Adeno-associated viral vector (AAV) has become a preferred gene therapy delivery modality; however, AAV manufacturing, and more specific the analytics, is not without its challenges, whether that is using crude samples, a lack of standardized methods or incompatible techniques.
This whitepaper highlights AAV analytics solutions, throughout the complete manufacturing process, based on label-free Biolayer Interferometry (BLI) technology. BLI provides highly sensitive, accurate and automated assays for integration into manufacturing processes.
Download this whitepaper to learn more about:
- BLI technology
- Rapid, label-free and easy to use AAV analytics solutions for capsid titer measurements across the complete manufacturing workflow
- AAV titer and empty/full capsid determination on one single platform, using crude samples
BLI is a label free detection method based on reflection of white light from the surface of a biosensor tip. It analyzes the changes in interference pattern of white light reflected from the tip when biomolecules bind to it. This change is recorded in real time and is expressed as nanometer shift. It is proportional to the number and size of biomolecules bound to the tip. Gator Bio’s AAV Analytics solution includes the BLI Instrument, biosensors coated with a nanobody that binds specifically to AAV capsids, reagents, and GatorOne software. What is BLI? Time Biosensor Tip Sample trays Shift (nm) Reflection beams Incident light Shift (nm) Time Biosensor tip ∆λ ∆λ BLI principlePage 3 www.gatorbio.com Page 4 Gator® Bio presents Next-gen Biolayer Interferometry (BLI)-based AAV Analytics suite of solutions that include capsid titer, empty/full ratio determination, and TAb and NAb assays of adeno-associated viral vector. AAV processing Cell banks and virus seed stocks Cell expansion Transfection Viral vector production Viral vector purification Viral vector formulation Clinical trial Downstream Upstream Fill & finish formulation HS AAV Kit HS AAV9 Kit AAV Ratio Kit AAVX Probe AAV Ratio Kit AAVX Probe AAV9 Probe AAV9 Probe NAb Assay Custom TAb Our AAV solutions include Next-gen BLI for AAV Analytics Custom TAb and NAb assays for clinical trials Kinetic characterization of AAV neutralizing antibodies AAV analytics during manufacturing process is challenging due to lack of standardized methods and crude matrix incompatible empty/full ratio determination techniques. Gator Bio’s AAV Analytics solutions offer crude sample compatibility, high sensitivity, accuracy, robustness, automation, reduced hands-on time, and fluidic-free instrumentation, making them suitable for integration into manufacturing processes. Automated and robust titer of AAV serotypes 1-10 and chimerics in the range 107 - 1013 vp/mL Automated empty/full determination with dynamic range of 0% - 100% full capsids with resolution of 5%Page 5 www.gatorbio.com Page 6 Gator® AAV solutions are based on simple single technology obviating the need for different types of instrumentation and expertise. The "dilute and dip" method for complex matrices simplifies the workflow that can be deployed in any laboratory supporting AAV processing. These methods have much shorter turnaround time compared to other methods. Gator® 26 min. 96 samples ELISA 240 min. Comparison of Gator® workflow Gator® ELISA 26 min. 240 min. Gator® BLI benefits Buffer prep Incubation Wash Wash Wash Detection Add substrate Finish Strep HRP Hydrate biosensor Assay Finish Gator® Titer Gator® E/F DLS/UV Mass photometry ELISA q PCR dd PCR AUC TEM 107 - 1013 107 – 109 105 – 1010 102 – 107 Capsid titer E/F ratio E/F ratio E/F ratio Capsid titer Genome titer Genome titer E/F ratio E/F ratio Fully automated Crude sample tolerant Dynamic range vp/mL CQA 0% - 100% full and resolution of 5%Page 7 www.gatorbio.com Page 8 Crude sample tolerant LoQ 107 vp/mL Single platform for titer and ratio Automated assay High precision What makes us different? One platform for titer and empty/full ratio determination in pure and crude matrices. Our kits Wide dynamic range - 107 vp/mL to 1013 vp/mL Single platform for titer and empty/full ratio determination Automated with minimal time Crude matrix compatibility Easy integration into manufacturing process High Sensitivity AAV Kit High Sensitivity AAV 9 Kit AAV Ratio Kit AAVX Probe AAV9 Probe High sensitivity titer of AAV 1 - 8, 10 and chimerics High sensitivity titer of AAV9 E/F ratio Binds serotypes AAV1 - 10 Binds specifically to AAV9 Titer Titer Ratio determination Titer Titer Dynamic range: 107 - 5 x 1010 vp/mL Dynamic range: 107 - 109 vp/mL Dynamic range: 0% - 100% full and resolution of 5% Dynamic range: 109 – 1013 vp/mL Dynamic range: 3 x 109 – 1013 vp/mL Upstream DownstreamPage 9 www.gatorbio.com Page 10 0 4.00E+07 8.00E+07 2.00E+07 6.00E+07 1.00E+08 2.0 3.0 3.5 1.0 0 Shift (nm) vp/mL R2 = 0.99 The Gator® HS AAV and HS AAV9 Kits enable high sensitivity titer of AAV serotypes 1 - 8, 10, chimeric serotypes, and AAV9 respectively in purified and crude samples. Powered by a patented signal amplification technology LoQ of 107 vp/mL Dilute