations of each method. The use of the rapid methods discussed above and in Table 1 should reduce the time needed for viral quantitation from weeks to hours, and for identification of an unknown contaminant in a sample from months (or years) to one or more days. This should greatly facilitate the monitoring of viral proliferation in manufacturing processes and the investigation of viral contamination events.

Porcine circoviruses, vaccines, and trypsin

2011-10-28T11:39:00.000-07:00

By Dr. Ray Nims

It has now been more than a year since the announcements by GlaxoSmithKline (GSK) and Merck of the presence of porcine circovirus (PCV) genomic material in their rotavirus vaccines.

The presence of the PCV viral sequences was, in both cases, provisionally attributed to the use of porcine trypsin during the culture of the cell substrates used in the manufacture of the vaccines. It has been reported that the genomic sequences were associated with low levels of infectious PCV in the GSK vaccine.

As mentioned in a previous posting, an expected outcome of these disclosures was heightened regulatory expectations, going forward, for PCV screening of porcine raw materials and of Master and Working cell banks which were exposed to porcine ingredients (e.g., trypsin) at some point in their development. In January of 2011, the European Pharmacopoeia (Ph. Eur.) chapter 5.2.3 Cell substrates for production of vaccines for human use was revised to include the following instruction: “Trypsin used for the preparation of cell cultures is examined by suitable methods and shown to be sterile and free from mycoplasmas and viruses, notably pestiviruses, <circoviruses> and parvoviruses.” The addition of circoviruses to the list of viruses of concern (previously, mainly bovine viral diarrhea virus and porcine parvovirus) in Ph. Eur. 7.2 was not unexpected, based on the rotavirus vaccine experience.

A more broad expectation going forward may also be that vaccine and biologics production cell banks be proactively screened for unexpected, perhaps previously undetectable, viruses using detection techniques such as the deep sequencing used initially to detect the PCV in the GSK rotavirus vaccine. A related technique referred to as massively parallel sequencing (Massively Parallel Sequencing (MP-Seq), a New Tool For Adventitious Agent Detection and Virus Discovery) has been adopted for detection of viral contaminants in cells and viral seed stocks and for evaluating vaccine cell substrates by the contract testing organization BioReliance.

The more important sequella of the porcine circovirus disclosures may therefore be the proactive use of these new and powerful virus detection techniques for ensuring the viral safety of production cell banks, going forward.

Ridding serum of viruses with gamma irradiation: part 2

2011-10-12T07:50:00.000-07:00

by Dr. Ray Nims

In a previous posting, we described the susceptibility of viruses from various families to inactivation in frozen serum treated with gamma irradiation (data from the literature). Gamma irradiation is a commonly employed risk mitigation strategy for biopharmaceutical manufacture, and indeed the European Agency for the Evaluation of Medicinal Products in its Note for guidance on the use of bovine serum states that some form of inactivation (such as gamma irradiation) is expected. The use of non-treated serum in the production of biologics for human use must be justified, due to the potential for introducing a viral contaminant through use of this animal-derived material.

But how effective is this particular risk mitigation strategy? To answer this, we expressed the susceptibility to inactivation by gamma radiation for a series of 16 viruses in terms of log10 reduction in titer per kiloGray (kGy) of gamma radiation. With this value in hand, one may easily calculate the log10 reduction in titer of a given virus which might be expected following irradiation of frozen serum at any given kGy dose (serum is typically irradiated at a dose of 25-40 kGy).

The next step is to try to understand the results obtain with this relatively limited series of viruses, so that we may extrapolate the results to other viruses. If we take a look at the viral characteristics that might confer susceptibility to inactivation by gamma irradiation, will we find what we are looking for?

Viral characteristics that have been postulated to contribute to susceptibility or resistance to inactivation by gamma irradiation include: 1) radiation target size (genome size, particle size, genome shape, segmentation of the genome); 2) strandedness (in double stranded genomes the genomic information is recorded in duplicate, so loss of information on one strand may not be as damaging as it would be in the case of a single stranded genome); 3) presence or absence of a lipid envelope (non-enveloped viruses are resistant to a variety of chemical and physical inactivation approaches); and 4) genome type (RNA vs. DNA). The characteristics for our series of 16 viruses are displayed in the following table (click on table to enlarge).

For evaluating the contribution of radiation target size, we are able to make use of quantitative values available for each virus for genome size (in nucleotides) and particle size (in nm). Plotting genome size vs. log10 reduction in titer per kGy yields the results shown below. The coefficient of determination obtained is just 0.32, suggesting that factors other than (or in addition to!) genome size in nucleotides must be important..

A somewhat better concordance is obtained between particle size and log10 reduction in titer per kGy as shown below. The fit line in this case is non-linear, with a coefficient of determination of 0.60.

The contributions of genome type (RNA vs. DNA), genome strandedness, and lipid envelope (present or absent) to susceptibility to inactivation by gamma irradiation appear to be minimal, within this limited series of viruses. As a result, we are left with the conclusion that the most clear, albeit incomplete, determinant of susceptibility to inactivation by gamma irradiation appears to be particle size. This probably explains the resistance to inactivation displayed by the extremely small viruses such as circoviruses, parvoviruses, and caliciviruses. It is less clear why the polyomaviruses (e.g., SV-40 at 40-50 nm) are so resistant to gamma irradiation while certain of the picornaviruses (25-30 nm) are less resistant to inactivation. More work in this area is needed to better understand all of the factors that contribute to susceptibility to gamma radiation inactivation in viruses and bacteriophage.

< This information was excerpted in part from Nims, et al. Biologicals (2011) >

Is Your Chromatography in Control, or in Transition?

2011-09-28T13:10:00.000-07:00

By Dr. Scott Rudge

While chromatography continues to be an essential tool in pharmaceutical manufacturing, it remains frustratingly opaque and resistant to feedback control of any kind. Once you load your valuable molecule, and insufferable companion impurities, onto the column, there is little that you can do to affect the purification outcome that waits you some minutes to hours later.

Many practitioners of preparative and manufacturing scale chromatography perform “Height Equivalent to a Theoretical Plate” testing prior to putting a column into service, and periodically throughout the column’s lifetime. Others also test for peak shape, using a measurement of peak skewness or Asymmetry. However, these measurements can’t be made continuously or even frequently, and definitely cannot be made with the column in normal operation. More over, the standard methods for performing these tests leave a lot of information “on the table” so to speak, by making measurements at half peak height, for example.

To address this shortcoming, many have started to use transition analysis to get more frequent snapshots of column suitability during column operation. This option has been made possible by advances in computer technology and data acquisition.

Transition analysis is based on fairly old technology called moment theory. It was originally developed to describe differences in population distributions, and applied to chromatography after the groundbreaking work of Martin and Synge (Biochem. J. 35, 1358 (1941)). Eugene Kucera (J. Chromatog. 19, 237, (1965) derived the zeroth through fifth moments based on a linear model for chromatography that included pore diffusion in resins, which is fine reading for the mathematically enlightened. Larson et al. (Biotech. Prog. 19, 485, (2003)) applied the theory to in-process chromatography data. These authors examined over 300 production scale transitions resulting from columns ranging from 44 to 140 cm in diameter. They found that the methods of transition/moment analysis were more informative than measurements of HETP and Asymmetry traditionally applied to process chromatography.

What is transition analysis, and how is it applied? Any time there is a step change in conditions at the column inlet, there will occur, some time later, a transition in that condition at the column outlet. For example, when the column is taken out of storage and equilibrated, there is commonly a change in conductivity and pH. Ultimately, a wave of changing conductivity, or pH, or likely both, exits the column. The shape of this wave gives important information on the health of the column, as described below. Any and all transitions will do. When the column is loaded, there is likely a transition in conductivity, UV, refractive index and/or pH. When the column is washed or eluted, similar transitions occur. As with HETPs, the purest transitions are those that don’t also have thermodynamic implications, such as those in which chemicals are binding to or exchanging with the resin. However, the measurements associated with a particular transition should be compared “intra-cycle” to the same transition in subsequent chromatography cycles, not “inter-cycle” to different transitions of different natures within the same chromatography cycle.

Since transition analysis uses all the information in a measured wave, it can be very sensitive to effects that are observed any where along the wave, not just at, for example, half height. For example, consider the two contrived transitions shown below:

In Case 1, a transition in conductivity is shown that is perfectly normally distributed. In Case 2, an anomaly has been added to the baseline, representing a defect in the chromatography packing, for example. Transition analysis consists of finding the zeroth, first and second moments of the conductivity wave as it exits the column. These moments are defined as:

These are very easy calculations to make numerically, with appropriate filtering of noise in the data, and appropriate time steps between measurements. The zeroth moment describes the center of the transition relative to the inlet step change. It does not matter whether or not the peak is normally distributed. The zeroth moments are nearly identical for Case 1 and Case 2, to several decimal places. The first moment describes the variance in the transition, while the second moment describes the asymmetry of the peak. These are markedly different between the two cases, due to the anomaly in the Case 2 transition. Values for the zeroth, first and second moments are in the following table:

	Case 1	Case 2
Zeroth moment	50.0	50.0
First moment	1002	979.6
Second moment	20,300	19,623

It would be sufficient to track the moments for transitions from cycle to cycle. However, there is a transformation of the moments into a “non-Gaussian” HETP, suggested by McCoy and Goto (Chem. Eng. Sci., 49, 2351 (1994)):

Where

Using these relationships the variance and non-Gaussian HETP are shown in the table below for Case 1 and Case 2:

Using this method, a comparative measure of column performance can be calculated several times per chromatography cycle without making any chemical additions, breaking the column fluid circuit, or adding steps. The use of transition analysis is still just gaining foothold in the industry, are you ahead of the curve, or behind?

A much improved Ph. Eur. Chapter 5.3.2

2011-09-22T11:52:00.000-07:00

By Dr. Ray Nims

Vaccine manufacturers intending to market in the EU should be aware of a recent change in the European Pharmacopoeia (Ph. Eur.) chapter 5.2.3 Cell substrates for production of vaccines for human use. This chapter addresses the characterization of vaccine cell substrates. The section on Test Methods for Cell Cultures within the chapter includes an instruction to perform a co-cultivation study. The language previously was as follows: “Co-cultivation. Co-cultivate intact and disrupted cells separately with other cell systems including human cells and simian cells. Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses. The cells comply with the test if no evidence of any extraneous agent is found.”

This section has been changed, as of Ph. Eur. version 7.2 effective in January of 2011, to the following: “Co-cultivation. For mammalian and avian cell lines, co-cultivate intact and/or disrupted cells separately with other cell systems including human cells and simian cells. For insect cell lines, extracts of disrupted cells are incubated with other cell systems, including human, simian, and at least 1 cell line that is different from that used in production, is permissible to insect viruses and allows detection of human arboviruses (for example BHK-21). Carry out examinations to detect possible morphological changes. Carry out tests on the cell culture fluids to detect haemagglutinating viruses, or on cells to detect haemadsorbing viruses. The test for haemagglutinating viruses does not apply for arboviruses to be detected in insect cells. The cells comply with the test if no evidence of any extraneous agent is found.”

So what is the big deal? Co-cultivation is a commonly employed technique for detecting infectious retrovirus in a cell bank. It is effective for this purpose because the chances for spread of infectious virus from test cell to indicator (host) cell are optimized by the cultivation of live cells of each kind in close proximity. The endpoint of the retrovirus assay, be it reverse transcriptase enzyme induction or rescue of an S+L- virus, is not interfered with by the presence of two cell types in one culture. The same is not always true for a co-cultivation of a test cell with an indicator (host) cell for detection of infectious virus when morphological changes (viral cytopathic effects) are one of the assay endpoints. The reason is that the diploid human cells (e.g., MRC-5 or WI-38) used as one of the indicator cells in such assays are rapidly displaced during co-cultivation with intact continuous cell lines used to produce vaccines, such as the simian cell Vero. The result of this is that within a short period of time in co-cultivation, the test culture is no longer predominated by the diploid cell but rather by the test cells and observation of the culture for cytopathic effects becomes problematic. Changing the language of this section to read “…co-cultivate intact and/or disrupted cells separately with other cell systems…” allows the user to eliminate the inoculation of intact test cells onto a diploid indicator cell.

The other useful modification to the language of this section is the following addition: “For insect cell lines, extracts of disrupted cells are incubated with other cell systems, including human, simian, and at least 1 cell line that is different from that used in production, is permissible to insect viruses and allows detection of human arboviruses (for example BHK-21).” Testing of insect cells for extraneous virus is only marginally effective when it is conducted per the usual method of inoculating another insect cell. Why? The insect cells that are available are most commonly suspension cultures, making observation for cytopathic effect problematic. The extraneous viruses that are of most concern for an insect production cell are the arboviruses (viruses transmitted via insect vectors). It has been known for some time that the Syrian hamster cell line BHK-1 is an excellent host cell for detecting arboviruses. The new language in this section of Ph. Eur. chapter 5.2.3 now clears the way for the use of the monolayer BHK-1 cell line to be used for the testing of insect cells for extraneous virus. In this regard the Ph. Eur. chapter is now more closely aligned with the World Health Organization’s 2009 Evaluation of cell substrates for the production of biologicals: revision of WHO recommendations. The latter has the following passage: "For instance, in the case of insect cell substrates, certain insect cell lines may be used for detection of insect viruses, and BHK cells may serve for the detection of arboviruses."

Taken together, the recent changes to Ph. Eur. Chapter 5.2.3 greatly improve the chapter and the viral safety testing of vaccine production cell banks specifically proscribed within it.

Ridding serum of viruses with gamma irradiation: part 1

2011-09-12T13:25:00.000-07:00

by Dr. Ray Nims

Blood serum, while at times required as a medium component for cell growth in vitro, is an animal-derived material that can introduce contaminating viruses such as Cache Valley virus, REO virus, vesivirus, and epizootic hemorrhagic disease virusinto a biological product. If animal serum must be used in upstream manufacturing processes, the risk of introducing a virus may be mitigated by gamma irradiation of the frozen serum prior to use. How effective is this treatment, and against which viruses?

To answer this question, I have surveyed the literature from the past two decades. A number of investigations have been conducted and the results are in the public domain. The most useful of these studies have examined the dose-response relationships for viral inactivation (rendering of the virus as non-infectious) by gamma irradiation.

In the table below, I have assembled the results obtained for 7 viruses, including four that might be expected to be found in bovine serum: (bovine viral diarrhea virus [BVDV], infectious bovine rhinotracheitis virus [IBR], respiratory-enteric orphan virus [REO virus], and parainfluenza type 3 virus [PI3]). The other three (canine adenovirus, porcine parvovirus [PPV], and mouse minute virus [MMV]), while perhaps not expected to be found in bovine serum, have been studied as model viruses for the adenovirus and parvovirus families (click on table to enlarge).

The efficacy of gamma irradiation for viral inactivation is reported as log10 reduction in titer per kGy, rather than the more commonly employed D10 (Mrad dose required to reduce the titer by 1 log10), as I find the former value to be more useful. To estimate the effectiveness of a given dose of gamma radiation for inactivation of a virus, just multiply the dose in kGy by the log10 reduction in titer per kGy value from the table. The result is the number of logs of inactivation estimated to be achieved for that virus at that radiation dose.

These data tell us that the mid- to large-sized viruses BVDV, IBR, PI3, REO, and CAV should be readily inactivated at the gamma radiation doses normally applied to frozen serum for risk mitigation (25-45 kGy). On the other hand, the two parvoviruses, PPV and MMV, are more difficult to inactivate, presumably due to their small size. Parvoviruses are often used to challenge viral removal and inactivation processes due to their size and lack of an envelope. Higher kGy dose levels may increase the effectiveness of inactivation for these viruses, although at such levels the performance of the animal serum being irradiated may be adversely impacted.

Gamma-irradiation can effectively mitigate the risk of introducing other potential contaminants of bovine serum, including Cache Valley virus, blue tongue virus, and epizootic hemorrhagic disease virus. Like the parvoviruses, however, other relatively small non-enveloped viruses of the calicivirus, picornavirus, polyomavirus, and circovirus families may represent cases where gamma irradiation is less effective at the doses normally applied. Other means of mitigating the risk associated with these viruses may need to be considered.

< This information was excerpted in part from Nims, et al. Biologicals (2011) >

Information sources: Daley et al., FOCUS 20(3):86-88, 1998; Wyatt et al. BioPharm 1993: 6(4):34-40; Purtel et al., 2006; Hanson and Foster, Art to Science 16:1-7, 1997; Hyclone Labs Art to Science 12(2): 1-6, 1993; Gauvin and Nims 2010; Plavsic et al. BioPharm 2001: 14(4):32-36.

Our take on process-specific vs. generic host cell protein assays

2011-08-26T10:49:00.000-07:00

By Drs. Ray Nims and Lori Nixon

The residual host cell protein (HCP assay) is used to determine the concentration, in process streams, of protein originating from the production cell (Chinese hamster, NS0, E. coli, etc.) used in the manufacture of a biologic. Host cell protein is considered to be a process-related impurity/contaminant. The most typical approach to quantitation of residual host cell protein in a test sample is the enzyme-linked immunosorbant assay (ELISA) platform.

Generic vs. process-specific assays. A question that often arises is whether a process-specific HCP assay is required, or whether a generic assay can be used. Although there is a spectrum of “process specificity” that can be described, in this discussion, we are considering a generic assay to be one that is developed from a broad set of antigens from the host strain or closely-related strains, but not necessarily under specific process conditions mimicking the actual production/purification process of the protein of interest. That is, the reagents for a generic assay could be developed in advance of a defined production process. For example, it is often possible to purchase a “generic” HCP ELISA kit from a commercial provider, such as Cygnus Technologies, selecting a kit that matches the production cell line. Practically speaking, most firms start with generic HCP methods in early development for an obvious reason: you can’t have a process-specific assay until you have a defined process. Even after the process is defined, the lead time for developing custom (process-specific) HCP reagents and an assay using these can be as long as 1-2 years.

It should be appreciated from the outset that there is no perfect HCP assay. Production cells can express thousands of proteins, with expression patterns differing under different growth conditions. At each successive stage of the recovery and purification process, the population of HCPs is altered. At the final drug substance stage, there may be only a few HCPs present that have co-purified with the product. If you attempt to design an assay that only selects those few remaining HCPs, then some will argue that you might miss HCPs that might make it through under other circumstances (such as a process deviation). If you attempt to design antibody reagents using the entire unpurified mixture of proteins from the production cell, the proteins of most interest may not elicit a good antibody response and you may have inadequate sensitivity to quantify HCP in your final drug substance. In fact, it has been reported that process-specific antibodies lead to HCP assays that are more sensitive for certain HCP species (and sometimes more broadly reactive) than the antibodies provided in a generic HCP assay.

In recent years, there has been some regulatory pressure setting an expectation that a process-specific HCP assay must be developed for product registration. In our opinion, this is unjustified as a blanket prescription. There are a multitude of approaches for creating antigen and antibody reagents, but the resulting assay should be judged primarily on its own merits.

Suitability of any HCP assay (whether generic or process-specific) must be evaluated on a case-by-case basis, and the assay validated for the specific product in question. One of the first characteristics to aim for is an assay that is able to readily quantitate HCP in the final drug substance. Without this sensitivity, it is difficult to get results that are meaningful to guide process optimization or ensure control of your product. If the available generic method(s) have limited sensitivity for the sample of interest (results are below the required range of quantitation), then it is a good idea to start planning the development of more process-specific reagents—which are likely to afford better sensitivity. Since the numerical values reported from these assays are semi-quantitative at best, sensitivity should be judged on the performance in actual samples rather than on the product specification. Of course there are other assay characteristics (non-reactivity to product, precision, dilutional linearity, spike recovery, etc) that are required to meet validation acceptance criteria. The antigen coverage should be determined, typically by 2D electrophoresis or 2D HPLC-ELISA. It is unrealistic to expect 100% coverage of all of the proteins present in the production cell; on the other hand, it is relatively more important to ensure response against bands that persist in the purification process. All other things being equal, of course greater coverage is more desirable.

Example of 2D electrophoresis of E. coli proteins, from Kendrick Labs

Whether a generic or process-specific method is implemented, it is vital to ensure a consistent reagent supply that will last throughout the commercial life of the product. If using vendor-supplied antigen and antibody, be aware that by nature these are “single-source” reagents with associated supply risks. If creating a custom reagent set, make enough supplies to last for years to decades.

As mentioned above, most firms start with a generic assay and may move to a process-specific assay in later stages of development. A close evaluation of the generic assay during its characterization and validation may lead to the conclusion that the generic assay is suitable for your product. In this case, be prepared to make a strong case in defense of your generic assay—based on empirical data with your product, rather than theoretical speculation.

Rapid Identification of Viral Contaminants, Finally

2011-07-25T16:38:00.000-07:00

By Ray Nims, Ph.D.

There was a time, not long ago, when it might take months to years to identify a viral contaminant isolated from a biological production process or from an animal or patient tissue sample. The identification process took this long because it involved what I have referred to as the “shotgun approach”, or it involved luck.

Let’s start with luck. That is probably the wrong term. What I mean by this is that there have been instances where an informed guess has led to a fairly rapid (i.e., weeks to months) identification of a contaminant. For instance, our group at BioReliance was able to rapidly identify contamination with REO virus (REO type 2 actually) and Cache Valley virus because we had observed these viruses in culture previously and because these viruses had unique properties (a unique cytopathic effect in the case of REO and a unique growth pattern in the case of Cache Valley virus). The time required to identify these viruses consisted of the time required to submit and obtain results from confirmatory PCR testing for the specific agents.

The first time we ran into Cache Valley virus, however, it was a different story. This was, it turns out, the first time that this particular virus had been detected in a biopharmaceutical bulk harvest sample. In this case, we participated in the “shotgun approach” that was applied to the identification of the isolate. The “shotgun approach” consisted of utilizing any detection technique available at the lab, namely, in vitro screening, bovine screening, application of any immunofluorescent stains available, and transmission electron microscopy (TEM). The TEM was helpful, as it indicated a 80-100 nm virus with 7-9 nm spikes. A bunyavirus-specific stain showed positive, and eventually (after months of work), sequencing and BLAST alignment was used to confirm the identity of the virus as Cache Valley virus.

The “shotgun approach” was subsequently applied to a virus isolated from harbor seal tissues, with no identity established as a result. After approximately a year of floundering using the old methods, the virus was eventually found to be a new picornavirus (Seal Picornavirus 1). How was this accomplished? During the time between the identification of the Cache Valley virus and the seal virus, a new technology called deep sequencing became available. Eric Delwart’s group used the technique to rapidly identify the virus to the species level. As this was the first time this particular picornavirus had ever been detected, deep sequencing is likely the only method that would have been able to make the identification.

