Emerging Viral Risks and Mitigation Strategies in Biologics Manufacturing
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The document discusses the risks associated with emerging adventitious viruses that may contaminate raw materials in vaccine and biological product manufacturing, emphasizing the importance of risk assessments and mitigation strategies. It highlights specific viruses such as porcine circovirus type 3, hepatitis E virus, and Schmallenberg virus, detailing their impact on animal health and potential threats to human health. Additionally, it advocates for the use of next-generation sequencing as a vital tool for identifying these viruses and ensuring the safety of raw materials.
Emerging Viral Risks and Mitigation Strategies in Biologics Manufacturing
- 1. Merck KGaA Darmstadt, Germany Merck KGaA Darmstadt, Germany Reginald Clayton, Ph.D. Principal Scientist Global Field Development Services
- 2. 2 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. 1. Risk assessments 2. Emerging adventitious viruses & mitigation ▪ Porcine circovirus type 3 ▪ Hepatitis E Virus ▪ Schmallenberg virus ▪ Zika virus ▪ Borna disease virus 3. Quality by Design and holistic screening of raw materials 3
- 4. 4 Viral safety Lot Release Testing Viral clearance or inactivation Characterization of cell lines and raw materials Risk Assessment Recent knowledge, Literature, Experience, Regulatory
- 5. 5 Emerging viruses are potential contaminants of raw materials used in the manufacture of vaccines and biologicals Challenge to the safety testing of products intended for human and veterinary use. Risk mitigation strategies are necessary to preserve the integrity of the manufacturing process and reduce the likelihood of impact upon animal and human health Risk assessments of emerging viruses with potential to contaminate raw materials should inform the evaluation of detection platforms that support and enable risk mitigation. Emerging viruses – a constant challenge
- 6. 6 Sources of recent viral contamination
- 8. Mitigating the Risk from Two Porcine Adventitious Viruses: 1. Porcine Circovirus Type 3 2. Hepatitis E Virus
- 9. 9 An Emerging virus of pigs: Porcine Circovirus type 3 • Porcine circovirus type 3 (PCV3) is a widespread contaminant of the herd. • Demonstrated to cause disease in pigs: porcine dermatitis and nephropathy syndrome, and reproductive failure (Palinsky et al 2017, J Virol 91;e01879- 16) • PCV3 is genetically divergent to PCV1&2, and diverse strains of PCV3 exist (Fux et al 2018 Virol. J. 15:25-34) • The plasma burden of PCV3 has been determined at ~4 logs/mL in infected animals, and in aborted fetuses at ~7 logs per gram of material. • Cap and Rep genes of PCV3 are genetically divergent from PCV1&2. • Attempts at in vitro isolation & propagation of PCV3 unfruitful so far. • PCV3 identified by Next Generation Sequencing of diseased primary material (skin lesions).
- 10. 10 PCV3 is distinct from known strains of PCV1/2 Source: Palinsky et al, 2017 • Genetically distal clustering of PVC3 genomes indicates divergence of strains over time • Phenotypes of PCV3 not yet established, but early data show PCV3 refractile to culture • PCR for risk mitigation
- 11. Porcine materials and Hepatitis E Virus Hepatitis E virus (HEV) is a known contaminant of Porcine species and causes disease in humans: • 20 million cases worldwide, 70,000 deaths/annum • Typical presentation; malaise, raised ALTs/GSTs, 6 weeks duration, self limiting • Often asymptomatic where patient is immunocompetent • Often fatal for fetus & mother where infection occurs in first trimester • One serotype of HEV, but four major genotypes; 1&2 associated with humans, 3&4 have zoonotic origin • HEV culture is difficult: virus isolation is possible by culture, but a sufficiently sensitive in vitro assay is not yet available. • Risk mitigation by PCR assay 11
- 12. Poll Question
- 13. Mitigating the Risk of Schmallenberg Virus Contamination in Raw Materials and Cell Lines
- 14. 14 Regulatory Background • Appropriate species specific tests should be performed depending on the passage history of the cell line...(ICH Q5A R1). • CPMP Note for Guidance on Production and Quality Control of Animal Immunolgobulins & Immunosera for Human Use (CPMP/BWP/3354/99) – this lists the viruses of concern and highlights which are classified as pathogenic for humans. • In the past two-three years, rigorous regulatory scrutiny has been noted in regard to the use of antibodies & antisera during cell line development.
