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Releasing Your AAV Therapy with Confidence: Regulatory Considerations and Key Assays
The document discusses regulatory considerations and testing strategies for the manufacturing process of adeno-associated virus (AAV) gene therapies by Merck KGaA, operating as MilliporeSigma. Key topics include viral risk mitigation, the necessity of characterizing and qualifying cell banks, and the testing of raw materials to ensure safety and efficacy of AAV therapies. It also outlines the importance of various assays and compliance with international guidelines to ensure the quality of gene therapy products.
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Releasing Your AAV Therapy with Confidence: Regulatory Considerations and Key Assays
- 1. The life sciencebusiness of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Releasing your AAV therapy with confidence – Regulatory considerations and key assays Steven McDade & Alfonso Lavorgna Ph.D.
- 2. The life sciencebusiness of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
- 3. Agenda Regulatory considerations throughoutthe AAV manufacturing process Steven McDade Review of the rcAAV and infectious titer assays Alfonso Lavorgna Ph.D. 1 2
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- 5. Viral Risk MitigationStrategy for gene & cell therapy Safe sourcing and testing of raw materials Verify absence of viral contaminants at appropriate stages Verify capacity of manufacturing process to remove or inactivate potential viral contaminants Difficult for enveloped viral vectors – but possible for AAV A virus safety risk assessment should be conducted (process indicated in European Pharmacopoeia 5.1.7) 5
- 6. Increase in regulationsfor cell and gene therapy observed in recent years FDA: Content and review of CMC information for Human gene therapy INDs Human somatic cell therapy INDs EMA: Guideline on human cell-based, gene therapy medicinal products EMA: GMP for ATMPs EU Draft : Annex 1 revision for sterile medicinal products PIC/S Annex 2A Draft guidance for the manufacture of ATMPs NIFDC. China: Quality Control of CAR-T Cell Therapy Products and Consideration for Non-clinical Research FDA: Potency tests for cellular and gene therapy products FDA Draft guidance on CMC and Retroviral testing guidance EMA Guideline on quality, non-clinical and clinical aspects of medicinal products containing genetically modified cells FDA: Finalized guidance Testing of retroviral vector-based human gene therapy products for replication competent retrovirus during product manufacture and patient follow-up. Chemistry, manufacturing and control (CMC) for human gene therapy INDs ChP: General Chapter of Gene Therapy Products for Human Use (draft) 2008 2011 2017 2018 2019 2020 6
- 7. Building a testingstrategy – what and when to test MCB WCB / CAL bioreactor purified drug substance final drug product raw materials plasmid plasmid stock Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Stability Container integrity Potency Identity Identity Biosafety Potency Identity Biosafety Potency Residuals Properties Residuals E.coli cell banks Identity Biosafety
- 8. Questions & answerson the principles of GMP for the manufacturing of starting materials of biological origin used to transfer genetic material for the manufacturing of ATMPs (EMA 2021) Principles of GMP apply to plasmid manufacturing For certain starting materials of biological origin, (such as e.g. linear DNA used as template for ex vivo transcription into mRNA, plasmids to generate viral vectors and/or mRNA, and vectors) used to transfer genetic material for the manufacturing of ATMPs it is, however, mandatory to comply with the principles of GMP.