Deep (massively parallel) sequencing is one of a few new technologies that will make virus isolate identification routine and rapid in the future. It has been adopted for detection of viral contaminants in cells and viral seed stocks and for evaluating vaccine cell substrates by BioReliance.The other is referred to as the T5000 universal biosensor. Houman Dehghani’s group at Amgen has been characterizing this methodology as a rapid identification platform for adventitious agent contaminations. Each technology has its advantages. Deep sequencing is more labor intensive, but has the ability to indicate (as described above) a new species. The universal biosensor can both serve as a detection method and as an identification method. Both can identify multiple contaminants within a sample.

Since identification of an adventitious viral contaminant of a biopharmaceutical manufacturing process is required for establishment of root cause, for evaluating effectiveness of facility cleaning procedures and viral purification procedures, and for assuring safety of both workers and patients, it is critical that the identification of a viral isolate is completed accurately and rapidly. Happily, we now have the tools at hand to accomplish this.

Can You Decide on CAPA?

2011-07-09T22:31:00.000-07:00

By Dr. Scott Rudge

When things go wrong in pharmaceutical manufacturing,consequences can be dire. Small changesin the quality of the pharmaceutical product can cause major consequences forpatients in ways too numerous to list in this blog. It can be surmised that the failure mode wasnot anticipated, so the manufacturer is wise to determine the cause of thefailure, correct it, and prevent it from happening again. This exercise is known as CAPA, Correctiveand Preventive Actions.

There are numerous tools for determining the root causes offailures, many of these are embedded fully in software, and ICH Q9, that helpsa manufacturer’s quality organization to track the resolution of CAPAs. Fault Tree Analysis and Root Cause Analysisare two common and popular examples of these tools. These tools help to trace the cause of afailure back to the basic characteristics of the failure. Once these basic characteristics have beenidentified, they can be remediated (eliminated).

When the root cause is found, it is eliminated, and theprocess goes on. The CAPA is closed andeveryone waits for the next crisis or problem that needs to be fixed. Right?

But root cause elimination, although a worthy corrective objective,does not appear to be sufficient as a preventive action. One should consider how the root cause is to be eliminated. And one should consider whether the mannerand means of root cause elimination might lead to other failure modes.

The first consideration, how to eliminate a root cause, is adecision. Decision analysis can be usedto improve the process of choosing between multiple remediation options. When there are multiple good options forfixing a problem, or perhaps a series of “less bad” options, it’s a good ideato do some analysis.

First, the decision should be defined. The required objectives that the remediationoption must meet should be listed. Theseshould be specific and measureable. Eachoption for addressing the problem should also be listed, and the “features” ofeach option listed, so there is little uncertainty about the option and itsimplementation. Each option must fullymeet all the required objectives, any option that partially addresses must beconsidered incomplete. Any option thatis incomplete should either 1) not be considered further, or 2) have elementsadded that allow the option to completely satisfy the required objectives. All options that meet each of the requiredobjectives is a “qualified option”.

There are often additional objectives that are less absolutethat should be considered in choosing a remediation option. These might be considered as differentiatingobjectives. For example, cost may be arequired objective (the remediation must be implemented within the currentbudget) and a differentiating objective (the least costly option, within thebudget, is preferred). Thesedifferentiating objectives should also be listed, and weighted according totheir relative importance. Theobjectives of all stake holders must be considered. The weightings may be controversial amongstakeholders, but with practice, an organization can learn to appropriatelyweight objectives.

Qualified options can be scored according to the weighted differentiatingobjectives. Each qualified option isranked according to how it fulfills these objectives, and the score ismultiplied by the weight. The weightedscores are summed, and a “most preferred” option is identified as aresult.

But wait, there’s more. A final check of failure modes should be made. The last thing we want is to implement aremediation option that introduces more vulnerabilities into our systems. A simple FMEA can be used for this. If your organization routinely uses FMEAs andhas standard criteria for scoring them, then you probably only need to performone FMEA, and ensure no resulting risk priority numbers exceed the establishedthreshold value for risk. If yourorganization doesn’t have this experience, then you may have to perform acomparative FMEA on two or more qualified options, to gauge relative risk.

If your decision making is already perfect, then there is noneed for this analysis. Such excellentseat of the pants decision making would be useful for the rest of us tosee! For the rest of us, when new issuesarise, the decision analysis can be revisited, and organizational assumptionsrevised, to improve the decision making in the future. And it might be useful to have documented theoptions you considered, and how you selected among them.

Small, non-enveloped viruses: number 1 threat to biologics manufacture

2011-06-10T15:46:00.000-07:00

by Dr. Ray Nims

Perhaps surprisingly, few types of viruses have infected biologics manufacture since the 1980s when the first recombinant proteins began to be produced in mammalian cells. While the list of contaminating viruses has included some relatively large enveloped and non-enveloped viruses (Reovirus type 2, epizootic hemorrhagic disease virus, Cache Valley virus, human adenovirus), by far the most problematic contaminants have been the small non-enveloped viruses. Why? For the most part, the contaminations involving the larger viruses have been attributed to the use of non-gamma irradiated bovine serum or to operators conducting open vessel manipulations. Remediating the manufacturing processes to include gamma irradiation of the serum (or elimination of the use of serum altogether), and eliminating wherever possible open vessel operations should mitigate the risk of experiencing these viruses.

Now we come to the small non-enveloped viruses, the real problem. Foremost among these has been murine minute virus (MMV). This 20-25 nm non-enveloped parvovirus has infected biologics manufacturing processes using Chinese hamster cell substrates on at least four occasions, affecting at least three different manufacturers (Genentech, Amgen, and Merrimack). In each case, the source of the contamination has been unclear, making remediation of the processes difficult. Due to the ability of these viruses to survive on surfaces and their resistance to inactivation by detergents and solvents, eliminating the agent from contaminated facilities may require drastic measures such as fumigation with vaporous hydrogen peroxide .

A second problem virus is the 27-40 nm non-enveloped calicivirus, vesivirus 2117. This is the virus that was found to have infected the Genzyme Allston manufacturing facility in 2009. The same virus had appeared already once in the past, at a manufacturing facility in Germany. Both of the infected processes involved Chinese hamster production cells and both involved the use of bovine serum at some point in the manufacturing process. Whether or not the animal-derived material was the actual source of the infection was not proven in either case. Unfortunately, if the source was the bovine serum, gamma irradiation probably would not mitigate the risk, as gamma irradiation is less effective for inactivating the smaller non-enveloped viruses. This is another virus that may be able to survive on facility surfaces. As in the case with MMV, ridding a manufacturing facility of vesivirus may require entire facility fumigation with vaporous hydrogen peroxide, as was done at Genzyme.

Another problem virus is the 17-20 nm porcine circovirus that was found to contaminate a rotavirus vaccine in 2010. This virus was thought to have originated in contaminated porcine trypsin used in the manufacturing process. Wouldn’t this contaminant have shown up in the raw material testing done for the trypsin, or in the extensive cell bank testing required for vaccine production substrates? The answer is no. The circovirus would not have been detected using the 9CFR-based detection methods used for trypsin at this time (and at present). And the required testing for cell banks used to produce vaccines would not have detected this particular virus. To make matters worse, gamma irradiation of the trypsin would not be expected to inactivate this virus. How can we mitigate the risk of this virus going forward? As described in a previous posting, manufacturers may need to apply specific nucleic acid tests for the circovirus as part of the raw material release process for trypsin.

These and other small non-enveloped viruses represent the greatest risk for biologics manufacturing because they are more difficult to inactivate in raw materials, and more difficult to eradicate from the facility once infected, and because the source of the infection is not always clear. There must be analogous small-non-enveloped bacteriophage lurking out there that represent, for the same reasons, special threats to the fermentation industry.

The Art of Bioreactor/Fermenter Scale-Up (or Scale-Down)

2011-05-31T15:42:00.000-07:00

by Dr. Deb Quick

Effective bioreactor or fermenter scale-up/down is essential for successful bioprocessing. During development, small scale systems are employed to quickly evaluate and optimize the process, but larger scale systems are necessary for producing commercial quantities at a reasonable cost. But how does one effectively transfer the process between scales so that the process performs the same?

In an ideal world, the physiological microenvironment within the cells/microorganisms will be conserved at the different scales, but with no direct measure of that microenvironment the scientist identifies relevant macroproperties to measure and control to ensure comparability. There are many macroproperties and operating parameters that define the process at each scale, and while the goal is to keep as many of those parameters constant between the scales, it simply isn’t possible to keep them all the same.

When using the same operating parameters at small and large scale is impractical, there are several correlations that are commonly used: mass transfer coefficient (kLa [the volumetric transfer coefficient, 1/hr] or OTR [oxygen transfer rate, mmol/hr]; volumetric power consumption (P/V, agitation power per unit volume); agitator tip speed; and mixing time.

Matching the kLa at different scales is generally considered the most important factor in scaling cell culture and microbial processes. The second most common approach is to match the power consumption. For both of these correlations, there are often multiple combinations of operating parameters that provide the same kLa or the same power consumption at the different scale. And herein lies the art of bioreactor and fermenter scale-up/down. Selecting the best combination of parameters to match process performance at different scales is an art. There is no magic combination that works best for all cell types and products.

To establish comparability at different scales, you’ll make your life significantly easier if you start with the same vessel design at the different scales, but this luxury is rarely reality. More often, the development lab has significantly different equipment than the manufacturing facility. But even with different reactor designs, comparable performance can be obtained at different scales through appropriate experimentation.

First, you’ll need to understand your equipment at all scales: measure the kLa and P/V of the different scales over a wide range of air flows, agitation rates, working volumes, and backpressures. It’s best to perform the testing in your process media, if possible. If you can find the time, it’s useful to evaluate different mixing schemes at small scale - different impeller styles and positions, baffles, and sparger styles and positions (particularly valuable if you already know the differences in these features between small and large scale systems available to you).
Second, you’ll need to understand how your product responds to the different operating parameters. Those dreaded statistically designed experiments (DoE) are particularly useful for understanding the effects and interactions of the many parameters that can be changed. Performing DoE experiments at small scale with your product to evaluate the effects of aeration, agitation, and volume will not only help you with scale-up, but will also provide useful information for setting acceptable ranges for the operating parameters at large scale. As with the kLa studies, it’s useful to study different mixing schemes at small scale if time allows. One set of experiments that is highly useful but rarely performed is the evaluation of the process performance at the same kLa (or P/V) obtained using different operating parameters.

Understanding your equipment and how your product responds to various operating conditions is the key to effective process scale-up and scale-down. Despite the historical and ongoing need for scaling bioprocesses up and down, there is no strategy that works in all situations. The art of successful scale-up lies in thoughtful experimental design and thorough data analysis in order to obtain the information that allows equivalent performance at all scales.

TFF Under Pressure

2011-05-06T10:27:00.000-07:00

By Dr. Scott Rudge

Are there scale up issues for cross flow filtration? In general, this step is overlooked as a scale up concern, and usually, given the primarily clean feed streams encountered in simple buffer exchange, this is warranted. However, forewarned is forearmed when scale up is concerned.

Primarily, there is just one scale up issue with cross flow filtration, and that is the path length on the retentate side of the filter. The flow on the retentate side of the filter is meant to continuously clean the filter surface, and prevent fouling, or at least limit it to a thin boundary layer. The shear rate created by the fluid at the filter surface increases as the square of the linear velocity of the fluid. The pressure drop through the filter module, from inlet to outlet, depends linearly on the length of the module, and also on the square of the linear velocity. In many cases, a manufacturing scale module is about a meter in length. However, on the lab scale, a module is likely to be closer to 10 cm. Therefore, the pressure drop from the inlet to outlet on the retentate side will be 10 times higher at constant linear velocity on scale up from lab to manufacturing. Since decreasing the flow rate will dramatically decrease the shear rate, the increased pressure will drive higher flux towards the membrane surface, increasing the thickness of the boundary layer and resulting in more surface polarization (fouling or gel formation, potentially).

One approach taken to this predicament is to keep the pathlength constant on scale up. This is analogous to maintaining constant bed height on chromatography scale up, an approach I disfavor. The result of this approach is a “horizontal” scale up, where more and more units of lab proportion are lined up side by side. This approach works, but is cumbersome and requires more and more manifolding for flow distribution, and other inconveniences. It also assumes that the length of filter the manufacturer provides is the best and only length for every application, which is absurd. However, this is an approach commonly pursued, and recommended by the filter manufacturers for its speed and certainty.

Another approach that is taken to this phenomenon is to increase the back pressure on the permeate. This slows down the permeate independent of changes on the retentate side of the filter. However, if the back pressure on the retentate side is greater than the pressure at any point along the filter on the retentate side, permeate will flow back to the retentate side. This is clearly inefficient, it means a particular fluid element will be filtered at least three times, crossing from retentate to permeate, then back to retentate, and then eventually back to permeate on a subsequent pass. This also means that the effective filtration area is decreased, as some portion of the filter is working in reverse, and another portion is working to correct the back flow. The negative flow counts against filter area that is filtering in the positive direction.

Finally, employing a constant pressure gradient along the retentate side is worth trying. Presuming the membrane geometry is essentially maintained on scale up (including spacers in the flow channel) maintaining constant pressure gradient along the retentate channel length means shear will be constant on scale up. Pressure drop from retentate to permeate will be higher at the retentate inlet, but if the shear is appropriate and the boundary layer controlled, this will only lead to higher flux, which may be preferred. This can be tested on the small scale by applying back pressure on the retentate and looking for leveling off of the flux vs. pressure curve. As long as flux vs. back pressure is increasing linearly, you can get improved performance at higher pressure. Then upon scale up, the pressure at the retentate inlet is held constant. It is certainly worth exploring longer path lengths on scale up, performance may improve!

In the end, either horizontal scale up will be used, or some reduction in retentate flow rate will probably be required. The result of the latter will be less shear at the membrane surface, but the payback will be in increased filtration efficiency. Some back pressure should be applied to the retentate side on the lab scale, as more pressure due to path length will almost surely need to be applied in manufacturing. Maintaining pressure drop on the retentate side with increased module length, along with back pressure on the permeate side usually results in successful scale up of a lab cross flow filtration procedure.

What's That in My Protein? Degraded Polysorbate Again?

2011-04-25T21:39:00.000-07:00

By Dr. Sheri Glaub

Mahler, et. al. have recently published a paper in Pharmaceutical Research entitled, “The Degradation of Polysorbates 20 and 80 and its Potential Impact on the Stability of Biotherapeutics.” (Subscription required.) As discussed in the paper, polysorbates are the most widely used non-ionic surfactants for stabilizing protein pharmaceuticals against interface-induced aggregation and surface adsorption.

Unknown Blogger uses a beater to induce aggregation in a protein solution

Concerns with polysorbate lot-to-lot variability, as well as potential degradation products prompted the authors to investigate the impact on four different monoclonal antibodies (mAbs). They performed an extensive characterization of polysorbate degradation products, both volatile and insoluble, which included a number of ketones, aldehydes, furanones, fatty acids, and fatty acid esters. They then examined the effect of degraded PS on these proteins.

They concluded that as long as threshold levels of PS20 and PS80 were present (in this case >0.01%), the stability of the four mAbs in pharmaceutically relevant storage conditions (2-8 °C) was maintained despite observed polysorbate degradation.

The authors also suggest during formulation development one evaluate carefully the amount of PS to be used, considering the shelf life and potential behavior during storage.

Getting a grip on prophage

2011-04-21T14:45:00.000-07:00

by Dr. Ray Nims

In a previous post, we discussed bacteriophage as a risk for the manufacture of biopharmaceuticals by bacterial fermentation. We mentioned briefly that bacteriophage may integrate within the genome of bacterial cells and that this may also represent a problem. Now we will explain why.

Bacteriophage are viruses that infect bacteria, and they have evolved two mutually exclusive strategies for survival. One involves a lytic growth cycle leading to death (lysis) of the host cell and release of progeny phage that may then infect additional host cells (so-called horizontal transmission). The other strategy is called lysogeny and involves integration of phage coding sequences into the host (bacterial) cell genome. The integrated phage is termed a prophage. This strategy for phage survival is referred to as vertical transmission since the phage genomic material is reproduced along with that of the host cell as the latter proliferates. Under certain circumstances, however, the integrated prophage can excise itself from the host cell chromosome in a process referred to as induction. The excised phage then may initiate a lytic infection of the host cell, causing all of the problems discussed in the previous post.

Illustration of a T4 phage infecting E. coli by Jonathan Heras

The relative success (i.e., from the perspective of the phage!) of the lytic vs. lysogenic survival strategies changes with the probability of host cell survival. Lysogeny appears to be a strategy that allows phage to persist during periods of low host cell availability or poor environmental (e.g., nutrient) conditions. Induction of prophage is an adaptation of the phage to host cell damage. This damage usually takes the form of a major stress to the host cell.

If stess can lead to prophage induction, the worry then becomes that some manipulation of a bacterial production cell during biopharmaceutical manufacture could lead to induction and initiation of a lytic phage infection. How can we assess and mitigate the potential for this to occur? There are two approaches: first, we can perform chemical or physical induction studies to determine the likelihood of encountering a prophage in a given production cell; and second, we can engineer the conditions of bacterial growth such that induction of a prophage is discouraged.

Phage induction studies may be performed on the bacterial production cell following initial engineering of the cell or during characterization of the cell bank. The inducing agent most often employed is mitomycin C. Other types of inducing agents (conditions) include carcinogens (such as the N-nitrosamines), hydrogen peroxide, high temperature, starvation, and UV radiation. The cells are treated with the inducing agent or condition, then one of various endpoints is used to detect the initiation of a lytic phage infection. These could include culture assays as well as molecular techniques such as PCR, microarray, or DNA chips.

Suppose you have an E. coli production cell harboring a problematic prophage. What can be done to discourage phage induction? Certain growth procedures have been shown to reduce spontaneous phage induction in E. coli cultures. These include using lower bacterial growth rates, replacement of glucose in growth medium with glycerol, and engineering the production cell through introduction of a plasmid conferring over-expression of the phage cI gene.

In summary, there are approaches that can identify the likelihood of encountering prophage induction from a bacterial production cell. The time to perform this type of testing is during development of the fermentation process (following the engineering of the production cell), or following banking of the production cell. If prophage induction appears to be a problem, bacterial growth procedures can help to reduce the potential. If this is not sufficient, the production cell may need to be re-engineered to produce a phage-resistant mutant.

Cold Facts About Dissolved Oxygen

2011-03-30T16:28:00.000-07:00

By Dr. Scott Rudge

What’s the solubility of oxygen in water? Everyone knows that the answer to this question is “low”, and that’s enough to know for many practical applications. But it’s high enough to rust unprotected metal surfaces, and high enough to grow cells, provided that it’s replenished at some rate. It’s easy to find a number on the internet, at a temperature and pressure that the author of the internet resource thinks is interesting. But my interesting condition always varies from the internet’s, and finding the constants that I need to calculate the actual value is always difficult.

Most references that you can find say that the solubility of oxygen in water follows Henry’s Law. Henry’s Law is a very simple expression that says the concentration of a substance in a liquid phase is related to the partial pressure of that substance in the gas phase by a “constant”. Constant is a relative term in thermodynamics, because the constant in this and most cases varies with temperature and other components in both phases. But we’ll go with it.

In this equation, k_H is Henry’s constant, p is the partial pressure of the substance in the gas phase and c is the corresponding concentration of that substance in the liquid phase. Partial pressure is simply the amount of the total pressure due to that substance. In a room full of air at sea level, the partial pressure of oxygen is approximately 21% of the atmospheric pressure, or 0.21 atmospheres (159.6 torr or mm Hg, or 3.1 psi). At 25°C, Henry’s constant for oxygen in pure water is 769.2 L*atm/mol.

To correct for temperature, an approximation using a reference temperature can be used, although the results will not be exact. The correlation is:

Where T_r is the reference temperature. Again, the use of these equations will give approximate values.

Adding salt to the water further decreases the solubility of oxygen, such that in sea water the solubility of oxygen is about 80% of that in pure water. In fermentation medium, the saturation concentration is probably even less.

For those who don’t like math, here’s the table for the dissolved oxygen concentration under air atmospheres containing 21% oxygen (dissolved oxygen concentrations in moles/L):

Lesson learned: Outsource but remain in control!

2011-02-25T09:30:00.000-08:00

By Dr. Ray Nims

In a previous posting, we described the responsibilities of the contract giver (contractee) and the contract acceptor (contractor) in outsourced pharmaceutical quality control testing. Our blog title: "Outsource it, and fuggedaboutit?" somewhat facetiously suggested that the outsourcing of quality control testing does not transfer quality control responsibility from the contract giver to the contract acceptor.

Elizabeth Meyers and I expanded upon this theme in a recent article in BioProcess International. Our conclusion in that article was more direct: “The use, by a pharma organization, of a contract testing lab to fulfill some or all of its Quality Control testing obligations does not absolve the contractee of its overall Quality responsibility of ensuring the safety, purity, identity, efficacy, and potency of its products.”

This point was illustrated nicely in a recent warning letter published on the FDA Website. The name of the firm involved is not important to this discussion. Among the other findings was the following:

“Your firm failed to properly evaluate a contract laboratory to ensure GMP compliance of operations occurring at the contract site.”

The FDA then provided the following detail: "...we are concerned about your firm’s fundamental understanding of what is required by your Quality Unit and the regulatory expectations for a firm that enters into agreements with contract testing laboratories. Although you have agreements with other firms that may delineate specific responsibilities to each party, you are ultimately responsible for the quality of your products and the reliability of test results. Regardless of who tests your products or the agreements in place, you are required to manufacture these products in accordance with section 501(a)(2)(B) of the Act to assure their identity, strength, quality, purity, and safety."

The take-home message from this is that in the outsourcing of quality control testing, responsibility for the outsourced testing is retained by the contract giver. Responsibilities of the contract giver include the following: selecting and qualifying the contract lab, ensuring the suitability of methods used (through qualification, transfer, verification, or validation); putting in place a Quality and business agreement; scheduling and submitting samples (including communicating expectations for sample results); providing in-life guidance; and monitoring of contract lab performance.

There is no denying that fulfilling these responsibilities requires a significant and ongoing effort on the part of the contract giver. In this respect, outsourcing of quality control testing is not so different from doing that testing in-house.

What's up with USP Chapter 1050?

2011-02-16T14:22:00.000-08:00

by Dr. Ray Nims

United States Pharmacopeia (USP) general chapter <1050> Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin originally appeared in supplement 10 to USP23-NF18 in May 1999 and was at that time essentially a verbatim adoption of the International Conference of Harmonization (ICH) Guideline Q5A (R1) having the same title.

