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Reducing Timelines & Increasing Titres by Host Cell Lines with Improved Characteristics

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Reducing Timelines & Increasing Titres by Identification of Host Cell Lines with Improved Characteristics

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Reducing Timelines & Increasing Titres by Host Cell Lines with Improved Characteristics

  1. 1. Cell line development: Reducing timelines and increasing titres by identification of host cell lines with improved characteristics Dr Alison Porter, Fujifilm Diosynth Biotechnologies, UK Cell Line Development and Engineering, 09 September 2014, Berkeley CA 1
  2. 2. FUJIFILM Diosynth Biotechnologies  EU and USA operations  Track record  ~950 staff  Over 1000 cGMP batches manufactured  5 Commercial Products  FDA & MHRA Inspection history  Extensive LSS application RTP (NC, USA) Contract Manufacturing & Process Development Billingham (UK)
  3. 3. Scope ►Aims and Objectives ►Host cell line development (CLD) • Directed evolution approaches • Results ►CLD run-through • Experimental design • Results ►Summary 3
  4. 4. Aims and Objectives ►To develop a mammalian expression platform which rapidly leads to efficient, robust and high quality biomanufacturing processes ►New host cell line ►New vector ►Optimised cell line development process ►Complementary medium and feed platform 4
  5. 5. 5 Host cell line development
  6. 6. Desirable features/attributes for a host cell line Adapted to chemically defined (CD) media Adapted to CD media Short doubling time 1 to 5 g/L titre for a model mAb Suitable product quality Rapid adaptation to production media CHO-based 6
  7. 7. How do we improve host cell functional capability? CELL LINE ENGINEERING? DIRECTED EVOLUTION? • Despite decades of research and a variety of different strategies to engineer CHO cells, outcomes remained mixed • May be a need to engineer multiple targets • May require a systems-based analysis for a tailor-made response • Utilise functional heterogeneity within CHO cell populations to identify better host cell lines • We already utilise this approach • Identify high producing recombinant clones during CLD • Adaptation to serum-free chemically defined media • Potential ability to manipulate multiple phenotypic outputs 7
  8. 8. Approaches taken to obtain a host cell line with improved features/attributes Isolate a cell line with more desirable attributes from a heterogeneous cell population Adaptation to chemically defined conditions Chemostat Subculture regime FACS Cloning Multiple rounds of sorting for cells with extended viability during batch shake flask screening Allows for evolution of rare mutants with desirable growth properties within a large population Continuous subculture (different regimes) and ability to grow in low [Gln] for lower [Amm] production 8
  9. 9. Derivation of multiple DG44 variants able to grow in chemically defined (CD) culture medium Static culture 100% MEM-α (+10% FBS) Suspension 100% MEM-α (+10% FBS) 50% CD-DG44 50% MEM-α (+10% FBS) 75% CD-DG44 25% MEM-α (+10% FBS) 90% CD-DG44 10% MEM-α (+10% FBS) C1 100% CD-DG44 Suspension 75% OPTI-CHO 25% MEM-α (+10% FBS) 80% OPTI-CHO 20% MEM-α (+10% FBS) 90% OPTI-CHO 10% MEM-α (+10% FBS) C2 100% CD-DG44 C3 100% CD-DG44 75% CD-DG44 25% OPTI-CHO 50% CD-DG44 50% OPTI-CHO 9
  10. 10. The long and winding road to CD media adaptation: The C2 story 1:100 anti-clump 1:700 anti-clump No anti-clump 0.2 x 106 seeding 0.3 x 106 seeding 0.2 x 106 seeding 3/4-day sub 3-day sub 85% OptiCHO Clumping issues Static 110 rpm 120 rpm 140 rpm 1.00 0.75 0.50 0.25 0.00 0 50 100 150 200 Growth rate (day-1) Days in culture 100% MEMα +10%FBS 75% OptiCHO 80% OptiCHO 90% OptiCHO 100% CD DG44 Desirable growth rate for a recombinant cell line assuming: • Seed = 0.2 x 106/mL • VCD obtained = 2.0 x 10 106/mL • Culture duration = 4 days • Allows a 1:10 split ratio during seed train
