Viral contamination in biologic manufacture and implications for emerging therapies

In the paper

“The lessons of the past have led to the current best practice, which relies on three pillars: the selection of appropriate starting and raw materials with a low risk of containing adventitious virus; testing of cell banks and in process materials to ensure they are free from detectable viruses; and finally, the incorporation of steps to remove and inactivate potential undetected adventitious and”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [9] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Q5A(R1). Viral Safety Evaluation Of Biotechnology Products Derived From Cell Lines Of Human Or Animal Origin (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 1999).
• Partly supported

  Split into 3 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 2 unverifiable. Expand the claim to see per-atom verdicts.

  cites [13] Grillberger, L., Kreil, T. R., Nasr, S. & Reiter, M. Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells. Biotechnol. J. 4, 186–201 (2009). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [14] Kozlowski, S. & Swann, P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv. Drug Deliv. Rev. 58, 707–722 (2006). source

In the paper

“At the end of Q3, 2019, more than 1,052 advanced therapy medicinal products (ATMPs) were being tested in phase I-III clinical trials (https://alliancerm.org/publication/q3-2019-data-report/) and several cell 45 and gene 46 therapies had received regulatory approval.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites https://alliancerm.org/publication/q3-2019-data-report/
• ?Not checked — no full text of the sourcecites [45] Murphy, M., Quesada, G. M. & Chen, D. Effectiveness of mouse minute virus inactivation by high temperature short time treatment technology: a statistical assessment. Biologicals 39, 438–443 (2011). source
• ?Could not verify

  The citation is attached to the statement that cell therapies had received regulatory approval; the Q3 2019 trial count is attributed to a separate URL. The Introduction and Discussion and conclusions address contamination and UV disinfection in biopharmaceutical manufacturing, not approvals of cell therapies. Searches for approved cell therapies and regulatory approval found no supporting passage in our copy. (LLM, conf=0.76)

  cites [46] Meunier, S. M., Sasges, M. R. & Aucoin, M. G. Evaluating ultraviolet sensitivity of adventitious agents in biopharmaceutical manufacturing. J. Ind. Microbiol. Biotechnol. 44, 893–909 (2017). sourceExperimental — accuracy not yet measured

In the paper

“In terms of safety implications, four (herpesvirus, human adenovirus type 1, parainfluenza virus type 3, and reovirus type 3) of the five viruses found to contaminate human and primate cell lines are known to be pathogenic in humans, whereas only one (Cache Valley virus) of the viruses found to contaminate CHO cell culture has been reported to cause disease in humans 36,37 .”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [36] Anonymous. A united front. Nature 472, 389–390 (2011).
• Partly supported

  The Conclusions report a fatal, culture-confirmed case of Cache Valley virus encephalitis in a human, supporting the claim that this virus can cause human disease. The paper does not establish that Cache Valley virus contaminated CHO cell culture, that it was the only such contaminant reported to cause human disease, or the comparison with viruses contaminating human and primate cell lines. (LLM, conf=0.86)

  cites [37] Campbell, G. L. et al. Second human case of Cache Valley virus disease. Emerg. Infect. Dis. 12, 854–856 (2006). sourceExperimental — accuracy not yet measured

In the paper

“Animal-derived raw materials (ADRMs), especially serum, carry a relatively higher risk of being contaminated with virus and are thus being replaced where possible throughout the industry 1,9,13 .”

What the sources say

• Partly supported

  The paper's introductory overview identifies contaminated raw materials as a route by which cell cultures acquire viruses, and its references include reports of viral contamination in calf serum. The passages retrieved do not establish that animal-derived materials pose a relatively higher risk than alternatives or that they are being replaced throughout the industry. (LLM, conf=0.73)

  cites [1] Merten, O. W. Virus contaminations of cell cultures — a biotechnological view. Cytotechnology 39, 91–116 (2002). sourceExperimental — accuracy not yet measured
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [9] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Q5A(R1). Viral Safety Evaluation Of Biotechnology Products Derived From Cell Lines Of Human Or Animal Origin (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 1999).
• Supported

  The paper’s opening summary says that concern about transmitting infectious agents through serum and other derived additives has driven their removal from recombinant-therapeutic manufacturing. It also says this practice is expanding throughout the industry; the claim’s reference to viral risk is consistent with its examples of hepatitis and HIV transmission. (LLM, conf=0.88)

  cites [13] Grillberger, L., Kreil, T. R., Nasr, S. & Reiter, M. Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells. Biotechnol. J. 4, 186–201 (2009). source

In the paper

“For recombinant viruses, master virus banks (MVBs) are generated and thoroughly characterized for contamination by adventitious viruses 52 .”

