Unveiling the Potential of your AAV Gene Therapy: Orthogonal methods to understand and define CQAs
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Watch the presentation of this webinar here: https://bit.ly/3pCCjPF Ensure your Adeno-Associated Virus (AAV) is safe throughout its entire drug development journey. Learn methods that will help you speed to clinic, potentially treating diseases sooner and with greater effectiveness. The potential of gene therapies to cure previously untreatable diseases has spurred the development of novel drugs, including those based on Adeno-Associated Virus (AAV). As with all biopharmaceuticals, it is important to identify and monitor the critical quality attributes (CQAs) of these products to ensure their safety and efficacy. In this webinar, we will present a range of orthogonal methods to understand and define the CQAs of AAV products. These include assays for the confirmation of capsid protein identity and quantity, as well as the characterization of important product-related impurities, such as aggregates. Together these methods represent a comprehensive analytical testing package to support the characterization and lot release of AAV products. In this webinar, you will learn: • How to identify and monitor the critical quality attributes (CQAs) of your AAV therapy • What assays to utilize to confirm capsid protein identity and quantity • Why you need look to product characterization to identify and remove important product-related impurities
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Unveiling the Potential of your AAV Gene Therapy: Orthogonal methods to understand and define CQAs
- 1. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Unveiling the Potential of your AAV Gene Therapy: Orthogonal Methods to Understand & Define CQAs Martin De Cecco, Ph.D. Development Services Manager, Product Characterization Piotr Kaczmarek, Ph.D. Principal Scientist
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
- 3. 3 Advanced medicinal therapeutic products Ex Vivo Gene Therapy – Retro/Lentivirus In Vivo Gene Therapy – AAV/Adenovirus Transgene
- 4. 4 Adeno-associated virus (AAV) 1:1:10 60 subunits Capsid Proteins - Viral Proteins (VPs) VP1 VP2 VP3 1 735 203 735 138 735 REP CAP >13 different serotypes 3.9 MDa (empty capsid) 20-25 nm in diameter Natively package ssDNA to ~ 4.7 kb
- 5. 5 Testing gene therapy products Identity Titer & Potency Purity Residuals
- 6. 6 Identity Viral Vector Characterization ELISA GOI PCR Identity Assay − Gene of Interest Platform − Product Specific Development − Validation AAV Titration ELISA − AAV 2, 5, 8, 9 − Detects viral particles − Quantitative (Titer) NGS − Virus identity test − Whole genome sequencing − Comparison to known reference NGS ID FDA Guidance for Industry (January 2020): “Uniquely identify” a product & distinguish it from others. Good practice to use different test methods, e.g. protein capsid analysis.
- 7. Serotype identity 7 K. Van Vliet et al. Journal of Virological Methods (2009) 159: 167–177 High sequence homology between serotypes AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV7 AAV8 AAV9 AAV10 AAV11 AAV12 AAV1 83% 87% 64% 58% 99% 85% 84% 82% 85% 68% 63% AAV2 83% 87% 61% 58% 84% 83% 83% 82% 84% 64% 62% AAV3 87% 87% 63% 59% 87% 84% 86% 84% 86% 65% 62% AAV4 64% 61% 63% 54% 64% 65% 64% 64% 65% 82% 79% AAV5 58% 58% 59% 54% 58% 59% 58% 57% 58% 55% 54% AAV6 99% 84% 87% 64% 58% 85% 84% 82% 85% 68% 62% AAV7 85% 83% 84% 65% 59% 85% 88% 82% 88% 68% 63% AAV8 84% 83% 86% 64% 58% 84% 88% 85% 93% 67% 63% AAV9 82% 82% 84% 64% 57% 82% 82% 85% 86% 65% 62% AAV10 85% 84% 86% 65% 58% 85% 88% 93% 86% 68% 63% AAV11 68% 64% 65% 82% 55% 68% 68% 67% 65% 68% 85% AAV12 63% 62% 62% 79% 54% 62% 63% 63% 62% 63% 85%
- 8. Capsid protein purity, ratio, identity 8 VP1 VP2 VP3 Separation of VP1, VP2, VP3 by SDS-PAGE In-gel digestion LC-MS/MS of unique peptide Purity, ratio Identity
- 9. LC-MS for capsid protein purity, ratio, identity 9 AAV5, RPLC-MS (C18): TIC VP3 VP3’ VP2 VP1 594490 80305 65270
- 10. LC-MS for capsid protein purity, ratio, identity… and PTMs 10 Phosphorylated AAV vectors have reduced transduction efficiency VP1 VP2 80305 65270 Phosphorylation +80 Da Phosphorylation +80 Da
- 11. Method optimization necessary for different serotypes LC-MS for capsid protein purity, ratio, identity, PTMs 11 X. Zhang et al. Waters (2020) 720006869en RPLC (C4):
- 12. 12 Testing gene therapy products Identity Titer & Potency Purity Residuals
