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.

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.

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.

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.