The chapter describes the methods of evaluating the viral safety of biotechnology pharmaceutical products that are manufactured using cell lines of human or animal origin.

In 2006, an ad hoc advisory panel was assembled by the USP and tasked with revision of this chapter. The goals were to update the chapter and, more specifically, to add greater detail in the viral clearance validation section. The hope was that a user following the recommendations set forth in the general chapter would have greater confidence that viral clearance validation data generated would prove acceptable to the regulatory agencies.

The organization of the revised chapter <1050> was not changed. It comprised the following main sections: 1) Introduction; 2)Potential sources of viral contamination; 3) Cell line qualification: testing for viruses; 4) Testing for viruses in unprocessed bulk; 5) Rationale and action plan for viral clearance studies and viruses tests on purified bulk; and 6) Evaluation and characterization of viral clearance procedures. The changes proposed for the initial 5 sections were minor and primarily reflected attempts to update the chapter and to align the chapter more closely with FDA guidance documents. The most extensive changes were to section 6, in keeping with the goals described above.

The revised chapter was published for public comment in Pharmacopeial Forum 36(3) in the fall of 2010. Comments received as a result of the public review apparently suggested that a more extensive update of the chapter was warranted. At any rate, the revised chapter was not made effective during the USP’s 2005-2010 revision cycle. A new ad hoc advisory panel now being assembled as part of USP's 2010-2015 revision cycle will take over the responsibility for moving the revision of this chapter forward.

Remember....bacteriophage are viruses too

2011-01-21T13:07:00.000-08:00

by Dr. Ray Nims

Are you using bacterial cells to produce a biologic? Do not make the mistake of thinking that your upstream process is safe from infection by adventitious viruses. True, you are not required to test for the usual viruses of concern using a lot release adventitious virus assay. But bacterial production systems are susceptible to introduction of viruses just as mammalian cell processes are. In this case, the viruses just happen to be referred to as bacteriophage. Other than this, the putative contaminants have the same nasty property exhibited by viruses that can contaminate mammalian cell processes, that is … their small size (24-200 nm) allows them to readily pass through the filters used to “sterilize” process solutions. So media, buffers, induction agents, vitamin mixes, trace metal mixes, etc. that are fed into the fermenter without proper treatment can introduce a bacteriophage. Especially worrisome in this regard are raw materials that are generated through bacterial fermentation (such as amino acids, antibiotics). A fermenter infected with a lytic phage exhibits a clear signal that the bacterial substrate is unhappy. The trick then is to discover where the phage originated and to mitigate the risk of experiencing it again.

How can you mitigate the risk of experiencing a bacteriophage infection? Many of the same strategies used to protect mammalian cell processes may be applicable to the bacterial fermentation world. Raw materials and/or process solutions may be subjected to gamma-irradiation, to ultraviolet light in the C range, to prolonged heating or to high temperature short time treatment, to viral filtration, etc. In addition, mitigation of risk of bacteriophage contamination may require filtration of incoming gasses using appropriate filters.

A sampling of the data available on inactivation of bacteriophage by various methods is shown in the table below. The literature is extensive, and as with viral inactivation, the inactivation of phage by certain of the methods (e.g., UVC, gamma-irradiation) may be dependent both upon the matrix in which the phage is suspended as well as the physical properties of the phage (e.g., genome or particle size, strandness, etc.). For fairly dilute aqueous solutions, gamma-irradiation, UVC treatment, or parvovirus filtration should represent effective inactivation/removal methods. HTST at temperatures effective for parvoviruses (102°C, 10 seconds) should be effective for most bacteriophage, although this is an area that needs further exploration.

Mitigating the risk of experiencing a bacteriophage contamination of a bacterial fermentation process is possible if one remembers that bacteriophage are similar to mammalian viruses. Strategies that are effective for small-non-enveloped mammalian viruses (i.e., the worst case for mammalian viruses) should also be effective for most bacteriophage.

A possible exception to this is prophage. In analogy with the presence of endogenous retroviruses in certain mammalian cells (i.e., rodent, human, monkey), there is a possibility of encountering integrated bacteriophage (prophage) in certain bacterial cell lines. Like endogenous retroviruses, prophage may result in the production of infectious particles under certain conditions. This phenomenon deserves some discussion, but this will have to be deferred to a future blog.

References: Purtel et al., 2006; Ward, 1979; Sommer et al., 2001.

Cell Culturists….Are your human cells authenticated?

2010-11-16T16:15:00.000-08:00

by Dr. Ray Nims

Until fairly recently, it has been common practice to authenticate human cell cultures using phenotypic status (e.g., receptor or protein expression) and animal species of origin testing. This level of authentication is better than none, but it is not sufficient to unambiguously identify a human cell culture. The result has been that we are still hearing about cases of misidentified human cells being used for biomedical research.

There are now methods available that are capable of rapidly and unambiguously identifying human cell lines, tissues, and cell preparations to the individual level. The recent demonstration of the potential utility of molecular technologies such as short tandem repeat (STR) and single nucleotide polymorphism (SNP) profiling for cell authentication has provided the impetus for development of a new standardized method for human cell authentication.

To this end, an ATCC Standards Development Organization workgroup with international representation has spent the past two years developing a consensus standard for the Authentication of Human Cell Lines through STR Profiling. The forthcoming Standard will provide guidance on the use of STR profiling for authenticating human cells, tissue, and cell lines. It will contain methodological detail on the preparation and extraction of the DNA, guidance on the appropriate numbers and types of loci to be evaluated and on interpretation and quality control of the results. Associated with the standard itself will be the establishment of a public STR profile database which will be administered and maintained by the National Center for Biotechnology Information (NCBI). The database primarily will contain STR profiles of commonly used cell lines.

STR Profiling of Hela Cells

An announcement that the Standard is now available for public 45-day review, comment, and vote was published in the October 22, 2010 issue of the ANSI newsletter Standards Action.

The benefits of the Standard will depend on the degree to which it is adopted and followed in the biomedical research and development and biopharmaceutical communities. Taking a broader view, it is hoped that funding agencies and journals will begin to use such authentication standards as important considerations for funding or publishing research employing human cells. The quality and validity of funded and published research should benefit greatly as a result of the reduction in frequency of use of misidentified human cells.

The deadline for comments is December 6, 2010. There is still time to review the draft Standard and to voice your opinions and concerns.

Fry those mollicutes!

2010-11-03T14:17:00.000-07:00

By Dr. Ray Nims

It is not only viruses that may be introduced into biologics manufactured in mammalian cells using bovine sera in upstream cell growth processes. The other real concern is the introduction of mollicutes (mycoplasmas and acholeplasmas). Mollicutes, like viruses, are able to pass through the filters (including 0.2 micron pore size) used to sterilize process solutions. Because of this, filter sterilization will not assure mitigation of the risk of introducing a mollicute through use of contaminated bovine or other animal sera in upstream manufacturing processes.

Does mycoplasma contamination of biologics occur as a result of use of contaminated sera? The answer is yes. Most episodes are not reported to the public domain, but occasionally we hear of such occurrences. Dehghani and coworkers reported the occurrence of a contamination with M. mycoides mycoides bovine group 7 that was proven to have originated in the specific bovine serum used in the upstream process (Case studies of mycoplasma contamination in CHO cell cultures. Proceedings from the PDA Workshop on Mycoplasma Contamination by Plant Peptones. Pharmaceutical Drug Association, Bethesda, MD. 2007, pp. 53-59). Contamination with M. arginini and Acholeplasma laidlawii attributed to use of specific contaminated lots of bovine serum have also occurred.

Fortunately, the risk of introducing an adventitious mollicute into a biologics manufacturing process utilizing a mammalian cell substrate may be mitigated by gamma-irradiating the animal serum prior to use. This may be done in the original containers while the serum is frozen. Unlike the case for viruses, in which the efficacy of irradiation for inactivation may depend upon the size of the virus, mollicute inactivation by gamma irradatiion has been found to be highly effective (essentially complete), regardless of the species of molicute. The radiation doses required for inactivation are relatively low compared to those required for viruses (e.g., 10 kGy or less, compared to 25-45 kGy for viruses). The gamma irradiation that is performed by serum vendors is typically in the range of 25-40 kGy. This level of radiation is more than adequate to assure complete inactivation of any mollicutes that may be present in the serum. For instance, irradiation of calf serum at 26-34 kGy resulted in ≥6 log10 inactivation of M. orale, M. pneumoniae, and M. hyorhinis. In the table below I have assembled the data available on inactivation of mollicutes in frozen serum by gamma-irradiation.

So, the good news is that gamma irradiation of animal serum that is performed to mitigate the risk of introducing a viral contaminant will also mitigate the risk of introducing a mollicute contaminant. If the upstream manufacturing process cannot be engineered to avoid use of animal serum, the next best option is to validate the use of gamma irradiated serum in the process. In fact, the EMEA Note for guidance on the use of bovine serum in the manufacture of human biological medicinal products strongly recommends the inactivation of serum using a validated and efficacious treatment, and states that the use of non-inactivated serum must be justified.

References: Gauvin and Nims, 2010; Wyatt et al. BioPharm 1993;6(4):34-40; Purtle et al., 2006

Risky Business

2010-10-27T13:23:00.000-07:00

By Dr. Scott Rudge

“Risk Analysis” is a big topic in pharmaceutical and biotech product development. The International Conference on Harmonization (ICH) has even issued a guidance document on Risk Analysis (Q9). Despite the documentation available and the regulatory emphasis, these tools remain poorly understood. They are used to justify limiting the extent of fundamental understanding that can be gained on a process, while simultaneously used as a cure-all for management challenges that we face in pharmaceutical process development.

Risk analysis focuses only on failure modes. Failure mode effect and analysis (FMEA) was developed by the military, first published as MIL-P-1629, “Procedures for Performing a Failure Mode, Effects and Criticality Analysis” in November 1949. The procedure was established to discern the effects of system failure on mission success and personnel safety. Since then, we have found a much broader range of applications for this type of analysis. The methodology is used in the manufacture of airplanes, automobiles, software and elsewhere. People have devised “design” FMEAs and “process” FMEAs (DFMEA and PFMEA). These are all great tools, and help us to design and build better, safer products with better, safer processes.

Where Risk Analysis Falls Down

The FMEA is such a great hammer, it can make everything look like a nail. And when regulatory authorities are encouraging companies to use Risk Analysis for product design and process validation, the temptation to apply it further can be overwhelming. In particular, risk analysis is used in three inappropriate ways, in my estimation:

Decision analysis
Project management
Work avoidance

Quite often, risk analysis tools are used to guide decisions. Here, the pros and cons of selecting a particular path are weighed, using the three criteria of risk analysis (occurrence, detectability and severity) as guides. However, not every decision path leads to a failure mode or an outcome that could be measured as a consequence. And detectability and occurrence may not be the only or most appropriate factors by which to weight a consequence. There are excellent decision tools that are designed specifically for weighting and evaluating preference criteria. A very simple tool is the Kepner Tregoe (KT) decision matrix. Decision analysis uses very detailed descriptions of the decision to model the potential outcomes. The KT decision matrix is sufficient for many decisions, large and small. But if you really want to study the anatomy of a decision, decision analysis is the most satisfactory method.

FMEAs are sometimes inappropriately applied in project management, to assign prioritization and order in which tasks should be completed. This may be handy in some instances, but is somewhat misleading or inappropriate in others. The riskiness of an objective should not be the sole determinant in its prioritization. Quite often, the fundamentals or building blocks need to be in place in order to best address the riskiest proposition in a project. Prematurely addressing the pieces of a project that present the greatest risk of failure may lead to that failure. On the other hand, being “fast to fail” and eliminate projects that might not bear fruit with the least amount of resources spent, is critical to overall company or project success. Project management requires consideration of failure modes, but also resource programming and timeline management. FMEAs can be an element of that formula, but should not be the focus.

Finally, and perhaps most painfully, FMEAs are used to justify avoiding work. Too often, risk analysis is applied to a problem, not to identify the elements that are most deserving of attention, but to justify neglecting areas that do not rank sufficiently high in the risk matrix. Sometimes, the smallest risks are the easiest to address, and in addressing them, variability can be removed from the process. Variability is the “elephant in the room” when it comes to pharmaceutical quality, as has been concisely pointed out by Johnston and Zhang.

The FMEA is a stellar tool, and it is applicable to problems in design, process and strategy across many industries. Its quantitative feel makes practitioners feel as though they are actually measuring something, and can make fine distinctions between risks that they were unable to articulate before. However, the FMEA can be applied rather too widely, or sometimes unscrupulously, yielding bad data and bad decisions.

National Chemistry Week

2010-10-14T21:14:00.000-07:00

By Dr. Sheri Glaub

Oct 17-23 is National Chemistry Week . This year’s theme is “Behind the Scenes with Chemistry”, which celebrates the chemistry in movies, set designs, makeup artistry and common special effects.

As exciting at that is, imagine a career working on life-saving therapies. Drug innovations in chemistry have helped increase life expectancy in the US by 30 years over the past century, and many chemical disciplines are involved. For example, medicinal and organic chemists isolate medicinal agents found in plants and create new synthetic drug compounds. Biochemists investigate the mechanism of a drug action; engage in viral research; conduct research pertaining to organ function; or use chemical concepts, procedures, and techniques to study the diagnosis and therapy of disease and the assessment of health. Analytical chemists use their knowledge of chemistry, instrumentation, computers, and statistics to solve problems in almost all areas of chemistry. Their measurements are used to assure compliance with regulations; to assure the safety and quality of food, pharmaceuticals, and water; to support the legal process; and to help physicians diagnose disease. Last, but not least, chemical engineers apply the principles of chemistry, math, and physics to the design and operation of large-scale chemical manufacturing processes. They translate processes developed in the lab into practical applications for the production of products such as plastics, medicines, detergents, and fuels.

This year, the Nobel Prize in Chemistry was jointly awarded to a trio of chemists for palladium-catalyzed cross couplings in organic synthesis. A recent article in Chemical and Engineering News quotes Stephen L. Buchwald, a chemistry professor at Massachusetts Institute of Technology. “This is a very exciting day for organic chemistry. This is a well-deserved award that is long overdue. It is hard to overestimate the importance of these processes in modern-day synthetic chemistry. They are the most used reactions by those in the pharmaceutical industry.”

The future of prescription drugs and medical devices depends on kids getting excited about science now. If you are in the Denver area, make plans to attend the Denver Museum of Nature and Science with your children on October 16 or 17 for the 7th Annual Demonstration and Outreach hosted by University of Northern Colorado Student Affiliate Chapter with advisor Dr. Kim Pacheco. If you are not in the Denver area, check with your local museum or your local ACS section to see what is planned.

Manufacturing Biologics with CHO Cells? What’s the Risk for Viral Contamination?

2010-09-22T14:24:00.000-07:00

by Dr. Ray Nims

Chinese hamster ovary (CHO) cells are frequently used in the biopharmaceutical industry for the manufacture of biologics such as recombinant proteins, antibodies, peptibodies, and receptor ligands. One of the reasons that CHO cells are often used is that these cells have an extensive safety track record for biologics production. This is considered to be a well-characterized cell line, and as a result the safety testing required may be less rigorous in some respects (e.g., retroviral safety) than that required for other cell types. But how susceptible is the cell line to viral contamination?

There are a couple of ways of answering this question. One way is to examine, in an empirical fashion, the susceptibility of the cell type to productive infection by model exogenous viruses. This type of study has been conducted at least three times over the past decades by different investigators. Wiebe and coworkers (In: Advances in Animal Cell Biology and Technology for Bioprocesses. Great Britain, 1989; 68-71) examined over 45 viruses from 9 virus families for ability to infect CHO-K1 cells, using immunostaining and cytopathic effect to detect infection. Only 7 of the viruses (Table 1) were capable of infecting the cells. Poiley and coworkers (In Vitro Toxicol. 4: 1-12, 1991) followed with a similar study in which 9 viruses from 6 families were evaluated for ability to infect CHO-K1 cells as detected by cytopathic effect, hemadsorption, and hemagglutination. This study did not add any new viruses to the short list (Table 1). The most recent study was conducted by Berting et al. This study involved 14 viruses from 12 families. The viruses included a few known to have contaminated CHO cell-derived biologics in the past two decades, and therefore did add some new entities to the list in Table 1. Still, the list of viruses that are known to replicate in CHO cells is relatively short.

Chinese hamster cells possess an endogenous retrovirus which expresses its presence in the form of retroviral particles, however these particles have been consistently found to be non-infectious for cells from other animals, including human cells. This endogenous retrovirus therefore does not present a safety threat (Dinowitz et al. Dev. Biol. Stand. 76:210–207, 1992).

A second way of looking at the question of viral susceptibility of CHO cells is to examine the incidence and types of reported viral contaminations of manufacturing processes employing CHO cell substrates. This subject has been reviewed a number of times, most recently by Berting et al. The types of viral contaminants fill a fairly short list (Table 2). In most cases, the contaminations have been attributed to the use of a contaminated animal-derived raw material, such as bovine serum.

Sources: Rabenau et al.1993; Garnick 1996; Oehmig et al., 2003; Nims Dev. Biol. 123:153-164, 2006; Nims et al., 2008; Genzyme 2009..

Considering the frequency with which CHO cell substrates have been used in biologics production, this history of viral contamination is remarkably sparse. This is further testament to the overall safety of this particular cell substrate.

FDA to viral vaccine makers: it's time to update viral testing methods

2010-09-08T09:58:00.000-07:00

By Dr. Ray Nims

If you have been following the recent (2010) unfolding of the discovery of porcine circovirus DNA contamination in rotavirus vaccines from GSK and Merck, you may not be surprised to hear that the FDA has asked viral vaccine manufacturers to outline, by October, their plans to update their testing methodologies to prevent future revelations of this type.

I had predicted earlier that biologics manufacturers would be asked to provide evidence, going forward, that their porcine raw materials (trypsin being the most common) are free of porcine circovirus. This testing has not been manditory in the past, but adding this to the porcine raw material virus screening battery moving forward is a prudent action in light of the recent rotavirus vaccine experience.

The FDA has appropriately gone a little farther in it's request to the viral vaccine manufacturers. The regulators would like to assure that the future will not bring additional discoveries of viral contaminants in licensed vaccines, and the best way to accomplish this at the moment appears to be to request implementation of updated viral screening methodologies. Does this mean that viral vaccine makers will need to employ deep sequencing on a lot-by-lot basis? Most likely not. It appears that reliance on the in vivo and in vitro virus screening methods which have been the gold standards since the 1980s will, however, no longer be sufficient. So what does this leave us with? What FDA appears to be asking for is a relatively sensitive universal viral screening method.

The in vivo and in vitro methods were, until now, the best option for this purpose. These methods detect infectious virus only and depend upon the ability of the virus to cause an endpoint response in the system (cytopathic effect, hemagglutination, hemadsorption, or pathology in the laboratory animal species used). So viral genomic material would not be detected, and the methods have had to be supplemented with specific nucleic acid-based tests for viruses which could not otherwise be detected (e.g., HIV, hepatitis B, human parvovirus B9, porcine circovirus).

Some options for sensitive and universal viral screening methods which might fit the requirements include DNA microarrays and universal sequencing methods performed on cell and viral stocks. The latter technology may be preferable, as microarrays are constructed to detect known viruses, while the desire is that the technology be universal in the sense that it detect both known and unknown viruses. Such a test will provide additional assurance that the virus and cell banks used to manufacture viral vaccines do not harbor a viral contaminant.

Other universal viral screening methods which are less labor intensive than the sequencing technologies may be developed in the near future and addition of one of these to the release testing battery for viral vaccine lots may need to be considered in satisfying the FDA's goals.

Is Clarence calculating clearance correctly?

2010-09-01T09:43:00.000-07:00

by Dr. Ray Nims

As pointed out by Dr. Rudge in a recent posting “Do we have clearance, Clarence?”, spiking studies conducted for the purpose of validating impurity clearance are often done at only one spiking level (indeed often at the highest possible impurity load attainable). This is especially true for validation of adventitious agent (virus and mycoplasma) clearance in downstream processes. The studies are done in this way in order to determine the upper limit of agent clearance (in terms of log10 reduction) by the process. Such log10 reduction factors from individual process steps are then summed in order to determine the overall capability of the downstream processes to clear adventitious agents. The regulatory agencies have fairly clear expectations around such clearance capabilities which must generally be met by biologics manufacturers.

The limiting factor in such clearance studies is typically the amount or titer of the agent that is able to be spiked into the process solution, which is determined by: 1) the titer of the stock used for spiking, and 2) the maximum dilution of the process solution allowed during spiking (typically 10%). Under these circumstances, as Scott points out, there is a possibility that the determined clearance efficiency (i.e., the percentage of the load which is cleared during the step) is an underestimate of the actual clearance that might be obtained at lower impurity loading levels.

Adventitious agent clearance is comprised of two possible modalities, removal and inactivation. Removal refers to physical processes designed to eliminate the agent from the process solution, usually through filtration or chromatography. Removal efficiency through filtration would not be expected to display variability based on impurity loading. On the other hand, chromatographic separation of agents (by, for example, ion-exchange columns) may display saturation at the highest loadings, and therefore use of the highest possible loading levels may result in underestimates of removal efficiency at lower (i.e., more typical) impurity levels.

Inactivation refers to physical or chemical means of rendering the agent non-infectious. Agent inactivation is not always a simple, first-order reaction. It may be more complex, with a fast phase 1 stage of inactivation followed by a slow phase 2 stage of inactivation. An inactivation study is planned in such a way that samples are taken at different times so that an inactivation time curve can be constructed. As with removal studies, the highest possible impurity levels are typically used to determine inactivation kinetics.

Source: Omar et al. Transfusion 36:866-872, 1996

While the information obtained through clearance studies of this type may be incomplete from the point of view of understanding the relationships between impurity loading levels and clearance efficiency, the results obtained are consistent with the regulatory expectation that the clearance modalities be evaluated under worst-case conditions. Therefore, at least in the case of adventitious agent clearance validation, I would say that Clarence is calculating clearance correctly!

Do We Have Clearance, Clarence?

2010-08-26T09:11:00.000-07:00

By Dr. Scott Rudge

As in take offs and landings in civil aviation, the ability of a pharmaceutical manufacturing process to give clearance of impurities is vital to customer safety. It’s also important that clearance mechanism be clear, and not confused, as the conversation in the classic movie “Airplane!” surely was (and don’t call me Shirley).

There are two ways to demonstrate clearance of impurities.

