Keeping the (Adventitious) Virus Out of the (Adeno-Associated) Virus
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Watch the presentation of this webinar here: https://bit.ly/2VRylbi How can you keep an adventitious virus from contaminating your gene therapy that is delivered by an adeno virus vector? As viral vector bioprocessing advances, regulatory requirements for viral safety will as well. Learn how to define your viral clearance strategy for AAV delivered gene therapies. How do you define a strategy for viral clearance for a process that inherently aims at purifying a virus? Gene delivery using AAV has received a boost from two major approvals and the nearly 300 programs in the clinic. Novel gene therapies using viral vectors enable companies to transform the lives of people living with certain rare and ultra-rare diseases where treatments are often not available currently. Amongst a multitude of challenges in viral vector bioprocessing, uncertainty in regulatory expectations is a major challenge to gene therapy developers. Regulatory requirements are evolving as the science and manufacturing matures with more stringent measures for viral safety assurance expected for future approvals. Learn how to implement techniques for adventitious virus removal in your viral vector process; we will focus on strategies for viral clearance along your journey towards commercial readiness of AAV-based processes. In this webinar, you will learn: • AAV process flows and focus areas for viral safety • Strategies for implementing viral clearance measures in bioprocessing • Case studies and data driven approaches on log reduction values (LRV) in a viral vector process • Best practices and evaluation roadmaps on conducting viral clearance studies Presented by: Ratish Krishnan, Senior Strategy Consultant, Novel Modalities Bioprocessing
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Keeping the (Adventitious) Virus Out of the (Adeno-Associated) Virus
- 1. Ratish Krishnan Sr Strategy Consultant Novel Modalities BioProcessing – Americas 12 Aug 2021 Keeping the (Adventitious) Virus out of (Adeno-associated) Virus! Approaches for Viral safety in AAV Processes
- 2. 2 The Life Science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. 1. Background 2. Risk Mitigation Strategy Prevent Remove Detect 3. Looking Ahead – Best Practices OUTLINE
- 5. High unmet need and potential cures 5 < 5% approved therapy Definition Data Population ~7000 Product Name Company Type of Therapy Imlygic® Amgen Oncolytic virus Yescarta® Kite (Gilead) CAR-T Kymriah® Novartis CAR-T Strimvelis® GSK Gene Therapy Luxturna® Spark Gene Therapy Zolgensma® AveXis (Novartis) Gene Therapy Tecartus® Kite (Gilead) CAR-T Breyanzi® BMS CAR-T Abecma™ BMS CAR-T Fastest growing sector in biotech; 1100+ active trials; $20B raised in 2020 Recent Approvals! Rare diseases: Significant number among population Source: FDA; ARM reports • Affects < 200,000 people in US • Primarily genetic in origin • No prior treatment regimen available 1 in 10 • Children impacted most • Survival rates are very low Approaches for Viral Safety in AAV processes | 12 Aug 21
- 6. Rushed PD is the New Normal Compressed Timelines for Process Decisions Years in pipeline 1 3 4 5 6 7 8 2 Phase III Process Characterization PPQ Control Strategy Submission Prep Facility & Equipment Readiness Phase I Phase II Process Development Phase IIa/III Process Characterization Control Strategy Submission Prep Facility & Equipment Readiness Process Development PPQ Years in pipeline 1 3 4 5 6 7 8 2 Phase I 6 Gene therapy manufacturers anticipate making post approval changes in the manufacturing process. FDA appears to be open* to such approach! * Bridging studies, repeat PPQ and comparability demonstration minimally required Approaches for Viral Safety in AAV processes | 12 Aug 21
