Legacy NVx/GSK HIV gp120.c/mf59 Vaccine Development. Fred Porter PhD GSK Vaccines HIV Env Manufacturing Workshop June 11 th, 2015

Transcription

1 Legacy NVx/GSK HIV gp120.c/mf59 Vaccine Development Fred Porter PhD GSK Vaccines HIV Env Manufacturing Workshop June 11 th, 2015

2 Legacy Novartis (now GSK) HIV program gp120/mf-59 Delivery of CTM material and technical information to P5 Two recombinant gp120 antigens expressed in CHO cells; TV1.C (chronic), 1086.C (transmitted founder) Target Product Profile (TPP): Delivery of 50,000 doses of each antigen to support Phase I through Phase IIb trials in a phase-specific manner A complete data package to support regulatory filings 2 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

3 HIV gp120 Structure and Properties 1086.C and TV1.C gp120 Parameters 1086.C TV1.C Subtype C C Disease stage isolated Transmitted founder Chronic Disulfide bond Amino acids (aa) pi (polypeptide) MW (polypeptide) PNGS (gp120) V1V2 length (aa) integrin binding Yes No C1-C5 constant regions V1-V5 Hyper variable regions; Chemical modifications within V1V2 regions can affect antigenicity of gp120. V3 region is also immunogenic. It is critical to co-receptor binding. Disulfide bond at the base of the V3 loop has a critical role in the function of the protein when it is oxidized or reduced 3 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

4 Technical Challenges gp120: challenging and complex antigens for technical development Yield from candidate CHO cell cultures was low (50-70 mg/l of cell culture media). gp120 is susceptible to clipping and other modifications during production - Variable loops are prone to clipping by host (CHO) proteases - Protein modification during expression and purification Developing appropriate analytical assays is key for process control and product release/stability Critical quality attributes (CQAs) to be maintained - Glycan profile (approx. 50% glycan by mass) - Disulfide bridging and dimer content - Low chemical modification due to protein stress (oxidation) - In-vitro functionality: CD4 binding, key antigenic epitopes in V1/V2 loops 4 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

5 Process and Product Development Objectives and Outcome Goals: Development plans are pre-defined following QbD (quality by design) approach Product and process development targets are defined to ensure program meets objectives Development Target 1086.C (500L) TV1.C (1000L) Attributes Unit Target Actual Actual s Upstream Titer mg/ L 100 >400 >150 Process Yield % 20 >50 >33 Purity % Clipping % Dimer content % Batch Yield g > NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015 Outcome: Exceeded all performance targets Cell culture titers improved 4-8-fold from candidate CHO cultures. Selected between two distinct CHO/media platforms to address titer and clipping for each antigen. High process yield: approx 40% TV1.C; 60%1086.C

6 Tech Transfer to Rentschler Biotechnologie Completed Cell Banking Master Cell Bank (MCB) preparation & Characterization Working Cell Bank (WCB) preparation & Characterization Process and Assay Transfer Upstream and downstream process transfer and scale up Establishment & qualification of analytical methods Drug Substance Full scale engineering runs GMP manufacturing Primary reference standard Stability study of Drug Substance (DS) & reference standards Ongoing Drug Product Fill & finish Stability study Labeling Ongoing Phase I complete 6 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

7 HIV gp120 Tech Transfer Overview 1086.C DS TV1.C DS Development Runs (Legacy NVx) Tranfer Runs/ Consolidation Runs (CMO) Engineering Run (CMO) GMP Runs NVx TD Rentschler Rentschler Rentschler 10L 10L 50L 500L 500L Tox and CTMs NVx TD Rentschler Rentschler Rentschler 50L 50L 250L 1000L 1000L Tox CTMs Tox DP CTM I DP CTM II DP 1086.C DP TV1.C DP Rentschler Rentschler Rentschler 5383 vials 8686 vials vials vials 4735 vials 8635 vials vials vials NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June

