Viral Risk Mitigation Strategies: Key Considerations in the Prevention and Detection of Viral Contamination
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Regulatory guidelines have defined industry best practices around adventitious virus contamination and risk mitigation in terms of patient safety. Today, the industry is taking a closer look at minimizing the business risk associated with viral contamination and is taking a more directed view of risk mitigation. This approach includes virus prevention and detection, in addition to removal. From cell culture seed train to final fill vial, this presentation will describe: -Potential risks associated with different areas of biotech processes -What can be done to minimize adventitious virus risk in those areas. The overarching strategy of risk mitigation will include evaluation of raw materials, modified expression systems, environmental controls, upstream and downstream processing, as well as testing and regulatory considerations.
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Viral Risk Mitigation Strategies: Key Considerations in the Prevention and Detection of Viral Contamination
- 1. Merck KGaA Darmstadt, Germany Alison A Armstrong, PhD Global Head of Field Development Services Key Considerations in the Prevention and Detection of Viral Contamination Viral Risk Mitigation Strategies
- 2. 2 The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. Viral Risk Strategies Content 2 1 Sources & Risks of Viral Contamination Complexity of biomanufacturing & regulatory requirements Developing a Biosafety Strategy Industry expectations and detection methods 3 Viral Risk Mitigation Prevent, Detect, and Remove 3 3
- 4. Sources & Risks of Viral Contamination
- 5. Sources & Risks of Viral Contamination Adventitious Agents Medicinal products during their development, and prior to being marketed, must meet strict criteria of Quality, Safety and Efficacy. Safety considerations are associated with contaminants in biological products including the following agents: Bacteria Fungi Mycoplasma Viruses Transmissible spongiform encephalopathies (TSE) 5
- 6. Growing Diversity/Complexity of Biologics Sources & Risks of Viral Contamination A biologic is a therapeutic substance that is produced through a biological process (often involving biotechnology methods), rather than chemical synthesis (as for traditional pharmaceuticals). Types of biologics include: recombinant protein, antibody, antibody-drug conjugate, cell component or derivatives, vaccines, gene therapies, and cell therapies. ► The manufacturing of biologics is complex. Raw materials Assembly at different sites Safety testing Timothy Moore, Senior Vice President, Global Head, Pharmaceutical Technical Operations Biologics Unit, Genentech https://www.gene.com/stories/how-hard-can-it-be 6 6
- 7. Case Studies of Microbial Contamination in Biologic Product Manufacturing Suvarna, K., Lolas, A., Hughes, P., Friedman, R. Biotechnology Manufacturing Team, Division of Manufacturing and Product Quality, Office of Compliance, Center for Drug Evaluation and Research, Food and Drug Administration Facility Equipment Process Materials Utilities Personnel Each source is a potential entry point for microbial contamination Acholeplasma laidlawii (<0.2um) Leptospira species (>5 um) Sources of Adventitious Agent Contamination Biosafety: A Challenge and Business Risk Minute virus of mice (MVM) ~18-24nm 7
- 8. Routes of Contamination Secondary Clarification Chromatography Protein A Column Protection Column Protection Final FillingFinal Sterile Filtration Concentration and Formulation Bulk Storage and Transport Viral Inactivation Column Protection Chromatography Purification Chromatography Polishing Virus Filtration Clearance TFF Bioreactor Column Protection Chromatography Purification Primary Clarification MCB WCB Seed Train Raw Materials Filtration 8 8