What the sources say

• Partly supported

  The risk-assessment discussion (p. 128) says master and working cell banks are extensively tested for viral contamination. It does not say that master virus banks are generated or characterized for recombinant viruses; cell banks and virus banks are distinct. (LLM, conf=0.78)

  cites [52] Shukla, A. A. & Aranha, H. Viral clearance for biopharmaceutical downstream processes. Pharm. Bioprocess. 3, 127–138 (2015). sourceExperimental — accuracy not yet measured

In the paper

“Additionally, determining if the contaminant identified in a nucleic acid based assay is biologically active may require a different method, though we would note that HTS of viral RNA has been used to demonstrate a virus is biologically active 61 .”

What the sources say

• ?Could not verify

  The Cells and Discussion sections describe CHO cell culture and growth, not viral testing. Searches of our full-text copy for HTS of viral RNA and high-throughput sequencing of viral contaminants found no passage addressing whether viral RNA sequencing can demonstrate biological activity. No page numbers were available in the indexed passages. (LLM, conf=0.78)

  cites [61] Miki, H. & Takagi, M. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media. Cytotechnology 67, 689–697 (2015). sourceExperimental — accuracy not yet measured

In the paper

“For GMP production, five of eight cell cultures preceding the reactor were originally identified as contaminated; one of six concurrent seed trains and no concurrent cell cultures for different products were also found to be contaminated.”

In the paper

“Despite this, the IVV assay was negative in four events and the contamination was detected by using another method (Table 3).”

In the paper

“Further, viral contaminations have been reported to lead to manufacturing interruptions (with a median plant downtime of one to two months, Fig. 3), loss of revenue, discarded batches, lawsuits against the company, a decrease in company reputation as reflected in stock value, as well as significant delays in product development.”

In the paper

“Nine out of the 20 responding companies (i.e., almost half of the involved industry respondents), reported having one or more adventitious virus contaminations in mammalian cell culture operations since 1985, totaling 18 virus contamination events reported to the CAACB.”

In the paper

“These events are rare; we are aware of only 26 virus contaminations over the past 36 years (Table 1) [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , 18 of which were reported directly as a part of this study.”

What the sources say

• ?Not checked — no full text of the sourcecites [18] Burstyn, D. G. Contamination of genetically engineered Chinese hamster ovary cells. Dev. Biol. Stand. 88, 199–203 (1996).
• ?Not checked — no full text of the sourcecites [19] Rabenau, H. et al. Contamination of genetically engineered CHO-cells by epizootic haemorrhagic disease virus (EHDV). Biologicals 21, 207–214 (1993). source
• ?Not checked — no full text of the sourcecites [20] Garnick, R. L. Experience with viral contamination in cell culture. Dev. Biol. Stand. 88, 49–56 (1996).

In the paper

“However, the number of contaminations reported to the CAACB per company does not correlate with total manufacturing volume, implying that a combination of circumstance, manufacturing controls in place, and prior lack of virus contamination disclosures may have contributed to this rate.”

In the paper

“Only after the viral load was increased to a detectable level, through concentration or biological amplification, was the virus detected in the raw material, and then only in three events (Fig. 1).”

In the paper

“Despite the fact that raw materials were determined to be the most likely source of the contamination in 11 events, testing those raw materials did not necessarily detect the contaminating virus.”

In the paper

“In comparison, for the human and primate cell lines, the manufacturing operators or cell line itself were suspected to be the source.”

In the paper

“The reported virus contaminations occurred at all stages of the product life-cycle, with three events occurring during pre-clinical non-GMP manufacture, two during clinical cGMP manufacture, and the remaining 13 occurring during commercial manufacture.”

In the paper

“Of the 20 companies completing the CAACB virus contamination survey, 45% of respondents had reported experiencing at least one virus contamination event between 1985 and 2018, which is higher than expected when the study began.”

In the paper

“However, in two events, there was no apparent change in cell culture performance and the contaminating virus was only detected by a virusspecific PCR assay.”

In the paper

“Because of this approach, these products have been safe for over 35 years, and to our knowledge, there has been no transmission of a contaminating virus to a patient from a therapeutic protein produced using recombinant DNA technology.”

In the paper

“The expense of such an investigation, as reported to the CAACB, was in the $1-10 million range, but in the worst cases the cost of investigating a contamination, implementing corrective actions, decontaminating the facility, and other ancillary costs could be in the hundreds of millions.”

