Delivering More Efficient Therapeutic Protein Expression Systems Through Cell Line Engineering Applications
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The document provides an overview of cell line engineering strategies for therapeutic protein manufacturing, highlighting the significance of genome editing technologies like ZFN and the advantages of using GS-/- host cell lines. It discusses approaches to improve clone stability, product yield, and efficiency in biomanufacturing processes through glycoengineering and targeted gene integration. The webinar emphasizes enhancing product quality and reducing regulatory complexities, ultimately leading to more effective therapeutic proteins.
Delivering More Efficient Therapeutic Protein Expression Systems Through Cell Line Engineering Applications
- 1. Trissa Borgschulte, PhD Head of Cell Line Development and Engineering Process Solutions Upstream R&D Applications in Therapeutic Protein Expression Systems ONLY FOR U.S. AND CANADA AUDIENCE
- 2. 2 Outline Biomarker Discovery Background ZFN Genome Editing Technology Glutamine Synthetase Cell Line Engineering Glycoengineering Targeted Gene Integration Webinar: Cell Line Engineering
- 3. 3 Industry feedback Webinar: Cell Line Engineering Support the use of concentrated feeds Provide for reduced analytical workload Support clone stability Maintain/enable favorable PQ attributes Reduce regulatory compliance costs Control/reduce metabolic waste Reduce product and process related impurities Reduce regulatory and risk considerations Increase process robustness Promote high product yield Support high growth rates and cell densities Scalable process Provide batch to batch consistency Support single use disposables Downstream process friendly Promote culture longevity Maintain product stability through harvest Reduce product heterogeneity What makes a robust therapeutic protein manufacturing process?
- 4. 4 Webinar: Cell Line Engineering Cell Line Development and Engineering Strategy Sources of Cell Line Engineering Targets Discovery Literature Customer requests Applications in Biomanufacturing Selection mechanisms Cell culture performance Anti-apoptotic, metabolic engineering Glycoengineering Host cell proteins Risk mitigation Viral resistance Targeted gene integration Understanding CHO Cell Biology Cell Line Engineering Media, Feed and Process Optimization
- 5. 5 Webinar: Cell Line Engineering Trait Stacking to Improve Therapeutic Protein Manufacturing Processes and Products TI Streamlined CLD Process and Enhanced Performance Homogeneity, stability More Effective Biologics Removal of immunogenic sugars, increased drug half-life Robust CLD Process and High Performing Recombinant Clones Reduced time lines, high titers, increased stability More Efficient Manufacturing Processes Viral resistant cell lines, removal of contaminating host cell proteins Strong Base Suspension adapted, CD media, bioreactor robust Glyco- engineering Risk Mitigation CHO GS-/- CHO K1
- 6. 6 Webinar: Cell Line Engineering Genome Editing with ZFNs Zinc Finger Nucleases Introduce double strand breaks at sequence specific genomic loci Limited off-target effects Cellular DNA Repair Mechanisms NHEJ – imperfect repair resulting in INDELS (gene KO) HR – “perfect” repair by copying off of a template (targeted integration) Cellular DNA Repair DNA Binding and Cutting
- 7. 7 Webinar: Cell Line Engineering Cell Line Engineering Workflow ZFN Transfection Activity Confirmation (Cel 1 assay) Single Cell Cloning Expansion and Further Characterization PCR and Seq Analysis Phenotypic Assay (if available)
- 8. 8 Webinar: Cell Line Engineering Metabolic Selection: Benefits of a GS-/- Host Cell Line GS-/- host cell lines lead to decreased CLD timelines and resources and enhanced manufacturing performance • Multiple rounds of amplification are not required • Fewer clones evaluated to identify high performers • Decreased CLD timelines and resources GS vs. DHFR • MSX not required • High performance in CD processes • Increased clone stability • Fewer clones evaluated to identify high performers • Decreased CLD timelines and resources GS-/- vs. GS+/+ • System saved 8 weeks of development time • Stable pool titers exceeding 1g/L • Clone titers averaging 2- 4.5 g/L • Significant increase in clone stability • Fewer clones evaluated to identify high performers GS-/- Industry Feedback