8 1086.C Engineering Run DS Purity and CD4 Binding Comparison to Tech Transfer Runs _A2682_SA #11 [modified by Sa_E] TR01 I60-PP 001 0_1 UV_VIS_1 150 mau WVL:215 nm 100 TR01 Drug Substance RP-HPLC-Purity 20, ,295 19, _A1317_SA #8 [modified by Sa_E] TR02 DS 0_1 UV_VIS_1 150 mau WVL:215 nm ,341 19,076 19,323 19, _A1439_SA #7 [modified by Sa_E] CR01 DS 0_1 UV_VIS_1 150 mau WVL:215 nm ,463 18,937 19,388 19, _A1423_SA #11 [modified by Sa_E] CR02 DS 0_1 UV_VIS_1 150 mau WVL:215 nm 100 TR02 Drug Substance CR01 Drug Substance CR02 Drug Substance 20,267 20,334 20,355 21,598 21,542 21,632 21,751 22,137 22,199 22,301 23,346 23,337 Run TR01 TR02 CR01 CR02 ER CD4 Binding (%) Good Good Good Good Low 18,462 19,177 19,414 19, _A1439_Sa #8 [modified by Sa_GMP] DS 192C _1 UV_VIS_1 150 mau WVL:215 nm 100 Engineering Run Drug Substance ~ 11% (Oxidized gp-120) 18,087 18,369 18,993 19,254 19, min 10,0 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 21,0 22,0 23,0 24,0 25,0 26,0 27,0 28,0 29,0 30,0 20,195 21,662 21,753 21,545 21,627 22,316 22,221 22,517 23,233 23,931 24,157 24,429 NVx/GSK 8 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

9 Glucose g/l Cell Density Viability 1086.C Engineering Run USP Higher than expected cell growth and nutrient depletion 500L Engineering Run - Glucose depletion - Product OOS Eng Run Eng Sat 10L ENG Satellite - Identical glucose depletion - Identical gp120 characteristics Glucose depletion coincided with decreased O 2 demand Leading peak was purified from DS and found to be oxidized 1086.C gp120 Eng Run Eng Sat NVx/GSK 9 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

10 HIV 1086.C Engineering Run Investigation Increase in oxidized species coincides with time of glucose limitation in the bioreactor ER Day 10 ER Day 11 ER Clarified harvest ER Satellite Day 10 ER Sattellite Day 11 NVx/GSK 10 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

11 HIV 1086.C ER Investigation Summary USP Purity and CD4 Binding RP-HPLC Purity Lead Peak CD4 Binding Run D9 D10 D11 Harvest D9 D10 D11 Harvest CR na Good Good Good na ER 10L na Good Good Low na ER 500L Good Good Low Low Key point: Purity and CD4 Binding both decrease between Day 10 Day 11 in Eng Run and ER Satellite USP 11 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

12 Purity HIV 1086.C ER Investigation: Small scale test runs Low purity enhanced in starved culture, mitigated with improved feeding strategy CR02 Enhanced feed Standard feed Eng Run ER Sat Depleted feed Key points: Investigation Run standard condition and forced depletion cultures show significant purity loss Day (as for Eng Run and ER Sat) Investigation Run with enhanced feed does not show Day purity loss 12 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

13 CD4 binding HIV 1086.C ER Investigation: Small scale test runs Low CD4 binding enhanced in starved culture, mitigated with improved feeding strategy Decreased CD4 binding Day Key point: ER 500L, ER 10L satellite, Investigation Run standard condition AND forced glucose depletion culture All show significant potency loss Day NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

14 TV1.C Process Scale-up Intermediate scale up at 250L prior to 1000L runs Tech Transfer to Rentschler at 50L Single Use Bioreactor Process Scale 2 runs for training 2 additional consolidation runs at 250L scale to prior to full scale up Process fit for scale up Scale up process to 1000L Single Use Bioreactor for Phase I/II clinical production 1 Engineering run cgmp runs to provide CTM for Phase I/II 14 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

15 Technical Challenges; TV1.C Investigation of Low titer at intermediate scale; restored productivity and quality 2 x Tech Transfer Runs, and 2 x Consolidation Run competed in 250L SUB completed 60% lower productivity observed in CR1 & CR2 (250L) compared to Transfer Run 1-2 Low titer investigation launched in parallel with CR02 10L satellite performed parallel to CR02 to evaluate scale up Two shake flask studies evaluating multiple criteria performed in less than a month Two single root cause identified - Glutamine source (SAFC vs Merck) - Duration of Temperature Shift (6 hours in 50L vs 16 hours in 250L) Consolidation Run 3 at 250L scale and 10L satellite performed with modification in GMP suite at Rentschler Consolidation Run 3 successfully harvested with desired product quality 15 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

