Bovine Virus Contaminationin Cell Banks used in Biopharmaceuticals and Vaccines
25th November 2011
By: Dr Daniel Galbraith, CSO
The risk of contamination of biopharmaceuticals and vaccines by Bovine viruses has been well recognized. Calf serum was, and in some instances still is, commonly used to supplement the growth media for cells used in production of many vaccines and some biologics. Although “serum-free” media is frequently used today the risk of exposure to bovine viruses is still present as production cells have been grown in bovine derived media supplements in the past and therefore have the virus risk.
The regulatory agencies in both Europe (EMA) and USA (FDA) have recognized the threat the contamination of bovine viruses cause. There are a number of guidelines which have required manufacturers to ensure that if cells have been exposed to bovine derived supplements during their history, tests have been done to ensure the safety of the products. The US Code of Federal Regulations (9CFR) was perhaps the first to specify testing for bovine viruses in serum used in production. These included Reo and Rabies virus, Blue tongue virus, Bovine adenovirus, Bovine respiratory syncytial virus and Bovine virus diarrhea virus (BVDV). BVDV is perhaps the most common virus detected in serum and is known to have contaminated many cell lines in the past. Recently there have been improvements in the selection and manufacture of the serum particularly since the bovine spongiform encephalitis issues in the 1990’s has meant that the serums used today are a very low risk of virus contamination. There are a number of viruses, not specified in the 9CFR, which are clearly a risk and have been shown to infect production batches of biologics. Vesivirus and some Arboviruses (Cache Valley virus) which have been widely reported to have infected production cells following contamination from a bovine source. Viruses which were missed by the traditional methods of detection and are now required to be tested for using other techniques, usually Polymerase Chain Reaction (PCR).
The traditional technique, applied by many contract testing companies, to detect bovine viruses has been by in vitro cultivation and immune-staining to identify specific viruses, essentially the CFR method (9CFR 113). While this technique has many advantages in the ability to detect many species of virus it is limited to be able to only detect those strains of virus which are able to grow well in cell culture. This is important to understand as many viruses grow poorly using the traditional method (as in the case of Bovine polyoma virus or Cache Valley virus) which can lead to an assay which can miss some viruses. The CFR method is designed as a test for raw materials, bovine serum, but is now being applied to test cell banks and other in-process materials. This 9CFR method uses 2 cell lines – Vero and BT and requires the test material to compose a percentage of the growth medium, something which is easy to do when the test material is a serum sample but is impossible if the material under test is a cell bank. The test itself is very complex in methodology, requiring a long period in culture and a number of virus spikes of the test material. One of the spikes is Rabies virus – something almost impossible to perform in the UK, although the CFR allows the omission of this test in such circumstances. The end point of the assay is haemadsorbtion and a specific immunofluorescence assay for a number of viruses, observed by microscopy. The reason for the spikes is to ensure that there is no inhibitory effect of the sample, which is understandable when the sample is serum but less so when the sample is a serum-free cell pellet and is perhaps above and beyond what is required for these types of samples, which frequently have not been in contact with any source of bovine material for a considerable time. An alternative technique is the use of the PCR to detect those viruses which are mentioned in the guidelines and those likely to be at high risk of contamination. This may extend the number to 10 viruses including Reo and Rabies virus, Blue tongue virus, Bovine adenovirus, Bovine respiratory syncytial virus and Bovine virus diarrhea virus, Bovine polyoma virus, Cache Valley virus, Bovine circovirus and Vesivirus. These tests are almost always performed along with a general in vitro assay when testing cell banks which has the ability to detect a wide variety of viruses not covered by the specific PCR assays. The detection of viruses by molecular techniques has become common place since the 1990’s and is frequently accepted in preference to other methods. It is more sensitive in many cases, much faster and cost effective than in vitro techniques. The drawback of PCR is that it detects genome rather than active virus and as a consequence may overestimate the amount of virus present particularly the amount of inactive virus. Both BVDV and Bovine polyoma virus sequences are commonly found in calf serum but seldom, if ever, as active virus. From a regulatory point of view the choice of method should be the most optimum for detection, and this would be PCR.
This, therefore, presents the manufacturer of a biologic with a conundrum: either to perform an assay which was not designed for the test material (9CFR) or to perform a PCR test for specific viruses and use the ability of the in vitro assay to detect any adventitious viral contamination. The guidance in this case is ambiguous and the application of regulations by the regulatory reviewer unpredictable. The 9CFR based assay is widely used and is perhaps the safest to choose but also supplemented with PCR to pick up viruses not easily identified using this method. There does need to be some clearer indications from the regulators to help manufacturers in this important matter.
1. EMA/CHMP/BWP/367751/2011 Concept paper for a guideline on the quality of porcine trypsin used in the manufacture of human biological medicinal products.
2. 9 CFR 113.53 Requirements for ingredients of animal origin used for production of biologics.
3. 9 CFR 113.53 Detection of extraneous viruses by the fluorescent antibody technique.