and dip: minimal hands on time compared to ELISA Automated assay steps including washes compatible with crude samples Samples such as cell culture media, clarified cell lysate, and cell lysis buffer Recovery of AAV spiked at high (108 vp/mL), medium (5.33 x 107 vp/mL) and low (107 vp/mL) concentration in various matrices HS AAV kit standard curve for AAV5 Cell Banks and Virus Seed Stocks Cell Expansion Transfection Viral Vector Production Viral Vector Purification Viral Vector Formulation Clinical Trial HS AAV Kit HS AAV9 Kit Downstream Upstream Fill & Finish Formulation AAV Ratio Kit AAVX Probe AAV Ratio Kit AAVX Probe AAV9 Probe AAV9 Probe NAb Assay Custom TAb Performance Advantages Titer solutions for upstream application 80 100 60 HEK293T lysate Cell lysis buffer Spent media High spike Medium spike Low spike 40 20 0 Recovery (%) LoQ Dynamic range Precision HS AAV 107 vp/mL 107 - 5 x 1010 vp/mL 100 min. <10% HS AAV9 107 vp/mL 107 – 109 vp/mL 100 min. <10% Assay runtime for 96 sampleswww.gatorbio.com Page 12 Page 11 Advantages Standard curve showing the dynamic range of AAVX probes using AAV2 Gator AAVX and ELISA titer correlation for AAV9 The Gator® AAVX and AAV9 probes enable direct capture and titer of AAV1-10, and AAV9 respectively in crude lysates, column eluates, cell lysates, and cell culture supernatants. These probes use proven CaptureSelect™ (Thermo Fisher Scientific) high affinity nanobodies. Faster than ELISA with minimal hands-on time Significantly cost effective because of reusable probes Compatible with crude samples such as cells lysates and media Excellent correlation with ELISA Cell Banks and Virus Seed Stocks Cell Expansion Transfection Viral Vector Production Viral Vector Purification Viral Vector Formulation Clinical Trial HS AAV Kit HS AAV9 Kit Downstream Upstream Fill & Finish Formulation AAV Ratio Kit AAV Ratio Kit NAb Assay Custom TAb AAVX Probe AAVX Probe AAV9 Probe AAV9 Probe Titer solutions for downstream application 0 2.0E+11 1.0E+11 3.0E+11 0.010 0.015 0.005 0 Binding rate vp/mL 0 5.0E+08 1.0E+09 1.5E+09 1.0E+09 1.5E+09 R2 = 0.99 5.0E+08 0 Gator vp/mL ELISA (vp/mL) Performance Serotype compatibility Dynamic range Regenerable Precision AAVX AAV1-10 and chimerics 109 – 1013 vp/mL 32 min. Yes <10% AAV9 AAV9 3 x 109 - 1013 vp/mL 32 min. Yes < 10% Assay runtime for 384 samples R2 = 0.99Page 13 www.gatorbio.com Page 14 Range Resolution AAV Ratio Kit 0% - 100% full 40 min. 5% Assay runtime for 8 samples Automated and fast workflow Easy integration into upstream process due to no sample preprocessing Single platform for empty/full determination Fast turnround time Low sample requirement Gator® AAV Ratio Kit detects single stranded DNA content in crude and purified AAV capsids. Unlike techniques like AUC that are only accurate above 40% full, the Gator method is accurate from 0-100% full. Cell Banks and Virus Seed Stocks Cell Expansion Transfection Viral Vector Production Viral Vector Purification Viral Vector Formulation Clinical Trial HS AAV Kit HS AAV9 Kit Downstream Upstream Fill & Finish Formulation AAV Ratio Kit AAV Ratio Kit NAb Assay Custom TAb AAVX Probe AAVX Probe AAV9 Probe AAV9 Probe Advantages AAV empty/full ratio determination Performance 3-step workflow Standard curve for AAV8 0 75 50 100 8 12 R2 = 0.99 4 6 10 2 0 Shift (nm) Full (%) 25 Affinity capture (crude samples) Lysis (ssDNA release) ssDNA assay Highly accurate % full ratio even below 40% full Biosensor TipPage 15 www.gatorbio.com Page 16 0 30 20 40 30 50 R2 = 0.95 10 20 40 0 Shift (nm) Full (%) 10 30 50 10 20 40 0 Shift (nm) Time (s) 37% 31% 24% 18% 11% 5% 0 200250 150 300 100 50 The AAV Ratio Kit crude sample compatibility is demonstrated by data using customer samples from different stages of AAV manufacturing process. The AAV Ratio Kit has an estimated Method Detection Limit (MDL) of 1.8% percent full with customer provided samples that range from 5% to 37% full. Cell Banks and Virus Seed Stocks Cell Expansion Transfection Viral Vector Production Viral Vector Purification Viral Vector Formulation Clinical Trial HS AAV Kit HS AAV9 Kit Downstream Upstream Fill & Finish Formulation AAV Ratio Kit AAV Ratio Kit NAb Assay Custom TAb AAVX Probe AAVX Probe AAV9 Probe AAV9 Probe Upstream samples Downstream samples AAV % full analysis MDL = 1.8% full Measured values and rank ordering is in line with gold standard method measurement AAV empty/full ratio determination Stage of manufacturing Crude sample Purified sample 9 12 2 5 2 27 4 13 1 12 22 33 2 3 15 6 9 40 14 24 28 19 12 5 13 18 4 10 8 9 18 17 3 0 8 11 6 8 13 19 11 38 10 19 17 19 12 3 1 2 3 4 5 6 7 8 90% 75% 60% 45% 30% 10% % Full Buffer Difference from expected values: <15% 15-25% >25% Difference between measured and expected % full values for eight different buffers utilized in chimeric AAV2 manufacturing % full ratio determination in crude customer samples AAV Ratio kit MDL determination in crude customer samples % full AAV standard curve ssDNA signal of lysed standards containing different % full capsids 5% (2) Blank (1.35) 0 1 2 ShiftPage 17 www.gatorbi