Multiplexing for Efficient Product Development

Transcription

1 Multiplexing for Efficient Product Development Susan Dana Jones, Ph.D. Presented at IC ioprocess International West Oakland Marriott, Oakland, C March 14 17, 2016 ioprocess Technology Consultants

2 Historical Perspective

3 Compared to Today Current Fast Track Product Development Timeline months Cell Line Development Cell banking and characterization Process Development & QC methods development Prepare tox lot Tox study GMP mfg IND

4 Cell Line Development cceleration CHEF1 5' flank MP CHEF1 promoter Intron pdef38.mb HindIII (4081) CHEF1 3' flank bp mbhc XhoI (5517) DHFR mblc Intron HindIII (8235) Expression Technologies XhoI (8958) CHEF1 5' flank CHEF1 promoter Robust Host CHO Cell Lines Rapid clone screening Micro-bioreactors Single use bioreactors High Throughput nalytics Figures courtesy of CMC iologics, Wikipedia, Molecular Devices, TP iosystems, and Günter Jagschies, GE Healthcare 4

5 VCD (x 10 6 cells/ml) Specific productivity (pg/cell.day) Harvest titre (g/l) Evolving Expression Technologies Reduce Risk FujiFilm Diosynth iotechnology (FD) pollo expression technology consists of optimized vectors, CHO host cell line, and media Pre-screening to identify a robust host CHO cell enables high titers and reduces scale-up risk C D Culture time (days) C D Data from pollo proof of concept work: Performance of four representative high producing cell lines Figures courtesy of 'FUJIFILM Diosynth iotechnologies Mammalian Cell Culture Team 5

6 Downstream Process Development Options Centritech Separation Systems Unifuge HTS systems (Tecan) with pre-packed plates, tips and columns to aide process design and process characterization (QD and DOE) Higher capacity, high resolution and high flow rate resins (Poros XS shown). Introduction of new modalities. e.g. the mixed mode resins and CaptCore700 Pre-packed columns and systems with disposable flow paths (shown is GE KT with Ready to Process column). Single use bags, filters and membranes are also available. Fully disposable plants exist Other areas of application and pursuit: ffinity chromatography capture steps for non Mabs Single-pass TFF Continuous chromatography PT

7 Historical Discovery and Development Path - Linear Target Discovery MO Screening & Lead Candidate Selection Process Development & Manufacturing Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2

8 New Discovery and Development Path - Integrated Target Discovery MO Screening & Lead Candidate Selection Process Development & Manufacturing Q1 Q2 Q1 Q2 Q3 Q4

9 Case Study #1 Progressing Multiple ntibody Candidates through Cell Line Development 9

10 ntibody Engineering and Development Company identified a murine antibody directed against its proprietary target cademic collaborator provided support for the development program ntibodies were humanized and affinity matured by ntitope Ltd. (Cambridge, UK) to obtain best-in-class lead therapeutic molecules Lead candidate termed b-01 in this presentation Development activities were initiated at CMC iologics S (Copenhagen) on eight candidates to de-risk candidate selection and accelerate process development Selected criteria for final b-01 lead selection Low nm to low pm affinity of whole IgG1 for human and primate target Good safety and PK in non-human primates Developability selection criteria Expression level of 1 g/l from pools and 2.5 g/l from clonal cell lines High melting temperature and stable to potential process conditions 10

11 Titer (mg/ml) Stable Pools using CHEF1 Expression Platform DN Sequence MP CHEF1 5' flank CHEF1 promoter Intron Transfection Transfection Pool Plasmid Construction CHEF1 3' flank pdef38.mb bp mbhc HindIII (4081) Selection XhoI (5517) CHEF1 5' flank DHFR CHEF1 promoter CHEF1 Expression Plasmid Enables Rapid Cell Line Development Transfection pool titers of >1 g/l can provide sufficient protein for early stage development activities Product from pools enables early evaluation in biologically relevant model systems Competitive early development titers (>2 g/l Mb), improved with Process Development mblc XhoI (8958) HindIII (8235) Intron Pool Titer Time (Days)

12 b-01 Evaluation and Lead Selection ctivities Stable pools produced > 30 mg of eight candidates for evaluation by functional assays including iacore and cell based assays Selection of four top candidates performed based on functional criteria To enable critical non-human primate assessments, > 2 g of top four candidates were produced from stable pools Provided test material and useful process information Stress stability studies performed on top four candidates Single cell cloning of all four candidates generated CHO production cell lines Single round of limiting dilution plus frequent imaging Day 0 Day 3 Day 7 Day 14 12