- 7. Regulatory Guidelines for Gene Therapies Multiple Reference Documents US FDA Guidance for Industry: Chemistry, manufacturing and control (CMC) for human gene therapy Investigational New Drug Applications (INDs). (2020) US FDA Guidance for Industry: Testing of retroviral vector-based human gene therapy products for replication competent retrovirus during product manufacture and patient follow-up. (2020) WHO Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks. TRS 978, Annex 3 (2013) ICH Q5A: Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. (1997) ICH Q5D: Derivation and Characterization of Cell Substrates used for Production of Biotechnological /Biological Products (1997) 1 3 2 4 CONSTANTLY EVOLVING Continuous Risk Assessment Introduce viral clearance measures Ongoing dialogue with agency Draft guideline on quality, non-clinical and clinical requirements for investigational advanced therapy medicinal products in clinical trials, EMA/CAT January 2019 EMA Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products. EMA/CAT/80183/2014 March 2018 European Commission Guideline on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products (2017) FDA EMA ICH WHO
- 8. FDA Guidance Document CMC Information for Human Gene Therapy INDs 8 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 10. Overview Generic Process for Viral Vector Production Raw materials Filtration Clarification Nuclease Treatment Concentration and diafiltration (UFDF) Purification by Chromatography (multiple steps) Concentration and diafiltration (UFDF) Final filtration Storage Virus production Virus filtration 10 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 11. Does size matter? Commonly used viruses Item Adenovirus Herpex Simplex Virus (HSV) Adeno- associated Virus (AAV) Lentivirus （Retrovirus） Genome ds DNA ds DNA ss DNA ss RNA Coat Naked Enveloped Naked Enveloped Genome Size 38-39 kb 150 kb 5 kb 9 kb Transgene Expression Transient Transient Potentially long- lasting Long-lasting Packaging Capability 7.5 kb >40 kb 4.5 kb 6 kb Immune Response High Low Very low Low Relative Viral Titer 10^11 in bulk 10^9 in bulk 10^7 in bulk 10^7 in bulk Relative Transduction Efficiency 100% 70% 70% 70% Adenovirus AAV HSV Lentivirus 11 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 12. Size does matter! 12 100nm 200nm 300nm Vaccinia virus 200nm x 300nm Adeno- associate Virus (AAV) 20nm Adenovirus 60-90nm Lentivirus or Retrovirus 80-120nm Sendai virus 260nm Herpes simplex virus (HSV) ~225nm 0.22 μm sterilizing grade filter Size increase by aggregation Biosafety HSV Approaches for Viral Safety in AAV processes | 12 Aug 21
- 13. Basics of Virus Inactivation and Removal Principles DNA Herpes 120 – 200nm Pseuodorabies (PRV) Retro 80 – 110 nm MuLV Reo 60 – 80 nm Reo 3 Parvo ~ 20 nm AAV Envelope Protein Lipid Layer Capsid Protein DNA RNA RNA Removal • Charge: IEX Chromatography • Size: Virus Filtration Inactivation • Low/High pH, Detergent, Heat Approaches for Viral Safety in AAV processes | 12 Aug 21 13
- 14. Biosafety Strategy Current Virus Reduction Technologies Virus Filtration: Size exclusion viral clearance Single Use Systems: • Gamma irradiated • Closed systems • Aseptic sampling Low pH/Solvent/Detergent: Viral inactivation Contract Labs: Validation, detection and characterization HTST or UV: Virus inactivation Virus Resistant Cell Lines: Prevent amplification in the bioreactor 14 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 15. Biosafety Millipore® Preparation, Separation Filtration & Monitoring Products BioReliance® Pharma & Biopharma Manufacturing & Testing Services SAFC® Pharma & Biopharma Raw Material Solutions Biosafety Strategy Tripod Approach for Complete Biosafety Assurance 15 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 16. Enveloped viral vectors; cell therapies Non-enveloped viral vectors Biosafety Strategy Modified Approach for Enveloped Viruses AAV Lentivirus Approaches for Viral Safety in AAV processes | 12 Aug 21 16
- 17. 02a PREVENT
- 18. • Risk assessment • Risk mitigation strategy Contamination risk TSE, bacteria, mycoplasma, fungi Viral Safety Residuals (DNA, HCP, etc.) • Demonstrate the control, elimination and/or risk mitigation strategy effectiveness 1 2 Biosafety Strategy Regulatory Expectations 18 Adherence to guidelines Approaches for Viral Safety in AAV processes | 12 Aug 21
- 19. Prevent Raw Material Risk Mitigation 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 19 Eliminating or Reducing the Load with a Layered Approach Approaches for Viral Safety in AAV processes | 12 Aug 21