The first is to track the actual impurity loads. That is, if an impurity comes into a purification step at 10%, and is reduced through that step to 1%, then the clearance would typically be called 1 log, or 10 fold.

The second is to spike impurities. This is typically done when an impurity is not detectable in the feed to the purification step, or when, even though detectable, it is thought desirable to demonstrate that even more of the impurity could be eliminated if need be.

The first method is very usable, but suffers from uneven loads. That is, batch to batch, the quantity and concentration of an impurity can vary considerably. And the capacity of most purification steps to remove impurities is based on quantity and concentration. Results from batch to batch can vary correspondingly. Typically, these results are averaged, but it would be better to plot them in a thermodynamic sense, with unit operation impurity load on the x-axis and efflux on the y-axis. The next figures give three of many possible outcomes of such a graph.

In the first case, there is proportionality between the load and the efflux. This would be the case if the capacity of the purification step was linearly related to the load. This is typically the case for absorbents, and adsorbents at low levels of impurity. In this case (and only this case, actually) does calculating log clearance apply across the range of possible loads. The example figure shows a constant clearance of 4.5 logs.

In the second case, the impurity saturates the purification medium. In this case, a maximum amount of impurity can be cleared, and no more. The closer to loading at just this capacity, the better the log removal looks. This would be the point where no impurity is found in the purification step effluent. All concentrations higher than this show increasing inefficiency in clearance.

In the third case, the impurity has a thermodynamic or kinetic limit in the step effluent. For example, it may have some limited solubility, and reaches that solubility in nearly all cases. The more impurity that is loaded, the more proportionally is cleared. There is always a constant amount of impurity recovered.

For these reasons, simply measuring the ratio of impurity in the load and effluent to a purification step is inadequate. This reasoning applies even more so to spiking studies, where the concentration of the impurity is made artificially high. In these cases, it is even more important to vary the concentration or mass of the impurity in the load, and to determine what the mechanism of clearance is (proportional, saturation or solubility).

Understanding the mechanism of clearance would be beneficial, in that it would allow the practitioner to make more accurate predictions of the effect of an unusual load of impurity. For example, in the unlikely event that a virus contaminates an upstream step in the manufacture of a biopharmaceutical, but the titer is lower than spiking studies had anticipated, if the virus is cleared by binding to a resin, and is below the saturation limit, it’s possible to make the argument that the clearance is much larger, perhaps complete. On the other hand, claims of log removal in a solubility limit situation can be misleading. The deck can be stacked by spiking extraordinary amounts of impurity. The reality may be that the impurity is always present at a level where it is fully soluble in the effluent, and is never actually cleared from the process.

Clearance studies are good and valuable, and help us to protect our customers, but as long as they are done as single points on the load/concentration curve, their results may be misleading. When the question comes, “Do we have clearance, Clarence?” we want to be ready to answer the call with clear and accurate information. Surely varying the concentration of the impurity to understand the nature of the clearance is a proper step beyond the single point testing that is common today.

And stop calling me Shirley.

Sizing Up Filters

2010-08-09T11:16:00.000-07:00

By Dr. Scott Rudge

Of all the unit operations used in pharmaceutical manufacture, filtration is used the most frequently, by far. Filters are used on the air and the water that makes its way into the production suite. They are used on the buffers and chemical solutions that are used to feed the process. They are used to vent the tanks and reactors that the products are held and synthesized in. But the sizing of the filters is largely an afterthought in process design.

Liquid filters that will be used to remove an appreciable amount of solid must be sized with the aid of experimental data. Typically, a depth filter is used, or a filter that contains a filtration aid, such as diatomaceous earth. A depth filter is a filter in which there are no defined pores, rather, they are usually some kind of spun fiber, like polyethylene, that serves as a matt for capturing particulate. You probably did a depth filtration experiment in high school with glass wool. Or you’ve used a depth filter in your home aquarium with the gravel (under gravel filter) or an external filter pump (where the fibrous cartridge you install is a depth filter, such as the "blue bonded filter pads" shown below).

A depth filter uses both its fiber mesh to trap particles, but also then uses the bed of particles to capture more particles. It is actually the nature of the particles that controls most of the filtration properties of the process.

Because of the solids being deposited onto the filter, the resistance of the filter to flow increases as the volume that has been filtered increases. Therefore, knowing the exact size of filter that will be required for your application can be complicated. The complication is overcome by developing a specific solids resistance that is normalized to the volume that has been filtered, and the solids load in the slurry. Once this is done, these depth filters can be sized by measuring the volume filtered at constant pressure in a laboratory setting. The linearized equation for filtration volume is:

By measuring the volume filtered with time at constant pressure, the two filtration resistances can be found as the slope and intercept of a plot of t/(V/A) vs. (V/A). The area of a depth filter is the cross section of the flow path. On scale up, the depth of a depth filter is held constant, and this cross section is increased. An example of the laboratory data that should be taken, and the resulting plots, is shown below:

As expected, the filter starts to clog as more filtrate is filtered. The linearized plot gives a positive y-axis intercept and a positive slope, which can be used to calculate the resistance of the filter and the resistance of the solids cake on the filter.

The resistance of the filter should be a constant and independent of any changes in the feed stream. However, the specific cake resistance, α, will vary with the solids load. It is important to know the solids load in the representative sample(s) tested, and the variability in the solids load in manufacturing. The filter then should be sized for the highest load anticipated. This will result in the under-utilization of the filter area for most of the batches manufactured, but will reduce or eliminate the possibility that the filter will have to be changed mid-batch.

Of course, reducing variability in the feed stream will increase the efficiency of the filter utilization, and reduce waste in other ways, such as reducing variability in manufacturing time, reducing manufacturing investigations and defining labor costs.

The nuts and bolts of retrovirus safety testing

2010-07-20T09:09:00.000-07:00

by Dr. Ray Nims

Retroviruses may integrate into the genome of host animals. For this reason they are often referred to as endogenous viruses. Viral particles may or may not be expressed in the host cell. Expressed viruses may be infectious or non-infectious, and infectious virus may have tropism for (ability to infect) the same or different animal species relative to the host cell of origin. Infection results from a process of reverse transcription of the viral RNA leading to proviral DNA. To accomplish this, retroviruses have a specialized enzyme known as reverse transcriptase. Through this process (see figure below), the infected cell may be enlisted to produce viral progeny. Certain of the retroviruses are known to be oncogenic (e.g., human T-lymphotropic virus 1, feline leukemia virus, Raus sarcoma virus, etc.). Other retroviruses are of concern as a result of disease syndromes caused in humans (e.g., human immunodeficiency virus 1 in acquired immunodeficiency syndrome, and the possible role of xenotropic murine leukemia virus-related virus in chronic fatigue syndrome). From a biosafety standpoint, there is a worry that under some conditions, integrated viruses in cell substrates employed to produce biopharmaceuticals which do not normally express their presence may be induced to produce infectious particles.

Source: AccessExcellence

Retrovirology safety testing for biologics manufacture can be confusing to those not familiar with the subject. Here is a brief overview.

Demonstrating retroviral safety typically involves a combination of the following three components:
• detecting infectious retrovirus through cell culture assays (XC plaque, cocultivation with mink lung or Mus dunni cells, etc.).
• measuring reverse transcriptase enzyme activities either through tritiated thymidine incorporation into templates, or through product amplification (PCR) techniques (PERT, etc.). This is not required if infectious retrovirus is detected.
• Visualizing and enumerating retroviral particles in supernatants or in fixed cells using transmission electron microscopy.

The various assays are applied during cell bank characterization (including end of production cell testing), during evaluation and validation of purification processes, and in some instances, as bulk harvest lot-release assays (results from 3 lots at pilot or commercial scale are submitted with the marketing application). For processes using well-characterized rodent cells known to contain endogenous retrovirus (CHO, C127, BHK, murine hybridoma), retroviral infectivity testing of the processed bulk is not required provided that adequate downstream clearance of the particles has been demonstrated.

Infectivity testing can be particularly confusing, due to the variety of cell-based assays employed. These include both direct and indirect assays. An example of a direct assay is the XC-plaque assay for ecotropic (a term meaning the virus is infectious for mouse cells) murine retroviruses. By definition, therefore, this would only be used to assay production cells of mouse origin.

Indirect assays are those in which a second endpoint is required to assess positive or negative outcome. Indirect assays include the various co-cultivation assays in which the test cells are co-cultivated with host cells such as mink lung, Mus dunni, and any of a number of human cells (see Table 4 within USP <1237> Virology Tests for a list of commonly used host cells). The indirect assays are performed to detect xenotropic retroviruses (retroviruses which are capable of infecting only animals other than the species of origin). The secondary endpoints used to assess outcome include reverse transcriptase activity, sarcoma virus rescue (S+L- focus formation assays), or enzyme immunoassay. The indirect assays are used in the retrovirus testing of mouse, hamster, monkey, and human production cell substrates. The selection of the host cell for the cocultivation assay is dependent upon the species of origin of the production cell, recognizing that cocultivation host cells from a species other than that of the production cells must be used. For production processes using rodent or other non-human cells, one or more human host cells are typically used for the cocultivation assay, as xenotropic retroviruses infectious for human cells are of obvious concern.

Still confused? Don’t worry. An individual with virology testing expertise can assist in designing the appropriate retrovirus testing battery for your biologic.

Assessing rapid microbial detection systems

2010-06-23T09:29:00.000-07:00

by Dr. Ray Nims

With each passing year, it seems that there are more options available for rapid microbial detection. These rapid systems come in a variety of “flavors”, that is - they differ with respect to a set of key attributes. For instance, how rapid is rapid? What is the sensitivity? What is the maximum sample volume that may be tested? Is it quantitative or qualitative? What units are the results given in? Is it destructive or non-destructive (i.e., can the organism, once detected, be identified)? When one considers the variety of applications for which rapid methods may potentially replace existing culture methods, it rapidly becomes clear that there may not be “one shoe that fits all”.

In order to select an appropriate rapid method for use in one of the many microbial detection applications, one must first assess the available rapid systems for the key attributes mentioned above. This then provides the opportunity to rule out systems which for one reason or the other will not suit the application. There may be some applications for which no rapid system currently meets all requirements. Those rapid systems which do appear to possess the attributes required may be further evaluated for cost and for performance capabilities using specific sample matrices.

In the table below, we have listed some of the currently available rapid microbial detection systems. These include only systems which are 48 hours in duration or less, and therefore some of the sterility replacement assays involving reduced incubation durations (e.g., BacT/ALERT®, Growth Direct™) are not listed.

The key attributes of these rapid systems are displayed in the table below. The systems are arranged by principle of detection, as in the table above. For certain methods (e.g., Micro Pro™) increased sensitivity can be gained through increasing the duration of the incubation time. For non-destructive methods, the ability to identify the organism(s) detected is facilitated by an additional incubation post-detection.

What is the regulatory position on rapid microbial detection methods? The U.S. FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing states that other suitable microbiological tests (e.g., rapid methods) may be considered for environmental monitoring, in-process control testing, and finished product release testing after it has been demonstrated that these new methods are equivalent or better than conventional (e.g., USP) methods. Additionally, the FDA Process Analytical Technology (PAT) initiative encourages the voluntary development and implementation of innovative approaches in pharmaceutical development, manufacturing, and quality assurance (from MJ Miller, PDA Journal 45: 1-5, 2002).

Are rapid methods being used in the pharmaceutical industry? ScanRDI was approved by the FDA for water testing at GSK and for sterility testing at Alcon; Pallchek has been approved by the FDA for bioburden testing at GSK; and Wyeth received approval for use of Celsis for microbial limits testing.

Like all methods proposed to replace existing “gold standards”, these rapid microbial detection systems must be demonstrated through comparability protocols to be equivalent to or better than the existing methods. The effort required should pay dividends in terms of shortened turnaround times and reduced costs.

Informing the FMEA

2010-06-16T23:18:00.000-07:00

By Dr. Scott Rudge
Risk reduction tools are all the rage in pharmaceutical, biotech and medical device process/product development and manufacturing. The International Conference on Harmonization has enshrined some of the techniques common in risk management in their Q9 guidance, “Quality Risk Management”. The Failure Modes and Effects Analysis, or FMEA, is one of the most useful and popular tools described. The FMEA stems from a military procedure, published in 1949 as MIL-P-1629, and has been applied in many different ways. The most used method in the health care involves making a list of potential ways in which a process can “fail”, or produce out of specification results. After this list has been generated, each failure mode in the list is assessed for its proclivity to “Occur”, “Be Severe” and “Be Detected”. Typically, these are scored from 1 to 10, with 10 being the worst case for each category, and 1 being the best case. The scores are multiplied together, and the product (mathematically speaking) is called the “Risk Priority Number”, or RPN. Then, typically, development work is directed towards the failure modes with the highest RPN.

The problem is, it’s very hard to assign a ranking from 1 to 10 for each of these categories in a scientific manner. More often, a diverse group of experts from process and product development, quality, manufacturing, regulatory, analytical and other stake holding departments, convene a meeting and assign rankings based on their experience. This is done once in the product life-cycle, and never revisited as actual manufacturing data start to accumulate. And, while large companies with mature products have become more sophisticated, and can pull data from other similar or “platform” products, small companies and development companies can really only rely on the opinion of experts, either from internal or external sources. The same considerations apply to small market or orphan drugs.

Each of these categories can probably be informed by data, but by far the easiest to assign a numerical value to is the “Occurrence” ranking. A typical Occurrence ranking chart might look something like this:

These rankings come from “piece” manufacturing, where thousands to millions of widgets might be manufactured in a short period of time. This kind of manufacturing rarely applies in the health care industry. However, this evaluation fits very nicely with the Capability Index analysis.

The Capability Index is calculated by dividing the variability of a process into its allowable variable range. Or, said less obtusely, dividing the specification range by the standard deviation of the process performance. The capability index is directly related to the probability that a process will operate out of range or out of specification. This table, found on Wikipedia (my source for truth), gives an example of the correlation between the calculated capability index to the probability of failure:

As a reminder, the capability index is the upper specification limit minus the lower specification limit divided by six times the standard deviation of the process. The two tables can be combined to be approximately:

How many process data points are required to calculate a capability index? Of course, the larger the number of points, the better the estimate of average and standard deviation, but technically, two or three data points will get you started. Is it better than guessing?

Riboflavin plus UVA irradiation: another inactivation approach to consider

2010-06-09T09:30:00.000-07:00

by Dr. Ray Nims

Short-wavelength ultraviolet irradiation (UVC) has been used for years to disinfect air, surfaces, and thin liquid films because it is effective in inactivating a variety of bacteria, protozoa, phage, and viruses. More recently, UVC (100-280 nm) irradiation has been shown to be useful for viral risk mitigation in biologics manufacturing. UVC-treatment of culture media and other liquid reagents has been demonstrated to inactivate potential adventitious viral contaminants, including those which are resistant to inactivation by other physical means (e.g., murine minute virus; calicivirus; and porcine parvovirus and SV-40 [Wang et al., Vox Sanguinis 86: 230-238, 2004]).

Another approach that has been used recently, especially in the ophthalmologic and blood products communities, is UVA (315-400 nm) in the presence of the photosensitizer riboflavin. Riboflavin interacts with nucleic acid and photosensitizes to damage by UVA leading to direct electron transfer, production of singlet oxygen, and generation of hydrogen peroxide. The treatment results in oxidation and ring-opening of purines and in DNA strand breakage. The advantage of riboflavin over other photosensitizers (e.g., methylene blue, psoralens, etc.) is that riboflavin (vitamin B2) is an endogenous physiological substrate.

The photosensitizer interacts with nucleic acids.

Upon irradiation, the results may include cross-linking, mutation, or strand breakage.
Source: Bryant and Klein

Riboflavin/UVA treatment has been explored in ophthalmology applications such as infectious keratitis and keratomycosis. The typical treatment paradigm involves application of a solution of 0.1% riboflavin (as riboflavin-5-phosphate) followed by irradiation using 365 nm light (5 to 10 J/ml). The approach has shown effectiveness against a variety of pathogenic bacteria, including drug-resistant strains. Effectiveness against fungal pathogens requires combination therapy with amphotericin B.

In the blood products community, photosensitizer/UV treatment is being explored for pathogen reduction. For instance, riboflavin/UV treatment is being evaluated for platelet and plasma pathogen reduction, for prevention of graft versus host reactions, and for pathogen reduction in whole blood products. In the proprietary application (Mirasol PRT®), blood product pools are combined with riboflavin (final concentration 50 µM) and the solutions are irradiated with 6.24 J/ml broadband (265-370 nm) UV light. The technology has been shown to be effective for a variety of pathogenic bacteria and viruses (see Table 3 in the review by Bryant and Klein).

Will this approach be useful in the biopharma industry? It appears so. Recently, irradiation with UVA (365 nm) light in the presence of 50 µM riboflavin has been evaluated for controlled inactivation of gene transfer (adenovirus, adeno-associated virus, lentivirus) virus preparations. Complete inactivation was obtained in each case within 90 minutes.

Are Generic Pills Harder to Swallow?

2010-05-28T15:29:00.000-07:00

By Ashley M. Jones

“What’s in a name? That which we call a rose, by any other name, would smell just as sweet” – It’s a good thing William Shakespeare lived much before the drug companies were around or he could never have come up with such an amazing statement. Today, there are drugs, and then there are drugs; and more than what’s inside or its efficacy, it’s the name that seems to matter. If you were asked to choose between a branded drug and its generic equivalent, given that their costs were almost the same, you would most probably go for the branded version. So why are generic pills harder to swallow than their branded counterparts?

Although generic medicines are much cheaper than branded drugs (they are priced lower because they can be manufactured only after the patent runs its course and so do not include the R&D costs that the big pharmaceutical companies incur when they manufacture new drugs), at an equal price, people prefer to buy the latter for various reasons.

For one, they may not want to call their doctor to verify that the generic equivalents are safe and the bioequivalent of the branded drug - the most important aspect to consider when you’re buying generic equivalents is to ensure that they are the bioequivalent of the branded drug you’ve been prescribed. Even if you know the active pharmacological ingredient in a drug, you may not be sure if the generic equivalent with the same active ingredient can be used as a reliable and safe substitute. For example, there are certain medicines that should not be substituted, like for example drugs that have the same active pharmacological ingredient but which are modified release and intermediate release pills respectively. These are different dosage forms, and hence not bioequivalent.

Also, some people may feel that generic drugs are not of the same quality as their branded equivalent. It’s not true of course, but perception is everything when it comes to drugs and medication. That’s why we find that even placebos work sometimes, because there are some diseases and illnesses that are cured by the power of the mind. So if you believe that a drug is inferior, your mind is going to block your recovery even though the drug is really efficient.

The key to choosing the best generic drugs is to go with those that your doctor prescribes or recommends. If you’re sure of the quality, generic pills become much easier to both swallow and digest.

This article is contributed by Ashley M. Jones, who regularly writes on the subject of Online Pharmacy Technician Certification. She invites your questions, comments at her email address: ashleym.jones643@gmail.com.

RMC Pharmaceutical Solutions welcomes guest posts related to pharmaceuticals, biotechnology, medical devices and other related topics.

Using porcine trypsin in biologics manufacture?

2010-05-19T11:44:00.000-07:00

by Dr. Ray Nims

On March 22, 2010, a press release from GlaxoSmithKline (GSK) announced that porcine circovirus 1 (PCV 1) DNA had been detected in their rotavirus vaccine. On May 6, Merck disclosed that it had found DNA fragments of both PCV types 1 and 2 in its rotavirus vaccine. The PCV 2 findings in Merck's vaccine may be of greater concern, due to the fact that this virus causes disease in pigs, while PCV 1 apparently does not. However, the relative amounts of PCV DNA found in the GSK vaccine appear to be much greater (the lab discovering the PCV DNA in the GSK vaccine did not detect any in the Merck vaccine), and the worry in this case is that some of the genomic material may be associated with infectious PCV 1 virus. In both cases, the presence of the PCV genomic material has been attributed to the use of porcine trypsin at some point in the vaccine manufacturing process.

The FDA convened an advisory committee meeting on May 7th to discuss the findings of PCV DNA in the two licensed rotavirus vaccines. What was the result of the advisory committee meeting? The advisory committee felt that the benefits of the rotavirus vaccines clearly outweigh the risks. This, added to the fact that there appears to be little human health hazard associated with these viruses, led to the FDA clearing the two vaccines for continued use on May 14th. The product labels will be updated to reflect the presence of the PCV DNA in these products. In the longer term, these products may need to be "reengineered" to remove the PCV DNA. This may involve the preparation of new Master and Working cell banks and thus will take some time.

Another likely outcome of the advisory committee’s meeting may be heightened expectations, going forward, for PCV screening of porcine raw materials and of Master and Working cell banks which were exposed to porcine ingredients (e.g., trypsin) at some point in their development. Porcine-derived raw materials which are used in the production of biologics are to be tested per 9 CFR 113.53 Requirements for ingredients of animal origin used for production of biologics for a variety of viruses of concern. In the case of ingredients of porcine origin, those viruses of concern are listed in 9 CFR 113.47 Detection of extraneous viruses by the fluorescent antibody technique. These include rabies, bovine viral diarrhea virus, REO virus, porcine adenovirus, porcine parvovirus, transmissible gastroenteritis virus, and porcine hemagglutinating encephalitis virus. While porcine circovirus may not be specifically mentioned in the 9 CFR requirements, it will be prudent to add a nucleic acid-based assay for detection of this virus to the porcine raw material testing battery going forward. Similarly, Master and Working cell banks exposed to porcine raw materials (e.g., trypsin) during their developmental history should be assayed for PCV prior to use.

Routine nucleic acid-based testing for PCV should detect the genomic sequences for this virus should intact infectious or non-infectious PCV be present in the test materials. Now that this virus is one of concern to the FDA and to the public, performing the appropriate raw material and cell bank testing for it will most likely become an expectation for vaccine and biologics manufacturers.

Bending the Curve

2010-05-12T11:11:00.000-07:00

By Dr. Scott Rudge

To understand the best ways to develop preparative and industrial scale adsorption separations in biotechnology, it’s critical to understand the thermodynamics of solute binding. In this blog, I’ll review some basics of the Langmuir binding isotherm. This isotherm is a fairly simplistic view of adsorption and desorption, however, it applies fairly well to typical protein separations, such as ion exchange and affinity chromatography.