- 15. 15 Schmallenberg virus (SBV): genus Orthobunyavirus. Simbu serogroup. SBV infects cattle, sheep & goats resulting in severe birth defects and still birth. Geographical dissemination of SBV was rapid through Europe during 2011-2013 and is a concern for agriculture. An emerging threat - Schmallenberg virus
- 16. 16 Spherical, enveloped virion, 80-120 nm diameter Infects livestock via transmission through insect vectors Can cross placenta and infect fetus An emerging threat - Schmallenberg virus
- 17. 17 The risk assessment for Schmallenberg virus Animal serum, particularly bovine and Ovine/Caprine serum Bovine serum is known to harbour SBV in infected animals, and is known to cross the placental barrier, therefore bovine adult and fetal serum have associated risk. Manufacturing processes using bovine serum (adult, or fetal) should be tested for SBV. Cell lines exposed to Ovine or Caprine serum (antiserum for clonal selection) should be tested for SBV.
- 18. 18 Detection of SBV: Detector cells were challenged with 5-1200 TCID50 SBV and monitored for CPE over 14 days DC line Inoculum (TCID50) Time to CPE (days) BHK-21 1200 NT 50 2 5 2 Vero 1200 1-2 50 2 5 2 CHO-K1 1200 1 50 2-3 5 2-3 MRC-5 1200 14 50 NT 5 NT 324-K 1200 5-6 50 6 5 6 FLK 1200 7-11 50 10-12 5 10-12 BPEK 1200 5-11 50 11 5 11 BT 1200 14 50 18-20 5 18-20 MDBK 1200 - 50 NT 5 NT Vero C1008/SBV 5 TCID50 3d PI NC NC FLK/SBV 1200 TCID50 11d PI (Ilchmann et al, 2017, Biologicals, 49; 28-32)
- 19. Poll Question
- 20. Mitigating the Risk of Zika Virus Contamination in Raw Materials and Cell Lines
- 21. 21 Zika virus & safety of substance of human origin Manufacturers have the responsibility to perform a risk assessment • US FDA Guidance for Industry Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells, Tissues, and Cellular and Tissue-Based Products & Revised Recommendations for Reducing the Risk of Zika Virus Transmission by Blood and Blood Components Guidance for Industry (2016) • European Centre for Disease Prevention and Control, Scientific Advice2016 Zika virus and safety of substances of human origin (2016) • Committee for Medicinal Products for Human Use (CHMP), EMA Report on viral safety of plasma-derived and urine-derived medicinal products with respect to Zika virus. (EMA/CHMP/BWP/596747/2016)
- 22. 22 Virus characteristics Enveloped, (+) ssRNA virus Mosquito-borne Flaviviridae family, genus Flavivirus Origin of the recent epidemic First identified in rhesus monkeys (1947) Spread through South East Asia to French Polynesia & Pacific islands Early 2015 spread through South & Central America Multiple strains: African & Asian origin Zika virus: an emerging pathogen of humans Image: Deutsche Welle
- 23. 23 • In 80% of cases infection is asymptomatic (healthy adults) • Symptomatic infection manifests with rash, joint pain, conjunctivitis, and fever • In rare cases infection can cause neurological complications: • encephalitis • meningo-encephalitis • Guillain-Barré syndrome • birth defects termed congenital Zika syndrome (including microcephaly) Image Source: CDC Zika virus: Impact on human health
- 24. 24 Zika virus & safety of Substances of Human Origin (Zmurko et al 2018, J Gen Virol 99, 2219-229) • Plasma • Platelets • Convalescent serum • Monocytes • Heterologous T-cells • Albumins • Coagulating factors • Immunoglobulins • Human chorionic gonadotropin (hCG) • Human menopausal gonadotropin or menotropin (HMG) • Follicle stimulating hormone (FSH) • Urokinase Blood & blood components Urine-derived medicinal products Stem cells
- 25. 25 In vitro adventitious virus testing Maintain for 14 or 28 days, examination for CPE Sample inoculated onto multiple detector cells Negative result Positive result Examination for CPE Haemagglutination assay Haemadsorption assay Sample Detector cells: MRC-5, Vero, others End point testing
- 26. Detection of Zika virus strain MR766 by in vitro assay End point testing - Cytopathic effect • retarded cell growth • Morphological changes in detector cells • cellular lysis • visualized under light microscope Figure Legend: Micrographs show observations made in detector cells following ZIKV MR766 inoculation (panels a-f). NC: mock-infected cells. Scale bar is 1000 µm. Zika virus is robustly detectable by in vitro assay (Zmurko et al 2018, J Gen Virol 99, 2219-229)26