- 20. Identification of a new virus in Sf cell lines in 2014 by CBER* Sf-Rhabdovirus Negative (RVN) cell line was developed following work from the Jarvis group and Glycobac technology The absence of any rhabdovirus sequence was proven Geisler, 2018** Sf-RVN exhibit the same characteristics as non RVN cell lines In 2018 Merck KGaA, Darmstadt, Germany obtained the exclusive distribution license for the Sf9-RVN cell line Prevent Sf-Rhabdovirus Negative Cell Line *Ma et al, JVI, 201Identification of a Novel Rhabodovirus in Spodopteral frugiperda Cell line **C. Gieser: A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production, BMC Biotechnology, 2018 Electron microscopy view of Sf-Rhabdovirus* 20 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 21. Prevent Emprove® Quality and Regulatory Support Emprove® Program Addresses existing and anticipated quality and regulatory requirements Emprove® Dossiers Supporting Qualification, Risk Assessment and Process Optimization Emprove® Suite 24/7 access Emprove® Portfolio Chemicals addressing different risk levels For Biologics: >400 different compendial raw materials in downstream/formulation 21 Value Vs Risk Approaches for Viral Safety in AAV processes | 12 Aug 21
- 22. 02B REMOVE
- 23. Remove Strategy for the Entire Process Raw material Clarification Benzonase® Endonuclease Remove residual DNA Concentration and diafiltration (TFF) Purification by Chromatography (multiple steps) Concentration and diafiltration (TFF) Millipak ® filter Final filtration Storage Virus production NFR (For AAV process) Remove helper/adventitious viruses Viresolve ® Barrier Remove virus, Bacteria, mycoplasma 23 Remove Approaches for Viral Safety in AAV processes | 12 Aug 21
- 24. Remove Viresolve® Barrier Filters Robust protection from adventitious agents • ≥ 3.0 log removal of parvovirus • ≥ 6.0 log removal of mycoplasma • Sterilizing-grade protection from bacteria • Filters 100% integrity tested in manufacturing Efficient filtration for cell culture media • No impact on media performance • Fast, high-flux virus filtration • Easy to implement, install and integrity test Chemically defined media only, Serum and hydrolysate are difficult to filter; Eg., OptiCHO™ CD Media 24 0 500 1000 1500 2000 2500 3000 0 100 200 Time (min) 300 400 Throughput (L/m 2 ) Viresolve® Pro filter Viresolve® NFP filter Downstream Filter 1 Downstream Filter 2 Downstream Filter 3 Downstream Filter 4 Viresolve® Barrier Approaches for Viral Safety in AAV processes | 12 Aug 21
- 25. Remove Viresolve® Barrier Filters Days No changes in pH, osmolarity, glucose, glutamate, lactate, or NH3 levels were seen (as measured by BioProfile® FLEX). No impact of virus filtration was observed on any cell culture attributes Cellvento® CHO 200 Media with Molecule A EX-CELL® Advanced™ Media with Molecule B 0 2 4 6 8 1 0 1 2 1 4 Viable Cell Density (cells x 10 6 /mL) Days 5 5 10 10 1 5 2 0 0 Virus Filtered 0.2 µm Filtered 0 15 20 - 2 4 6 8 10 12 14 Viable Cell Density (cells x 10 6 mL) Virus Filtered 0.2 µm Filtered 25 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 26. FDA – Cellular & Gene Therapy Guidance – Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy: […] Gene therapy is a medical intervention based on modification of the genetic material of living cells. Cells may be modified ex vivo for subsequent administration to humans, or may be altered in vivo by gene therapy given directly to the subject. […] Recombinant DNA materials used to transfer genetic material for such therapy are considered components of gene therapy and as such are subject to regulatory oversight. […] 1.Tests of drug substance (bulk product not necessarily in final formulation): a. Purity (21 CFR 610.13) […] iii. Test for contamination with RNA or with host DNA, e.g. gel electrophoresis, including test with bacterial host-specific probe. […] • FDA requires DNA/RNA digestion (reduce the risk for the patients) • Higher virus yields • Drug Master File type II available (Benzonase® Endonuclease treatment is FDA approved process, DMF could be cited by our customer in their drug filing) • Full EMPROVE® dossiers Literature on the use of Benzonase® Endonuclease in Cell Therapy and Gene Therapy: Templated Process using Benzonase® Endonuclease: Bioprocess Int. 2012 February ; 10(2): 32–43. Production of CGMP-Grade Lentiviral Vectors Lara J. Ausubel et al. Benzonase® Endonuclease used in 7 of 11 described trials: Methods & Clinical Development (2016) 3, 16002; doi:10.1038/mtm.2016.2 Manufacturing of recombinant adeno-associated viral vectors for clinical trials Nathalie Clément and Joshua C Grieger 26 Remove DNA Removal in Gene Therapy Approaches for Viral Safety in AAV processes | 12 Aug 21
