Biosafety Testing Solutions for Cell & Gene Therapy
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This document discusses biosafety testing for cell and gene therapies performed by BioReliance, a testing services division of Merck KGaA. It outlines the comprehensive testing performed at various stages of development, including testing of cell banks, viral vectors, and final drug products. Testing evaluates important product attributes like identity, purity, potency and residuals to ensure safety and quality. A wide range of assays are used to characterize products and identify potential contaminants.
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Biosafety Testing Solutions for Cell & Gene Therapy
- 1. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Biosafety Testing solutions for Cell & Gene Therapy Munehisa Masuda Business Development Manager - Japan 6 Oct 2021
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
- 3. 3 Munehisa Masuda MSc. of Bioscience, Nara Institute of Science and Technology, Japan 2020- Business Development Manager, Biosafety testing services, Merck Japan 2017-2020 Business Development Manager, Drug Discovery and CDMO, Curia (Ex-AMRI) 2015-2017 Senior Sales Specialist, Actives and Formulations, Sigma Aldrich Japan under Merck Group 2014-2015 Senior Marketing Specialist, Gene Silencing Products, Sigma Aldrich Japan 2003-2014 Assistant Manager, Peptide Synthesis Services, Eurofins Genomics (Ex-Operon Biotechnologies)
- 4. Agenda Introduction of BioReliance® Testing Services Biosafety Testing for Cell Therapy Biosafety Testing for Gene Therapy 1 2 3
- 6. BioReliance® Services include a full complement of services BioReliance® pharma and biopharma manufacturing and testing services Pharma and Biopharma Testing Services Biomanufacturing Services Biosafety Testing Product Characterization Validation Services End-to-End Solutions Viral Vector Manufacturing Cell Banking & Testing Viral Clearance Lot Release Testing Analytical & Bio- analytical Testing Analytical Chemistry Binding Potency Physical Testing Chemical Testing Microbiology testing 6
- 7. 7 BioReliance® Services Biosafety Testing Product Characterization Services Validation Services Viral and Gene Therapy Manufacturing Viral and Gene Therapy Testing End-to-End Solutions Sales Offices Legend
- 8. Focus on 4 elements for gene therapy testing services Identity Purity Potency Residuals 8
- 9. 9 Comprehensive biosafety testing portfolio for cell and gene therapies Identity Purity Potency Residuals CO1 Barcode STR analysis Karyotyping Mycoplasma Sterility In vitro adventitious viruses In vivo adventitious viruses PCRs specific to viruses Endotoxin rcAAV TCID50 of viral vector Genomic titer Expressing protein Custom potency assay development • Residual host cell DNA • Residual DNA size distribution • Host cell protein, Residual BSA • Residual AAV Affinity Ligand • Purity by SDS PAGE • Empty:Full Capsid
- 10. Biosafety Testing for Cell Therapy
- 11. 11 Overview for allogeneic and autologous cell therapies
- 12. 12 Testing for stem cell therapy Identity Microbiology Adventitious Viruses Expressed Retroviruses Specific Human Pathogens Cell properties Bovine and Porcine Viruses
- 13. 13 Testing for Stem Cell Therapy Identity • For biologics production, CO1 on species level is accepted. • For cell therapies, an unambiguous method for cell line authentication, to the level of the individual, must be used. Cytochrome C oxidase subunit 1 (CO1) barcoding assay on species level Short Tandem Repeat (STR) assay on individual level AGAT AGAT 5X repeats 3X repeats 3X repeats 5X repeats 3X repeats 4X repeats Cell properties
- 14. 14 Testing for stem cell therapy Microbiology Microbiology Adventitious Viruses Expressed Retroviruses • Sterility • Mycoplasma • Mycobacterium • in vitro • in vivo • NGS • QPERT • TEM • Detection or enumeration, and identification of microbial contaminants • Normal culture based assay by direct inoculation • Rapid testing assay for of living cells (BacT® system/Myco PCR) • in vitro co-culture with 3 detect cell lines within 28 days • in vivo testing with suckling mice, adult mice and embryonated eggs • NGS may be used to reduce consumption of limited MCB material • Quantitative Fluorescent Product Enhanced Reverse Transcriptase (QPERT), Test RT activity of retroviruses in biologics • Visible virus particle will be counted in Transmission electron microscopy BacT/ALERT® Rapid Sterility Testing
- 15. 15 Testing for Stem Cell Therapy Bovine and Porcine Viruses Specific Human Pathogens • Polyomavirus • Bocavirus • Metapneumovirus • Syphilis • Neisseria gonorrhoeae • Papillomavirus • Varicella Zoster virus • West Nile Virus • Zika virus • COVID?? Real time PCR for human specific pathogens Considered Bovine and Porcine original component in vitro • Co-culture assays with detector cell Real time PCR • Polyomavirus • Parvoviruses • Circovirus • Herpesvirus • Bornavirus • Hepatitis type E virus
- 16. 16 Testing for Stem Cell Therapy *Tumorigenicity or TPD50 in adult nude mice Autologous Allogeneic WCB MCB Cells at Limit of In Vitro Cell Age (LIVCA) DP Identity Adventitious Viruses Expressed Retroviruses Bovine and Porcine Viruses Specific Human Pathogens * Cell properties Microbiology
- 17. Biosafety Testing for Gene Therapy