In the paper

“In 67% of reported events, the manufacturing platform was Chinese hamster ovary (CHO) cells, whereas the other 33% of events involved human or primate cell lines.”

In the paper

“When a viral contaminant matched a PCR target, this was effective at preventing contamination of the entire manufacturing facility.”

In the paper

“In the other eight contamination events, virus testing of raw materials was negative and the source of the contamination was only identified using indirect evidence.”

In the paper

“It is important to note that in all cases reported to the CAACB no drug substance produced from a contaminated cell culture was ever released for human use, and all such drug substance was subsequently destroyed.”

In the paper

“Our study showed that the control of raw materials by direct testing had limited value (Fig. 1), either due to virus concentrations in the raw material being below the assay detection limit, or because the contaminating virus was not homogeneously distributed in the raw material.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Fig. 1

In the paper

“The CAACB study has identified five viruses that have been shown to contaminate CHO cell culture, and four viruses that have contaminated cell culture with human or primate cells.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Table 2

In the paper

“Reovirus / Unknown 34 events, the contamination was contained in the cell culture, for 6 events the contamination was spread to downstream purification operations, and for 5 events the extent of contamination was unknown.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Fig. 2

In the paper

“Whereas learning from these events has historically been a challenge, the work presented here represents the first comprehensive collection and analysis of previously unpublished industry wide viral contamination information.”

In the paper

“Despite this excellent safety record, viral infection of mammalian cell culture is a real risk with severe consequences.”

In the paper

“This included the contamination of poliovirus vaccine with Simian Virus 40 (SV40) 3 , for which the health impacts were not fully known for many decades 4 .”

What the sources say

• ?Not checked — no full text of the sourcecites [3] Shah, K. & Nathanson, N. Human exposure to SV40: review and comment. Am. J. Epidemiol. 103, 1–12 (1976). source
• ?Not checked — no full text of the sourcecites [4] Stratton, K., Alamario, D. A. & McCormick, M. C. Immunization Safety Review: SV40 Contamination of Polio Vaccine and Cancer. (National Academies Press, Washington, DC, 2003).

In the paper

“The implication is that different host cells may require the consideration and management of different virus contamination risks, with human and primate cell lines being more susceptible to contamination from operators.”

In the paper

“Data collected as a part of the CAACB study indicate that current manufacturing controls used to prevent the spread of a potential contaminant within manufacturing facilities are effective as no cross-contamination of other concurrent manufacturing operations was reported.”

In the paper

“This result is not unexpected as CHO cells are the most commonly used host cells by the recombinant biologic-industry with published reports indicating that approximately 70% of approved biotech products are manufactured using CHO 12 .”

What the sources say

• ?Not checked — no full text of the sourcecites [12] Walsh, G. Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32, 992–1000 (2014). source

In the paper

“The IVV assay also takes 14 to 28 days-much too long to assess the reactor for contamination prior to processing downstream for purification.”

In the paper

“The in vitro virus (IVV) assay is a cell-based assay used to test harvest cell culture samples for potential viral contaminants 38 .”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Sexton, D. J. et al. Life-threatening Cache Valley virus infection. N. Engl. J. Med. 336, 547–549 (1997). source

In the paper

“Minute virus of mice is especially challenging as a potential contaminant.It is shed from ever present wild mouse populations, may not be detectable even with established rodent control, and can persist in the environment and in raw materials long after being shed.”

In the paper

“These data imply that the safety of biologic products should not rely on testing alone (including orthogonal methods) but be assured by multiple controls (including prevention, detection, and viral clearance) throughout the process.”

In the paper

“In the case of living cell-based therapies, viral clearance would be required to remove or inactivate viruses in the cell culture supernatant as well as separating or destroying infected cells, which harbor virus, from any cells not infected with virus.”

In the paper

“The three main risks for viral contamination in cell culture for therapeutic production are cell sources, materials utilized in cell culture, and exposure of the cell culture process stream to the operator/environment.”

In the paper

“However, due to the scale of their manufacture, ATMPs may rely much more on open cell culture transfers than recombinant proteins and vaccines.The result is an increased possibility of virus contamination from open operations for these types of products.”

In the paper

“Despite the effectiveness of treating raw materials, we note that some animal-or human-derived materials may be sensitive to heat, radiation, or UV exposure, which may ultimately affect cell growth and performance.”

In the paper

“Additionally, new detection technologies, such as high-throughput sequencing (HTS), have detected adventitious viral contaminants in the presence of virus product 54 and are being explored for use in the industry 55,56 .”

