Viral clearance studies …. are they needed for proteins produced using bacterial or yeast fermentation processes?

Dr. Ray Nims

In E. coli or Pichia pastoris-based bioproduction of recombinant proteins, there are no suitable host cells for amplification of viruses that are infectious for humans. Bacteria such as E. coli and yeast such as P. pastoris can only support the growth of certain bacteriophage or yeast viruses, respectively. These types of viruses are not infectious for humans or animals.

The potential for viral infection of a bacterial cell process or a yeast cell process used for production of a recombinant protein is therefore a business risk, not a patient safety risk. This is the reason why case studies of bacteriophage infection of bacterial fermentation processes or of viral contamination of yeast cell processes have not appeared in the literature (or in the news). Unlike virus contamination of animal cell-based production processes, the events involving bacterial or yeast do not have patient safety implications. When a bacteriophage or yeast virus contamination occurs, manufacturers quietly go about the business of restarting the fermentation. Remediation and prevention of future occurrences is driven primarily by business concerns, as opposed to regulatory concerns.

Manufacturers of recombinant proteins produced using bacterial or yeast cell substrates are not required to conduct cell line viral testing (though phage induction studies may be performed as part of bacterial cell line characterization – to mitigate business risk!), nor are they required to conduct lot-by-lot viral testing of bulk harvest samples. Such requirements are mandated for production processes using animal or human cells. As the title of the document “Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin” indicates, the ICH Q5A (R1) guidance mandating such testing is clearly directed toward manufacturing processes utilizing the types of cells which may support growth of viruses infectious for human or animal cells. This ICH document also provides guidance on the conduct of viral clearance studies.

Viral clearance studies are intended to provide evidence of the ability of downstream purification steps of a manufacturing process to remove or inactivate viruses that potentially may contaminate the process, make their way into final product, and represent a health risk to patients. As such, the evaluation typically involves both relevant viruses (i.e., viruses that are known to contaminate these kinds of processes) and model viruses (i.e., viruses representative of the types of viruses that could contaminate these processes). In the case of bacteria- and yeast-based manufacturing processes, the relevant viruses (bacteriophage and yeast viruses, respectively) are not infectious for humans or animals. In addition, there are no model viruses that are capable of infecting these kinds of cells that are of concern for humans or animals. Therefore, viral clearance studies are typically not required or conducted.

Are there any circumstances where a bacterial or yeast fermentation process could be expected to harbor a virus capable of infecting humans? The only that I can imagine is a process utilizing primary or secondary animal-derived raw materials in rather large quantities. The worry would not be that viral amplification might occur, but that some carryover of surviving animal viruses to the final product might be possible. The use of such animal-derived materials by a manufacturer of a therapeutic protein must be justified based on risk analysis. If substantial risk is introduced by the use of such raw materials, then perhaps a raw material treatment approach would need to be validated.

In the absence of the use of animal derived materials or plant materials not subject to processing steps that would inactivate contaminating animal viruses, incorporation and validation of viral clearance steps into protein production processes using bacteria or yeast cell substrates is not expected, nor would this be of practical value in assuring patient safety.

Moving Past the Bottleneck

By Dr. Scott Rudge

Is there a bottleneck in Downstream Processing? The membrane chromatography vendors certainly want you to think so.

The problem is in the efficiency of chromatographic purification.  Without a doubt, chromatography is slow and inefficient. A typical protein loading for commercial scale chromatography is 25 to 40 g/L, and a typical cycle is on the order of 8 hours.  So the productivity of a chromatography column is 3 to 5 g/L/hr.  Compare this to an aggressive microbial fermentation, which produces 10 g/L in a 40 hour fermentation (0.25 g/L/hr) or a very aggressive cell culture which produces 10 g/L of antibody in seven days (0.06 g/L/hr). Clearly, even with the inefficiency of chromatography, there is plenty of volumetric productivity to keep up with modern cell culture and fermentation.

As was pointed out in a previous blog, there is no total capacity difference between typcal chromatography resins and derivatized membranes, and the dynamic binding capacity, where membrane chromatography should be superior, is also not different.So membrane chromatography does not appear to be the answer.

One technology that could intensify the performance of chromatography is “simulated moving bed” chromatography. With simulated moving beds (or SMB), the non-productive volumes of the chromatography column are put into use. This is done by segmenting the column, or making a series of much smaller columns, each of which can be operated differently at any given time.  For example, a section of the column near the classic “inlet” would be regenerated after the product has passed through it.

A section of the column downstream of the product front would be equilibrated just before the product front entered it.

In its simplest form, the SMB column is thought of in four sections, one for feed, one for product, one for regeneration, and one for raffinate, as shown in the figure below:

(from Imamoglu, S., Advances in Biochemical Engineering/Biotechnology, Vol. 76, Springer-Verlag, Berlin, p 212 (2002).)