A chemical solution that is brought into contact with a resin that has binding sites for that chemical will partition between the solution phase and the resin phase. The partitioning will be driven by some form of affinity or equilibrium, that can be considered fairly constant at constant solution phase conditions. By “solution phase conditions”, I mean temperature, pH, conductivity, salt and other modifier concentrations. Changing these conditions changes the equilibrium partitioning. If we represent the molecule in solution by “c” and the same molecule adsorbed to the resin by “q”, then the simple mathematical relationship is:

If the capacity of the resin for the chemical is unlimited, then this is the end of the story, the equilibrium is “linear” and the behavior of the adsorption is easy to understand as the dispersion is completely mass transfer controlled. A example of this is size exclusion chromatography, where the resin has no affinity for the chemical, it simply excludes solutes larger than the pore or polymer mesh length. For resins where there are discrete “sites” to which the chemical might bind, or a finite “surface” of some kind with which the chemical has some interaction, then the equilibrium is described by:

and the maximum capacity of the resin has to be accounted for with a “site” balance, such as shown below:

Where S_tot represents the total number of binding sites, and S₀ represents the number of binding sites not occupied by the chemical of interest. The math becomes a little more complicated when you worry about what might be occupying that site, or if you want to know what happens when the molecule of interest occupies more than one site at a time. We’ll leave these important considerations for another day. Typically, the total sites can be measured. Resin vendors use terms such as “binding capacity” or “dynamic binding capacity” to advertise the capacity of their resins. The capacity is often dependent on the chemical of interest. The resulting relationship between c and q is no longer linear, it is represented by this equation:

When c is small, the denominator of this equation becomes 1, and the equilibrium equation looks like the linear equilibrium equation. When c is large, the denominator becomes K_eqc, and the resin concentration of the chemical is equal to the resin capacity, S_tot. When c is in between small and large, the isotherm bends over in a convex shape. This is shown in the graph below.

There are three basic conditions in preparative and industrial chromatographic operations. In the first, K_eq is very low, and there is little or no binding of the chemical to the resin. This is represented by the red squares in the graph above. This is the case with “flow through” fractions in chromatography, and would generally be the case when the chemical has the same charge as the resin. In the third, K_eq is very high, and the chemical is bound quantitatively to the resin, even at low concentrations. This is represented by the green triangles in the graph above. This is the case with chemicals that are typically only released when the column is “stripped” or “regenerated”. In these cases, the solution phase conditions are changed to turn K_eq from a large number to a small number during the regeneration by using a high salt concentration or an extreme pH. The second case is the most interesting, and is the condition for most “product” fractions, where a separation is being made. That is, when the solution phase conditions are tuned so that the desired product is differentially adsorbing and desorbing, allowing other chemicals with slightly higher or lower affinities to elute either before or after the desired product, it is almost always the case that the equilibrium constant is not such that binding is quantitative or non-existent. In these cases, the non-linearity of the isotherm has consequences for the shape of the elution peak. We will discuss these consequences in a future blog.

In a “Quality-by-Design” world, these non-linearities would be understood and accounted for the in the design of the chromatography operation. An excellent example of the resulting non-linearity of the results was shown by Oliver Kaltenbrunner in 2008.

Relying on linear statistics to uncover this basic thermodynamic behavior is a fool’s errand. However, using basic lab techniques (a balance and a spectrophotometer) the isotherm for your product of interest can be determined directly, and the chromatographic behavior understood. This is the path to process understanding!

Epizootic hemorrhagic disease virus: a future troublemaker?

2010-05-06T14:41:00.000-07:00

by Dr. Ray Nims

Epizootic hemorrhagic disease virus (EHDV) is a double-stranded RNA virus of family Reoviridae, genus Orbivirus. This is a non-enveloped virus of approximately 60-80 nm size. This arbovirus is transmitted by a biting midge of genus Culicoides, and is closely related to another Orbivirus, the bluetongue virus. Two serotypes are endemic to cattle in North America (EHDV-1 and EHDV-2); the infections caused tend to be subclinical (asymptomatic) and therefore may go undetected.

Infections in cattle are more prevalent in areas of widespread infection within the local deer population. As shown in the figure below, the geographic distribution of infection of deer populations with EHDV and bluetongue virus includes areas within the high plains and mountain states in which bovine serum production is high (Utah, Kansas, etc.).

From Daniel Mead, Risk of Introduction of New Vector-borne Zoonoses

There have been recent outbreaks of epizootic hemorrhagic disease in cattle in Indiana (2006) as well as other states; in Israel (2006); and in Turkey (2007).

Basis of Concern: EHDV has been isolated previously from a biologics manufacturing process employing a Chinese hamster ovary (CHO) cell substrate (Rabenau et al. Contamination of genetically engineered CHO-cells by epizootic haemorrhagic disease virus (EHDV). Biologicals 21, 207-214, 1993). The infection was presumed, but not proven, to originate from use of a contaminated bovine serum in the manufacturing process.

Regulatory Expectations. EHDV is not mentioned specifically in 9CFR 113.47 (Detection of extraneous viruses by the fluorescent antibody technique as a virus of concern for raw materials of bovine origin), although this regulation requires testing for the closely related bluetongue virus. EHDV would be expected to cause cytopathic effects in Vero cells, one of the detector cells used in the 9CFR 113.47 assay, therefore this assay should detect the virus in grossly contaminated bovine sera.

Mitigating Risk. Elimination of animal-derived materials (esp. bovine sera) from the manufacturing process should reduce the risk of experiencing this virus. If this is not possible, treatment of the sera should be considered. Gamma-irradiation of the frozen serum at the dosages normally used should be effective, judging from results obtained with REO virus, another member of the family Reoviridae (Gauvin, 2009).

Conclusions. EHDV has been found previously to contaminate a biologics manufacturing process employing a CHO-cell substrate. It is therefore a virus of concern for the biopharmaceutical industry. Risk of infection of biological products with EHDV through use of bovine-derived materials such as bovine sera may increase in the event of future outbreaks of this disease in cattle from serum-producing regions of North America or Australia. Risk may be mitigated through implementation of gamma-irradiation of bovine sera and of viral purification processes capable of removing and inactivating non-enveloped viruses such as MMV and REO.

FDA regulation of “combination” products

2010-04-28T13:30:00.000-07:00

by Dr. Ray Nims

The FDA’s Office of Combination Products (OCP) was established in 2002 to shepherd combination products, those comprised of a combination of drug, biological, and/or device, through the review and regulation process. An example of a combination product is the drug-releasing stent (see figure below). The OCP does not conduct the reviews, but is responsible for: assigning the product to the appropriate FDA center; coordinating reviews involving more than one center; and working with agency centers to develop guidance and regulations to make the regulation of combination products “as clear, consistent and predictable as possible”.

An example of a drug-releasing stent.

From Wikipedia

The complexity of combination products arises because the efficacy and safety of the individual constituent components (i.e., the drug, biologic, or device) must be considered alone, as well as within the context of the combination product. Because of this complexity, there is no single development paradigm for all combination products. The guidance recommends that combination product developers consider any prior approval/clearance of the constituent parts, as well as how their testing may be influenced by the interaction of the components. Factors that should be considered (from the guidance) include:

• Are the constituent parts already approved for an indication?
• Is the indication for a given constituent part similar to that proposed for the combination product?
• Does the combination product broaden the indication or intended target population beyond that of the approved constituent part?
• Does the combination product expose the patient to a new route of administration or a new local or systemic exposure profile for an existing indication?
• Is the drug formulation different than that used in the already approved drug?
• Does the device design need to be modified for the new use?
• Is the device constituent used in an area of the body that is different than its existing approval?
• Are the device and drug constituents chemically, physically, or otherwise combined into a single entity?
• Does the device function as a delivery system, a method to prepare a final dosage form, and/or does it provide active therapeutic benefit?
• Is there any other change in design or formulation that may affect the safety/effectiveness of any existing constituent part or the combination product as a whole?
• Is a marketed device being proposed for use with a drug constituent that is a new molecular entity?
• Is a marketed drug being proposed for use with a complex new device?

As with individual constituent drugs, biologics, and devices, the FDA will require that the combination products be manufactured according to current good manufacturing practices. In most cases, a single investigational application (IND or IDE) is submitted to enable the clinical trials planned for the combination product. The science and technology associated with the combination products should drive the selection of statistical approaches, sample sizes, study endpoints, and methods for active principle measurement and for evaluating possible interactions between components. It may be best to involve the FDA in these decisions.

Complexity for the regulation of combination products also stems from the fact that separate manufacturing processes may exist for the various constituent parts. Potential changes in any of the component manufacturing processes, subsequent to initiating clinical trials or post-market, will need to be evaluated for possible effects on the safety and efficacy of the combination product.

Combination products represent therapeutic modalities with great promise for advancing health care. We expect to see more and more pharmaceutical activity in this area going forward.

Is There Ever a Good Time for Filter Validation?

2010-04-23T10:59:00.000-07:00

By Dr. Scott Rudge

What is the right time to perform bacterial retention testing on a sterile filter for an aseptic process for Drug Product? I usually recommend that this be done prior to manufacturing sterile product. After all, providing for the sterility of the dosage form for an injectable drug is first and foremost the purpose of drug product manufacturing.

But there are some uncomfortable truths concerning this recommendation

1. Bacterial retention studies require large samples, liters

2. Formulations change between first in human and commercial manufacturing, requiring revalidation of bacterial retention

3. The chances of a formulation change causing bacteria to cross an otherwise integral membrane are primarily theoretical, the “risk” would appear to be low

On the other hand

1. The most frequent sterile drug product inspection citation in 2008 by the FDA was “211.113(b) Inadequate validation of sterile manufacturing” (source: presentation by Tara Gooel of the FDA, available on the ISPE website to members)

2. The FDA identifies aseptic processing as the “top priority for risk based approach” due to the proximal risk to patients

3. The FDA continues to identify smaller and smaller organisms that might pass through a filter

Is the issue serious? I think so, risk of infection to patients is one of the few direct consequences that pharmaceutical manufacturers can directly link between manufacturing practice and patient safety, which is one of the goals of Quality by Design. Is the safety threat from changes to filter properties and microbe size in the presence of slightly different formulations substantial? I don’t think so, especially not in proportion to the cost to demonstrate this specifically. But the data aren’t available to demonstrate this hypothesis, because the industry has no shared database to demonstrate a range of aqueous based protein solutions have no effect on bacterial retention. There is really nothing proprietary about this data, and the only organizations that benefit from keeping it confidential are the testing labs. Sharing this data should benefit all of us. An organization like PDA or ISPE should have an interest in polling this data and then making a case to the FDA and EMEA that the vast majority of protein formulations have been bracketed by testing that already exists, and that the revalidation of bacterial retention on filters following formulation changes is mostly superfluous.

In the meantime, if you don’t have enough product to perform bacterial retention studies, at least check the excipients, as in a placebo or diluents buffer. A filter failure is far more likely due to the excipients than the active ingredient, which is typically present in much smaller amounts (by weight and molarity). By doing this, you are both protecting your patients in early clinical testing, and reducing your risk with regulators.

Oops, adventitious viral DNA fragments in a vaccine

2010-04-14T13:05:00.000-07:00

by Dr. Ray Nims

On March 22, 2010, a press release from GlaxoSmithKline (GSK) announced that DNA from porcine circovirus had been detected in their rotavirus vaccine. According to GSK, the DNA ”was first detected following work done by a research team in the US using a novel technique for looking for viruses and then confirmed by additional tests conducted by GlaxoSmithKline”. As a result of this finding, the FDA “is recommending that US clinicians and public health professionals temporarily suspend the use of Rotarix as a precautionary measure. The FDA have also stated that they intend to convene an advisory committee, within approximately four to six weeks, to review the available data and make recommendations on rotavirus vaccines licensed in the USA. The FDA will also seek input on the use of new techniques for identifying viruses in vaccines.” The EMEA, on the other hand, does not appear to consider this finding to be a safety concern, citing the fact that porcine circovirus is not infectious for human cells and does not cause disease in humans.

Porcine circovirus

Source: Meat and Livestock Commission, UK

What is porcine circovirus? Porcine circovirus (PCV) is a member of the family Circoviridae, among the smallest of the known animal DNA viruses. It is approximately 17 nm in diameter, non-enveloped, with icosahedral symmetry. The virus was originally identified as a noncytopathic contaminant of the PK-15 porcine kidney cell line (Tischer et al., Zentralblatt Bakt Hyg A 226:153-167, 1974). Like many very small, non-enveloped viruses, PCV represents a challenge for removal and inactivation.

Why is this finding coming to light now, or stated another way, why wasn’t the PCV DNA detected when the vaccine was initially tested and approved for human use? The press release wasn’t specific as to method used. It has subsequently been revealed, however, that these fragments were detected using sequence-independent amplification (deep sequencing; or as Eric Delwart calls it, metagenomics). The resulting library of amplified sequences is characterized by BLAST searching using identification algorithms. Confirmation in this case was obtained using microarray and PCR analyses. The sequencing techniques have been available for some time, and have proven useful for identification of viruses in an academic setting, though they have not been applied to safety testing of biopharmaceuticals until fairly recently due to the relatively high costs associated with the analyses.

The finding of viral DNA should not be equated with detecting the infectious virus in the product. The sequence-independent amplification, microarray, and virus-specific PCR assays used can detect viral nucleic acid, but as normally performed do not indicate whether infectious virus is present. Generally, with the possible exception of transforming viruses, it is the infectious virus that is of concern, not its DNA. Efforts to assess the presence of intact viral genomic material and of infectious porcine circovirus in this vaccine are most likely underway at this time.

The presence of the PCV viral sequences has provisionally been attributed to the use of porcine trypsin during the culture of the Vero cell substrate in which the vaccine is manufactured. The trypsin used had been gamma-irradiated to inactivate adventitious viruses prior to use. While it would be comforting to believe that the PCV DNA may simply have reflected carryover of non-infectious, lethally-irradiated PCV1 from the trypsin, the fact is that gamma-irradiation is not very effective at inactivating this small, non-enveloped virus (Plavsic and Bolin. Resistence of porcine circovirus to gamma irradiation. Biopharm Int. 14:32-36, 2001). In the case of porcine circovirus, there is little evidence to indicate that the virus is infectious or pathogenic for humans. So regardless of the outcome of the various ongoing studies, it is likely that the use of the GSK rotavirus vaccine will be re-instated after the FDA convenes and discusses the implications of this finding.

I predict that there will be more and more of this kind of revelation in the future as the sequencing techniques display stretches of viral or other contaminant DNA within samples of biopharmaceuticals. I would hate to see revelations like this impede the use of the sequencing technologies going forward, as these technologies are going to be very useful to the industry as rapid detection methods for contaminant identification.

What's Your Velocity?

2010-04-07T21:04:00.000-07:00

By Dr. Scott Rudge

With the development of very high titer cell culture and fermentation processes, downstream processing has been identified as a new bottleneck in biotechnology. The productivity of chromatography in particular, has become a bottleneck. There are two schools of thought for scaling up chromatography: in one, linear velocity (flow rate divided by column cross sectional area) is held constant; in the other, the total (volumetric) flow rate divided by the volume of the column is held constant. In the former method, the length of the column has to be held constant. In the latter method, the geometry of the column is not important, as long as the column can be packed efficiently and flow is evenly distributed. This makes the latter method more flexible, and accommodating of commercially available off the shelf column hardware packages. But does it work?

In my experience, holding flow rate divided by column volume constant between scales works very well. There is plenty of theoretical basis for the methodology as well. Yamamoto has published extensively on the reasons that this technique works. This method is also the basis for scale up described in my textbook. Here, briefly, and using plate height theory, is the theoretical basis:

The basic goal in chromatography scale up is to maintain resolution. “Resolution” is a way to describe the power of a chromatography column to separate two components. It depends on the relative retention of the components, which is fixed by the thermodynamics of the column and remains constant as long as the chemistry (the resin type, the buffer composition) remains constant. It also depends on the peak dispersion in the column, which is a function of the transport phenomena, and is only related to the chemistry by the inherent diffusivity of the molecules involved. Otherwise, it is dependent on mass transfer, flow rate, temperature, flow distribution. Treating the thermodynamics as constant, we can say:

where Rs is Resolution, and N is the number of theoretical plates. N is the ratio of the column length L to the plate height, H, so

In liquids, H is approximately a linear function of linear velocity v, according to van Deemter, as discussed in a previous post. So we can say that H = Cv (where C= the van Deemter constant). Now, the linear velocity is the flow rate divided by the cross sectional area of the column, A, and the column volume, V, is the cross sectional area times the column length. The total flow rate (F) divided by the column volume is held constant between scales, we’ll call this constant “f”.

This bit of mathematical gymnastics says that Resolution only depends on two fundamental properties of the scale up, van Deemter’s term “C”, which considers the dispersion caused by convection relative to mass transfer to and from a resin particle, and “f”, the flow rate relative to the column volume that is chosen. There is no need to hold column length or linear velocity constant as long as the flow rate relative to column volume is held constant. You might also notice from the math that doubling the column length has the same effect on resolution as dropping the flowrate by half. However, doubling the length costs more in terms of resin, and consumes more solvent than decreasing the flow rate (that’s for you, Mike Klein!) and increases the pressure.

The plate count analysis is very phenomenological, but it does hold up under practice (otherwise it would be abandoned). And the more delicate mathematical models predict the same performance, so confidence in this scale up model is high.

One common mistake made by those using the constant linear velocity model is in adding extra column capacity. Since most people are unwilling to pay for a custom diameter column, but base their loadings on the total volume of resin, they add bed volume by adding length. But since they are unwilling to change the linear velocity, they end up decreasing the productivity of the column (because, for example, the resolution they achieved at a smaller scale in a 12 cm long column at 60 cm/hr is now being performed in a 15 cm long column, at 60 cm/hr, therefore taking 25% more time).

If the less well known constant F/V model is used for a process involving mammalian cells, it would be imperative to explain and demonstrate this model in the scale down validation that is a critical part of the viral clearance package.

But how can you get even more performance out of your chromatography? Treating the unit operation as an adsorption step, and scaling up using Mass Transfer Zone (MTZ) concepts will be treated in a future posting.

How Do You Set Endotoxin Specs on API?

2010-03-31T13:00:00.000-07:00

By Dr. Lori Nixon

For parenteral drug products, setting a specification limit for endotoxin is a relatively straightforward exercise. If you know the maximum hourly dose and route of administration, the endotoxin limit can be readily derived from the equations set out in USP<85>.

But how do you determine an appropriate limit for your API? Don’t make the mistake of applying the same limit as product (in terms of EU/mg of active). And don’t make the mistake of blindly applying the same limit as a similar API, that may be dosed differently. Since the drug product is typically a combination of active ingredient along with excipients, solvents and container components—each of which may potentially contribute to the total endotoxin level—you should account for other potential contributions and set your drug substance limits accordingly. By setting the appropriate limit in API, you avoid the risk of an endotoxin failure that doesn’t show up until your precious API has been formulated and filled into vials—a business catastrophe!

Consider the following example:

Drug product at 50 mg/mL, intended for iv delivery in adults.

For this product, the maximum human dose/hour is 250 mg, delivered as a 5 mL volume.

From USP<85>, the endotoxin limit for iv delivery is 5 EU/kg. For a 70 kg adult, this corresponds to 350 EU maximum, or 70 EU/mL.

Tabulate the potential endotoxin contributors from your drug product formulation, container and manufacturing process. Look up the endotoxin limits (specifications) for each component you listed. In some cases it’s not possible to get a hard number, but you can at least estimate or list a “worst-case”. Some components will have negligible contribution based on their low ratio of surface contact to total formulated volume (such as the needle/tubing in the fill line); or low overall volume contribution (such as an acid/base used in a titration step). In this scenario, the sum of endotoxin contributions from all components (including API) cannot exceed 350 EU. “Solve” for the allowable endotoxin contributions from the API (X, in the table).

*Notice that in the table, drug substance is shown as 60 mg/mL (as powder) even though the formulation strength was listed as 50 mg/mL. Recognize that endotoxin measurements are usually based on powder weight rather than active ingredient weight. In this example let’s assume that the API has a moisture specification of NLT 95% and purity specification of NLT 88%; so 50 mg/mL of active (in the worst-case) actually corresponds to 60 mg/mL of API powder. So a 5 mL dose may contain as much as 300 mg of API powder.

You won’t be able to nail down everything exactly, and should recognize that relying on release specifications for some of the components above will clearly result in overestimations for endotoxin contribution. For example, the specification limit for the vials may not take into account additional reduction from depyrogenation; filters would be pre-rinsed and any subsequent endotoxin contributions would be largely diluted within the total product volume; and so forth. Nonetheless, this can be a convenient and systematic way to start your evaluation from a “worst-case” standpoint.

The allowed endotoxin contribution from the API can be calculated by subtracting the sum of the other component contributions (far right column of the table) from the total of 350 EU. In this case, X = 235.7 EU. Divide by the largest amount of drug substance powder that could go into the maximum dose (in this case 300 mg), and you have the drug substance endotoxin limit (0.79 EU/mg).

Now, do a reality check: If this drug substance limit looks like it may be problematic for any reason (analytical or process capabilities), look again at the table to see other possible ways to reduce the overall endotoxin (or revisit some of the worst-case assumptions that were clearly overly exaggerated). In the example, Salt 2 appears to be a large potential contributor, with a fairly loose specification for endotoxin. It may be possible to specify a different grade of this excipient (low-endotoxin or parenteral grade). Remember that compendial grades don’t necessarily mean low-endotoxin grades or even endotoxin-controlled grades.

Innovators and Perfectionists

2010-03-23T10:14:00.000-07:00

by Dr. Ray Nims

In a previous posting, Leah Choi described her frustration over the lack of specific training, received during her undergraduate schooling, in aspects germane to the realities of employment within the biopharmaceutical industry. Employment in the highly regulated world of biopharmaceutical manufacturing, Quality, and Quality Control (biopharmaceutical operations) requires different skills and employee temperaments compared to employment within the research and development (R&D) world. The academic institutions would do well to consider this during the preparation of students for eventual life in the working world.

What do we mean by different skills and employee temperaments? Most of us are familiar with the Myers and Briggs personality typing instrument which looks at attibutes like intro/extraversion, sensing, intuition, etc. This instrument provides interesting and revealing information about how different people deal with the world. In addition to the qualities addressed by Myers & Brigg, however, there is a personality spectrum which I will refer to as Innovation ↔ Perfection.

In the R&D world, technical knowledge, mastery of the literature concerning a subject, and most importantly, innovation are the attributes which are essential for success. A researcher must long to travel untraveled paths, to uncover new ground, to learn and often develop methods where such may not have existed previously (i.e., to go where no man has gone before). Those who are well suited to this environment we refer to as innovators. The academic institutions are pretty good at fostering these attributes in students.

