Update: New USP General Chapter 1050.1

By Dr. Ray Nims

There is a new general chapter being prepared for inclusion in the United States Pharmacopeia (USP). It will be entitled “Design, Evaluation, and Characterization of Viral Clearance Procedures” and will be numbered 1050.1 to associate it with the current General Chapter <1050>.

A little history is called for to make this association more clear. Chapter <1050> first appeared in supplement 10 of USP23-NF-18 in 1999. It was, and still is, a verbatim copy of the International Conference on Harmonisation (ICH) document Q5A R1. In fact, it has an identical title: “Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin”.

In many respects, U5P Chapter <1050> (and ICH Q5A R1) is similar in content and philosophy to the 1993 US FDA Points to Consider (PTC) document entitled "Characterization of Cell Lines used to Produce Biologicals".  Like the 1993 PTC, USP Chapter <1050> expresses an overall safety paradigm composed of three orthogonal approaches. These approaches may be summed up as consisting of testing of raw materials and cell banks, testing of unprocessed bulk harvest, and evaluation of viral clearance steps used during purification. Also like the 1993 PTC, Chapter <1050> describes potential sources of viral contamination, including raw materials and the cell substrate. The scopes of the documents are similar, applying to products derived from cell lines. Transmissible spongiform encephalopathy agents and live or inactivated intact viral vaccines and gene therapy vectors are out of scope and are covered by other guidance documents.

Due to the potential for introducing a viral contaminant through use of an infected cell substrate (e.g., SV40 in polio vaccine, or more recently PCV-1 in rotavirus vaccine), USP Chapter <1050> and the 1993 PTC address the concept of cell banking and the extensive viral testing that is required for cell banks at the Master Bank, Working Bank, and Limit of Cell Growth levels. The second approach of the safety paradigm is the requirement for lot-to-lot testing of the unprocessed bulk harvest. Testing is done at this level as opposed to purified product since purification is designed to eliminate viruses and therefore might preclude the manufacturer from discovering a viral infection. It is the intent of the regulatory agencies that potential opportunities for introduction of a viral contaminant be investigated and remediated, so testing is done prior to purification. The viral testing that is required for unprocessed bulk harvest is a subset of the tests done on the cell banks.

Both USP Chapter <1050> and the 1993 PTC discuss the limitations of viral testing as a sole means to assure viral safety, and this provides a segue for discussion of the need for viral clearance steps and the validation of the ability of such steps to clear viruses from the product. In fact, USP Chapter <1050> does a good job of providing the fundamentals of viral clearance evaluation, however there was a feeling expressed by some in the industry (and especially Mike Rubino, a member of the original ad hoc advisory panel for this chapter) that more detailed guidance on experimental design was needed. This, by the way, is true in general about Chapter <1050> (and IQH Q5A Rl upon which USP <1050> was based) that it is strong on philosophy but weak on specific methodological detail.

The original USP ad hoc advisory panel for Chapter <1050> revision was assembled with the purpose of updating the chapter to include this missing experimental detail. The panel went through each section of the existing chapter and added the detail that was felt to be lacking. This was done with the overall mandate to avoid introducing any new language that might conflict with the original language (and ICH Q5A Rl). The revised Chapter <1050> was published in the Pharmacopeial Forum in 2010. Responses obtained indicated that there was reluctance to modify in any way the language of this chapter without also modifying ICH Q5A. The situation as of February 2011 was described in an earlier posting.

As a result, the USP decided to keep Chapter <1050> intact and identical to ICH Q5A Rl.  A new ad hoc advisory panel was assembled with the purpose of producing a new chapter in the general information series that would be a companion to the existing Chapter <1050>. The new chapter received the number 1050.1 and provided the vehicle for adding the desired methodological detail.

The proposed new General Chapter <1050.1>, entitled "Design, Evaluation, and Characterization of Viral Clearance Procedures" consists of some background information on process evaluation and process characterization, then launches into experimental design for evaluating both inactivation and removal steps. The latter section includes some specific experimental design flow charts addressing virus removal by filtration and chromatography and virus inactivation. Along with the flow charts describing the design of the studies are description of the methods used to assess potential cytotoxicity or interference caused by the process materials themselves.

The subheadings of the background information are shown in the text box below.

The information presented under these subheadings reflects the panel's understanding of current regulatory expectations. Regulatory input obtained during the public comment period will assure that the panel's perceptions were correct. Discrepancies will be corrected as required.

Finally, a goal of the new chapter was to provide an updated list of the types of viruses that have been and may be used in viral clearance evaluations. For studies enabling clinical trials, it is common for evaluations to use a parvovirus such as MMV and a retrovirus such as X-MuLV as models. This provides a small non-enveloped virus to challenge filtration steps, as well as an enveloped virus to assess inactivation steps designed for lipid enveloped viruses. For BLA enabling studies, a few additional viruses may be selected from this updated list, keeping in mind that the viruses should represent a diversity of characteristics (envelope status, genome type, size, etc.).

The intention of the new chapter <1050.1> is that manufacturer’s may use the methods as appropriate to their own processes and will be able to cite the guidance in their descriptions of the study design. At the present time, no guidance having sufficient experimental design descriptions is available to cite in this respect. The planned date for publication of the proposed chapter in the Pharmacopeial Forum is January 2013.

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.

Getting a grip on prophage

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.

Cell Culturists….Are your human cells authenticated?

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.

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.

What cell line is this anyway?

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.