- 9. • “Should becapable of maintaining a level of cell viability upon reconstitution which is both consistent and adequate for production use” • “The design and performance of specific tests for adventitious microbial agents and adventitious cellular contaminants should take into account the properties of the banked cell, the likely contaminants based upon scientific literature, source, methods and materials used for cultivation and other organisms present in the lab” • “Visual examination of the characteristics of well isolated colonies is suggested, using several microbiological media” • “Analysis of growth on selective media is usually adequate to confirm host cell identity at the species level” • “Either phenotypic or genotypic characteristics may be used in identity testing” • “Consistency of the coding sequence of the expression construct should be verified” • “Restriction endonuclease mapping or other suitable techniques should be used to analyse the expression construct for copy number, for insertions and deletions” • “For extrachromosomal expression systems, the percent of host cells retaining the construct should be determined” Key elements of E.coli cell bank characterization Identity Purity Stability Viability ICH Q5B & Q5D plus Ph. Eur. 5.14 apply to address the characterization & genetic stability of E.coli cell banks
- 10. Overview of E.colicharacterization assays Description Viability Determination of cell viability in the sponsor’s cell bank Purity Qualification of purity testing for microbial cell banks Purity testing of microbial cell banks: Presence of bacterial and fungal contaminants Determination of the purity of the sponsors bacterial strain by Gram's staining Detection for the presence of bacteriophage in material derived from a cell bank of E. coli or material used in the propagation of E. coli cultures. Detection of lysogenic bacteriophage in test material derived from Escherichia coli cell banks Identity Confirmation of the identity of an E. coli strain by RAPD analysis Identification of Enterobacteriaceae and other Gram negative rods using the API® 20 Identification system Determination of the partial genotype of the sponsor's E. coli; Ampicillin Resistance Kanamycin resistance Chloramphenicol Resistance Tetracycline Resistance Nalidixic Acid Resistance Streptomycin Resistance Determination of the partial genotype of the sponsor's E. coli; Determination of gal genotype Determination of lac genotype Determination of met genotype Determination of thi genotype Determination of recA-1 genotype Determination of supE genotype Determination of F genotype Genetic Stability Retention of Expression Construct in a Bacterial Cell Bank Nucleotide sequence analysis of recombinant plasmid expression vector Genetic Stability and Plasmid Identity by restriction enzyme map analysis Design and qualification of a droplet digital PCR assay (ddPCR) for the determination of plasmid copy number Determination of Plasmid Copy Number in a Microbial Cell Bank by droplet digital PCR (ddPCR)
- 11. Plasmids used forvector production Ph. Eur. 5.14 Gene transfer medicinal products for human use Plasmid intermediates used for vector production − Information needed (identification, source, means of isolation, nucleotide sequence, source and function of promoters, origin of replication, selection marker genes) Bacterial cell bank used to generate plasmids must comply with the requirements of the “Bacterial cells used for the manufacture of plasmid vectors” section Recommended testing for plasmids: − Identity: Sequencing or PCR − DNA concentration − DNA forms: HPLC or CE − Supercoiled, multimeric, relaxed monomer and linear forms − Residual host cell DNA, RNA and host cell protein − Sterility, endotoxin − Appearance, pH, extractable volume, residual moisture (if freeze dried) 11
- 12. Building a testingstrategy – what and when to test MCB WCB / CAL bioreactor purified drug substance final drug product raw materials plasmid plasmid stock Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Stability Container integrity Potency Identity Identity Biosafety Potency Identity Biosafety Potency Residuals Properties Residuals E.coli cell banks Identity Biosafety
- 13. Human cell linecharacterization CMC Information for Human Gene Therapy IND applications (2020) “For biologically sourced reagents including human, bovine and porcine derived materials we recommend that you also refer to FDAs Guidance for Industry “Characterization and Qualification of Cell Substrates and other Biological Materials used in the Production of Viral Vaccines for Infectious Disease indications (2010)” ICH Q5D is also referenced − Ensure absence of microbial contamination − For cell lines used for production of viral vectors, test for retroviral contamination (TEM and RT assay) − Adventitious agents including bovine/porcine viruses if exposed to serum/trypsin − Species specific pathogens (CMV, HIV, HTLV, HHV 6,7,8, JC, BK, EBV, B19, HBV, Papillomavirus, HCV, Circovirus) − Confirm identity (Short Tandem Repeat, Karyology) − Tumorigenicity 13
- 14. Cell line characterization(e.g. HEK293 cells) MCB Master cell bank WCB Working cell bank CAL Cells at limit of in vitro cell age Identity: CO1 barcode, STR analysis, karyology Microbiology: Bacteria, fungi, mycoplasma, mycobacterium Adventitious viruses: in vitro, in vivo, PCR* Retroviruses: TEM, reverse transcriptase, retrovirus infectivity Genetic stability (if stable transfection) Identity: CO1 barcode, STR analysis, karyology Microbiology: Bacteria, fungi, mycoplasma, mycobacterium Adventitious viruses: in vitro Identity: CO1 barcode, STR analysis, karyology Microbiology: Bacteria, fungi, mycoplasma, mycobacterium Adventitious viruses: in vitro, in vivo, PCR Retroviruses: TEM, reverse transcriptase, retrovirus infectivity Tumorigenicity: tumor formation in nude mice *HIV I & II, HTLV I & II, HAV, HBV, HCV, CMV, EBV, HHV 6, 7, 8, B19, HSV I & II, human polyoma viruses, Bocavirus, Metapnuemovirus, etc.