- 9. 9 Webinar: Cell Line Engineering CHO K1 GS-/- Cell Line Generation and Characterization Genotype Confirmation •Genome sequencing •qRT-PCR •Western Blot •Glutamine sensitivity Phenotype Characterization •Robust growth/scalability in CD media •Metabolic Profiling •Transfectability •Transient r-protein expression •Cloning efficiencies Performance Validation •Stable r-protein expression •High pool and clone titers •Long term stability •Complex N-glycans 20 clones 3 clones
- 10. Transfection -Gln Stable Pool Selection (minipools) Stable Pool Expansion and Characterization Single Cell Cloning of Lead Pools Clone Expansion and Characterization 10 Webinar: Cell Line Engineering GS-/- Recombinant Cell Line Generation 7–9 weeks 7–9 weeks
- 11. 11 Webinar: Cell Line Engineering GS-/- IgG Producing Stable Pools and Clones: Productivity Performance Fed Batch TPP 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Stable Pools - Fed Batch Clones - Fed Batch g/L Titer Range of Top Expressing GS-/- Pools and Clones
- 12. 12 Webinar: Cell Line Engineering GS-/- IgG Stable Clones: Stability Performance 8 out of 10 top clones maintain >70% titer over 60 generations
- 13. 13 Webinar: Cell Line Engineering Glycoengineering for Enhanced Product Quality
- 14. 14 Webinar: Cell Line Engineering N-Glycan Biosynthesis in CHO Modified from: Hossler et al., (2009) Glycobiology GGTA
- 15. 15 Webinar: Cell Line Engineering Engineering a Mgat1- Cell Line for Recombinant Proteins with High Man5 Glycoforms Sources: 1. Betting, et al. (2009) Vaccine 2. Lam, et al. (2005) J of Immunology Targeting applications for therapeutic proteins Cerezyme® targets the mannose receptor on macrophages Carbohydrate remodeling required Increased efficacy of mannose receptor targeted vaccines • Insect cell derived antigens are more effective for targeting mannose receptors on APCs1 • Antigens produced in Pichia Pastoris are more potent at inducing CD4+ T cell proliferation2 X-ray crystallography • Increased homogeneity in protein structure
- 16. 16 Webinar: Cell Line Engineering Mgat1- Cell Line Engineering in a Recombinant IgG Producing CHO Clone Lectin Enrichment of ZFN Transfected Pools Ricinus communis agglutinin-I (RCA-I) Lectin isolated from castor bean seeds Binds to terminal galactose residues Toxic to CHO cells with WT glycan profiles Ladder Mock ZFN DNA ZFN RNA ZFN RNA ZFN DNA Percent Modified 0 3.8 5.5 35.7 33.2 -RCA +RCA
- 17. 17 Webinar: Cell Line Engineering Man5 Glycoform is the Predominant Species in Lectin Enriched Pools Mock-transfected RCA-I enriched
- 18. 18 Webinar: Cell Line Engineering Man5 Glycoform is the Predominant Species in Lectin Enriched Pools 0 10 20 30 40 50 60 70 80 90 100 PercentGlycanSpecies Mock Mgat1 ZFN DNA Mgat1 ZFN RNA Mgat1 ZFN DNA + RCA-I Mgat1 ZFN RNA + RCA-I
- 19. 19 Webinar: Cell Line Engineering Mgat1- Clones Have Similar Growth and Productivity to WT Clones 0 1 2 3 4 5 6 7 8 9 0 2 4 6 8 10 12 14 VCD(10e6cells/ml) Days in Culture Original WT1 WT2 KO1 -RCA KO2 -RCA KO3 +RCA KO4 +RCA KO5 +RCA KO6 +RCA 0 200 400 600 800 1000 Original WT1 WT2 KO1 - RCA KO2 - RCA KO3 +RCA KO4 +RCA KO5 +RCA KO6 +RCA IgG(mg/L)
- 20. 20 Webinar: Cell Line Engineering Mgat1- Clones Have Man5 as Predominant Glycan Species 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 PercentGlycanSpecies Original WT1 WT2 KO1 -RCA KO2 -RCA KO3 +RCA KO4 +RCA KO5 +RCA KO6 +RCA
- 21. 21 Webinar: Cell Line Engineering Mgat1- Cell Line Engineering in a CHO K1 GS-/- Host Cell Line: Growth Performance 0 2 4 6 8 10 12 0 2 4 6 8 10 12 VCD(10e6cells/ml) Days in Culture WT AB4 BC9 BD7 BH11 CG10
- 22. 22 Webinar: Cell Line Engineering GS-/-/Mgat1- IgG Producing Stable Pools: Productivity Performance 7 day terminal TPP 0 50 100 150 200 250 300 350 400 GS-/- GS-/-/Mgat1-/-