- 27. Detection of Zika virus strain PE243 by in vitro assay (Zmurko et al 2018, J Gen Virol 99, 2219-229) End point testing - Cytopathic effect • retarded cell growth • Morphological changes in detector cells • cellular lysis • visualized under light microscope Figure Legend: Micrographs show observations made in detector cell lines following ZIKV PE243 inoculation (panels a-f). NC: mock-infected cells. Scale bar is 1000 µm. Zika virus is robustly detectable by in vitro assay 27
- 28. A Largely Ignored, But Not Forgotten Adventitious Virus: Borna Disease Virus (BoDV)
- 29. 29 BoDV discovered in Borna, Germany, late 1800s Responsible for deaths and behavioural changes in horses Strong CNS tropism No classical symptoms (no raised ALTs, no rash), but psychological disturbances, and encephalitis in humans Documented infections in many species, with distribution of virus in CNS (Kinnunen et al J Gen Virol 2013, 94, 247- 262) Recent cases of transmission and fatalities highlight risks of human infection BoDV is a mononegalovirus Borna Disease Virus
- 30. HEV BoDV antigens in CNS of squirrels & Patients by IHC analysis. Viral antigen in neurons (black arrows), glial cells (black arrowheads). Recent cases highlight the BoDV risk • Several fatalities from zoonotic BDV infection (Hoffman et al 2015 NEJM 373;2 p154) • Fatalities following donor organ transplantation (European Centre for Disease Prevention and Control). Acute encephalitis associated with infection with Borna disease virus 1 – Germany, 2018. 26 March 2018. Stockholm: ECDC; 2018) (Hoffman et al 2015 NEJM 373;2 p154)30
- 31. 31 Mitigation of risk from BoDV • Potential sources of BoDV include domestic cats, rodents, and many other animals, therefore the risk profile may include diverse types of BoDV. • In vitro isolation and propagation of BoDV is relatively inefficient, and in vitro phenotypes of variant strains remain unclear; a molecular assay is best suited to detect BoDV. • Cell banks of human origin (Stem cells, ATMPs) should be screened by PCR. • Cell banks & raw materials with exposure to equine serum should be screened. • Risk profile: Substances of Human Origin must be considered at risk for BoDV contamination • Allogeneic stem cell banks, urine-derived products, plasma-derived products • ATMPs with substances of human origin (e.g. stem cell banks) must be screened for BoDV
- 32. Poll Question
- 33. Quality by Design and Holistic Screening of Raw Materials
- 34. 34 Emerging viruses are a constant. Diligence is required. Next Generation Sequencing (NGS; Massively Parallel sequencing, Deep sequencing) identifies all adventitious viruses, mycoplasma and bacteria NGS has enabled the detection of novel viruses but in biotechnology applications it also provides reassurance to regulators and sponsors that their product is safe! Applications for vaccines: Virus identification during contamination events Raw material testing Cell substrate characterization Virus seed characterization
- 35. 35 Next Generation Sequencing Advantages No pre-selection of nucleic acids: agnostic technique Can identify contaminants with known genome sequences but also organisms with only 50% homology with known sequences Depth of sequencing may allow construction of whole genome of a contaminant Will be quicker than many classical infectivity assays Considerations Detects nucleic acids and not infectious particles Requires powerful bioinformatics algorithms Careful interpretation is required to avoid false positives Need experienced virologist to interpret data
- 36. 36 Raw material qualification: Holistic assessment enabled by Next Generation Sequencing (NGS) • Raw materials batch-batch variation • Novel production substrates • Bespoke custom media components • Novel risk profiles • Viruses not yet discovered.
- 37. 37 Summary Risk assessments evolve over time: new knowledge informs risk assessments Specific viruses may require targeted PCR assays to enable testing In vitro assays enable sensitive and robust detection of many emerging viruses Next Generation sequencing enables the identification of novel risks, and therefore informs the risk assessment of raw materials. We have not seen the last of emerging viruses, and vigilance must be constant.
- 38. CVR Medical Research Council University of Glasgow Centre for Virus Research Prof. Alain Kohl Dr Claire Donald Alison Armstrong, Ph.D. Jo Zmurko M. Sc. Anne Ilchmann Ph.D. Principal Scientist, Field Development Services (EMA) [email protected] ACKNOWLEDGEMENTS REGINALD CLAYTON, PHD The vibrant M and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Thank you for your attention. Questions?