On the other hand, the highly regulated areas of biopharmaceutical operations require individuals who are capable of following set instructions time after time, documenting their work in a precise and strictly controlled manner. Innovation, improvisation, and experimentation with mature methodologies and standard operating procedures are not encouraged. A mind-set which is compatible with achieving perfect compliance with documented procedures is the key to success in this environment. Such individuals we refer to as perfectionists, as they are motivated by the desire (or if not desire, at least the requirement) to conduct their work exactly as proscribed. It is this particular set of attributes which many academic programs fail to address adequately. This leaves employers with the task of training their entry-level staff in such matters, and (as Leah mentioned in her posting) students with the sometimes shocking revelation that they are poorly prepared for this type of employment.

Are there individuals who can be successful both as “innovators” and as “perfectionists”? Undoubtedly so! It is more likely, however, that most people fit within a spectrum falling between the two temperaments. I, for instance, have always regarded myself more of a perfectionist than an innovator, happily conducting the same assay the 100th time and still trying to do a better job than the last time. I know of others who, as soon as they learn a method, are bored with it and anxious to move on to something new. These temperaments may be determined by our personalities and may not be subject to alteration. It would appear to be valuable for academic programs to try, therefore, to determine the temperaments of their students, and to provide training suitable and appropriate for both the innovators and the perfectionists. Both the students as well as the biomedical industry would benefit from a little temperament triage and curriculum adjustment done at the undergraduate level.

Assessing rapid mycoplasma detection systems

2010-03-10T14:10:00.000-08:00

by Dr. Ray Nims

The European Pharmacopoeia chapter 2.6.7 Mycoplasmas, begining with version 5.8, has provided a mechanism for replacement of the current 28-day culture method for detection of mollicute (mycoplasma and acholeplasma) contaminants in biopharmaceutical bulk harvest samples with more rapid, nucleic acid-based, methods. The US FDA has yet to provide formal guidance on this topic, although it has become clear that the agency is willing to consider such methods, provided that they are shown to be equivalent to or superior to the current approved methods.

For biopharmaceuticals, a satisfactory outcome in a mycoplasma detection assay which is compliant with European Pharmacopoeia 2.6.7 or the 1993 FDA Points to Consider guidance is required on a lot-by-lot basis. Of the various lot-release assays performed on each given lot of a biopharmaceutical, this particular test is typically the most lengthy. Expediting the lot-release process through replacement of the 28-day approved culture test with a rapid mycoplasma detection test is therefore a strong motivating factor for the biopharmaceutical industry.

Figure. The MicroSEQ Mycoplasma assay provides a level of detection less

than 10 CFU/ML.

What options are now available to the industry? Several contract testing laboratories have recently announced the availability of validated rapid mycoplasma assays suitable for biopharmaceutical lot-release. For instance, BioReliance offers a hybrid culture/quantitative polymerase chain reaction (qPCR) assay, Charles River Laboratories offers a reverse transcriptase (RT)-PCR assay (BioProcess Int. April 2009, 30-42), Vitrology offers a qPCR assay, and WuXi AppTec offers a “touchdown” PCR assay.

In addition, several vendors are now offering mycoplasma detection kits which will allow biopharmaceutical entities to perform rapid mycoplasma testing in-house. For example, Life Technologies offers the MicroSEQ® Mycoplasma Detection Assay, Roche Applied Science offers the MycoTool™ PCR test and Millipore offers the MilliPROBE® mycoplasma detection system.

It is incumbent upon the biopharmaceutical company to demonstrate comparability between the rapid mycoplasma method and the current approved culture method for each product matrix for which a rapid method is proposed. Guidance on such comparability testing is provided in the European Pharmacopoeia chapter 2.6.7. Comparability studies for rapid methods intended to satisfy the US FDA should be discussed with that agency, as no formal guidance has been published.

What attributes should be considered when selecting a rapid mycoplasma detection method?

1. Sample volume. The current approved culture methods test at least 10 mL of sample. A rapid method intended to replace the current methods should ideally be able to test an equivalent volume of sample. It may be difficult to gain FDA approval for nucleic acid-based methods which can test only microliter amounts of sample.
2. Duration. Hybrid culture/PCR systems may take as long as 14 days to complete, while direct nucleic acid-based methods should be completed within a week or less.
3. Specificity. European Pharmacopoeia 2.6.7 specifies that the nucleic acid test must be able to exclude closely-related bacterial species.
4. Sensitivity. FDA indicates that the rapid method should be equivalent to or better than the approved culture method in terms of sensitivity (limit of detection), based on comparability studies using viable mycoplasma organisms.
5. Orthogonal endpoints. Having two or more orthogonal endpoints is desirable to allow one to discriminate between low level positive and negative signals.
6. Validation status. For contract methods, has the method been validated per European Pharmacopoeia 2.6.7? For kit methods, has the vendor validated the method per European Pharmacopoeia 2.6.7?
7. Drug Master File. For kit methods, has the vendor submitted a drug master file to the FDA for the method?

These considerations should help in deciding among the various options now available for implementing rapid nucleic acid-based mycoplasma testing for biopharmaceutical lot release applications.

QbDer, Know Thy Model!

2010-03-04T11:35:00.000-08:00

By Dr. Scott Rudge

Resolution in chromatography is critical, from analytical applications to large scale process chromatography. While baseline resolution is the gold standard in analytical chromatography, it is seldom achieved in process chromatography, where “samples” are concentrated and “sample volumes” represent a large fraction of the column bed volume, if not multiples of the bed volume. How do you know if your resolution is changing in process chromatography if you can’t detect changes from examining the chromatogram?

Many use the Height Equivalent to a Theoretical Plate technique to test the column’s resolution. In this technique, a small pulse of a non-offensive but easily detected material is injected onto the column, and the characteristics of the resulting peak are measured. The following equation is used:

Where H is the “height” in distance, t_r and t_w,1/2 are the retention time and time of the width of the peak at ½ height, L is the length of the column and N is the “number” of theoretical plates in the column. The lower the H, the smaller the dispersion, the greater the resolution.

It is well established that the flowrate and temperature affect the plate height. In fact, when the plate height is plotted against flow rate, we generate what is typically called the “van Deemter plot”, after Dutch scientists (van Deemter et al., Chem. Eng. Sci.,5, 271 (1956)) who established a common relationship in all chromatography (gas and liquid) according to the following equation:

Where A, B and C are constants and v is the linear velocity (flow rate divided by the column cross sectional area) of fluid in the column. It was later proposed by Knox (Knox, J., and H. Scott, J. Chromatog., 282, 297 (1983)) that van Deemter plots could be reduced to a common line for all chromatography if the plate height was normalized to resin particle size, and linear velocity was normalize to the resin particle size divided by the chemical’s diffusivity. While this did not turn out to be generally true, it is very close to true. Chemical engineers will recognize the ratio of linear velocity to diffusivity over particle size as the Peclet number, “Pe”, a standard dimensionless number used in many mass transfer analyses.

Since diffusivity is sensitive to temperature, it is logical that Pe is also sensitive to temperature, decreasing temperature decreases the diffusivity, and increases Pe. Thus, Pe is inversely proportional to temperature. We measured plate height as a function of linear flowrate and temperature in our lab on a Q-Sepharose FF column, using a sodium chloride pulse, and found the expected result, shown in the graph below.

We can easily use this graph as a measurement of van Deemter’s parameters A and C, and find the dependence of the diffusivity of sodium chloride on temperature. Based on these two points, we find the Peclet number proportional to 0.085/T where T is in degrees Kelvin. We also find the dependence of plate height on linear velocity, and we can predict that resolution will deteriorate in the column as flow rate increases.

We can also use Design of Experiments to find the same information. Analyzing the same data set with ANOVA yields significant factors for both flow rate and temperature, as shown in the following table:

Term	Coef	SE Coef	T	P
Constant	0.09756	0.05604	1.74	0.157
Temp	-0.007935	0.001858	-4.27	0.013
v	0.0497	0.02147	2.32	0.082

But since the statistics don’t know that the temperature dependence is inverse, or that the Peclet number is a function of both temperature and flowrate, the model yields no additional understanding of the process.

It is possible to use analysis of variance to fit a model other than linear, as the van Deemter model clearly is. But one must know that the phenomenon being measured behaves non-linearly in order to use the appropriate statistics. Using Design of Experiments blindly, without knowing the relationship of the factors and responses, leads to empirical correlations only relevant to the system studied, and should only be extended with caution.

Hot Tubs and Bioreactors

2010-02-25T10:39:00.000-08:00

By Dr. Ray Nims

Contaminating organisms which most commonly are under the radar for biopharmaceutical manufacturing operations include bacteria, mollicutes (mycoplasmas and acholeplasmas), and viruses. Various in-process and lot-release detection assays are mandated by the FDA and the International Conference on Harmonisation to ensure that such contaminants are detected in bulk harvests and/or final products as part of assuring patient safety (specified in ICH Q5A R1 and the 1993 Points to Consider in the Characterization of Cell Lines used to Produce Biologics). There is, however, an additional group of organisms which may threaten biologics production (one which is not normally associated with such manufacturing activities) namely, the Mycobacterium fortuitum complex.

The what?? The fortuitum complex is a group of relatively rapid-growing (non-tuberculosis) mycobacteria which is more typically associated with hot tub disease, and the contamination of industrial cutting fluids and foot baths used for pedicures. The group includes M. fortuitum, M. chelonae, M. abscessus, M. immunogenum, M. mucogenicum, M. peregrinum, and a few others. These mycobacteria, as well as other groups of non-tuberculosis mycobacteria, can be pathogenic in humans, even those who are immuno-competent. The organisms of the fortuitum complex represent a potential risk to the biopharma industry due to their propensity for forming biofilms, their ability to proliferate in water under relatively low nutrient conditions, their resistance to typical water disinfection methods, and their relatively slow growth in nutrient media.

Mycobacteria growing at the liquid/air
interface of a growth medium.

These characteristics render the organisms capable of existing in water piping and other surfaces in contact with water or aqueous media. Their slow growth in nutrient media may result in these agents being overlooked in biopharmaceutical manufacturing operations, especially when surveillance methods such as short-term bioburden assays are employed. A few cases of contaminated vaccines and tissue extracts have been reported in the literature (Mycobacterium chelonei in abscesses after injection of diphtheria-pertussis-tetanus-polio vaccine. Am. Rev. Respir. Dis. 1973 Jan; 107:1-8; Abscesses due to Mycobacterium abscessus linked to injection of unapproved alternative medication. Emerg. Inf. Dis. 1999; 5: 681-687).

Are there other examples? It is, unfortunately, difficult to estimate the frequency of occurrence of mycobacterial contamination in biologics manufacturing, since many episodes may lead to premature bioreactor termination, with little evidence to implicate a mycobacterium. It is also likely that episodes may have occurred without being reported in the literature.

Can't See Your Team?

2010-02-18T14:06:00.000-08:00

By Dr. Scott Rudge

The world is shrinking and collaborations growing. If you’re like me, you’re getting less sleep as collaborations go global. It’s all made possible by the internet, but is it good or bad?

I don’t think that it is either good or bad, it’s a new and evolving reality, and figuring out how to collaborate effectively using the internet is a job skill that everyone needs these days. In this blog, there are three tips for collaborating on the internet with your colleagues, local and distant.

Set rules, enforce them, obey them. Every good collaboration needs a good set of rules for how collaborations are to take place. Very few teams take time to communicate the rules, which fails as a strategy for getting people to obey the rules. An example is the use of a shared document repository. Folder names and structure should be thoughtfully laid out. The status of a particular piece of work should be clear from its file name. If the repository features check-in / check-out capability, it should be used religiously. Once set, collaborators should refrain from making additional folders, unless the workspace has been created for brainstorming, rather than, say, assembling a regulatory filing. Once the rules have been set, enforce them. If a team member doesn’t know how to use the technology, teach them; resist the temptation to do file management for them, for example. If you are not leading the collaboration, but contributing, learn the rules and follow them. If you’re confused or uncertain, ask for help. Be a good citizen in your collaboration community.

Don’t use too much email. Email is great for quick communication, horrible for collaborating on technology development. The fastest way to lose critical comments and revisions to your work is to use email to distribute it. You will get as many versions back as you’ve sent out, and collecting all the information back in to one piece of work is laborious and prone to error. Furthermore, emails cross in the ether, and are not always copied to everyone (and when they are copied to everyone, they become even more painful). It is much better practice to post your work in a repository and email a link to it, to let everyone know that your part is done, or in progress, and that they are free to look at it and contribute. Free up your inbox, start posting!

Be transparent. The old days when you can walk down the hallway and get a quick status update from everyone are over. And status updates are needed by management with very little notice. You want your update to be accurate and reflect the true progress of your collaboration, but you don’t have time to call everyone, and quite often it’s inappropriate to do so, because of time zone differences. To make updates easy and accurate, it is again critical to make use of the internet repository for all your “updateable” work. In addition, creating a tracking sheet that is also available in the internet repository is a good idea. Furthermore, it should be the responsibility of every member of the collaboration team to update the tracking sheet, at a frequency mandated by the collaboration leader (see “rules”, above). If you keep your work posted, and your status updated on the tracking sheet, you will be valued as a collaborator, and your work will be appropriately communicated outside the team.

There will be future tips, but these three should get you started towards being an excellent collaborator, much valued by your team. If you are a collaboration leader, you can get better results with less heartache. Good luck!

USP 63 Mycoplasma Update

2010-02-10T15:09:00.000-08:00

By Dr. Ray Nims

The United States Pharmacopeia’s (USP) new chapter <63> Mycoplasma Tests was planned to become effective on May 1, 2010 as part of USP 33. This new chapter was intended to fill a void in the USP for mycoplasma testing, which had been addressed previously within the FDA’s 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals and the European Pharmacopoeia (EP) chapter 2.6.7 Mycoplasmas (a separate document applying only to mycoplasma testing of live and inactivated viral vaccines, 21 CFR 610.30 will not be considered here). Since there were some methodological differences between the FDA guidance and the EP chapter, it was hoped by the industry that the USP guidance would serve to harmonize mycoplasma testing as much as practically possible.

Indeed, a quick look at the new USP chapter <63> indicates that the chapter was based in large part on EP chapter 2.6.7. A comparison of the three documents (USP, FDA, and EP) reveals methodological differences in only a few areas. These include the assessment of nutritive properties of the solid growth media (agar) used for mycoplasma testing, the assessment of inhibitory substances in the test material, the incubation temperature ranges to be used, and the number of positive controls to be used.

The EP chapter 2.6.7 states that “The solid medium complies with the test if adequate growth is found for each test micro-organism (growth obtained does not differ by a factor greater than 5 from the value calculated with respect to the inoculum)”. There is a different requirement within USP chapter <63>: “The solid medium complies with the test if a count within a 0.5-log unit range of the inoculate amount is found for each test microorganism”. Assuming an inoculate of 100 colony forming units (CFU), the acceptable ranges for the recovered organisms would be 32-316 CFU for the USP version vs 20-500 CFU for the EP version. The USP version is therefore more stringent in this respect.

Similarly, for the assessment of inhibitory substances, EP chapter 2.6.7 states that “…if plates directly inoculated with the product to be examined have fewer than 1/5 of the number of colonies of those inoculated without the product to be examined” there are inhibitory substances in the test material. The USP version indicates that there are inhibitory substances “…if plates directly inoculated with the test article/material are not within a 0.5-log unit range of the number of colonies of those inoculated without the test article/material.” So the USP version is again more stringent in this respect.

Minor differences in incubation temperature for test cultures exist between the documents (36 ± 1°C for the USP and PTC documents vs 35-38°C for the EP chapter). The USP and PTC documents specify the number and types of positive controls to be used in the assays: at least two known Mycoplasma species or strains should be included as positive controls (one a dextrose fermenter and one an arginine hydrolyzer). The EP chapter specifies that at least one of the six Mycoplasma species listed in the chapter be used as a positive control.

The USP chapter <63> differs from EP chapter 2.6.7 also in that the former does not provide requirements for validation of a nucleic acid-based test for mycoplasma. The USP chapter mentions the possibility of replacing the culture method with an alternative (nucleic-acid or enzymatic) method, stating that the alternative method must be validated and shown to be comparable to the agar/broth and cell culture methods. The EP chapter laid the foundation for validation of a nucleic acid-based mycoplasma detection test for the first time in version 5.8 (effective July 2007). This provided the industry with expectations for implementation of a rapid alternative test to the approved culture test for mycoplasma, which is 28 days in duration. Similar guidance is not yet forthcoming from the FDA or USP.

The issues of nutritive properties and inhibitory substances are not addressed within the FDA’s 1993 Points to Consider guidance. In order to now be compliant with the FDA and EP requirements as well as the new USP chapter, testing labs will have to make adjustment within their protocols to account for the stricter USP criteria for assessing nutritive properties and inhibitory substances. Due to errors within some of the monographs to appear in the issuance of the USP to become effective May 1, 2010, this issuance of USP 33, including chapter <63>, was retracted in January of 2010. However, it will be re-issued in March 2010 with an official date six months after reissue, and the methodological differences may need to be accounted for in testing protocols used by quality control laboratories for which USP compliance is applicable.

Once this chapter becomes effective, the mycoplasma test methods described will be considered compendial, meaning that labs following the methods outlined in the chapter will not be required to perform method validation. The labs will only be required to perform method verification for each test sample type (matrix qualification) per USP <1226>.

Mixed Up?

2010-02-03T09:44:00.000-08:00

By Dr. Scott Rudge

Determining and defending mixing time is a common nuisance in process validation. There are rarely data existing from process development, and there is rarely time or enthusiasm for actually studying the tank dynamics to set mixing times appropriately. Although there typically are design criteria for tank and impeller dimensions, motor size and power input into the tank, these design criteria are rarely translated to process development and process validation functionaries.

http://www.chemineer.com/mixing_technology.php

There are resources for mixing time determination if the basic initial work has been done. A very elegant study is included in the recent PQLI A-Mab Case Study produced by the ISPE Biotech Working Group. This study shows how to scale mixing from a lab scale 50 L vessel where a correlation between power and Reynolds number has been developed, to mixing vessels of 500 and 1500 L scales. The study requires that two critical dimensional ratios remain nearly constant on scale up, the diameter of the impeller divided by the diameter of the tank, and the height of the fluid level to the diameter of the tank. The study shows very close agreement between predicted mixing time and actual mixing time. The basis for the scale up is that the power input per unit mixing volume should be constant from scale to scale.

When the dimensional ratios cannot be kept constant, there are still rules for scale up. For example, as shown in the chart below (from Perry and Chilton’s Chemical Engineers’ Handbook, 5th edition, 1973), as the impeller diameter increases relative to the tank diameter, the relative power requirement declines, but the torque required to turn the impeller increases. This correlation can be used to adjust the power requirement to the scale up condition.

Additionally, there is “general agreement that the effect of mixer power level on mass-transfer coefficient is greater before than after off-bottom motion of all particles in a solute-solvent suspension is achieved (op.cit.)”.

In other words, once particles have been fluidized off the bottom of the vessel, whether they are carried all the way to the top of the vessel or not is not so important when it comes to predicting complete dissolution of the solids. At that point, the mass transfer coefficient is related only weakly to the power input, as shown below. Mass transfer coefficients for the dissolution of solids can be easily determined in the lab, and do not have to be determined again and again for new processes.

Knowing the minimum power requirements for particle suspension and the mass transfer coefficients for the solids being dissolved allows estimation of mixing times required for preparing a buffer. Knowing the mixing time allows the manufacturer to schedule buffer or medium preparation more precisely, eliminating over-processing or incorrect processing (a principle of lean manufacturing) and helps to guarantee a quality reagent/intermediate is produced each time, on time, and ready to implement in the next manufacturing step.

Eliminating those Pesky Viruses

2010-01-28T11:10:00.000-08:00

By Dr. Ray Nims

As part of mitigating the risk of introducing viral contaminants into a product during manufacturing, biopharma companies must assess the overall risk from a variety of sources (cell substrate, animal-derived raw materials, upstream and downstream processes, etc.) and consider options for reducing such risk. For global submissions, this requirement is formalized within EP 5.1.7 Viral Safety. For domestic submissions, such risk assessment and mitigation is consistent with the philosophy of the US FDA as formalized within the 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals; and ICH Q5A (R1) Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin.

A photomicrograph of the HIV virus from the CDC

The following options are available for reducing the risk of introducing a virus during manufacture of a biological product:

• Selection of a cell substrate with low inherent viral risk, and adequate characterization of the manufacturing cell substrate will reduce the risk associated with this important reagent.

• Elimination of the use of animal-derived raw materials and excipients will greatly reduce the risk of introduction of a virus, but of course this is not always possible.

• Where it is necessary to use an animal-derived material (ADM) in the manufacturing process, the following steps should be taken: (1) evaluate the viral risk associated with the ADM; (2) mitigate the viral risk through sourcing strategies, quality control testing at the source and/or at the biopharma, and implementation, where possible, of viral inactivation treatment (e.g., gamma-irradiation) of the ADM.

• Once an ADM has been incorporated into a reagent such as a culture medium, the reagent itself may be subjected to viral inactivation strategies such as UVC-treatment or high-temperature short-time (HTST) treatment.

• Avoidance of the use of open-vessel operations during upstream processes, as they provide entrance points for viruses.

• Implementation of in-process and lot release viral detection tests to provide early indications of a viral infection in an upstream process.

• Implementation and characterization of robust and efficacious viral purification strategies during downstream processing of the biologic.

It is an expectation of the regulatory agencies that each biopharma will employ a combination of the above options in order to assure the viral safety of their biological products. If a viral contamination event should occur during a manufacturing run, it should be thoroughly investigated, with the aim of identifying the source(s) of the contamination. The information learned during the course of investigation should be used to eliminate the source of the contamination, and mitigate the risk of any future similar recurrences of the contamination.

Re-educating Leah

2010-01-21T11:34:00.000-08:00

By Leah Choi

Four grueling years of chemistry, biology, math, and physics barely prepared me for life after college. As I entered America’s workforce on November 13th, 2006, I was equipped with nothing more than a general knowledge of what was to come.

Within my first few weeks at RMC Pharmaceutical Solutions, I quickly realized my inadequacies. GMP? GLP? NDA? IND? The acronyms alone could have driven me to near insanity. Likewise, I had spent four years at the University of Colorado learning the theory behind chromatography even putting it into practice on an ancient gas chromatography system. Yet this was no match for the advanced chromatography systems used in today’s biotech industry. To complicate matters further, I did not fully understand the intricacies of working in a regulated environment. What did it mean to follow a standard operating procedure? To evaluate and qualify the design, installation, performance, and operation of an instrument? What did it mean to document deviations? To perform a corrective and preventative action? Each new client and each new project presented a fresh set of unfamiliar issues. I often questioned if I would ever be able to bridge the gap between the theories of my college education with the applications of my working world.