The flow of mobile phase moves countercurrent to the direction of switching of the columns, and the velocity of switching the columns is in between the velocity of the product and the next fastest or slowest contaminant.  In the configuration shown above the raffinate moves more quickly than the product (extract), and as the solid moves counterclockwise, the extract moves backwards to the elution zone.  Meanwhile, the fast moving raffinate is allowed to exit the loop to waste.  Column segments in Zone IV are regenerated and re-equilibrated to a condition where the extract is bound but the raffinate continues to travel down column. SMB can increase the productivity of chromatography resin by a large factor.  In the simplistic diagrammatic example, the productivity could be increased by a factor of 4.  Depending on the length of the zone required for separation, the increase can be much higher.

SMB has been used for the production of amino acids, enantiomers and many other small molecules. More recently, it has been used for purification of proteins such as albumin, antibodies and some artificial demonstration mixtures such as myoglobin/lysozyme.  Innovations such as the application of gradients to SMB have been developed.  This technology has the potential to reduce cycle times and increase efficiency by smarter use of existing resources.

UV-C versus small, non-enveloped viruses

By Dr. Ray Nims

Small, non-enveloped viruses (especially the circoviruses, parvoviruses, picornaviruses, caliciviruses, and polyomaviruses) and bacteriophage with similar characteristics represent a special challenge to the biologics industry.

In fact, contamination events have occurred with each of these virus families; in some cases more than once. Within the Circoviridae, the primary concern has been the porcine circovirus discovered in rotavirus vaccine. At least four contamination events involving the parvovirus, mouse minute virus, have been reported. The calicivirus vesivirus 2117 has contaminated biologics manufacturing processes on at least two occasions. The picornaviruses are a threat which has yet to be realized in the manufacturing context; but a viral safety test conducted for a biologics cell bank was the scene of a contamination with equine rhinitis A virus. The polyomavirus SV40 was found to have survived the formaldehyde inactivation used in the preparation of polio vaccines and therefore made its way into the doses of vaccine delivered to millions of individuals during the decade between 1955 and 1965 (being born in 1952, it is possible that the author received such tainted vaccine!).

The contamination events described above are, at least in part, a reflection of the ability of these viruses to withstand conditions that would lead to inactivation of other types of viruses. Inactivation strategies that are typically employed for viral safety include chemical (low pH, high pH, disinfectants, solvents, detergents, etc.) and physical (heat, irradiation, pressure, etc.) means. In a previous posting, the efficacy of gamma irradiation for inactivating various types of viruses within frozen animal serum was discussed. The use of UV-A in combination with riboflavin has also been discussed previously.

It is perhaps fortunate that the efficacies of different inactivation approaches are in many cases complementary. For instance, it appears that the efficacy of gamma irradiation for inactivation of viruses decreases as viral particle size decreases (although this relationship is not strictly linear). The outcome of this is that gamma irradiation does not appear to be particularly effective for inactivating small, non enveloped viruses from the circovirus, parvovirus, and polyomavirus families. On the other hand, ultraviolet radiation in the C range (254 nm is the most commonly employed wavelength) appears to be more effective for the inactivation of smaller viruses than for larger viruses (or bacteria).

The table below is a compilation of the UV-C inactivation constants (K, defined as the log₁₀ reduction in titer per mJ/cm² fluency) for various families of small, non-enveloped viruses. These K values represent inactivation of the viruses in a variety of matrices, ranging from water to protein-containing matrices such as albumin or complete culture medium). These results suggest that UV-C irradiation may be a viable approach for inactivating many of these problem viruses in raw materials or process intermediates used in biologics manufacture.

references for K values: * Lytle and Sagripanti 2005, ¶ Kowalski et al. 2009; † Maier 2007. The other values are the mean K values assembled by the author from the inactivation literature.

The one exception appears to be the polyomaviruses, which appear to be relatively resistant to the inactivating effects both of gamma irradiation and UV-C irradiation. This family of viruses may best be inactivated using high-temperature short-time treatment (HTST), though the efficacy of this approach for the polyomaviruses has yet to be demonstrated empirically.

Assessing rapid viral enumeration/detection systems

By Dr. Ray Nims

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

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.

A much improved Ph. Eur. Chapter 5.3.2

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.

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

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.

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

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.

Remember....bacteriophage are viruses too

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.

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

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

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?

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!

The nuts and bolts of retrovirus safety testing

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

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.

Riboflavin plus UVA irradiation: another inactivation approach to consider

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.

Using porcine trypsin in biologics manufacture?

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.

Epizootic hemorrhagic disease virus: a future troublemaker?

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.