16 HIV TV1.C gp120 DS Analytical Testing Results Tox specifications based on 7 runs, ER met all the criteria. Assay Method Lot# Novartis Vaccines (NVx) Rentschler Biotechnologie (RB) Dev R1 Dev R2 Dev R3 TR01 TR02 CR01 CR03 ER1 Appearance Clarity Clear Clear ph Antigen Content (µg/ml) Intact TV1.C gp120 (%) in vitro Potency (% of ref std) Potentiomet ry RP-HPLC Purity (%) RP-HPLC Monomer (%) SE-HPLC Reduced SE-HPLC CD4 binding by Biacore Residual DNA (ng/100 µg of gp120) qpcr <LOQ <LOQ <LOQ <0.001 <LOQ <LOQ nd <LOQ CHO HCP (ng per µg of gp120) ELISA Endotoxin (EU/ 1000 µg of gp120 DS) NA not tested 2.2 <LOQ <LOQ nd <LOQ Protease NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

17 Technical Challenges; TV1.C Particulate formation observed in drug substance; solution identified Particulates first observed in DS after thaw, during DP development using material from RB lab run CR03 Particulate was not TV1.C Components of isolated particles found to be membrane associated HCPs by LC-MS. Present in trace amounts << 0.1% total protein Solved by addition of a flow-through chromatography step to remove HCP source Implemented with no change to buffer system, protein concentration Therefore no impact subsequent DP steps or stability program, Further processing step added to the current process without issue 17 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

18 Remedial Action Investigation Flow through chromatography works to reduce particulate formation Success in reducing particulate formation in Technical Run and Consolidation run material Particles Seen After (days) Control - unprocessed 4 DS Remains Clear (Ongoing) After (days) Resin 1 Processed 1CV load >38 Resin 1 Processed 3CV load >38 Control - unprocessed 4 Resin 2 Processed 1CV load >38 Resin 2 Processed 3CV load >38 18 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

19 Summary 1086.C and TV1.C Investigations 1086.C Observed lower purity and CD4 binding from Engineering Run DS at RB Upstream process modifications were made that resolved glucose limitation at high cell density and harvest pump limitations. Product quality was successfully restored. TV1.C Low titer and high HCP seen at 250L scale during CR01 were successfully resolved. Particulate formation was observed in DS. Further processing step is required to address this challenge. Solution has been identified and will be implemented for Engineering and GMP run DS. 19 NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

20 CTM Production GMP DS runs for 1086.C and TV1.C met all performance targets Upstream Process Performance Attributes Units Success Criterion Working Cell Bank Viability at Thaw Production Final Viability 1086.C TV1.C GMP Run Success Criterion GMP Run % % Production Final Titer mg/l Harvest Recovery % Harvest HCP Content ng/µg of in Harvest gp120 Harvest DNA Content ng/ml Downstream Process Performance Attributes Units Success Criterion 1086.C TV1.C GMP Run Success Criterion GMP Run Yield % Amount of protein g HCP Reduction from Harvest % N/A 98 N/A 95 DNA Reduction from Harvest % N/A >99.9 N/A > NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

21 1086.C Tox and CTM I/II Drug Products 1086.C DP lots passed all release criteria Attribute Analytical Method Tox DP CTM I DP CTM II DP (1 st Lot) Concentration RP-HPLC (mg/ml) In vitro Potency (CD4 Binding) Biacore (% to Ref Std) 93% 90% 98% Purity RP-HPLC 97.9% 98.0% 97.9% Monomer SE-HPLC 96.0% 95.5% 96.0% Intact gp120 Reduced SE-HPLC 82.7% 83.3% 85.8% Identity Slot Blot Positive for 1086.C gp120; negative for TV1.C gp120 Positive for 1086.C gp120; negative for TV1.C gp120 Positive for 1086.C gp120; negative for TV1.C gp120 Endotoxin LAL 1.3 EU/1000 µg < 1.23 EU/1000 µg <1.3 EU/1000 µg Clarity Clear Clear Clear Appearance Color Colorless Colorless Colorless Visible Particles Free of visible particles Free of visible particles Free of visible particles ph ph Osmolality Compendial 578 mosm/kg 581 mosm/kg 577 mosm/kg Sterility Membrane filtration Sterile Sterile Sterile Light obscuration Subvisible particles/container particles/container particles/container 1. Particles 10µm: particles 2. 1 particles/container 2. 1 particle/container 2. 1 particle/container 2. Particles 25µm: Uniformity of Mass variation 11.3% 4.7% 5.1% dosage unit Volume Extractable volume 0.44 ml 0.44 ml 0.44 ml NVx/GSK 21 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