Am I an isolated incident or do current biotechnology educational programs lack the necessary curriculum to develop entry-level employees? According to a recent survey conducted by AAPS (American Association of Pharmaceutical Sciences) and published by the National Institute of Pharmaceutical Technology and Education (NIPTE), 35% of respondents believe that current training for entry-level pharmaceutical development scientists is inadequate, 60% believe that there is a shortage of suitable candidates and nearly 70% asserted that there is an inadequacy in the number of US colleges focusing on industrial needs. According to this survey, academic programs training the majority of pharmaceutical product development scientists have declined substantially in recent years due to the emphasis in professional pharmacy programs on patient care rather than product knowledge. Additionally, these programs lack research funding in basic physical sciences supporting development and manufacturing.

With so much on the line, what is currently being done to address these nationally recognized problems? Organizations such as NIPTE have implemented plans with hopes of providing the “highest caliber entry-level scientists/engineers for the pharmaceutical and biopharmaceutical industries”. These plans include training students in degree programs using shared curricula materials, summer training programs and industrial internships via a network of industrial and institutional collaborators. The curriculum is based on “the precepts of interdisciplinary approaches strongly advocated by the National Academy of Sciences and constructivist learning theories important in the development of modern engineering and science higher education”. Presently, only ten universities have signed onto this plan: Duquesne University, Illinois Institute of Technology, Purdue University, Rutgers University, University Puerto Rico San Juan/Mayaguez, University of Connecticut, University of Iowa, University of Kansas, University of Kentucky, University of Maryland-Baltimore, and the University of Minnesota.

Other initiatives include the National Science Foundation (NSF) which currently provides $16.3 million in support of biotechnology programs through its Advance Technological Education (ATE) program. NSF regularly brings together scientists, educators, and other stakeholders to share their opinions on official issues such as biotechnology workforce development. Panelists share their opinions about how the biotechnology industry will grow during the next five years, the skills that technicians will require to meet workforce needs, and their experiences with promising educational practices. April 2008 conference recommendations include

1. instruction in written and verbal communication and “soft skill” such as team work and time management;

2. core curriculum courses that transfer and articulate from high school to two-year and four-year degree programs;

3. a strong theoretical understanding of the entire manufacturing process encompassing upstream and downstream process;

4. the introduction of immerging technologies in basic biotechnology courses; and

5. the redesign of standard microbiology and biology curricula to include applications in industrial and environmental biotechnology.

The limitations of ordinary degree programs spurred Montgomery College in Maryland to develop curriculum specifically aimed at preparing students for life after college. By soliciting information from industry personnel, coordinators of the program have developed and continually update courses that meet the current skill sets expected from entry level employees. Students acquire real life experience through internships engineered by the program. The success of this program depends highly on the continual collaboration between working professionals and academic faculty. Without a doubt, this synergistic dynamic allows both sides to benefit: educators gain valuable input for relevant curriculum while industry gains practical, productive entry level employees. Programs like these emphasize the absolute necessity for industry involvement.

I realize the learning curve is steep but not unconquerable. To all those recent graduates or better yet, those who are still in school the best advice I can give, is to get involved. Build your own bridge, by participating in professional groups and societies. Attend local events and seminars. Organizations such as the PDA (Parental Drug Association) AAPS (American Association of Pharmaceutical Scientists) and ISPE (International Society of Professional Engineers) often provide students with special benefits and discount memberships. As a PDA Student Member, you receive access to numerous benefits which provide you with the most current scientific and technical information. You receive access to Student Programs which provide grant funding and career growth resources, subscriptions to the PDA Journal of Pharmaceutical Science and Technology, including PDA Technical Reports which offer expert guidance and opinions on a variety of important scientific and regulatory topics pertaining to pharmaceutical and biopharmaceutical production.

Begin building contacts as early as possible. Make your first professional contacts at a career fair. Collect business cards and follow up. Professional social networking sites such as LinkedIn, Xing, Plaxo, and Spoke make these connections simple and instantaneous. Seek and invite professionals to speak at your school’s student group and find other means of collaborating with industry experts. Build a bond with someone who can act as a mentor. Having a mentor can be a great way to develop your career for the long term.

Take the opportunity to educate yourself on current topics and those of interest. Earlier this year, I was afforded the opportunity to gain valuable knowledge by earning a Certificate of Good Laboratory Practices/Good Manufacturing Practices from the University of Denver. We spent 10 weeks focusing on the regulations surrounding device manufacturing and use, specifically 21CFR820 and 21CFR58. My thirst for knowledge did not end there. In the same months I earned a Yellow Belt Training Certificate from the Colorado Association of Manufacturing and Technology. For six weeks my classmates and I studied yellow belt topics, specifically in the areas of Six Sigma Root Cause Analysis, 8D Problem Solving and Statistical Process Controls. Both of these courses were free, local and most importantly, offered helpful insights into contemporary subjects.

I commend unique programs and organizations such as NIPTE, NSF and Montgomery College for offering much needed curriculum that unites academia with industry. With increasing awareness and initiatives, my hope is that future graduates will be endowed with the basic education and ability to hit the ground running within this rapidly emerging industry.

Is Bovine Polyoma Virus Getting You Down?

2010-01-14T12:00:00.000-08:00

By Dr. Ray Nims

Bovine polyomavirus (BPyV) is a double-stranded DNA virus of genus Polyomavirus. It is non-enveloped and 40-50 nm in diameter, and is a member of the same genus as SV40.

Basis of Concern. The Polyomavirus genus was so-named due to the ability of the viruses to cause tumors in susceptible host animals. Genomic sequences for the potentially oncogenic bovine polyomavirus have been detected with high frequency in bovine sera, regardless of geographic region of origin (Shuurman et al., J. Gen. Virol. 72: 2739-2745, 1991; Wang et al., New Zealand Vet. J. 53: 26-30, 2005).

Regulatory Expectations. Bovine polyomavirus is not mentioned specifically in 9CFR 113.47, Detection of extraneous viruses by the fluorescent antibody technique, as a virus of concern for raw materials of bovine origin. As a result, BPyV is not specifically probed for during most 9CFR 113.53-based raw material viral infectivity testing, and this test most likely would not be capable of detecting BPyV if present in the test material. The EMEA Note for Guidance on the use of Bovine Serum in the Manufacture of Human Biological Medicinal Products (CPMP/BWP/1793/02) states that sera users are “encouraged to apply infectivity assays for BPyV and to investigate methods for inactivation/removal of BPyV in order to limit or eliminate infectious virus from batches of serum”.

Mitigating Risk. In actual practice, the available infectivity assays for BPyV involve numerous passages using a bovine detector cell such as MDBK and are somewhat lengthy and insensitive, though more sensitive assays are under development. Cell-based infectivity testing for BPyV is not always being performed by users for each batch of bovine serum. The lack of a rapid and sensitive infectivity assay also means that viral inactivation studies for BPyV are not practically possible. While another polyomavirus such as SV40 could be used in viral inactivation/removal studies as a proxy for BPyV, in actual practice the murine parvovirus MMV (mouse minute virus) is more typically used as a worst-case model virus for such studies since it is non-enveloped and even smaller than BPyV. The few studies performed with SV40 indicate that gamma-irradiation at the dosages normally employed is not effective at inactivating this virus, as might be expected for a virus of this relatively small size (e.g., Gauvin, 2009). On the other hand, it has been shown (Wang et al., Vox Sanguinis 86: 230-238, 2004) that UVC treatment is effective in inactivating SV40. Note: since originally authoring this blog, I have come across a great number of UV-inactivation papers which indicate that polyomaviruses, and SV-40 in particular, appear to be relatively resistant to UV inactivation. The Wang et al. result may represent an outlier. I will address this in a future blog. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for BPyV.

Conclusions. At the present time, infectivity screening of bovine sera for BPyV is not always being performed, and it is believed that the high frequency of detection of genomic material in bovine sera may not reflect a similarly high frequency of infectious BPyV. Risk of infection of biological products with BPyV through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UVC- or HTST-treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.

Quality by Design: Dissolution Time

2010-01-07T14:25:00.000-08:00

By Dr. Scott Rudge

In a previous post, I discussed the prevalence of statistics used in Quality by Design. These statistical tools are certainly useful and can provide (within their limits of error) prediction of future effects of excursions from control ranges for operating parameters, specifically for Critical Quality Attributes (CQA’s). The limitations of this approach were discussed in the previous blog. In the next series of blogs on Quality by Design, I will discuss opportunities for increasing quality, consistency and compliance for biotechnology products by building quality from the ground up.
While active pharmaceutical ingredient (API) manufacture by biosynthesis is a complicated and difficult to control prospect, there are a number of fundamental operations that are imminently controllable. Media and buffers must be compounded, sometimes adjusted or supplemented, stored and ultimately used in reactors and separators to produce and purify the API. These solutions are fairly easy to make with precision. Three factors come immediately to mind that can be known in a fashion that is scale independent and rigorous, 1) the dissolution rate, 2) the mixing power required and 3) the chemical stability of the solution.

The dissolution rate is a matter of mass transfer from a saturated solution at the dissolving solid interface to the bulk solution concentration. If particle size is fairly consistent, then the dissolution rate is represented by this equation:

where k is the mass transfer coefficient, provided the dissolving solid is fully suspended. It is easy to measure this mass transfer rate in the laboratory with an appropriate measure of solution concentration. For example, for the dissolution of sodium chloride, conductivity can be used. We conducted such experiments in our lab across a range of volumes salt concentrations, and found a scale independent mass transfer coefficient of approximately 0.4 s^-1. An example of the results is shown in the accompanying figure.

With the mass transfer coefficient in hand, the mixing time can be precisely specified, and an appropriately short additional engineering safety factor added. If the times are known for dissolution, and mixing is scaled appropriately (as will be shown in future blogs) then buffers and other solutions can be made with high precision and little wasted labor or material. In addition, the properties of the solutions should be constant within a narrow range, and the reproducibility of more complicated unit operations such as reactors and separators, much improved.

A design based on engineering standards such as this produces predictable results. Predictable results are the basis of process validation. As the boiling point of water drives the design of the WFI still, we should let engineering design equations drive Quality by Design for process unit operations.

Leah Choi contributed to this work

Can Cache Valley Virus Trash Your Manufacturing?

2009-12-30T15:02:00.000-08:00

By Dr. Ray Nims

Cache Valley virus is a single-stranded RNA virus of family bunyavirus, genus Bunyavirus. It is enveloped and nominally 80-120 nm in diameter. Cache Valley virus was first isolated in Utah in 1956 and is carried by mosquitoes. It has since been found to be a widespread virus, having been isolated in Texas, Michigan, North Carolina, Indiana, Virginia and Maryland, for example.

source: Nims et al., BioPharm. Int. 21: 89-94, 2008

Basis of Concern. Cache Valley virus is known to infect livestock, causing birth defects. There have been two reports of encephalitic disease in humans attributed to Cache Valley virus. This virus has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions from 2000 to 2004 (Nims et al., BioPharm. Int. 21: 89-94, 2008). The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated bovine serum is considered to be the most likely source. Thus far the virus has only been known to infect manufacturing processes employing bovine serum.

Regulatory Expectations. Cache Valley virus is not mentioned specifically in any regulatory guidance, as the detection of this virus in biologics production has been reported only within the past decade. It is the intent of the guidance, however, that occurrences of viral contamination in biologics manufacturing be dealt with through implementation of specific testing methods as required to assure detection of future recurrences (e.g., ICH Q5A R1). In addition, it is expected that the route of entry of the virus be established and that the process be remediated so that future recurrences are prevented where possible (e.g., 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use).

Mitigating Risk. Although many Contract Testing laboratories offer rapid nucleic acid-based detection assays for Cache Valley virus, raw material screening for this virus using such assays does not appear to be a viable means of eliminating risk. The industry experience thus far indicates that Cache Valley virus may be a low level, non-homogeneous contaminant of bovine serum. Viral screening performed on one bottle out of a large lot of serum therefore is no guarantee that this virus will not be encountered. Elimination of animal-derived materials (esp. bovine serum) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the serum or serum-containing media should be considered. Gamma-irradiation has been demonstrated to be effective in inactivating this virus in bovine serum (Gauvin, 2009). UVC treatment of media containing bovine serum also appears to be quite effective at inactivating Cache Valley virus (Weaver, 2009).

Conclusions. Cache Valley virus infects livestock and has been found to contaminate biologics manufacturing processes employing bovine serum. It is a virus of concern for biologics manufacturers employing bovine serum which has not been gamma-irradiated. Risk of infection of biological products with Cache Valley virus through use of bovine serum may be mitigated through implementation of gamma-irradiation of the serum, or UVC- or high-temperature short-time (HTST) treatment of media containing the serum and of viral purification processes capable of removing and inactivating enveloped viruses.

What cell line is this anyway?

2009-12-23T09:21:00.000-08:00

By Dr. Ray Nims

For about as long as scientists have been using cell cultures in biomedical research, there have been cases of cell line misidentification. This has been especially true for continuous cell lines, with the increased probability over time of mislabeling or cross-contamination. The primary cross-contaminant historically has been HeLa, a human cervical carcinoma cell which, given the opportunity, could outgrow most other cells in culture. More recently, the use of feeder cells for the propagation of human stem cells, and the use of xenografting for the propagation of human tumor cells, has provided additional opportunities for cell line cross-contamination and misidentification.

In the past, confirmation of cell line species of origin has been the main approach for authenticating cell lines. This was done initially by karyotyping or by immunological techniques, but more recently it has been done through the technique of isoenzyme analysis. An example of an isoenzyme analysis is shown below for Peptidase B and Aspartate Aminotransferase. These agarose gels show a positive control, a negative control (this is the band that does not line up with the others), the test article and a standard extract. These gels confirmed the identity of the test article as mouse derived, as expected.

Isoenzyme analysis has the advantage that it is rapid, not very technically demanding, and may be used not only to confirm species of origin but also to detect the presence of a cross-contaminating cell if the latter is present in the culture at 10% or greater (Nims et al., Sensitivity of Isoenzyme Analysis for the Detection of Interspecies Cell Line Cross-Contamination. In Vitro Cell. Dev. Biol.-Animal 34:35-39, 1998). In fact, isoenzyme analysis is currently the primary method employed within the biopharmaceutical industry for cell line authentication in satisfaction of 1993 Points to Consider and ICH Q5D guidance.

Recent advances in molecular diagnostic techniques have made possible the authentication of human cell lines to the individual level. DNA fingerprinting technologies have matured to the point that some of them, especially single nucleotide polymorphism (SNP) typing and single tandem repeat (STR) profiling, are now considered to be viable options for standardizing human cell authentication (see ATCC SDO newsletter article, page 5. For both human and animal cells, DNA fingerprinting provides a means of determining authenticity to the individual level. However, the primary drawback is that the fingerprinting techniques as routinely performed will be less or not at all useful for detecting interspecies cocultivations or cross-contaminations. For this purpose, it may be necessary to retain isoenzyme analysis as part of the authentication armament even when the molecular technologies become the definitive authentication practices for human and animal cell lines.

Got Animal-Derived Materials? Part 3

2009-12-18T13:37:00.000-08:00

By Dr. Ray Nims

The assessment of viral and transmissible spongiform encephalopathy (TSE) risk for animal-derived materials (ADM) used in the manufacture of biologics, which we have described in previous blogs, is just one component of an overall ADM program that should be in place at each organization producing biologics.

A formal ADM program at a biologics manufacturer ideally should be driven by an overriding SOP or policy document. This should address the procedures in place for minimizing the use of ADM, for procuring ADM with a view to minimizing viral and TSE risk, and for assessing the viral and TSE risk associated with the ADM that are used. There are specific sourcing requirements for ADM that are intended to minimize TSE risk (EMEA/410/01 Rev. 2 October 2003), and these must be followed or justification provided if deviated from. The evaluation of ADM for the presence of viruses of concern is addressed in the Code of Federal Regulations, Title 9 Part 113.53. ADM viral and TSE risk assessments should be conducted according to a formalized procedure by teams of individuals with education, training, and/or experience appropriate for these tasks. The composition of the risk assessment teams and the qualifications of their members should be described in revisable controlled documents. The risk assessments themselves should be recorded in controlled documents which may revised as new information becomes available from the ADM suppliers. The ADM information that is used as part of the risk assessment process should be archived in a manner tying it to the risk assessment itself.

The existence of a formalized ADM program, qualified risk assessment teams, as well as reports documenting the individual ADM risk assessments may be the subject of regulatory scrutiny during periodic inspections or inspections tied to a new product application. This is especially likely if the product is intended for global distribution, as these ADM issues are specifically mentioned in EP (Chapter 5.1.7) and EMEA (EMEA/410/01 Rev. 2 October 2003) guidance.

Advantages of Compendial Methods

2009-12-14T21:26:00.000-08:00

By Dr. Lori Nixon

When you are developing a new product specification, it is usually recommended to rely on the appropriate compendial method for applicable “generic” quality characteristics such as pH, residual solvents, trace metals, bioburden, etc. By compendial method, we mean methods that are described as chapters in the United States Pharmacopeia (USP) or others that may be applicable for a specific regulatory region. The three main compendia include the USP, European Pharmacopoeia, Japanese Pharmacopeia (USP, PhEur, JP); these are the “tripartite” bodies that are involved in the International Conference on Harmonization (ICH).

photo of USP laboratories from DPR Construction Inc.

Why rely on compendial methods rather than just using your own? It is generally recommended to refer to compendial methods where applicable. The advantage to the drug sponsor is a reduced requirement for validation supporting such methods (the methods themselves are considered validated, and may only require product-specific verification in the particular testing lab). Compendial methods are “familiar” to regulatory reviewers; they are also generally expected. If you propose your own method as an alternate method, you will need to justify why your own method is equivalent or better. For the testing lab, there is some advantage in having methods that can be applied to multiple products (avoiding a multiplicity of similar methods) and where the change process is relatively well-defined and publically communicated. You may also find it simpler to transfer testing between different labs.

To reference the compendial method in your specification, you may refer simply to the test by attribute and chapter, along with the associated limit for your product. For example, your specification may include a limit of 10 EU/mL for bacterial endotoxin as measured by USP<85>. The general expectation here is that at the time of testing, the current version of USP is used. Clearly, this will require that your testing lab is aware of any potential changes to the USP and can prepare for such changes accordingly. As with any other change to an analytical method, changes to compendial methods can impact training, internal procedures, product-specific re-validation/verification, etc.

In practice, labs often rely on additional internal descriptive procedures in order to execute the compendial methods (i.e., rather than just directing analysts to follow the chapter directly). This is usually a good idea, for several reasons. It can be easier to train analysts according to a standard documentation format, and it is often necessary to describe details that may be specific to the particular lab, equipment, instrumentation, reagents, reporting requirements, etc. Again, even if there is a lab-specific procedure, it is usually best to refer directly to the compendial method (USP<85>, e.g.) in the sponsor’s product specification.

Be aware of compliance with compendial testing requirements when you are outsourcing testing. For example, almost any chemical testing lab with have a method for pH, but that doesn’t necessarily mean that it will comply with USP<791>. For example, in this case the USP method describes measuring the sample temperature within a certain range; often “generic” lab methods for pH do not specify control of the sample temperature. There are additional requirements such as the calibration standards chosen, etc that must also be considered. When reviewing vendor methods, check the following:

- Does the method purport to comply with any compendia? (should be clearly stated in the vendor’s procedure if so)

- Which compendia?

- Check details of the method to ensure that it does indeed comply with the current compendial procedure(s) in question

- Does the lab have a mechanism to stay current with upcoming compendial changes?

- Do they have appropriate change control system to ensure that they can prepare for method changes and associated re-validation, etc?

- Consider what verification/validation is required to ensure that the vendor method provides reliable results for your particular product/sample type.

Of course, the downside of compendial methods is that they are region-specific, and one region may not recognize the compendia of another region. There have been efforts in recent years towards harmonizing methods (ICHQ4B for bioburden testing, for example), but this process is slow and far from complete.

If you intend to market or perform clinical trials in more than one region, you may need to ensure compliance with multiple compendia. In this case, consider the following:

• Has method been harmonized through the ICH process?

• Is it possible to create an internal “harmonized” method that meets the requirements of all relevant compendia?

• Is it possible to meet the requirements of all by following the “most stringent” compendial procedure?

• Will you need to test by multiple procedures to generate results acceptable to each region?

Should we care about…Vesiviruses?

2009-12-02T10:03:00.000-08:00

By Ray Nims

Vesiviruses are single-stranded RNA viruses of family calicivirus, genus Vesivirus. They are non-enveloped and 30-40 nm in diameter, and the genus includes feline calicivirus, vesicular exanthema of swine virus, rabbit vesivirus, and San Miguel sea lion virus, as well as vesivirus isolate 2117.

source: Stewart McNulty, Queens University, Belfast, UK

Basis of Concern. Vesivirus 2117 has been isolated from biologics manufacturing processes employing Chinese hamster cell substrates on a number of occasions, the first being reported in 2003 (Oehmig et al., J. Gen. Virol. 84, 2837-2845, 2003), and additional occurrences being reported in 2008 and 2009.
The susceptibility of relevant manufacturing cell lines of different animal species to infection by this virus appears to be limited to the Chinese hamster. When infected, these cells undergo a relatively rapid lytic infection. The route of entry of the virus into biologics production processes has not been established with certainty, although the use of contaminated animal-derived materials, such as bovine sera, is considered to be the most likely source.

Regulatory Expectations. Vesivirus is not mentioned specifically in any regulatory guidance, as the detection of the 2117 isolate in biologics production has been reported only within the past decade. It is the intent of the guidance, however, that occurrences of viral contamination in biologics manufacturing be dealt with through implementation of specific testing methods as required to assure detection of future recurrences (e.g., ICH Q5A R1). In addition, it is expected that the route of entry of the virus be established and that the process be remediated so that future recurrences are prevented where possible (e.g., 1997 Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use).

Mitigating Risk. At least three Contract Testing laboratories have announced rapid nucleic acid-based detection assays for vesivirus isolate 2117 within the past year. These assays are available for raw material screening and for in-process testing of biologics bulk harvest samples. Elimination of animal-derived materials (esp. bovine sera) from the manufacturing process may help to reduce the risk of experiencing this virus. Should this not be possible, treatment of the sera or sera-containing media should be considered. Studies on the inactivation of caliciviruses indicate that UV treatment may be effective (Duizer et al., Appl. Env. Microbiol. 70, 4538-4543, 2004; de Roda Husman et al., Appl. Env. Microbiol. 70, 50989-5093, 2004). Gamma-irradiation at the dosages normally used does not appear to be effective, as might be expected for a virus of this relatively small size. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for vesiviruses in general.