- 15. Building a testingstrategy – what and when to test MCB WCB / CAL bioreactor purified drug substance final drug product raw materials plasmid plasmid stock Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Identity Biosafety Stability Container integrity Potency Identity Identity Biosafety Potency Identity Biosafety Potency Residuals Properties Residuals E.coli cell banks Identity Biosafety
- 16. 16 Characterization of viralvectors Identity Biosafety Titer/Potency Residuals Product • Ph. Eur. 5.14 Gene Transfer Medicinal Products for Human use • FDA CMC for Human Gene Therapy INDs (2020) 16
- 17. • Identity: Geneof interest (GOI) • Biosafety: • Sterility or Bioburden • Mycoplasma • Mycobacterium • Adventitious viruses (in vitro and in vivo) • Replication competent AAV (rcAAV) • Potency: Infectivity, genomic titer Testing bulk and final lots: AAV vectors Unprocessed bulk Purified bulk • Identity: GOI, ELISA, vector genome • Biosafety: Sterility, endotoxin, rcAAV • Potency: infectivity, genomic titer, expressed protein level/function • Residuals: • Residual host cell DNA • Residual plasmid DNA • Host cell protein, residual BSA, residual Benzonase® endonuclease • Purity by SDS-PAGE • Empty:full capsid ratio • Identity: GOI • Biosafety: Sterility, endotoxin • Potency: infectivity, genomic titer, expressed protein level/function • Product properties: • Vector aggregates • Osmolality • pH • Extractable volume • Appearance • Particulates Formulated and vialed final lots 17
- 18. 18 BacT® Alert 3Drapid microbial detection system Rapid sterility testing Scan Load Inoculate Samples are automatically monitored and read every 10 minutes Sensor in broth bottle monitors CO2 production, indicative of the presence of microbial growth Presence of turbidity caused by debris/cellular material does not interfere with interpretation of results Non-destructive technology allows for subculture of positive broths for identification The BacT/ALERT® 3D system can detect most common contaminants within 48 to 72 hours
- 19. Mycoplasma assay comparison:Cultivation vs QPCR Historical compendial method Established alternative method Method largely manual Automated, reproducible Detection limit: ≤ 100 Colony Forming Units Detection limit: 10 Colony Forming Units 15 ml of UBH required 4 ml of UBH required Assay turnaround time: 28 days Assay turnaround time: 3 - 7 days The use of PCR for the detection of mycoplasma, supported by comparability data, has been used within industry for several years. This has included the testing of starting materials through to clinical material for a range of biopharmaceuticals and many submissions have been made to the US and European regulatory authorities over this time. Cultivation QPCR
- 20. Stability studies • Demonstrateshort and long-term stability of a product after exposure to various environmental conditions − Temperature − Humidity − Light • Analytical testing programs are designed by industry-experienced scientists to suit your needs • Accelerated stability and forced degradation studies are available • State-of-the-art instrumentation and cGMP capabilities provide results you can trust • Reliable service with fast turnaround delivers data on time Timepoint Infectious Titer
- 21. Viral clearance studiesfor AAV therapies FDA CMC for Human Gene Therapy IND (2020) references “In some instances, robust viral clearance studies may be necessary to remove and inactivate adventitious agents”. If safety can be built in to a process, it should be. Clearance steps should be evaluated and be aligned with ICH Q5A expectations for study design. Draft of ICH Q5A revision 2 expected to be issued in 2022. Viral safety strategy includes prevention, detection, and if possible, removal/inactivation of viruses AAV are small, non-enveloped viruses Focus on inactivation and filtration methods. Target reduction of large, enveloped viruses and medium sized non-enveloped viruses 21
- 22. Review of thercAAV and infectious titer assays
- 23. Assays Replication competent AAV(rcAAV) assay AAV Infectivity TCID50 assay 1 2
- 24. Detection of replicationcompetent AAV (rcAAV) • Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), dated April 2008 “…to determine the amount of replication competent AAV present in the final vector product and report the results in your IND” • Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), dated January 2020 “We recommend that you test for rcAAV, which could potentially replicate in the presence of helper virus, and report these results.” 24