- 13. 13 Titer/Potency Viral Vector Characterization AAV TCID50 TCID50 − Infection of HeLaRC32 cells with serial dilutions of test sample − Virus is detected using CMV promoter based PCR − Scoring of +/- wells − Spearman/Karber Calculation of TCID50 Genomic Particle Count − Droplet Digital PCR based − CMV promoter for AAV − Client Specific GOI − Absolute quantitation − No need for standard curve Particle Count ELISA AAV Titration ELISA − AAV 2, 5, 8, 9 − Detects viral particles − Quantitative (Titer)
- 14. AAV titer by UHPLC 14 R² = 0.999 R² = 0.998 0 200 400 600 800 1000 0.0E+00 1.0E+13 2.0E+13 Peak area AAV concentration (vp/mL) 1.6E13 vp/mL 8.0E12 vp/mL 2.0E12 vp/mL AAV5 AAV5 AAV8
- 15. 15 Testing gene therapy products Identity Titer & Potency Purity Residuals
- 16. Empty/full capsid analysis 16 ▪ Empty capsids (containing no vector genome) potentially impact AAV product safety and efficacy ▪ Need to be characterized during process development and monitored ▪ Several possible analytical approaches: ▪ UV Spectroscopy (A260/A280) ▪ qPCR (vg) / ELISA (vp) ▪ Transmission Electron Microscopy (TEM) ▪ Analytical Ultracentrifugation (AUC) ▪ Ion exchange (IEX) chromatography… ▪ A combination of orthogonal approaches is recommended Empty Full FDA Guidance for Industry (January 2020): Defective or empty capsid particles should be measured & reported.
- 17. Empty/full capsid analysis by AEX-HPLC 17 H. Yang et al. Waters (2020) 720006825en Anion exchange chromatography: separation by differences in surface charge ▪ High throughput, low sample consumption ▪ Serotype dependent AAV8
- 18. Analytical Ultracentrifugation: sedimentation velocity analysis ▪ High resolution separation ▪ Serotype independent Empty/full capsid analysis by AUC 18 empty partial full aggregates?
- 19. Dynamic Light Scattering: ▪ Fast analysis ▪ Serotype independent Aggregates by DLS AAV2 AAV2 (aggregated) FDA Guidance for Industry (January 2020) Aggregates: key characteristic
- 20. Aggregates by SEC Size Exclusion Chromatography provides an orthogonal approach to other techniques (DLS, AUC) AAV8 Monomer 99.7% LMW 0.1% HMW 0.2%
- 21. Aggregates by SEC 21 HMW 4.5% AAV2 Monomer 94.7% LMW 0.8% AAV4 Monomer 94.8% HMW 2.6% LMW 2.6% AAV9 Monomer 98.3% HMW 1.1% LMW 0.7% AAV (DJ) Monomer 94.5% HMW 2.8% LMW 2.6%
- 22. Aggregates by SEC 22 S. M. Koza and W. Chen, Waters (2020) 720006812en Extended characterization by Multi-Angle Light Scattering M. Chen and A. Purchel, Wyatt (AN1617)
- 23. 23 Biosafety Viral Vector Characterization NGS-AAT Sterility Sterility − BACT/ALERT® 3D − Detects changes in pH due to bacterial growth − Real time sample monitoring − Objective readout Adventitious Virus − Detection and identification of adventitious agents − Circumvents toxicity and neutralization issues − Can be combined with ID test Replication Competent AAV − HEK-293 cell-based culture − Three rounds of amplification − qPCR endpoint Mycoplasma − PCR − Equivalent sensitivity and specificity to compendial method − GMP and EP 2.6.7 compliant − TAT and sample requirements better suited for cell therapies Mycoplasma rcAAV http://www.med.upenn.edu/gtp/images/e20_ aav_med_full.jpg
- 24. 24 Testing gene therapy products Identity Titer & Potency Purity Residuals
- 25. 25 Residuals Viral Vector Characterization Process Related Impurity Nature of Test Used Host cell protein ELISA using HCP specific antibodies (quantification), 2D LC-MS (characterization) Host cell DNA qPCR to target generic host cell sequences or specific sequences of concern Residual plasmid DNA qPCR targeting plasmid sequence (non-vector) Residual cell culture related components, e.g. BSA, HSA ELISA Residual process reagents e.g. benzonase, chromatography ligands ELISA Residual transfection agents (e.g. PEI) and surfactants (e.g. PS-20 / PS-80) UHPLC-CAD, UV-visible spectrophotometry
- 26. Analysis of gene therapy products Conclusions 1 There is increasingly clear regulatory guidance to ensure product quality and safety 2 A package of orthogonal analytical approaches is recommended 3 Analytical methods and technologies are improving, challenges remain 26
- 27. Development Services Manager, Product Characterization martin.de-cecco@merckgroup.com Martin De Cecco, 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. © 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Principal Scientist piotr.kaczmarek@milliporesigma.com Piotr Kaczmarek, PhD