- 9. Regulatory Requirements and Guidance Sources & Risks of Viral Contamination ► To ensure the quality, safety and efficacy of biotherapeutic products intended for use in humans ► Guidelines are based on experience gained over three decades in this technically demanding field 9 9 • FDA: U.S. Food and Drug Administration • FDA: Center for Biologics Evaluation and Research • European Pharmacopoeia • CFDA: China Food and Drug Administration • World Health Organization • USP • NIST: National Institute of Standards and Technology • FDA: Center for Drug Evaluation and Research • ICH • European Medicines Agency • Pmda: Pharmaceuticals and Medical Devices Agency • ChP • JP: Japanese Pharmacopoeia • Ministry of Food and Drug Safety
- 10. ICH Q5A: Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (Apr 1997 to Feb 2000) ICH Q5B: Analysis of the expression construct in cells used for production of rDNA derived protein products (Dec 1995 to Jan 1998) ICH Q5C: Stability testing of biotechnological / biological products (Dec 1995 to Jan 1998) ICH Q5D: Derivation and characterisation of cell substrates used for production of biotechnological/biological products (Sept 1997 to July 2000) ICH Q5E: Comparability of biotechnological / biological products subject to changes in their manufacturing process (Dec 2004 to June 2005) Regulatory Requirements and Guidance Sources & Risks of Viral Contamination 10
- 11. FDA Guidance for Industry: Characterization and Qualification of Cell Substrates and other Biological Starting Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases (Draft 2006, Final 2010) EMA Guideline on Virus Safety Evaluation of Biotechnological Investigational Medicinal Products (EMEA/CHMP/BWP/398498/2005) July 2008 World Health Organization recommendations for the Evaluation of Animal Cell Cultures as Substrates for the Manufacture of Biological Medicinal Products and for the Characterization of Cell Banks. 2011 TRS 978 Annex 1 Regulatory Requirements and Guidance Sources & Risks of Viral Contamination 11
- 13. Cell Type Viral Contaminant Source of Virus Various BVDV (non-cytopathic) Bovine serum BHK/CHO Reovirus Bovine serum CHO EHDV Bovine serum (not screened) CHO Cache Valley Virus Bovine serum (not screened) CHO Minute Virus of Mice Components of media CHO Calicivirus 2117 Bovine serum? Vero Porcine circovirus Porcine trypsin Vero Bluetongue virus ? Rhesus monkey kidney SV40 Primary cell line Various SMRV Other cell lines Insect Tn5 (High Five) Nodavirus Latent infection of cell Insect Sf9, Sf21 Rhabdovirus Latent infection of cell Contamination of Production Processes 13
- 14. Minute Virus of Mice (MVM) A Threat Even in Animal Component Free Processes Parvovirus (family Parvoviridae) Small (< 20 nm) non enveloped virus with 5-kb linear, ssDNA genome Extremely virulent One viral particle per liter of culture sufficient to generate a contamination event Difficult to remove from processes Present in animal component free processes Resistant to inactivation via chemical or physical means 14 14
- 15. Three Complementary Approaches Viral Risk Mitigation ✓ Selecting and testing of starting/raw materials Cell line Culture media, sera, supplements & reagents Drug substance/component ✓ Testing the product intermediates at appropriate steps of production MCB, WCB, EOPC Bulk harvests ✓ Assessment of the capacity of USP and DSP to clear viruses Low pH inactivation Chromatography Nanofiltration 15 15
- 16. You may not find what is truly there… Sensitivity of assay limits detection All assays have a LOD Sample volume limitations Cell lines may not be permissive for some known or novel viruses Interference/matrix effects Anti-virus antibodies in FCS used in in vitro assays Cytotoxicity of indicator cells Inhibition of PCR assay enzymes You only find what you are looking for…. Screening assays are designed to assess known/past pathogens Adapted from K Brorson, US FDA Detection Issues with Detection Assays 16
- 17. • Cell banks should be prepared, stored and tested under GMP conditions • Reduces risk of contamination “During the establishment of the cell bank, no other living or infectious material (e.g. virus, cell lines or cell strains) should be handled simultaneously in the same area or by the same persons.” “Following the establishment of cell banks, quarantine and release procedures should be followed. This should include adequate characterization and testing for contaminants.” “Cell banks should be stored and used in such a way as to minimise the risks of contamination (e.g. stored in the vapour phase of liquid nitrogen in sealed containers)” • EU Guidelines for GMP for Medicinal Products for Human and Veterinary Use, Annex 2, Manufacture of Biological Active Substances and Medicinal Products for Human Use. Production of Cell Banks under GMP Conditions 17