- 27. 27 DNA Digestion Processing Challenges Caused by Nucleic Acids BENEFITS Frees virus from DNA Protects against DNA fouling Reduces viscosity Yield loss due to virus-nucleic acid complexes Changes in virus / protein characteristics Unpredictable purification (shift in pI, retention times etc.) Low recovery and purity Fouling of downstream equipment Shortened lifetime of chromatography columns Reduced capacity of filters Viscosity increase Impedes liquid handling Reduced efficiency of separation methods Easy Removal Animal-Origin Free (Benzonase® Endonuclease Safety Plus) PATIENT SAFETY …minimize the biological activity of any residual DNA associated with your vector… limit the amount of residual DNA for continuous non-tumorigenic cells to less than 10 ng/dose and the DNA size to below approximately 200 base pairs. Source: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) Draft Guidance for Industry – FDA Approaches for Viral Safety in AAV processes | 12 Aug 21
- 28. Benzonase® endonuclease Safety Plus EMPROVE® Expert Specifications Standard Benzonase® Endonuclease Benzonase® Endonuclease Safety Plus EMPROVE® Expert Lot release testing for Mycoplasma No Yes Lot release in-vitro test for absence of adventitious viruses (3 cell lines) by BioReliance® Services No Yes Chemically defined fermentation media to claim non-animal origin No Yes (animal origin-free) Microbial testing < 10 CFU/100,000 U < 10 CFU/100,000 U Endotoxins (LAL) < 0.25 EU/1,000 U < 0.25 EU/1,000 U FDA Drug master file (DMF) Yes Yes Shipment with temperature strips No Yes Tailgate Samples No Yes (with 5M unit size) Product Availability Both Benzonase® Endonuclease products will stay in portfolio 28 ASGCT Poster May 2020 – Ratish Krishnan Remove Approaches for Viral Safety in AAV processes | 12 Aug 21
- 29. Virus Safety Strategy Helper/Adventitious Virus Removal 29 Viresolve® Normal Flow Retrovirus (NFR) filter Pleated Retropore® membrane ≥ 6 log removal of retroviruses > 98% recovery of protein (up to 700 kD) 100% integrity tested in manufacturing Air-water diffusion integrity testable SIP/autoclave compatible Viresolve® NFR filter Helper / Adventitious virus retained AAV passes through Data presented at ACS 2019, Orlando FL Study Summary • Capacity > 2e16 vp • Throughput 115 L/m2 • Recovery > 90% Approaches for Viral Safety in AAV processes | 12 Aug 21
- 30. Remove Maximizing Yield with Millipak® Filter • Unique stacked design allows minimal hold-up volume and particle shedding • Constructed with Durapore® PVDF membranes for high flow rates and throughputs • Various stack sizes for optimum flexibility Filter EFA Total Volume (mL) Downstream Volume (mL) Hold up Volume (mL) Millipak® 200 Filter 1,000 cm² 130 40 9 Opticap® XL 2 Capsule 900 cm² 225 30 15 Benefits • Minimum hold up volume and protein binding • Maximum product recovery and cost savings • Fully supported with different formats and documentation Millipak® 200 Filter VS Opticap® XL 2 Capsule 30 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 31. Perform normal 0.22μm sterilizing filtration at final filling step Optimize process parameters, in-process buffer and final filling solution to control aggregation Perform normal 0.22μm sterilizing filtration at final filling step Use sterilized raw materials to prevent potential contamination Implement closed processing Adapt appropriate in-process bioburden control method (e.g., 2 x 0.45μm filters) Remove Sterilization Strategies for Viral Vectors Small to Medium Virus Medium Aggregating Virus Large Virus (> 0.22μm) Adeno-associate virus (AAV) Adenovirus (AdV) Retrovirus (RV) Lentivirus (LV) Herpes simplex virus (HSV) Sendai virus Vaccinia virus 31 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 32. 02C DETECT