- 25. Detection of replicationcompetent AAV (rcAAV) • To detect replication competent AAV, if present, in unprocessed bulk/purified bulk and report the results. • Test material includes: AAV drug substance or cell banks modified with AAV vector. • What subtype/serotype of AAV can be tested? HEK293 cell line is used for the amplification phase and it can support replication subtype 1, 2, 3, 6, 8 and 9. Other subtypes will need to be evaluated case by case. • Share Rep2 sequence to confirm qPCR target compatibility. Assay Purpose Key Questions 1 2 25 • Cell culture allows amplification of rcAAV present in the test material at low level. • Endpoint qPCR is targeting Rep2 gene, which should be present in rcAAV viruses but not in the replication incompetent AAV. • Limit of detection of the assay is 20 IU/inoculum. • Qualification required? Y • Sample type: drug substance or cell bank • Sample Volume: 0.5-1.0mL • Compliance: GMP Key Assay Info 4 Method / Technology 3
- 26. Cell transduction efficiency TestSystem HEK 293 Ellis et al. Virology Journal 2013, 10:74 26
- 27. 27 Detection of replicationcompetent AAV (rcAAV) – How the assay is performed TEST CONDITIONS PSQ • Cell Control • Negative Control • Test Article D1* • Test Article D2* • Test Article D3* • Positive Control 20 IU • Test Article D1+ PC 20 IU GMP • Cell Control • Negative Control • Test Article • Positive Control 20 IU • Test Article + PC 20 IU * - dilutions (doses) of TA 3 days Detection (Rep2 qPCR) 3 days 3 days Sampling Inoculation (max 1.0E11 vg) Cell Passages (HEK293) P2 P3 P1 Sampling R2 R3 R1
- 28. Assay essentials: PositiveControl, Test System and Assay End-Point Test system End-point rep2-qPCR Positive Control Virus Client-specific assay Development Test system Platform-based validation: ➢ Simplified validation protocol and report ➢ Document templates ➢ Limited number of tests for validation Assay platform Generic assay Test system HEK293 Ad5 End-point Rep2-qPCR Positive Control Virus AAV2 Detection of replication competent AAV (rcAAV) From generic to client-specific assays 28
- 29. AAV Infectivity: TCID50 Content/Potency (ICH Q2(R1)) Parameters included in validation: accuracy, precision, specificity, linearity and range AVV-based gene therapies require high concentrations of viral vectors to be effective. That is because such treatments often need to be administered in low volumes in smaller areas of the body. An accurate titration is critical for the success if the product. Assay essentials: Reference Material, Test System and Assay End-Point Reference Standard: universal AAV Reference Standard Material (AAV RSM), which is an AAV sample that has been quantified by 16 labs around the world and can be used to validate their qPCR assay. HeLaRC32: modified cell line that can amplify many AAV subtypes. Assay endpoint: qPCR for CMV promoter which is applicable to many vectors. 29
- 30. • The genericassay has been fully validated • The Product Specific Qualification (PSQ) is recommended: a. to establish a serial dilution range for testing b. to assess some of the parameter assessed in validation • GMP testing Deliverables: Technical specification, Certificate of Analysis, Laboratory Records (QA Audited) • NRT testing Deliverables: Unaudited Final Report, Raw data summary if required. 30 AAV Infectivity: TCID50
- 31. 31 AAV infectivity: TCID50 1ml etc… H G F E D C B A N 1 -0.7 2 -1.4 3 -2.1 4 -2.8 5 -3.5 6 etc. 7 8 9 10 11 12 1 ml 1 ml 1 ml 1 ml 4 ml 4 ml 4 ml 4 ml Virus Dilution Proportion of positive wells 1 1 0.75 0.5 0.375 0.125 0 0 0 0 0 0 Wells are scored as + or - 50% Infection < 50% Infection > 50% Infection Infect replicate wells with serial dilutions of sample Virus Detection by PCR
- 32. Assay essentials: PositiveControl, Test System and Assay End-Point Client-specific assay Development Platform based validation: ➢ Simplified validation protocol and report ➢ Document templates ➢ Limited number of tests for validation Assay platform Generic assay Test system HeLaRC32 Ad5 End-point CMV-qPCR Positive Control Virus AAV2 Detection of replication competent AAV (rcAAV) From generic to client-specific assays Test system End-point GOI-qPCR Positive Control Virus provided by Sponsor 32
- 33. Summary 33 Holistic approach Assay versatility Riskassessment Regulatory considerations 1 2 3 4
- 34. Operations Manager, VirologyServices Senior Technical Specialist, Field Technology Management Alfonso Lavorgna Ph.D. Steven McDade Thank you! 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. © 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.