Conclusions. Vesivirus isolate 2117 preferentially infects Chinese hamster cells and has been found to contaminate biologics manufacturing processes employing this cell substrate. It is now a virus of concern for the biopharmaceutical industry. Risk of infection of biological products with vesiviruses through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UV or HTST treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.

The Good Buffer

2009-11-10T22:58:00.000-08:00

By Scott Rudge

“A Good Buffer” has a number of connotations in biochemistry and biochemical engineering. A “good buffer” would be one that has good buffering capacity at the desired pH. The best buffering capacity is at the pK of the buffer of course, although it seems buffer salts are rarely used at their pK.

Second, a good buffer would be one matched to the application. Or maybe that’s first. For example, the buffering ion in an ion exchange chromatography step should be the same charge as the resin (so as not to bind and take up resin capacity). For example, phosphate ion (negative) is a good choice for cation exchange resins (also negatively charged) like S and CM resins.

Another meaning of a “Good” buffer is a buffer described by Dr. Norman Good and colleagues in 1966 (N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa and R. M. M. Singh (1966). "Hydrogen Ion Buffers for Biological Research". Biochemistry 5 (2): 467–477.). These twelve buffers have pK’s spanning the range 6.15 to 8.35, and are a mixture of organic acids, organic bases and zwitterions (having both an acidic and basic site). All twelve of Good’s buffers have pK’s that are fairly strongly temperature dependent, meaning that, in addition to the temperature correction required for the activity of hydrogen ion, there is an actual shift in pH that is temperature dependent. So, while a buffer can be matched to the desired pH approximately every 0.2 pH units across pH 7 ± 1, the buffers are expensive and not entirely suited to manufacturing applications.

In our view, a good buffer is one that is well understood and is designed for its intended purpose. To be designed for its intended purpose, it should be well matched to provide adequate buffering capacity at the desired pH and desired temperature. As shown in the figure, the buffering capacity of a buffer with a pK of 8 is nearly exhausted below pH 7 and above pH 9.

It’s easy to overshoot the desired pH at these “extremes”, but just such a mismatch between buffering ion and desired pH is often specified. Furthermore, buffers are frequently made by titrating to the desired pH from the pK of the base or the acid. This leads to batch to batch variation in the amount of titrant used, because of overshooting and retracing. In addition, the temperature dependence of the pK is not taken into account when specifying the temperature of the buffer. Tris has a pK of 8.06 at 20°C, so a Tris buffer used at pH 7.0 is already not a good idea at 20°C. The pK of Tris changes by -0.03 pH units for every 1°C in positive temperature change. So if the temperature specified for the pH 7.0 buffer is 5°C, the pK will have shifted to 8.51. Tris has 3% of its buffering capacity available at pH 7.0, 5°C, it’s not well matched at all.

A good buffer will have a known mass transfer rate in water, so that its mixing time can be predicted. Precise amounts of buffering acid or base and cosalt are added to give the exact pH required at the exact temperature specified. This actually reduces our reliance on measurements like pH and conductivity that can be inexact. Good buffers can be made with much more precision than ± 0.2 pH units and ± 10% of nominal conductivity, and when you start to make buffers this way, you will rely more on your balance and understanding of appropriate storage conditions for your raw materials, than making adjustments in the field with titrants, time consuming mixing and guessing whether variation in conductivity is going to upset the process.

Enzyme induction…done pharmacodynamically

2009-11-06T13:07:00.000-08:00

By Ray Nims

Pharmacodynamics is the study of a specific effect of a drug as related to drug concentration at the putative active-site for that effect. Pharmacodynamics is sometimes used to model quantitatively the effect of a drug over time as drug concentration at the active-site rises and falls. Another type of pharmacodynamic study entails exposing the animal or in vitro system to graded doses of a drug and monitoring the effect associated with each active-site concentration. From the latter type of study, one is able to estimate both potency for the effect (given in terms of the active-site drug concentration at the half-maximal effect for that drug, or EC50) and its efficacy (given in terms of percentage of maximal response compared to other drugs causing the same effect through the same mechanism). In receptor theory, EC50 is considered to reflect the affinity of the drug for a receptor, while efficacy is a measure of the bound drug’s ability to cause the specific response.

The induction of drug-metabolizing enzymes, such as the cytochromes P450, may be considered to represent an effect of a drug or xenobiotic. It is common for investigators to measure such induction at one or a few dose levels and to compare the resulting enzyme induction with that of a prototype inducer. These comparisons are sometimes described in terms of the test xenobiotic causing “strong” (“potent”) or “weak” induction in comparison with the prototype inducer. As already pointed out quite elegantly by D. A. Smith and coworkers (Letter to the Editor: The Time to Move Cytochrome P450 Induction into Mainstream Pharmacology is Long Overdue. Drug Metab. Dispos. 35:697-698, 2007; http://dmd.aspetjournals.org/cgi/content/full/35/4/697), such statements are both misleading and inaccurate. As with any drug effect, enzyme induction must be described in terms of both potency and efficacy. It is possible for an inducer to be very potent but to display little efficacy. In fact, a xenobiotic having high potency and little or no induction efficacy might represent a competitive inhibitor for this effect. In contrast, there may be inducers which are very effective, but not very potent.

It is possible for efficacy and potency for enzyme induction to be estimated on the basis of studies using intact animals, provided that certain assumptions are made (e.g., that total plasma drug concentration is a suitable proxy for drug concentration at the induction active site, which cannot be sampled directly). An example of such a study is that of R.W. Nims and coworkers (Comparative Pharmacodynamics of Hepatic Cytochrome P450 2B Induction by 5,5-Diphenyl- and 5,5-Diethyl-substituted Barbiturates and Hydantoins in the Male F344/NCr Rat. J. Pharmacol. Exp. Therap. 270: 348-355, 1994; http://jpet.aspetjournals.org/cgi/content/abstract/270/1/348). A more straightforward approach is offered through in vitro enzyme induction studies, in which enzyme induction can be related to drug concentration in the culture medium (e.g., Kocarek and coworkers: Differentiated Induction of Cytochrome P450b/e and P450p mRNAs by Dose of Phenobarbital in Primary Cultures of Adult Rat Hepatocytes. Mol. Pharmacol. 38:440-444, 1990; http://molpharm.aspetjournals.org/cgi/content/abstract/38/4/440).

Measurement of the induction of the cytochromes P450 and other drug-metabolizing enzymes following drug treatment in animals and humans is an important aspect of drug characterization. The studies should be performed and reported in a manner consistent with other drug effects, that is, in a manner consistent with the principles of pharmacology.

Got animal-derived materials? Part 2

2009-10-29T12:39:00.000-07:00

By Ray Nims

As part of a formal animal-derived materials program, biopharma companies must assess the viral and TSE risk associated with materials derived from animals or which have been in contact with animal-derived materials at some point. We have addressed the viral risk within a previous blog installment. Now let’s consider the TSE risk.

TSEs (transmissible spongiform encephalopathies) are fatal diseases believed to result from exposure of a few animal species (including various ruminants, cervids, ungulates, cats, minks, and humans) to “infectious” prion proteins. The infectious proteins (PrP^Sc) are capable of interacting with and altering the normal prion proteins (PrP^c) within the brain and spinal cord, changing the normal proteins to the abnormal form. Since humans have contracted prion disease as a result of consuming tissues from cattle with bovine spongiform encephalopathy (mad cow disease), there is concern about the use of bovine materials and materials from other “relevant species” in the manufacture of biopharmaceuticals.

The EMEA has provided a guidance document (EMEA/410/01 Rev. 2 October 2003; http://www.emea.europa.eu/pdfs/human/bwp/TSE%20NFG%20410-rev2.pdf) describing the requirements for the use of animal-derived materials from relevant animal species (cattle, sheep, goats, and other animals which are naturally susceptible to TSEs but not including humans or non-human primates) in the manufacture of medicinal or veterinary products. The guidance applies to active substances, excipients and adjuvants, raw and starting materials and reagents, and materials which come into contact with products or equipment used to make product. The major point of the guidance is that TSE risk can be minimized, but in many cases not entirely eliminated. Where TSE risk cannot be avoided through elimination of animal-derived materials completely, the guidance provides principles for the minimization of TSE risk which include: the use of low-risk (non-relevant) animal species, the geographical sourcing of relevant species from low-risk regions, the use of low-risk tissues, the use of appropriate slaughter techniques to reduce potential for contamination of low-risk tissues with high-risk tissues, the appropriate oversight of manufacture of the animal products through Quality Assurance and self and external auditing and Quality Control testing, and the implementation of process designs to remove or inactivate the abnormal prion proteins.

Biopharma companies using animal-derived materials are instructed to perform risk assessments on those materials, which take into account the factors described above. The risk assessments must be completed as part of a formalized animal-derived materials program, documented procedurally and executed by staff that are experienced and trained to conduct such assessments. EMEA inspectors will expect to see this formal program in place. Risk assessments for individual raw materials may then be rolled up into a risk assessment for the biopharma product. The rolled up risk assessment may also consider manufacturing steps at the biopharma which may remove or inactivate the abnormal prion proteins, though these, if cited, may need to be validated. Finally, it is expected that companies will conduct a benefit/risk evaluation to assure that any benefits realized by the patient taking the product will outweigh and justify the risk associated with the use of materials derived from relevant animal species.

Got Animal Derived Materials?

2009-10-23T13:37:00.000-07:00

By Ray Nims

Most biopharma manufacturing processes utilize a few raw materials (including cell substrates, excipients, materials which come into contact with the product, etc.) derived from animals or which have been in contact with animal-derived materials at some point. As part of a formal animal-derived materials program, the biopharma must assess the viral and TSE risk associated with such materials. Let’s consider the viral risk first. From a viral safety standpoint, it is important for each biopharma to consider such ingredients and to be aware of the inherent risk of transmitting virus into the product via the materials. Why? As Genzyme discovered in the spring of 2009 (http://www.genzyme.com/corp/media/GENZ%20PR-061609.asp), viruses can infect the upstream manufacturing processes with results devastating to both the biopharma and to the patients its products are intended to treat. Viral risk mitigation, and regulatory guidance (e.g., EP Chapter 5.1.7: Viral Safety), require that viral risk assessments be performed for animal-derived materials used to manufacture biologics. In this context, raw materials include also excipients, growth media, column packing resins, and cell substrates.

For each product, manufacturers should list the animal-derived materials utilized, and should perform a viral risk assessment for those materials. This assessment considers the animal species and tissue, the processes used to manufacture the raw material, the quality control testing performed on the raw material, and in some cases, the manufacturing process in which the raw material is to be used at the biopharma. Inspectors from the EMEA will not only expect the risk assessments to have been performed and documented, but will expect that the assessment process be formalized into a business practice or standard operating procedure. The staff performing the assessments should be qualified for this task and the assessment team should include staff knowledgeable in virology, viral inactivation and removal, and the manufacturing and purification processes employed for the specific product at the biopharma.

Viral risk assessments completed for individual raw materials may eventually be rolled up into a viral safety assessment for the product per EP Chapter 5.1.7. This product evaluation will also consider other factors, such as the patient profile and route of administration, the cell substrate, the types and pathogenicities of viral contaminants found in the cell substrate and the manufacturing process, the amount of bulk material required for a human dose, and the viral inactivation and removal capabilities of the manufacturing downstream processes.

Outsource it, and fuggedaboutit?

2009-10-13T16:18:00.000-07:00

By Ray Nims

Much has been written about the rationales and advantages for outsourcing of manufacturing and/or testing services; about the selection of outsourcing partners; and about the optimization of the pharma/contractor relationship. In any pharma/contractor relationship, there are responsibilities associated with the pharma as well as contractor responsibilities. These include both business as well as compliance responsibilities. The business realities and regulatory expectations associated with the use, by a pharma company, of a contract testing organization must be considered when the decision is made to outsource. A contract testing organization desiring to provide services for a pharmaceutical must be aware of the expectations and responsibilities associated with such a partnership. The optimal and most defensible programs will be those in which the various practices to be described below are formalized within internal Quality Systems, policies, and/or standard operating procedures as well as Quality Agreements.

Responsibilities falling upon the pharmaceutical partner include: 1) the selection of the contract testing lab; 2) commissioning and providing test samples of raw materials and products for method verification (compendial methods) and method qualification (non-compendial methods); 3) instituting of a Quality Agreements, business agreement, and/or confidentiality agreement with the contractor; 4) scheduling and shipping of test samples in accordance with the requirements of the testing lab and the test system; 5) providing in-life guidance and oversight of investigations of unexpected and out of specification results; and 6) ongoing monitoring of the performance of the contract lab and its methods.

Responsibilities primarily falling upon the testing lab include: 1) attaining and maintaining GLP or GMP compliance as appropriate for the intended use of the method; 2) providing assurance that the methods offered will be available to the client over the long term; 3) responsiveness to the sponsoring pharma and adherence to the terms of the Quality and/or business agreements; 4) method validation, verification, and or qualification as appropriate for the intended use of the method; 5) control of reagent, raw material, control, and standard inventory and quality; 6) assuring secure and retrievable data archiving; and 7) retention of staff possessing the appropriate expertise for direction of operators and the methods.

Do You Use Risk Assessments in Auditing?

2009-08-04T12:02:00.000-07:00

Audits are a critical component of quality systems, but are they guided by formal assessments of risk to your products? In this world of ICH Q9, can you offer even a semi-quantitative justification for your audit priorities? We have spoken to many people in the industry, and almost all mention a risk assessment being undertaken prior to an audit. But we have not found many people that formalize that risk assessment, or keep it updated from audit to audit. Even fewer communicate their scoring of risk to either their internal clients or the vendor that has been audited.

A new trend in auditing is to use a form of risk assessment both before and after the audit. A popular form is the Failure Modes and Effects Assessment, or FMEA (see, for example, http://www.sre.org/pubs/Mil-Std-1629A.pdf). In a traditional FMEA, risks of failure are identified in a detailed fashion, and scored in three categories related to the failure’s probability, detectability and severity. Scoring is done on a semi-quantitative or relative basis using an arbitrary scale such as 1-10. For an audit, you might use the same categories as they relate to a particular vendor's (or department's) ability to deliver a product or service, failure free. You could organize your FMEA according to the critical quality attributes of the product or service being delivered or according to a list of requirements from a guideline or the CFR's. Your FMEA should receive input from affected departments, and should be used for prioritization of audit items. You should have the FMEA in mind as you conduct your audit, and remember why various items received high prioritization. You may change ratings for probability or detectability based on what you observe. If instead, you confirm your evaluation, you should probe remediations that decrease your firm's primary concern. A remediation that addresses detectability, when the issue was probability, likely won’t mitigate the risk of failure.
When you return from your audit, rescore the FMEA with assessments based on your observations and data that you collected. Make sure that you share your analysis with the stakeholders. And monitor the performance of the vendor until the next audit; the data will help inform your next FMEA.

Do you already use FMEA's in audit preparation and reporting? Let us know your practices.

Why is Quality by Design so heavy with statistics?

2009-06-23T11:02:00.000-07:00

Why is the literature on Quality by Design so laden with statistics and experimental design space jargon? After all, the definition of the term “design” doesn't seem to include the analysis of messy data leading to rough correlations with results that are valid only over a limited range. So what gives?

The idea behind QbD was to use mathematical, predictive models to predict process outcomes. This concept can be applied directly to simple unit operations, such as drying, distilling, heating and cooling. However, unlike in the petrochemical business, the thermodynamic properties of most active pharmaceutical ingredients are not known and are difficult to measure. The unit operations used to manufacture common biotechnology products, such as cell culture, chromatography and fermentation have been modeled, but the models are very sensitive to unknown or unmeasureable adjustable parameters. The batch nature of these operations also makes their control difficult, as classical control theory relies on the measurement of an output to make an adjustment to an input to correct the output back towards the design specification.

Since there does not appear to be a clear path to using models, an approach has been chosen that emphasizes getting as much phenomenological information from as few experiments as possible. This is the Design of Experiments approach, where input conditions or operating parameters are systematically varied over a range and the process outputs measured, with statistics used to deconvolute the results. The combined ranges tested become the “design space”, and the process performance outputs with the variations closest to the process failure limit become the critical performance parameters. The results are useful, but only within the design space, and only with the certainty that the statistics report. Also, since the results are phenomenological, the effect of scale is often unknown.

The statistical approach is acceptable, and for the immediate future it's probably the best that we can expect. But the focus on this approach seems to drown out the more pressing need for good process models and physical properties data. These are the elements that allowed the petrochemical and commodity chemicals industries to scale up processes with assurance that quality specifications would be met. There are countless models available for bioprocessing's more complicated unit operations, but they have parameters that we don't know and can't calculate from first principles. There is no question that we need to find ways to collect this data, and a commitment to publish or share it. There are also simpler unit operations that we can model, scale up and scale down with complete assurance. These include operations such as mixing and storing solutions, filtration, diafiltration, centrifugation and some reactions. We shouldn't let the more complicated operations that still require statistical DOE approaches prevent us from applying the true principles of QbD to our simpler unit operations.

Ten Steps to Choosing your Contract Manufacturer

2009-06-03T08:44:00.000-07:00

For many young companies, choosing a contract manufacturer, or CMO, for their lead pharmaceutical candidate is critical. Choose the wrong contractor, and you could be faced with delays and cost overruns with which your investors and patients won’t be very sympathetic. While there is no guarantee that you will always make the right decision, here are some tips that can help you make your choice in an organized, thoughtful, meaningful and objective way:

1) Make a list of all the possible suppliers. Such lists may be purchased, but they are also easily assembled from internet searches. In fact, you can do a little pre-screening with your own internet search.

2) Screen potential suppliers with a phone call. You will probably speak with a business development or sales person representing the contractor, but usually these people are quite knowledgeable about their company’s capabilities, and common problems encountered in the industry. We recommend you not reveal too many details about your project, and be prepared mostly to listen. However, you should have three or four key capabilities or proficiencies that you are looking for in all of the potential vendors. If possible, try to rule out vendors who do not meet your “must-have” requirements at this stage. Stay tuned for a blog on how to establish your showstopper list, it’s a critical exercise, and may extend beyond key capabilities and proficiencies.

3) Keep a matrix, and record the date you first contacted the vendor, when they responded to you, the status of any confidentiality agreements, and all the contact information that you can gather (email addresses, cell phone numbers, main switchboards and extension numbers). Also note the responses that each contact had relative to your three or four show-stopper criteria.

4) Meet with your team, and select three to five potential vendors to request a proposal from. We don’t recommend more than five: getting good, comparable proposals is a lot of work, like 2n times the work, where n = the number of proposers. Not to mention the work that contract manufacturers go through to read your RFP and prepare a proposal. Your three or four showstopper criteria should help you limit the number of proposers; if necessary you can begin to narrow down based on “nice-to-have” criteria as well. You may also deliberately choose to look at a range of vendors that represent different strengths/weaknesses (for example, do you prefer a “one-stop shop” that is convenient, but maybe not the best at everything, or a “specialty” vendor that provides higher levels of expertise, but will require you to select and manage multiple vendors?).

5) Solicit proposals. Most contract manufacturing seekers have a Request for Proposals (RFP) process that includes a document. These RFP documents vary from one page requirements descriptions, to lengthy, legalistic documents that require a team, and a month, to respond to. You should do what is comfortable for your organization. There needs to be enough information so that the vendor is able to respond with a meaningful proposal. There is some legal danger, particularly with intellectual property, so it’s not a bad idea to get your RFP reviewed by your legal counsel. And you should only send an RFP to a vendor after they have signed a mutually agreed confidentiality agreement.

6) Score your proposals. Find some basis to make apples to apples comparisons. RMC uses a modified Kepner Tregoe analysis, but many forms of analysis will work. You should have determined how you will assess and weight qualitative data before you begin. And in doing so, you should not under-estimate intangible factors: the ability to communicate, time zone differences, good references (you’ve checked, right?) are some examples. At this stage you should be ranking and scoring on “nice-to-have” criteria as well as comparing cost/timeline, capabilities, capacity, and viability of the business. You might form your opinion of the viability of the business by reading annual reports and press releases, and by assessing how busy the manufacturing area and support labs look during your visit.

7) Visit the top two or three vendors in person. Vendors may not allow a formal quality audit prior to signing a contract, but be sure to bring your quality representatives even for an informal “technical visit”. If the project is large, you may take the resistance to a pre-use quality audit as a red flag. Again, spend as much time as possible listening, rather than talking. Get a tour, and copies of all presentations. Ensure that you have a meeting between the decision makers for both sides as well as aligning discussions between key technical and quality personnel. If there are disputes or further work to do, your decision makers must have a good working relationship.
Your visit is also your best opportunity to break past the business development group and take a true measure of the business. Chat with the people in the lab or production area if you can. Look over the state of the equipment, the cleanliness of the facility and the stock in the warehouse. Check their flexibility-- what can they make happen for you, vs. what will have to be run past someone in another building, or another city? Ultimately, you should think about hiring a contract manufacturer similarly to how you hire an employee, by hiring for expertise as well as fit with your team.

8) Consider entering contract negotiations with at least two vendors. Things can go wrong in negotiations, and your position is stronger if you can legitimately walk away. We typically don’t let any of the final three candidates off the hook until the ink is dry on the final contract. If your budget can justify it, having a second contractor performing development work and verifying the primary contractor’s results is an excellent idea. It may ultimately spread your risk in supply chain, and give you leverage in negotiating commercial supply agreements later on.

9) Revisit your analysis tool. You may learn new things in your contract negotiations that cause you to adjust your evaluation. Don’t be afraid to be frank if you feel like terms have been changed since the selection was made. This is a good reason to keep a back up vendor.

10) You must now manage the project according to the criteria by which you selected your supplier. Hold your supplier and yourself accountable to these criteria. For example, after selecting a vendor because they can meet a very aggressive timeline, do not put the project timeline at risk by failing to order back up critical path supplies, in case the primary order doesn’t arrive, or fails to meet specifications on arrival. We will have more to say about managing a contract manufacturer in a future blog.

Choosing the right manufacturing partner is critical for your success as a drug developer. Spend the time required to make a good decision. This time should be spent gleaning as much tangible and non-tangible information on all your options as possible, and then objectively comparing it. You should have an idea about how you’re going to evaluate and weight non-tangible factors into your decision. And once you have made the choice, manage according to your criteria. Although everyone has their own methods for vendor selection, these are some suggestions that have worked well for us and our clients. If you have questions or comments, please visit http://www.rmcpharma.com/ or email us at info@rmcpharma.com.