What the sources say

• ?Not checked — no full text of the sourcecites [54] Wirth, T., Parker, N. & Ylä-Herttuala, S. History of gene therapy. Gene 525, 162–169 (2013). source

In the paper

“These are filter sterilized before use, typically with 0.1µm rated sterilizing-grade filters through which most viruses will pass.”

In the paper

“Tests to assure that the derived cells are free of adventitious virus generally cannot be completed prior to initiating cell therapy manufacturing and the process generally proceeds at risk.”

In the paper

“This is never more true than when faced with a previously unknown emerging virus, such as SARS-CoV-2, where the capacity of the virus to infect production cell lines or be detected in existing assays is not initially known.”

In the paper

“Animalderived (Table 2) and human-derived components (e.g., serum, growth factors), which carry a higher risk of virus contamination than other media components 47 , are commonly added to media for ATMP production 48 .”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [47] Mundt, W. et al. Virus filtration of cell culture media. Patent WO2013192009A1 (2003).
• ?Not checked — no full text of the sourcecites [48] Mullard, A. Second anticancer CAR T therapy receives FDA approval. Nat. Rev. Drug Discov. 16, 818 (2017). source

In the paper

“This can be a challenge for some ATMPs, especially those where nutrient requirements are not well understood, such as primary cell cultures, or which may have variable starting cells, such as autologous cell therapies.”

In the paper

“Cell culture processes employed for the manufacture of biopharmaceutical products and ATMP products all utilize a variety of basal media formulations comprised of a mixture of more than 50 essential nutrients (e.g., amino acids, vitamins, trace elements) and other chemicals.”

In the paper

“High-throughput sequencing provides a greater breadth of detection than PCR and is seeing widespread interest from the vaccine and recombinant protein industry 56 .”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [56] US Food and Drug Administration. Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (US Food and Drug Administration, 2010).

In the paper

“Viral-vectored gene therapy products utilize plasmids or recombinant viruses to initiate production 51 .”

What the sources say

• ?Not checked — no full text of the sourcecites [51] Brindley, D. A. et al. Peak serum: implications of serum supply for cell therapy manufacturing. Regen. Med. 7, 7–13 (2012). source

In the paper

“As noted above, virus-risk mitigation for biopharmaceutical manufacturing uses three complementary approaches: 1) preventing virus entry by selecting low risk starting and raw materials and utilizing manufacturing controls, 2) testing of in process materials to ensure they are free of virus and enable lot rejection and 3) clearance of virus (inactivation and/or removal) from the product 9,14 .”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [9] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Q5A(R1). Viral Safety Evaluation Of Biotechnology Products Derived From Cell Lines Of Human Or Animal Origin (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 1999).
• ?Not checked — no full text of the sourcecites [14] Kozlowski, S. & Swann, P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv. Drug Deliv. Rev. 58, 707–722 (2006). source

In the paper

“A delay in administering treatment, such as due to contamination of a production process, could negatively impact patients, possibly leading to their death.”

In the paper

“These differential clearance strategies, coupled with an understanding of the most likely viral risks, potential sources of virus, and the host cell line susceptibility to those viruses, could enable the development of a virus removal strategy.”

In the paper

“This is often more challenging in ATMP manufacturing as there are more open manipulations that provide opportunity to introduce an environmental contaminant and many small lots may be manufactured at once.”

In the paper

“Additionally, new detection technologies, such as high-throughput sequencing (HTS), have detected adventitious viral contaminants in the presence of virus product 54 and are being explored for use in the industry 55,56 .”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [55] US Food and Drug Administration. Draft Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) (US Food and Drug Administration, 2018).
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [56] US Food and Drug Administration. Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (US Food and Drug Administration, 2010).

In the paper

“Virus contamination during ATMP cell-culture manufacturing processes by environmental sources must be strictly avoided and can be done so by the use of functionally closed systems, which often employ single-use, disposable equipment.”

In the paper

“While challenging to test for adventitious viruses in the presence of recombinant viral stocks, approaches to develop successful virus tests have been developed and used 53 .”

What the sources say

• ?Not checked — no full text of the sourcecites [53] Ye, G. J. et al. Herpes simplex virus clearance during purification of a recombinant adeno-associated virus serotype 1 vector. Hum. Gene Ther. Clin. Dev. 25, 212–217 (2014). source

In the paper

“However, current HTS sample preparation approaches and bioinformatic pipelines are not as rapid as PCR and can take 7-10 days 55 .”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [55] US Food and Drug Administration. Draft Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) (US Food and Drug Administration, 2018).

In the paper

“Many of the virus clearance unit operations used during purification of therapeutic proteins described earlier are not suitable for use with, or have not been widely applied to, ATMPs.”