- 18. Virus-Based Delivery System Eye • REP 65 Spark Neurology • SMA Avexis Liver • Hemophilia • GPC3 Lungs • CRSP/VRTX Heart • BMY Infectious Disease • AIDS Oncology • NHL Kite/NVS Hematology • LentiGlobinTM BLUE Rare Diseases • Glybera® uniQure Musculoskeletal • DMD Type Approach Delivery Example Pros/Cons Current Uses Future Enhancements In vivo Inject corrected gene Viral vector AAV +Produces missing or fixed protein -Virus supply Severe Disease Repeat dosing Ex vivo Reinfuse enhanced cells Viral vector+cell infusion CAR/TCR +Efficacy in last line cancer setting -Virus supply, manufacture time Cancer Allogeneic Organ AAV Serotype Eye 2,4,5,6,7,ChR2 Liver 3.8.9.rh10 Blood Lenti, 5 Muscle 1,5,8,9 CNS 2,4,5,6,9,10 Heart 1,2,9 Pancreas 8 18
- 19. Potential production impurities Virus Production Process Impurities • Residues of starting materials • Manufacturing additives Raw materials, adventitious and leachable and extractables Product Impurities • Host-cell components/ contaminants • Empty/partial empty capsid Extractable & Leachable Resin residual Microorganisms Extractable & Leachable Extractable & Leachable Extractable & Leachable Benzonase® nuclease Salty solution Extractable & Leachable HCP, HCD Plasmid DNA Empty capsid Detergent PEI Porcine/Bovine components Porcine/Bovine /Human virus Sterility etc. Banking & Seeding Cell Culture Lysis Clarification Incubation Pre Capture Capture Concentration Sterile Fill & Finish
- 20. 20 Cell Bank Preparation Virus Production Produced to GMP **“Establishment of cell banks should be performed under circumstances which are demonstrably appropriate” **Information on individual process steps and minimize the risk of microbiological contamination • Air classification for rooms • Equipment calibrated and maintained * CMC Information for Human Gene INDs 2020 FDA **Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products 2018 Starting Material *MCB information as cell substrates • Cell source • Bank generation procedure • Testing to characterize the bank • Genetically modification if applicable **Flow diagram, and Source, quality and control • Identity • Purity • Cell number • Viability • Strain characterization • Genotyping/phenotyping **Measuring viability of cells over time after cryopreservation Biorepository **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
- 21. 21 Cell Bank Testing Virus Production Maximum Population Doubling Cell Bank Working Cell Bank Master Cell Bank Identity • CO1 barcode • DNA footprint Microbiology • Sterility • Mycoplasma Adventitious viruses • Cell-based assays • In-vivo testing Specific Virus • Human sourcing • Porcine/Bovine sourcing Cell properties Identity • CO1 barcode • DNA footprint Microbiology • Sterility • Mycoplasma Adventitious viruses • Cell-based assays • In-vivo testing Identity Microbiology Adventitious viruses Specific Virus
- 22. 22 Unpurified and Purified Bulks Adeno-Associated Virus Production Purification Bulk Harvest ➢ Target viral vector • Full capsid • Partial full capsid • Empty capsid • Replication competent* ➢ Helpers • Plasmids • Viral vector ➢ Host cell components • DNA • Protein ➢ Microorganisms • Bacteria • Fungi • Mycoplasma • Adventitious viruses ➢ Start materials/additives • Chemicals • E&L from consumables • Benzonase® endonuclease • ….. ➢ Target viral vector • Full capsid • Partial full capsid • Empty capsid • Replication competent* Potency Testing Residuals Testing Identity Testing
- 23. 23 Unpurified and Purified Bulk Testing Virus Production Purification Unpurified Bulk Purifed Bulk Identity • GOI Titer • TCID50 of viral vector Purity • Bioburden • Mycoplasma • Mycobacterium • Adventitious viruses (in vitro & in vivo) • Replication competent AAV (rcAAV) Identity • GOI • ELISA • Vector genome Titer • TCID50 of viral vector • Genomic Titer Potency • Expressed protein • Function Purity • Sterility • Endotoxin • rcAAV Residuals • Residual host cell DNA • Residual DNA size distribution • Host cell protein, Residual BSA • Residual AAV Affinity Ligand • Purity by SDS PAGE • Empty: Full Capsid
- 24. Formulated Final Lots Virus Production 24 Product Characteristics Identity Purity Potency GOI Sterility, Endotoxin TCID50 of viral vector Genomic titer Expressing protein Vector aggregates Osmolality pH Extractable volume Appearance Particulates Format Endotoxin Upper Limit* intrathecally 0.2EU/kg/hour intraocularly 2EU/dose/eye ophthalmic 0.5EU/ml Others 5 EU/Kg/hour Endotoxin claim in different format* * CMC Information for Human Gene INDs 2020 FDA • Dosing units • Genotypic or phenotypic variation • Particle number and size • Aggregation state • Infectivity • Specific activity (ratio of infectious to non-infectious particles or full to empty particles) • Biological activity • Potency • Immunological activity *Parameters relevant to the performance of the DP
- 25. 25 Summary Gene Therapy Testing Virus Seed* Cell Banks Plasmids Unprocessed Bulk Harvest Purified Bulk Harvest Final Lot Sterility Identity Titer Adventitious Agents Cell Properties Vector Concentration Expression of Gene Residuals Product Characteristics Endotoxin rcAAV Full/Empty BacT® System Adventitious Virus Custom…
- 26. The vibrant M, Benzonase, 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. © 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.