13 Cell Line Development Overlaps with Production CHO pools generated for multiple candidates (4 months) Single cell cloning and selection of 180 clones per antibody candidate (2 months) Evaluation of top 30 clones and selection of 2-4 cell lines per antibody (2 months) Variant Single cell Cloning Yes Top Clone Expression level 2.5 g/l Yes 3.2 g/l Yes 2.9 g/l Yes 3.6 g/l 13

14 Case Study #2 Screening for Lead Candidate using CHO Stable Pools and Platform Purification 14

15 ffimed Transforming Immuno-Oncology Using Next-Generation Immune Cell Engagers Nasdaq-listed company with headquarters in Heidelberg, Germany and offices in the U.S. Engineers targeted immunotherapies, seeking to cure patients by harnessing the power of innate and adaptive immunity (NKand T-cells) Pipeline based on proprietary, next-generation bispecific (Tandbs ) and trispecific antibodies Tandbs are designed to recruit immune effector cells (NK-cells or T-cells) and direct them to tumor cells resulting in their destruction Their adequate safety profile makes them suitable for monotherapy as well as for combination therapies with other drugs (e.g. CPIs) ffimed s bispecific NK-cell and T-cell engagers are currently in Phase 2 and Phase 1 clinical trials, respectively 15

16 ffimed s Tandb Platform is ased on the Variable Domains Tandbs are characterized by their unique tetravalent structure (four binding sites), which enables higher specificity and stronger binding 16

17 F o l d i n g p a t h w a y Tandb ntibodies Gene Organization and Intermolecular Pairing ) Gene organization: ) Tandb Formation: 5 V L V H V L V H 3 Variant 1 5 V L V H V L V H 3 L1 L2 L3 L1 L2 L3 Variant 2 Variant V H V L V H V L L1 L2 L3 V L V V V H L H 3 3 L1 V L V H L2 V L L3 V H non-functional monomer L1 L2 L3 Variant 4 5 V H V L V H V L 3 L1 L2 L3 V L L1 V H L2 V L L3 V H L1 L2 L3 Linker Fv-1 Fv-2 Variable antibody fragment NH 2 COOH V H L3 V L V H L1 V L non-covalent dimerization (head-to-tail) NH 2 Fv-1 Fv-2 Fv-2 Fv-1 L1 L2 L3 Tetravalent functional homodimer (~110 kda) L3 L1 17

18 Tandb Generation and Lead Candidate Selection Established Selection Process Tandb construction and generation Selection of variable fragments (Fvs) in accordance with antigen specific target profile parameters Source: phage or yeast display libraries, rabbit batch humanization (partner daughter company: bcheck) Construction of Tandb mini-libraries (up to 200 molecules) by combining Different target antibodies Different immune cell engaging antibodies (e.g. anti-cd3, anti-cd16) Variable linker lengths Variable linker compositions Established processes allow fast screening for Tandb candidates which qualify for further development; generic and project specific selection criteria apply, well balanced for: Expression and biophysical characteristics (incl. manufacturability) Functionality Safety 18

19 Screening Scheme for Tandbs Library screening and identification of target-specific scfv ffinity maturation Selection of scfv Formatting of target specific domains into Tandbs using anti-cd3 or anti-cd16 effector domain 1 st Selection criteria Stability inding Expression and purification of Tandbs from stable cell pools 1 st selection of Tandbs Identification of Lead candidates 2 nd selection of Tandbs Development and backup candidate 3 rd selection of Tandbs GMP manufacturing 2 nd Selection criteria Expression Homodimer content ctivity (binding) 3 rd Selection criteria Purity ccelerated & thermal stability Low ph stability ctivity in vitro (binding, cytotoxicity) Safety in vitro (proliferation, cytokines, act. markers) Characterization in vitro (specificity, internalization, etc.) 4 th Selection criteria Confirmation of stability / activity USP / DSP feasibility Pharmacodynamics in vivo 19