- 33. Safety testing strategy Virus safety risk assessment Regulation & guidance documents Regulatory updates Detect Building a Robust Safety Testing Strategy 33 GOAL: Confirm the identity of the vector, ensure its purity and potency and verify that any process impurities have been removed. Novel Biological Therapeutic Raw Materials Cells Unpurified Bulk Process Clearance Purified Bulk Final Product Approaches for Viral Safety in AAV processes | 12 Aug 21
- 34. Detect Cell Banks Biosafety Testing MCB WCB CAL* Sterility: Bacteria, fungi & mycoplasma Adventitious viruses: In vitro, in vivo & PCR Retroviruses: EM, RT & infectivity Species specific viruses: (PCR), ex: CMV, HIV, EBV, HCV Sterility: Bacteria, fungi & mycoplasma Adventitious viruses: In vitro Sterility: Bacteria, fungi & mycoplasma Tumorigenicity: Nude mice in vivo tumor formation Adventitious viruses: In vitro, in vivo Retroviruses: EM, RT & retrovirus infectivity Species specific viruses: (PCR) *Maximum use cells / Cells at Limit of in vitro cell age 34 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 35. Detect Recommended Testing Options 35 AAV Lenti/Retrovirus Adenovirus Identity • PCR for Genomic Region of Interest • AAV Serotype • Vector Sequence (NGS) Titer • TCID50 Assay • Genomic Titer by Droplet Digital PCR Potency • r-AAV Expressed Protein Purity • Sterility (also rapid method) • Mycoplasma/Spiroplasma (also rapid method) • Endotoxin • In Vitro Adventitious Viruses • In Vivo Adventitious Viruses • Replication Competent AAV Process/Product Residuals • PCR for helper viruses or transfected plasmids • Host Cell DNA • Host Cell Protein • DNA Size Distribution • Empty/Full Capsid by AUC • Residual AAV Ligand • Residual Benzonase® Endonuclease • Residual BSA Final Product Characterization • Vector Aggregates by DLS • Osmolality • pH • Appearance • Particulates Identity • PCR for Genomic Region of Interest • Vector Sequence (NGS) Titer • TCID50 Assay • Genomic Titer by Droplet Digital PCR Potency • r-LV/RV Expressed Protein Purity • Sterility (also rapid method) • Mycoplasma/Spiroplasma (also rapid method) • Endotoxin • In Vitro Adventitious Viruses • In Vivo Adventitious Viruses • Replication Competent LV Process/Product Residuals • PCR for transfected plasmids • Host Cell DNA • Host Cell Protein • DNA Size Distribution • Residual Benzonase® Endonuclease • Residual BSA Final Product Characterization • Vector Aggregates by DLS • Quantitation of LV Particles Using Virus Particle Counter • Osmolality • pH • Appearance • Particulates Identity • PCR for Genomic Region of Interest • Vector Sequence (NGS) • Restriction Endonuclease Analysis for ID of Purified Adenovirus Vector Titer • FFU Assay • Genomic Titer by Droplet Digital PCR Potency • r-Adeno Expressed Protein Purity • Sterility (also rapid method) • Mycoplasma/Spiroplasma (also rapid method) • Endotoxin • In Vitro Adventitious Viruses • In Vivo Adventitious Viruses • Replication Competent Adenovirus Process/Product Residuals • PCR for transfected plasmids • qPCR Detection of AAV • Host Cell DNA • Host Cell Protein • DNA Size Distribution • Residual Benzonase® Endonuclease • Residual BSA Final Product Characterization • Vector Aggregates by DLS • Osmolality • pH • Appearance • Particulates Approaches for Viral Safety in AAV processes | 12 Aug 21
- 36. Human 293 producer cells Virus stock Vector bulk harvest Vector purified MCB WCB Absence of microbial contamination Sterility assay Mycoplasma assay Absence of adventitious viruses In-vitro assay (3 detector cell lines, 28 days) In-vivo (embryonated eggs suckling & adult mice Transmission electron microscopy PCR/RT-PCR assays for human viruses PERT assay Bovine virus assay Porcine virus assay Porcine circovirus Absence of replication competent vectors Other tests (biosafety related) Host cell DNA Residual plasmids Endotoxin Detect Testing Regimen Recap 36 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 37. Detect Next Generation Sequencing: An Additional Tool “Is the biologic system what is believed to be”? “ Is the system contaminated or impure? “ Genetic properties of the MVSS (identity/purity/stability) Adventitious agent testing Raw material qualification In process testing Lot release testing • EP is recommending reducing in vivo virus detection assays and supplementing classical virus detection and identity assays with new technologies such as NGS analysis 37 1. Sample Processing Nucleic Acid Extraction & Library Construction 2. Sequencing 3. Data Analytics / Bioinformatics • Personalized Medicine and Clinical Applications • Biosafety Testing Approaches for Viral Safety in AAV processes | 12 Aug 21 MVSS – master virus seed stock