- 18. Virus Contamination Prevention Options: Detection Secondary Clarification Chromatography Protein A Column Protection Column Protection Final FillingFinal Sterile Filtration Concentration and Formulation Bulk Storage and Transport Viral Inactivation Column Protection Chromatography Purification Chromatography Polishing Virus Filtration Clearance TFF Bioreactor Column Protection Chromatography Purification Primary Clarification MCB Pretreated Raw Materials FiltrationHTST γ Radiation UV-CRaw Materials Virus Resistant Cell Line Seed Train WCB = Routes of contamination = Upstream Virus Prevention = Downstream Virus Removal = Points for testing 18 18
- 19. Rigorous Screening of Cell Banks Viral Risk Mitigation • Starting material for the whole of the production process • Full characterisation for microbial and viral contaminants • One time testing MCB • Small number of passages beyond MCB • Reduced package of testing on cells directly from 1st WCB • Full characterisation for subsequent WCBs WCB • “Worst case” for amplification of contaminants • Full characterisation, one time testing at production scale • For “well characterized cell lines” may not be required at early clinical development stages EPC/CAL 19 19
- 20. Broad Range Virus Detection Approach Viral Risk Mitigation viruses detected by cytopathic effects (CPE), haemadsorption, haemagglutination, IFAIn vitro virus assay •Not all viruses produce CPE •Can only use a limited number of detector cells •Not all viruses grow well in tissue culture injection into suckling mice, adult mice, embryonated eggs (allantoic cavity and yolk sac) and (Guinea pigs)In vivo virus assay •Viruses detected by morbidity and mortality •Classical virus isolation methods that may detect viruses that do not grow well in tissue culture •Ongoing discussion about the sensitivity and usefulness of this assay detects intracellular virus particlesElectron microscopy •No amplification potential •Required specialized resources capable of detecting a broad range of microorganisms including the unknownsNext generation sequencing •Use is not yet specifically defined for most applications, but may be inferred under use of ‘state of the art’ technologies •US FDA have suggested using NGS for the analysis of gene therapy viral vectors •Required specialized resources 20
- 21. A Powerful Tool for Detection of Adventitious Agents Next Generation Sequencing ✓ Tool to detect contaminants ✓ No selection of nucleic acid prior to sequencing ✓ Depth of sequencing may allow construction of whole virus genome Sample Processing Sequencing Bioinformatics Next Generation Sequencing (NGS) 21 21
- 22. • Adventitious Agent Testing • Raw Material Qualification • In-process Testing • Lot Release Testing • MVSS Identity Genetic Purity • Cell Line Identity/Purity • Engineered Culture Purity • Genetic Stability A powerful tool for the detection of unknown adventitious agents Applications in Use Today Next Generation Sequencing 22 22
- 23. Regulatory Acceptance Next Generation Sequencing ✓ Recent WHO and European Pharmacopoeia documents have indicated the potential usefulness of NGS for viral vector identification/stability, recombinant cell line genetic stability and adventitious agent detection ✓ Draft EP 5.2.14. Substitution of in vivo method(s) by in vitro method(s) for the quality control of vaccines ‒ Novel, sensitive molecular techniques with broad detection capabilities are available, including deep sequencing or high throughput sequencing methods. ‒ The use of these new broad molecular methods has highlighted the gaps with the existing testing strategy by identifying previously undetected viral contaminants in final product, the cell banks from which it was produced and intermediate manufacturing stages. ‒ The implementation of such new broad molecular methods as substitutes for existing methods requires a comparison of the specificity (breadth of detection) and the sensitivity of the new and existing methods. ✓ Companies have used NGS for adventitious agent detection and submitted the data to regulatory agencies in support of the conventional testing also performed 23 23