22 TV1.C Tox and CTM I/II Drug Products TV.C DP lots passed all release criteria Attribute Analytical Method Tox DP CTM I DP CTM II DP (1 st Lot) Concentration RP-HPLC µg/ml µg/ml µg/ml In vitro Potency (CD4 Binding) Biacore (% to Ref Std) 98% 102% 102% Purity RP-HPLC 99.5% 99.5% 99.4% Monomer SE-HPLC 87.8% 88.1% 88.0% Intact gp120 Reduced SE-HPLC 92.6% 90.9% 91.2% Identity Slot Blot Positive for TV1.C gp120; negative for 1086.C gp120 Positive for TV1.C gp120; negative for 1086.C gp120 Positive for TV1.C gp120; negative for 1086.C gp120 Endotoxin LAL < 1.30 EU / 1000 µg < 1.27 EU / 1000 µg < 1.26 EU / 1000 µg Clarity Clear Clear Clear Appearance Color Colorless Colorless Colorless Visible Particles Free of visible particles Free of visible particles Free of visible particles ph ph Osmolality Compendial 578 mosm/kg 583 mosm/kg 581 mosm/kg Sterility Membrane filtration Sterile Sterile Sterile Light obscuration Subvisible particles/container particles/container particles/container 1. Particles 10µm: particles 2. 1 particles/container 2. 1 particle/container 2. 1 particle/container 2. Particles 25µm: Uniformity of Mass variation 4.6% 3.1% 3.9% dosage unit Volume Extractable volume 0.44 ml 0.43 ml 0.43 ml NVx/GSK 22 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

23 P5 Collaboration: What are the key project learnings? Aggressive timelines meant very large cross functional team of SMEs at Legacy NVx & at CMO 2 tiered governance assured visibility to all data & technical decisions ( bi-weekly TCs with PP slides). This business practice also included in MSA with CMO. Senior level governance needed as escalation mechanism for the technical team to manage business issues ( Legal agreements) and manage expectations Strategic gap in Legal agreements ( QAG & CTA) existed at the beginning of the project. Need to clarify Funder expectations for Regulatory filings, labeling text of CTM, and mechanism for document review/approvals earlier. Future programs would benefit from more coordinated planning between all key functions (Research, Technical development & Clinical) and funder counterparts prior to project initiation to align on all elements of the project. NVx/GSK 23 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

24 Summary Team has successfully completed the product and process development for both HIV TV1.C and 1086.C gp120 antigens. Processes and analytical methods have been successfully transferred to Rentschler Biotechnologie for GMP production. Productive processes with good yield for challenging targets Process scale of >300,000 doses/batch Yield of approx. 100 g of each gp120 product CQA monitored by well-established analytical assay panel to ensure consistent production of drug products for Tox and CTMs In-depth characterization of the antigens has been conducted to provide a robust package of supporting data. Phase I CTM delivered and clinical trial started Phase II delivery by the end of NVx/GSK HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015

25 Acknowledgements TD- Viral DS Brandon Medeiros Abishek Chandrashekhar Tiffany Teske Selen Karaca Kate Henry Swetha Sridhar Jurgen Mullberg Lauren Crumpler Chanel Marshall Matt Tyson Dennis Yu Chris Dadd Nathan Alden Akshit Gupta Brenda Kellogg Dipak Thakur Luis Maranga Pooja Balchandani Eric Garr James Leverone TD- Viral DS Philippe Gilbert Mark Wilson Fred Porter TD- Drug Product Antu Dey Adora Padilla Johanna Dutton Jonathan Anderson TD- Analytical Development Amy Yi Kwadwo Caesar Vaneet Sharma Maggie Liu Sang-woon Myung Henry Kha Changyun Xiong Rich Buckholz Ying Zhang Kingman Ng NVx/GSK 25 HIV gp120/mf59 Vaccine Program Fred Porter 11 June 2015 Program Management Raghu Shivappa Paula Keith Research Susan Hilt Mark Wininger Priyanka Ramesh Yingxia Wen Andrea Carfi Susan Barnett GSK Vaccines Legacy GSK Vaccines Marguerite Koutsoukos and team. Rentschler Biotechnologie Funding: NIH & BMGF