Slide 1: Developability assessment of novel AAV capsids and payloads at early preclinical stage
Developability assessment of novel AAV capsids and payloads at early preclinical stage to enable development of AAV gene therapies Matteo Placidi, Hima Ramachandrareddy, Michael Grannan, Jeffery Thompson, Joseph Clement, David Alvarez, Kumar Dhanasekharan CONFIDENTI AL
Slide 2: Developability assessment as a de-risking tool
Developability assessment as a de-risking tool Critical Quality Role of Attributes Developability • A physical, chemical, • Applied as a tool to biological or microbiological filter out weaker characteristic/property candidates within an appropriate range to ensure the desired • Earlier intervention
Slide 3: Roadmap for established biologics
Roadmap for established biologics CQA/CDA Stress/Degradation Predictive Analytical Tool Rationale Aggregation, fragmentation, ΔTemp, ΔpH, F/T, high salt, SEC(MALS), DSF, CESDS, RP- Stability predictor, impact of viral Purity/ hydrophobicity, charge ionic strength HPLC(MS), HIC, CIEF, IEX, ζ- inactiv
Slide 4: Roadmap for established biologics
Roadmap for established biologics CQA/CDA Stress/Degradation Predictive Analytical Tool Rationale Aggregation, fragmentation, ΔTemp, ΔpH, F/T, high salt, SEC(MALS), DSF, CESDS, RP- Stability predictor, impact of viral Purity/ hydrophobicity, charge ionic strength HPLC(MS), HIC, CIEF, IEX, ζ- inactiv
Slide 5: Standard approach to stage appropriate analytics
Standard approach to stage appropriate analytics Narrowing of Increasing Development Toolkit Candidates Sophistication Detailed Characterization Initial Release Methods ATTRIBUTE METHOD ATTRIBUTE METHOD Vector titer ddPCR Vector titer ddPCR Genome sequence PacBio Sequencing/NGS Genomic integrity Aga
Slide 6: Advanced analytics to assess capsid integrity
Advanced analytics to assess capsid integrity SEC-HPLC AUC CESDS CESDS Capsid Identity AUC (LOC/ HOC) (%HMW) %(Full/Partial/Empty) (VP3:VP2:VP1) %Purity AAVa.TG1 9.9 53/ 10/ 14 11/ 12 7:1:1 77% AAVb.TG1 12.3 54 / 12 / 20 0 / 14 9:1:1 86% AAVc.TG1 13.2 62 / 8 / 17 8 / 15 6:1:1 98% AAVa.TG2 4.6 80 / 3
Slide 7: Capsid integrity of batches pre/post optimization
Capsid integrity of batches pre/post optimization Capsid Identity SEC-FLD (%HMW) SEC-MALS (%Full) AUC (% Full/ Partial/ Empty) AUC (LOC/ HOC) AAVa.TG1, high conc., 2-8 °C 9.9 69 53/ 10 / 14 11 / 12 AAVa.TG1, lower conc., -80 °C 1.8 83 82 / 5 / 4 1/9 AUC: Capsid Occupancy CESDS: VP Ratio & %Purity Fu
Slide 8: AUC as a tool to examine fragments and aggregates
AUC as a tool to examine fragments and aggregates AAVa.TG1 AAVa.TG1 Capsid fragments, DNA with associated DNA species (HOC) (LOC 1) Overfilled (LOC 2) • UV A260 and interference (IF) collected on LOC 1 LOC 2 Empty Partial Full HOC the same sample, data analyzed using SEDFIT Signal (Area) (Area) (Are
Slide 9: Transgene sequencing: NGS and PacBio
Transgene sequencing: NGS and PacBio PacBio long read: Transgene Sequence NGS: Short sequence and variants AAVa.TG1 Population mapped % Consensus similarity to TG1 to reference Reference AAVa 93.5% 100 AAVb 89.1% 100 AAVc 94.7% 99.93 AAVa (opt) 94.7% 100 Average length: 2.4 kb, 3% variants in GOI •
Slide 10: Can early in vitro assays be used to screen outcomes?
Can early in vitro assays be used to screen outcomes? Capsid.Transgene (LogIC50) Relative Potency: Functional Output AAVa.TG1 5.22 120 AAVa.TG1 100 AAVb.TG1 5.05 % Analyte Knockdown AAVb.TG1 80 AAVd.TG1 AAVd.TG1 5.71 60 AAVe.TG1 40 AAVa.TG1 Optimized AAVe.TG1 4.96 20 0 10 1 10 2 10 3 10 4 10 5 10 6
Slide 11: LCMS peptide mapping to establish baseline PTMs
LCMS peptide mapping to establish baseline PTMs Other PTMs (Oxidation, methylation, phosphorylation) were examined, focus on deamidation • Majority high deamidations contain NG motif1 • Average for each construct has ≤ 5% • Changes at key deamidation sites have the potential to impact stability and
Slide 12: Early process comparison with advanced analytics
Early process comparison with advanced analytics AUC SEC-FLD CESDS Construct Source %(Full/Partial/Empty) %HMW %Purity Process 1 Process 1 70 / 7 / 5 4.6% 98% AAVd.TG3 AAVa.TG2 Process 2 78 / 2 / 1 0.1% 98% Process 1 76 / 9 / 5 4.3% 97% AAVa.TG3 Process 2 84 / 2 / 1 0.1% 98% Process 1 68 / 11 / 9 7.
Slide 13: Conclusions
Conclusions • To de-risk early selection activities recommend the inclusion of key analytics prior to in vivo studies • Essential to correlate structural data and in vivo activity to continually assess and build analytical toolkit • Need to bring in earlier manufacturability/developability assessmen
Slide 14: Acknowledgements
Acknowledgements Voyager • The Research Team – Collaboration and guidance • Process Analytics Team – SEC-MALS, AUC & CESDS data • Vector Production & Pilot Plant Team – Steadfast providers of high-quality material BioAnalysis • AUC and LCMS analysis Azenta • NGS and PacBio analysis 14 |