- 25. A Multi-tiered Approach Viral Risk Mitigation Detect Virus Risk Mitigation Raw material control or barrier technology Process’s ability to reduce potential risk Ensure that the approach to risk works Plavsic, M. BioPharm International, May 2016. p.40 25 25
- 26. Sources of Contamination Exist at Multiple Stages Viral Risk Mitigation Facility registration (ISO, cGMP, FDA), component grade, component concentration rodent attractant Origin and Source Raw Materials Manufacturing Process and Facility Repacking of Material Shipping and Warehousing Geographical origin, synthetic or mined, fermented, plant or animal derived Added step to raw materials manufacturing Warehouse control, lack of transport vehicle control 26 26
- 27. Genetic Engineering of MVM Virus Resistance in CHO Cells Targets to Reduce MVM Uptake and Propagation Create a CHO parental cell line (CHOZN® cell line background), that is resistant to (or cannot propagate) an MVM infection while maintaining robust biomanufacturing performance. Infection initiation and entry - Capsid-mediated binding to one or more surface receptor(s) - Receptor-mediated endocytosis Transfer into/through the cytoplasm - Affected by a capsid-borne phospholipase Entry into the host nucleus - Replication 27 27
- 28. Raw Material Risk Mitigation Approach Viral Risk Mitigation Eliminating or reducing the load with a layered approach “Point-of-origin” • Low risk vendors • Pretreated products (HTST pre-treated glucose or gamma irradiated serum) • Material origin (serum sourcing for BSE mitigation) “Point-of-use” • Point of origin prevention will not mitigate all components • Virus barrier technologies • Mitigate contamination from shipping 28 28
- 29. Contamination Profile & Risk Profile Vary throughout the Process Focus Upstream Secondary Clarification Chromatography Protein A Column Protection Column Protection Final FillingFinal Sterile Filtration Concentration & Formulation Bulk Storage & Transport Viral Inactivation Column Protection Chromatography Purification Chromatography Polishing Virus Filtration Clearance TFF Bioreactor Column Protection Chromatography Purification Primary Clarification MCB WCB Seed Train Filtration Raw Materials 29 29
- 30. Preventing Contamination Adventitious Agent Safety of Upstream Raw Materials Consider virus resistant cell lines •Centinel® technology, first commercially available gene editing tool to confer Minute Virus of Mice (MVM) resistance to CHO cell lines Replace animal derived components in media • Recombinant proteins (r-Insulin, r-Trypsin, etc.) • Serum alternatives Source lower risk animal derived components • Source from lower risk geographies • Irradiation of raw material • Viral testing (CFR9) of raw material by manufacturer Adopt of chemically defined animal derived component-free media • Most conservative approach 1 2 3 4 30 30
- 31. Upstream Prevention Technologies Prevention Strategies 102°C for 10-15 seconds 25 – 40 kGy exposure 254 nm wavelength Size exclusion removal HTST Pasteurization Gamma Radiation UV-C Radiation Microbiological or Virus Barrier FiltrationPretreatment option Pretreatment option Pretreatment option 31 31
- 32. Preventing Contamination Raw Material Pre-Treatment Technology Robust Clearance Media Compatibility Point of Use Scalability Cost Effective HTST (~102C ~10 sec) Yes Component dependent Yes Large Scale Yes at Large Scale UV-C (254 nm) Organism dependent Component dependent Yes Challenging at large scale Yes but challenges at large scale Gamma – Radiation Yes Component dependent No Small batches Yes Upstream Virus Barrier Filtration Yes by size exclusion. Consistent LRV Yes Yes Yes Yes upstream virus filters 32 32
- 33. Prevention Strategies Virus Barrier Filters (Media Nanofiltration) Specifically designed for media filtration Broadly effective against adventitious agents Low/no capital Easy to scale (robust scale-down model) Easy to validate Small system footprint No impact to cell culture media Low development & validation costs High operating expenses Media dependent throughput 33 Benefits Limitations 33
