Technology Highlights Adam Adler, Ph.D. LakePharma, I nc. October 4, 2013 LakePharma
Outline of Technology Highlights Hybridoma cloning • Work with complicated hybridoma samples Stable Cell Line Generation • Achieve high yield stable cell lines in a short time frame Maxcyte Electroporation • Improve methods for high viability, high yield transient protein production (and stable cell generation) Recombinant Protein Production • Rapid protein production at small-scale • DNA sequence to gram scale production in under 6 weeks Antibody Humanness Score • Developed method to quantify the humanness of monoclonal antibodies LakePharma
Hybridoma Cloning Have successfully cloned I gG hybridoma V H and V L from multiple species Have successfully cloned I gM hybridoma V H and V L from multiple species Have successfully cloned I gG B-cell V H and V L Success rate is > 95% (from more than 400 samples) Turnaround time is < 7 days LakePharma
Case Study: Customized Cloning of a Hamster Hybridoma with a Dominant Aberrant Light Chain Standard cloning method Customized cloning method QPPLVSVALGQKATITCSGD... QPPLVSVALGQKATITCSGD... QPPLVSVALGQKATITCSGD... LakePharma
Case Study: Cloning of a Mouse Hybridoma with Multiple Heavy and Light Chains Standard cloning method Relative affinity Customized analysis procedure LakePharma
Outline of Technology Highlights Hybridoma cloning • Work with complicated hybridoma samples Stable Cell Line Generation • Achieve high yield stable cell lines in a short time frame Maxcyte Electroporation • Improve methods for high viability, high yield transient protein production (and stable cell generation) Recombinant Protein Production • Rapid protein production at small-scale • DNA sequence to gram scale production in under 6 weeks Antibody Humanness Score • Developed method to quantify the humanness of monoclonal antibodies LakePharma
CHO Stable Cell Line Generation - Antibiotic System Generates Pools in 2 Weeks Serum-free suspension cell culture process Multiple selection markers can co-express multiple genes – Zeocin, puromycin, and hygromycin B Pool generation: 2 weeks, yield range is 50-200 mg/L 1 day 12-14 days 12-14 day production run in shake flasks Transfection Apply Cell banking; selection Ready for production run Clone generation: 5 weeks, yield range is 100-500 mg/L 1 day 12-14 days 4 days 9 days 5 days 12-14 day production run in 96 well plates Transfection Apply Transfer Transfer to 100 mL in Cell banking; selection to 24 well 50 mL tube shake flask Ready for production run Growth VCD & titer Titer LakePharma
CHO Antibiotic Stable Cells - Comparing Pool and High Performing Clone Antibody stable pool generated and Viable cell density robust clonal lines selected Highest performing clones show significant titer improvement over pool Cell viability Titer 1000 200 LakePharma
Case Study: I ncrease Expression of a Low Producing Antibody with CHO Antibiotic Stable A mouse IgG2b antibody produced at 4 mg/L in a CHO chemical transient transfection – 10-fold increase for electroporation or stable pool – 45-fold increase for stable clone Production yield Conditioned media Purified sample 4C4 6B5 6C4 Reducing Non-red Individual Clones LakePharma
Outline of Technology Highlights Hybridoma cloning • Work with complicated hybridoma samples Stable Cell Line Generation • Achieve high yield stable cell lines in a short time frame Maxcyte Electroporation • Improve methods for high viability, high yield transient protein production (and stable cell generation) Recombinant Protein Production • Rapid protein production at small-scale • DNA sequence to gram scale production in under 6 weeks Antibody Humanness Score • Developed method to quantify the humanness of monoclonal antibodies LakePharma
MaxCyte STX™ Electroporation Key advantages – > 95% transfection efficiency in CHO – > 95% viability after transfection – Scalable to 1 liter transfection LakePharma and MaxCyte are partners – To improve process – To further increase yield LakePharma
Case Study: High yield transient production using MaxCyte Antibody transiently transfected into CHO cells with MaxCyte electroporation – Reached 500 mg/L within 7 days of production Viable cell density (E6/mL) Cell viability Titer (mg/L) 500 LakePharma
