CHO-GSN PLATFORM STABLE CELL LINE GENERATION. NR v5

Transcription

1 CHO-GSN PLATFORM STABLE CELL LINE GENERATION NR v5

2 Highlights of LakePharma s CHO-GSN Cell Line Platform 2 LakePharma proprietary technology Complete cell line lineage and clear path to commercialization, freedom to operate Robust performance demonstrated CHO-K1 GS knockout line that enables stronger GS selection, resulting in high titer quickly Pool can reach >1 gram per liter in shake flasks Does not require extensive clone screening to obtain clones good enough to move forward Start to finish of the RCB in 6 months Compatible with many chemically defined commercial media High tolerance with shear (foam and tip speed) Generational stability >80 generations LakePharma technical team has significant experience expressing difficult proteins

3 CHO-GSN Stable Cell Line Generation Process 3 Clone selection by Ambr15 High titer clones selected Chemically defined, commercially available media used Ensured monoclonality Tolerance to shear force & other bioreactor conditions tested

4 Example 1: Titer of CHO-GSN Pool and Clones 4 mab Titer Profile This antibody had an expression level of 0.24 g/l in transient production in HEK293 cells The pool titer was 1.3 g/l. All 3 clones had varying levels of increased productivity. The best clone (1B6) had a titer of 4.5 g/l.

5 Example 2: CHO-GSN can achieve significant better yields than CHO-K1 5 Fc-Fusion Protein Titer Day CHO-K1 pool titer GSN is the LakePharma adapted suspension cell line. CHO-K1 is the parental cell line (native, suspension adapted). After transfection, selection and recovery of the GSN pool, single cell cloning was performed to obtain clones GSN 1A3 and GSN 1C1. The CHO-K1 cells were also transfected with the same DNA. There is a significant increase in titer when using the GSN line and an even higher increase when single cell cloning is performed.

6 Example 3: CHO-GSN Producing A Bispecific Antibody 6 Bispecific A Titer GSN Pool GSN 4C2 GSN 4C2 3A6 A bispecific antibody was produced in the GSN platform Characteristics: Non-Fc Product related variants presented challenges in single cell cloning and downstream process development Through method development and custom screening approach, LakePharma was successful in generating a cell line expressing the bispecific antibody

7 Example 4: Confirmed Monoclonality With Cell Imaging 7 Day 0 Day 1 Day 2 Day 7 Day 14 Stable Subclone Generation Using a Cell Imager. A stable clone was generated through limited dilution. To obtain a top subclone, the Solentim Cell Metric CLD was used. The top subclone can be clearly recorded to have been derived from a single cell at day 0.

8 Example 5: Clone Screening Using Sartorius Ambr15 Instrument 8 VCD Top clones: 5E7, 30H4, and 1B3 were top three clones selected by Qp and clone characterization Antibody Titer

9 Example 6: Cell Culture Process Optimization Using Ambr15 9 (Feeding strategy and ph control) Peak VCD reach to 35X10^6 cell/ml Feed F has better performance in general High dose feeds have detrimental impact on the cell line Higher ph has better cell growth profile and higher titer One round process optimization results more than 2.6 folder titer increase

10 Example 7: Gene Copy Number Correlates Well with Production Level 10 Antibody Productivity Correlates with Gene Copy Numbers of Both Heavy and Light Chains Standard Curve with Reference Gene Slope: R 2 : 0.999

11 RCB Characterization and Process Development 11 Release Testing Stability Studies Process Development Scale-up Productions Sterility Mycoplasma Copy Number Viral testing Develop and qualify analytical assays for stability and lot release Characterize Reference Materials; Demonstrate GLP/GMP batch comparability Cryopreservation and stability study over 80 generations Scalability in small scale bioreactors Generate sufficient stability data to support shelf-life of clinical material Process optimization Scale Down Scale up Develop a highperformance process suitable for clinical supply Transfer process to CMO and generate GMP Manufacturing Production Records Formulation Development WAVE bag production Medium scale bioreactor Produce GLP Drug Substance and produce/support GMP Drug Substance and Drug Product