- 34. Contamination Profile & Risk Profile Vary Throughout the Process Focus: Downstream Secondary Clarification Chromatography Protein A Column Protection Column Protection Final FillingFinal Sterile Filtration Concentration and Formulation Bulk Storage and Transport Column Protection Chromatography Purification Chromatography Polishing Virus Filtration Clearance TFF Bioreactor Column Protection Chromatography Purification Primary Clarification MCB WCB Seed Train Raw Materials Viral Inactivation Filtration 34 34
- 35. Preventing Contamination Adventitious Agents Downstream Employ single-use systems • Closed systems and sterile connectors minimize the chance for microbial ingress from the environment and personnel • Eliminates need for CIP SIP, sanitization and storage Utilize sound process design for multi-use systems • Minimization of dead legs • Proper pipe sloping • Understand impact of temperature differentials • Verify torque settings Understand the risk profile of process chemicals • Chemicals for buffers, cleaning, sanitization, inactivation, and storage should be of suitable quality for pharmaceutical GMP production 1 2 3 35 35
- 36. Downstream Virus Clearance Technologies Removal Strategies Parvovirus LRVa 1-3 Retrovirus 1-4 Parvovirus LRVb > 4 Retrovirus > 5.8 Parvovirus LRVb > 4 Retrovirus > 6 Virus Flow-Through Protein A Chromatography Virus Inactivation Low pH Solvent/Detergent Virus Binding Chromatography Anion* IEX Size Exclusion Virus Nano-Filtration* Parvovirus LRV a 3.25 Retrovirus LRV 4.22 a) Remington et. al. Viral Clearance by Protein A, Anion Exchange and Cation Exchange Chromatography Steps, 2015 American Pharmaceutical Review. b) Merck KGaA, Darmstadt, Germany data * Sterilizing-grade microfiltration used for bioburden reduction at various points downstream36 36
- 37. Process Step Clears Enveloped Virus Clears Non- Enveloped Virus Robustness of Step Ease of Downscale Typical Clearance Low pH Inactivation Yes No Yes; at or below pH 3.5 Easy ≥ 4 logs Detergent Inactivation Yes No Yes Easy ≥ 4 logs Anion Exchange/Anion Mixed Mode Chromatography Potential Exists; Virus Dependent Potential Exists; Virus Dependent No More Difficult 1 to ≥ 4 logs Other Chromatography Potential Exists; Virus Dependent Potential Exists; Virus Dependent No More Difficult 1 to 3 logs Virus Reduction Filtration Yes Yes Yes; for viruses above filter pore size Easy (with vendor small- scale device) ≥ 4 logs Removal Strategies Downstream Virus Clearance 37
- 38. Build a Robust Safety Strategy • Source low risk raw materials • Consider virus resistant cell lines • Choose appropriate chemicals for • GMP Production • Implement Single Use Systems Detect Virus Risk Mitigation • Consider implementation of upstream virus barrier filtration • Understand benefits and limitations of upstream pre-treatments • Develop a robust virus clearance process • Understand limitations of methods • Use PCR and other rapid methods for process monitoring38 38
- 39. Summary Virus Risk Mitigation 4 3 2 1 Sourcing low risk raw materials • Moving towards serum free, chemically defined media • Evaluate production culture for adventitious agents early in production cycle to mitigate product loss and spread to facility Developing a robust testing strategy • Tailor sampling plans based on achievable viral clearance • New genomic detection methods can improve the breadth of virus detection Implementation of viral clearance technologies • Incorporate robust viral inactivation steps and virus removing filter in product purification Overall Strategy • Must be a strategic application of the three steps (highly inter-related) • Optimization possible to drive risk down to the lowest theoretical level possible 39 39
- 40. Alison Armstrong, PhD Global Head of Field Development Services Life Science business of Merck KGaA, Darmstadt, Germany +44 141 576 2440 alison.armstrong@sial.com © 2017 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The Vibrant M is a trademark 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. Contact Questions?