AAV Purification Techniques To Advance Gene Therapy

Learn more at thermofisher.com/captureselect Bioprocessing INTRODUCTION Adeno-Associated Virus (AAV) has become the vector of choice in many gene therapies. Recent advances in chromatography resin development for viral vectors have demonstrated that AAV purification can be scalable and efficient, achieving high purity and yield in a single step. With an extensively growing pipeline of gene therapy clinical trials, it is evident that scalable production solutions are needed. Here we outline the benefits of implementing affinity chromatography in the downstream purification of viral vectors. Chantelle Gaskin2, Kelly Flook2, Pim Hermans1, Alejandro Becerra2 , Frank Detmers1, Mike Coleman2 1. Thermo Fisher Scientific, Leiden, the Netherlands 2. Thermo Fisher Scientific, Bedford, MA Accelerating advancement in gene therapy by improving downstream purification of viral vectors TRADEMARKS/LICENSING © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. Intended use: For research use or further manufacturing. Not for use in diagnostic procedures. POROS™ CAPTURESELECT™ AAVX RESIN: A TRUE PLATFORM FOR AAV PURIFICATION Fig.1 CaptureSelect™ ligands are VHH fragments (single domain antibody fragments – sdAb), the smallest antigen binding molecule. The small size of VHH fragments (15kD) allows binding at difficult to reach epitopes. Overall, VHH fragments offer high specificity, affinity and stability. Harvest Clarification Cation Exchange Anion Exchange (Flow through) Anion Exchange Tangential Flow Filtration Sterile filtration Harvest Clarification Affinity Concentration Sterile Filtration Customer testimonial: “Process yield improvement from 20% to 60% & cost reduction by a factor of 6” CaptureSelect™ Paradigm •Affinity capture = fewer chromatography steps •Simplified process •Lower cost & speed to market •Fewer steps = higher yield AFFINITY SOLUTIONS FOR VIRAL VECTOR PURIFICATION Thermo Scientific™ resin Binding Capacity (vg/mL) Serotype Affinity POROS™ CaptureSelect™ AAV8 >1013 AAV8 POROS™ CaptureSelect™ AAV9 >1014 AAV9 POROS™ CaptureSelect™ AAVX >1014* AAV1, AAV2, AAV3, AAV4, AAV5. AAV6, AAV7, AAV8, AAV9, recombinant & chimeric vectors • Affinity through antibody selectivity (CaptureSelect™ technology) : technology based on single domain [VHH] antibody fragments. • Animal origin free production process (Saccharomyces cerevisiae) • Combined with the large through-pore POROS backbone * viral genomes per millilitre (vg)/mL, binding capacity will vary based on serotype, feed stream, additives, and mutations to parent serotypes ü Reduce the number of steps in a purification procedure without compromising product purity and yield ü Increase process flexibility and throughput ENABLING INDUSTRIAL SCALE DEVELOPMENT OF AAV VECTORS PURITY SDS-PAGE purity profile of AAV9 Vector recovery using POROS CaptureSelect AAV9 at various scales Fig. 3 Vector purity. A 1-step Comparible purity, as determined by SDS page, was obtained between a 1-step AAV9 affinity step en 3 IEX steps. Fig. 4 Vector recovery. Yields > 80% were obtained from 10 L tot 200 L scale. YIELD Fig. 2 Reduced number of process steps through AAV affinity chromatography resins ü Combining antibody-based selectivity and process robustness in unique AAV affinity resins ü Broad selectivity to both natural and synthetic capsids ü High dynamic binding capacity ü High elution recovery at different flow rates ü Robust, with less process optimization % breakthrough vg loaded per ml of resin Fig.6 AAVX resin serotype specificity with a large variety of serotypes. Experimental settings. Static binding mode experiment: resin was mixed with AAV serotype in tube – no flow properties performed. vg was determined by qPCR * Data kindly provided by Massachusetts Eye and Ear 97.8 98.33 98.05 97.88 98.93 98.37 97.76 98.43 96.28 99.29 98.51 77.99 98.39 0 20 40 60 80 100 AAV2 AAV2_HSPG AAV4 AAV5 AAV6.2 AAV7 AAV8 AAV9 AAVrh10 AAVrh32.33 AAV9PHPB Anc80 AAV7m8 POROS™ CaptureSelect ™AAVX resin: Serotype specificity vg distribution (% - eluate) % binding - eluate * ü To date, the AAVX ligand has shown affinity towards all serotypes tested Fig. 7 AAVX Breakthrough analysis. Breakthrough as a function of vg loaded per ml of resin. The breakthrough stayed below 0.5% up to 2e13 VG/ml of resin. Fig. 8 Elution recovery. Elution recovery was above 80% at each of the three different flow rates used. Recovery was not influenced by flow rate or residence time. ü High capacity and a high degree of process design flexibility ü High elution recovery at different flow rates CONCLUSIONS POROS™ CaptureSelect™ AAV resins address the current challenges involved with viral vector purification. Use of these resins will: • Simplify your purification process and increase process design flexibility • Increase purity and yield • Allow for scalable purification of multiple AAV serotypes in a single platform In addition we have demonstrated that the AAVX resin can be an effective viral clearance step in the downstream process of AAV manufacturing Fig. 9 Purification yield is comparable at various scales. Vector recovery in eluate after using POROS™ CaptureSelect™ AAVX resin for the purification of two different serotypes at various production scales. Use of AAVX at various scales shows: ü Robust resin scale up ü Comparable recoveries at various scales ü Consistency in resin performance 0% 20% 40% 60% 80% 100% 120% 12 L 500 L1000 L 3 L 7 L 10 L 200 L Serotype A Serotype C % Recovery UNDERSTANDING THE VIRAL CLEARANCE POTENTIAL Table 1. Clearance of model viruses. Log reduction values (LRV) of enveloped (env) and non-enveloped (nonenv) model viruses using the AAVX resin. ü The AAVX resin can be an effective viral clearance step in the downstream process of AAV production AAVX SEROTYPE SPECIFICITY BREAKTHROUGH ANALYSIS AND ELUTION RECOVERY DEMONSTRATING SCALABILITY RNA env DNA non-env DNA env RNA non-env Run Description XMuLV MVM Reo-3 HAV PRV HSV-1 Manufacturing process conditions > 6.4 4.4 2.7 > 4.9 4.0 3.1 Higher load ratio + residence time (worst case scenario) 4.6 3.6 2.5 5.0 3.8 3.6 Clearance Effective Effective Contributing Effective Effective Contributing ≥ 4 LRV Effective 1 - 3 LRV Contributing < 1 LRV Negligible Viral clearance data from an AAV8 clinical production process using the AAVX resin.