- 39. Viral Clearance Template Evaluation Roadmap 39 Detergent Treatment Low pH Hold Nanofiltration Nuclease Digestion Chromatography (multiple) Leverage •Past templates •Other modalities Analyze •Current Process •Midstream and Downstream Unit Operations Experiment •Non-GLP study •Preliminary results Confirm •GLP study •Representative Material Approaches for Viral Safety in AAV processes | 12 Aug 21
- 40. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 20 40 60 80 100 Log Reduction Value Time (min) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 20 40 60 80 100 Titer (logTCID 50 /mL) Time (min) 0.5% TX-100 0.5% TDAO Indicates no detectable virus Indicates no detectable virus TDAO detergent meets ASTM E3042-16 assurance of ≥4 log10 of infectious rodent retrovirus (xMuLV) inactivation obtained with Triton™ X-100 treatment within E3042-16 hold time of 60 min. No virus was detected in samples treated with TDAO detergent within 5 min. 40 Virus Titer LRV [Detergent]: 0.5% v/v pH 8 Hold temp: 21-24°C xMuLV in Clarified Harvest Virus inactivation: Deviron™ C16 detergent vs. Triton™ X-100 0.5% Triton ™ X-100 0.5% TDAO Approaches for Viral Safety in AAV processes | 12 Aug 21
- 41. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 20 40 60 80 100 Titer (logTCID 50 /mL) Time (min) 1.0% TX-100 1.0% TDAO 0.5% TX-100 0.5% TDAO TDAO detergent is as effective as Triton™ X-100 in the inactivation of other enveloped viruses such as Pseudorabies Virus (PRV). No active virus was detected after 5 min incubation at RT. Indicates no detectable virus Indicates no detectable virus 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 20 40 60 80 100 Log Reduction Value Time (min) 41 [Detergent]: 1.0% v/v 0.5% v/v pH 8.0 Hold temp: 21-24°C Virus Titer LRV Virus inactivation: Deviron™ C16 detergent vs. Triton™ X-100 PRV in Clarified Harvest 0.5% Triton ™ X-100 0.5% TDAO 1% Triton ™ X- 100 1% TDAO Approaches for Viral Safety in AAV processes | 12 Aug 21
- 42. LRV data Evidence of viral load reduction 42 Log10 Virus Reduction of BACV Process Step Log10 Virus Reduction of VSV 3.48 Low pH Inactivation 1.16 ≥5.15 Detergent Inactivation ≥4.41 ≥6.70 Chromatography 1 ≥6.08 2.67 Chromatography 2 3.93 1.82 Chromatography 3 3.84 ≥4.41 Nanofiltration ≥4.83 ANALYSIS • Low pH can achieve inactivation of enveloped viruses; reduction is dependent on the pH and the susceptibility of individual viruses to low pH. • Detergent can achieve effective inactivation of enveloped viruses. • Reduction by chromatography is dependent on the resin, the conditions under which the chromatography step is run and the virus itself. • Nanofiltration provides another useful assurance to viral vector processes. Approaches for Viral Safety in AAV processes | 12 Aug 21 24.23 24.25 19.42 19.82
- 43. Take Away Messages Conclusion Strategy: Develop a multi-faceted contamination risk mitigation strategy when developing a viral gene therapy Prevent: Ensure safety of raw materials Detect: Implement rigorous testing procedures for raw materials, cell banks, virus seed stocks, bulk harvests and drug substances Remove: Wherever possible implement virus, DNA & bacterial contaminant clearance technologies Solution: Partner with experts to develop your biosafety assurance strategy 43 Approaches for Viral Safety in AAV processes | 12 Aug 21
- 44. ACKNOWLEDGEMENTS 44 Sarah Sheridan Technical Consultant Trish Greenhalgh Integrated Marketing
- 45. ratish.krishnan@milliporesigma.com RATISH KRISHNAN THANK YOU! The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada © 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. MilliporeSigma, the vibrant M, Benzonase, Millistak, Opticap, Emprove, Virsolve, Millipak, Cellvento, EX-CELL, Durapore, and Deviron 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.