20 CCKEL_10 CHO/T100 Fast and Parallel Stable Cell Pool Generation and Production of Tandb Candidates for Screening Flp-In CHO host cell adapted to suspension (serum-free) Parallel processing of multiple constructs Small cell culture volumes during selection phase Recovery of stable pools within 2-3 weeks Transfection Selection Expansion for Production & Cryopreservation Selection Transfection Cryo. Production 20

21 Fast and Parallel Stable Cell Pool Generation and Production of Tandb Candidates for Screening Stably transfected CHO cell cultures are used for Tandb production Fed batch cultures in Shake flasks reach high cell densities, maintain high viabilities and accumulate high Tandb titers in the cell culture supernatant Production at variable scales (<10ml to >5L) reproducibly supplies high quality material for all phases from early screening to preclinical development 21 21

22 Purification Scheme for Tandb ntibodies Generic purification scheme is used to produce research grade material for all kind of testing during the Tandb selection process Generic purification process is used for process development at the CMO enabling attractive timelines in process development and manufacturing Clarified harvest ffinity chromatography Evaluation of homodimer content Virus inactivation Low ph stability Size exclusion chromatography ccelerated stability study Tandb lot XYZ Freeze/thaw stability study 22

23 Stability ssessment of a Tandb Candidate Stability assessment: Early ph-dependent thermal stability as well as accelerated stability testing using HPLC-, electrophoretic, as well as differential scanning techniques 23

24 ffimed Tandb ntibodies ffimed is developing next generation bispecific (Tandb) and trispecific antibody therapeutics Established screening process identifies suitable Tandb development candidates which comply with predefined functional, safety and developability criteria Major achievements Fast and reliable system for the production of Tandbs starting with the initiation of a Tandb project Defined product quality for all Tandbs during the entire development process Scalable production to supply different steps in discovery and preclinical development Developability assessment and established purification schemes Ensures developability at CMO Shortens process development at CMO Timelines of ~3 years from project initiation to IND is feasible 24

25 Moving on to Production Emerging Technologies Continue to Reduce Scale-up Risk and Timeline 25

26 High Throughput Process Development Workflow Plan Evaluate Predict 1 X X1 1 X3 1-1 Optimize Perform nalyze Optimize Courtesy Günter Jagschies, GE Healthcare 26

27 Single Use Technology Reduces Scale-up Time and Risk Smaller, single use bioreactors can produce product for tox and Phase 1 studies more rapidly and reliably than older technology Reproducible geometry and product contact surfaces facilitate scale-up to GMP Disposable technologies reduce suite set-up and turnover time Courtesy Günter Jagschies, GE Healthcare 27

28 Conclusions Significant advances in expression and purification technologies, miniaturization of process unit operations, robotics, and analysis have reduced timelines and risks for developing the initial manufacturing process Multiplexing, defined as overlapping discovery and development activities, can be applied more widely due to these technical advances in development Multiplexing enables superior candidate selection and accelerated timelines Requires additional resources to accomplish a non-linear development pathway Two case studies demonstrate application of multiplexing to facilitate the end of discovery and transition to development Parallel development of multiple antibody candidates facilitated by the industry s product class knowledge ffimed has developed a fast and reliable system for the production of Tandbs starting with the initiation of a Tandb project Multiplex approach allows lead candidate selection to be based on process considerations as well as biology De-risking product selection and future processing activities 28

29 cknowledgements Rob Holgate Project Management rron Hearn ntibody Engineering Tim Jones ntibody Engineering gata Oruba nnabelle Herrington-Symes Dawn embridge Estelle ona Laura Perry Tiffany Coget Torben Frandsen, VP PD Ulrika ndersson, Project Management Poul Rasmussen, Cell Line Development Christian Müller Upstream PD Simon ergmann Downstream PD Peter Thorsted nalytical Helle Wendelboe Manager, nalytical Kristina Ellwanger Protein Expression and Cell Engineering Michael Weichel Protein Chemistry Stefan Knackmuss Preclinical Development Claudia Wall PM, Regulatory ffairs & Q Erich Rajkovic, Research Operations & IP Shalaka Purohit, Program Management Joe Siemiatkoski nalytical Frank Riske Downstream PD Frank Castillo Cell culture PD and production ndreas Woppmann Quality 29

30 Thank You! Susan Dana Jones ioprocess Technology Consultants, Inc. 12 Gill Street, Suite 5450 Woburn, M 01801

31 Questions? Comments? Opinions? 31