- 23. 23 Webinar: Cell Line Engineering Man5 Glycoform is the Predominant Species in GS-/-/Mgat1- IgG Stable Pools GS-/- IgG Stable Pool 1 GS-/-/Mgat1- IgG Stable Pool 1 GS-/-/Mgat1- IgG Stable Pool 2
- 24. 24 Webinar: Cell Line Engineering Engineering CMAH-/- and GGTA-/- Cell Lines for Decreased Biotherapeutic Immunogenicity CMAH CMP-Neu5Ac hydroxylase Conversion of Neu5Ac to Neu5Gc sialic acid N-acetylneuraminic acid (NANA or Neu5Ac) N-glycolylneuraminic acid (NGNA or Neu5Gc) Neu5Gc is not expressed in humans and can be recognized as a foreign epitope GGTA N-acetyllactosaminide 3-α-galactosyltransferase-1 Immunogenic α-gal moiety Expressed in murine cells but not in human Humans have circulating antibodies to alpha-gal Some CHO cell lines express functional Ggta1
- 25. 25 Webinar: Cell Line Engineering CMAH-/-/GGTA-/- Cell Line Engineering in a CHO K1 GS-/- Host Cell Line GS-/- Host CMAH ZFN Transfection SCC and Genotyping 5 KO Clones Clone Expansion and Characterization Clone Banking GS-/-/CMAH-/- Host GGTA ZFN Transfection SCC and Genotyping 1 KO Clone Clone Expansion and Characterization Clone Banking
- 26. 26 Webinar: Cell Line Engineering GS-/-/CMAH-/-/GGTA-/- Host Cell Line Growth Characterization 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 VCD(10e6cells/ml) Days in Culture 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 0 2 4 6 8 PercentViability Days in Culture
- 27. 27 Webinar: Cell Line Engineering GS-/-/CMAH-/-/GGTA-/- IgG Producing Stable Pools: Productivity Performance 7 day terminal TPP 0 50 100 150 200 250 300 350 400 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 IgG(ug/ml) GS-/- GS-/-/CMAH-/-/GGTA-/-
- 28. 28 Webinar: Cell Line Engineering Targeted Gene Integration for Biomanufacturing Reduced variability/enhanced cell line performance • Decreased integration site side effects • Clone to clone consistency • Molecule to molecule consistency Decreased cell line development timelines • More homogeneous stable pools • Decreased clone screening Increased pool and clonal stability • Use of well characterized safe harbor sites • Remove stability from the critical path
- 29. 29 Webinar: Cell Line Engineering Targeted Gene Integration Project Focus Areas 321 • Short RFLP donors • GFP reporters • IgG and Fc r-proteins • Decreasing NHEJ • Increasing HR • Tagged ZFNs • Optimized donor designs • TI landing pads • Reverse engineering of high expressing clones • Transciptomis analysis of constitutively expressed genes Increased EfficiencyFeasibility Hot Spot Identification
- 30. 30 Webinar: Cell Line Engineering Targeted Integration of GFP at the Actin Locus DonorOnlyZFN+Donor 1.2% GFP+ 2.4% GFP+ decreased scaling actual GFP Reporter HAHA Actin GeneGFP Reporter Actin Gene
- 31. 31 Webinar: Cell Line Engineering Targeted Integration of GFP at the Actin Locus Mock DonorOnly Donor+ZFN-Lsort Donor+ZFN-HsortGFP Reporter Actin Gene
- 32. 32 Webinar: Cell Line Engineering Targeted Integration on IgG at a Safe Harbor Site Transfection • CHO SH1 ZFN RNA • IgG/GS Plasmid Donor Stable Pool Generation • GS Selection • FACS Enrichment SCC Generation • jPCR/Seq, qPCR • G&P • Stability Targeted Integration SCC Random Integration SCC
- 33. 33 Webinar: Cell Line Engineering Summary 4 3 2 1 Genome editing is a valuable tool for enhancing biomanufacturing expression systems GS-/- host cell lines can lead to more efficient cell line development processes and higher performing recombinant pools and clones Glycoengineering can lead to more efficient manufacturing processes and the production of more effective therapeutic proteins Targeted integration can result in increased homogeneity and stability of recombinant stable pools and clones 5 Full extent of cell line engineering applications in biomanufacturing remains to be discovered
- 34. Questions? 34
- 35. Erika Holroyd Product Manager, Cell Line Development and Engineering CHOZN® Program erika.holroyd@sial.com CONTACT Trissa Borgschulte, PhD Head of Cell Line Development and Engineering CHOZN® Program | Process Solutions Upstream R&D trissa.borgschulte@sial.com Contact