Case Study: I mprove stable cell line generation process with MaxCyte system Results – Same DNA, same CHO cells, same selection procedure, different transfection method – Cell viability after transfection is higher with electroporation – 2-3 fold more expression with electroporation Stable Pool Comparison # 1 [Antibody] Stable Pool Comparison # 2 [Fc-fusion] Hypothesis – More copies of DNA are introduced to the cells by electroporation LakePharma
Outline of Technology Highlights Hybridoma cloning • Work with complicated hybridoma samples Stable Cell Line Generation • Achieve high yield stable cell lines in a short time frame Maxcyte Electroporation • Improve methods for high viability, high yield transient protein production (and stable cell generation) Recombinant Protein Production • Rapid protein production at small-scale • DNA sequence to gram scale production in under 6 weeks Antibody Humanness Score • Developed method to quantify the humanness of monoclonal antibodies LakePharma
High-Throughput Small Scale Antibody Production Standard order [10-100 antibodies per week]: 3-4 weeks 10-14 days 2 days 5 days 2 days Start DNA Constructs complete; Transfection Purification SDS-PAGE, construction DNA scale-up [50-100 mL] QC Rush order [3-5 antibodies]: ~ 2 weeks 7 days 5 days 2 days Start DNA Constructs complete; Purification SDS-PAGE, construction DNA scale-up; QC transfection Example: – Produced 4 antibodies for a client in 12 days • Started from DNA sequence: synthesis through purification • Included an international holiday – Obtained ~ 1 mg of each antibody LakePharma
[Low-Throughput Large Scale] Gene Synthesis to Gram Scale Antibodies I n 6 Weeks Gene synthesis and cloning are integrated; can be completed in 1 week Multiple production routes Route Yield range Gene Pilot Stable pool Scale up Total (mg/ L) synthesis test run generation production/ time + cloning purification 293 transient 50 – 500 1 week 1 week 2 weeks 4 weeks N/A CHO transient 50 – 500 1 week 1 week 3 weeks 5 weeks N/A (MaxCyte EP) CHO stable 50 – 500 1 week N/A 2 weeks 3 weeks 6 weeks LakePharma
Case Study: High yield 293 transient production Transient transfection of antibody into 293 cells: Viable cell density Cell viability Titer 5.0 120 700 600 600 100 4.0 500 80 1E6/mL 3.0 mg/L 400 % 60 300 2.0 40 200 1.0 20 100 0.0 0 0 d0 d1 d2 d3 d4 d5 d6 d7 d0 d1 d2 d3 d4 d5 d6 d7 d0 d1 d2 d3 d4 d5 d6 d7 Timeline: < 3 weeks 10 days 9 days kDa Start DNA DNA complete; QC’d purified 150 construction transfect cells protein is ready At 600 mg/ L, a 2 liter 100 75 production would be After second production run: 4 weeks total 50 sufficient for gram scale 37 9 days 25 20 New QC’d purified 15 transfection protein is ready 10 LakePharma
Case Study: Using a Stable Pool For Rapid Production Make CHO stable pool immediately followed by production run: Timeline: 5 weeks 7 days 14 days 14 days Start DNA DNA complete; Start QC’d purified construction electroporate; production run protein is ready apply selection for stable Viable cell density Cell viability Titer 300 At 300 mg/ L, 4 liter production would be sufficient for gram scale [or 10 liter WAVE would produce 3 grams] LakePharma
Case Study: Purify 1 Gram of a Low Producing Ab Antibody was low producing in transient: ~ 1.5 mg/L CHO stable clone was developed: ~ 50 mg/L Two 10 L production runs (WAVE) were performed: Viable cell density Cell viability Titer 1,126 mg antibody was purified I f speed was priority, same goal could be achieved with stable pool and lager volume production LakePharma
Outline of Technology Highlights Hybridoma cloning • Work with complicated hybridoma samples Stable Cell Line Generation • Achieve high yield stable cell lines in a short time frame Maxcyte Electroporation • Improve methods for high viability, high yield transient protein production (and stable cell generation) Recombinant Protein Production • Rapid protein production at small-scale • DNA sequence to gram scale production in under 6 weeks Antibody Humanness Score • Developed method to quantify the humanness of monoclonal antibodies LakePharma
T20 Score Analyzer to Quantify Ab Humanness We developed a tool to quantify the humanness of the variable regions of monoclonal antibodies – Based on sequence alignment to 1000s of sequenced human antibodies LakePharma
T20 Score Analyzer to Quantify Ab Humanness Heavy (variable) Light (variable) T20 Score Analyzer T20 score From 0-100; Higher score = More human-like LakePharma
Recommend
More recommend
