BVDV in commercial bovine serum...still?

by Dr. Ray Nims

One of the animal-derived materials (ADM) most commonly utilized for cell culture and for production of biologicals manufactured using cell cultures is bovine serum (most typically calf serum or fetal bovine serum). There is an inherent risk of introduction of adventitious contaminants (viruses and molllicutes) associated with the use of culture media containing serum. In fact, most of the viral contaminants that have been isolated from biologics bulk harvests (including REO type 2, Cache Valley virus, epizootic hemorrhagic disease virus, and vesivirus 2117) are believed to have been introduced via contaminated bovine serum. Another potential contaminant that may be introduced via bovine serum is the pestivirus bovine viral diarrhea virus (BVDV).

BVDV is a medium-sized (40-70 nm), enveloped, single-stranded RNA virus of the Flavivirus family. The regulatory requirements pertaining to the use of bovine materials for manufacturing biologics (9CFR113.47) contain specific instructions related to the detection of BVDV contamination. EMEA regulations require not only the testing of bovine sera for infectious BVDV, but also assessment of the presence of antibodies to BVDV. Neutralizing antibodies for BVDV are of concern since their presence could theoretically interfere with the detection of the virus in testing done for release of the serum. Although testing of bovine serum to be used for manufacturing biologicals is a regulatory expectation, experience has indicated that such testing is fraught with  false negative results. The relatively large volumes of serum that comprise a given batch are obtained by pooling large numbers of individal serum draws, and there is a chance of non-homogeneous contamination from a limited number of BVDV-infected draws.

How frequently has infectious BVDV been detected in commercially available bovine serum? What percentage of serum lots has been found to contain neutralizing antibodies to BVDV? Has BVDV genomic RNA invariably been found in bovine serum when tested by RT-PCR? These questions have been addressed by various authors over the past four decades.

Infectious BVDV continues to be detected in fetal bovine serum samples up to the present time. This reflects the fact that BVDV is distributed in cattle worldwide, subclinical infections with non-cytopathic BVDV are common in herds, and large serum pools are likely to be non-homogeneously contaminated with BVDV-infected serum. Over the past four decades, 667 lots of commercial fetal bovine serum have been examined for the presence of infectious BVDV in studies reported in the literature. Positive results have been reported for 29% of the lots examined, although the variability in frequency of detection has been quite large, as indicated by the range in the values that have been obtained in the various studies. The percentage of isolates comprising non-cytopathic BVDV has ranged from 98-100%, reflecting the continuing predominance of this variant over the cytopathic strains in cattle herds.

The frequency of detection of neutralizing anti-BVDV antibodies has ranged from 61% to 98% of fetal bovine serum lots. The overall number of commercial fetal bovine serum lots that have been evaluated for neutralizing antibodies to BVDV is 182, with antibodies being detected in 70% of these lots. 

Genomic RNA for BVDV has been detected in 79% of the 155 commercial fetal bovine serum lots that have been evaluated since 1996, when the RT-PCR methodology was initially applied to this question.

The risk of introducing infectious BVDV through contaminated FBS may be mitigated through gamma-irradiation of the FBS. BVDV is relatively sensitive to inactivation by this treatment. It is therefore unusual to detect infectious BVDV in gamma-irradiated serum, though in one case in the literature, a single lot of irradiated serum out of 9 lots tested contained infectious BVDV. This inactivation strategy used to mitigate risk of introducing infectious BVDV is not expected to reduce the frequency of detecting neturalizing anti-BVDV antibodies or genomic RNA for BVDV in the treated serum lots.

See Nims and Plavsic. The Pervasiveness of bovine viral diarrhea virus in commercial bovine serum.  BioProcessing Journal Winter 2012 /2013, 19-26 for the individual study results used to prepare the table shown above. 

Is Bovine Polyoma Virus Getting You Down?

By Dr. Ray Nims

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

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

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

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

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

Got Animal-Derived Materials? Part 3

By Dr. Ray Nims

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

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

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