  11. 11. Batch shake-flask screening of CD variants 15 10 5 30 20 10 ► Assess growth and metabolite profiles during an 8-day batch shake-flask screen (at which point viability < 50%) • Indication of a cell line’s potential ability to grow during extended culture • Ideally have a limited accumulation of waste metabolites (e.g. ammonium and lactate) 11 0 C1 C2 C3 2B-1 2B-2 IVC (x 106 cell∙day/mL) 0 C1 C2 C3 2B-12B-2 Metabolite Concentration (mM) Amm Lac
  12. 12. Higher transfection efficiency and transient titre obtained with cell line C2 80 60 40 20 0 C1 C2 C3 2B-1 2B-2 Transfection efficiency (%) 4 3 2 1 ► Assess transfectability (using GFP) and overall expression/secretion capacity (using a model mAb) • Comparison of percentage GFP positive cells 24 hours after transfection • Comparison of secreted mAb concentration 5 days post-transfection ► Higher transfection efficiency and transient titre obtained with cell line C2 C2 progressed to directed evolution stages due to superior performance during evaluation 12 0 C1 C2 C3 2B-1 2B-2 Transient mAb expression (mg/L) DG44-2B in ACF
  13. 13. Comparison of host cell lines obtained by directed evolution Growth rate Adapted to CD media during subculture IVC obtained in batch shake-flask screen Colony survival in semi-solid medium Waste metabolite (Amm and Lac) production in batch shake-flask screen Transient expression of mAb 1 and mAb 2 13
  14. 14. 0 5 10 15 20 25 30 DG44-2B C2 Sort-1 Sort-2 Sort-3 Sort-4 Sort-5 Sort-6 Clone-7 Clone-11 Clone-26 Clone-27 Clone-36 Clone-46 Clone-56 Clone-59 Stat-1 Stat-2 Stat-3 Stat-4 Stat-5 Stat-6 8 mM 6 mM 4 mM 2 mM 1 mM Doubling time (h) Doubling time during subculture 14 ► Improved doubling time with C2 compared to DG44-2B: ~27hrs to ~21hrs ► Directed evolution approaches had little effect on doubling time
  15. 15. 0 2 4 6 8 10 12 14 DG44-2B C2 Sort-1 Sort-2 Sort-3 Sort-4 Sort-5 Sort-6 Clone-7 Clone-11 Clone-26 Clone-27 Clone-36 Clone-46 Clone-56 Clone-59 Stat-1 Stat-2 Stat-3 Stat-4 Stat-5 Stat-6 8 mM 6 mM 4 mM 2 mM 1 mM Batch shake-flask screen harvest IVC (x 106 cell∙day/mL) IVC obtained during batch shake-flask screening 15 ► Potential to increase IVC from directed evolution approaches ► Higher IVC for DG44-2B may be media dependent (presence of hydrolysate)
  16. 16. 0 1 2 3 DG44-2B C2 Sort-1 Sort-2 Sort-3 Sort-4 Sort-5 Sort-6 Clone-7 Clone-11 Clone-26 Clone-27 Clone-36 Clone-46 Clone-56 Clone-59 Stat-1 Stat-2 Stat-3 Stat-4 Stat-5 Stat-6 8 mM 6 mM 4 mM 2 mM 1 mM mAb 2 transient expression (mg/L) mAb 1 transient expression (mg/L) 0 2 4 6 8 10 12 DG44-2B C2 Sort-1 Sort-2 Sort-3 Sort-4 Sort-5 Sort-6 Clone-7 Clone-11 Clone-26 Clone-27 Clone-36 Clone-46 Clone-56 Clone-59 Stat-1 Stat-2 Stat-3 Stat-4 Stat-5 Stat-6 8 mM 6 mM 4 mM 2 mM 1 mM Transient expression 16 ► Improved titre obtained from C2 compared to DG44-2B ► Directed evolution had variable effect: up to 3-fold increase compared to DG44-2B
  17. 17. % Colony survival in semi-solid media 0 20 40 60 80 100 120 DG44-2B C2 Sort-4 Sort-5 Sort-6 Clone-7 Clone-11 Clone-26 Clone-27 Clone-36 Clone-46 Clone-56 Clone-59 Stat-1 Stat-2 Stat-3 Stat-4 Stat-5 Stat-6 8 mM 6 mM 4 mM 2 mM 1 mM Growth in semi-solid media 17 ► Improved colony survival obtained from C2 compared to DG44-2B ► Directed evolution approaches have a variable effect on colony survival
  18. 18. 0 2 4 6 8 10 12 14 16 18 Clone 11 Sort 5 Sort 4 1 mM Clone 27 2 mM Stat 6 Clone 56 Sort 6 6 mM Clone 26 Stat 2 Stat 5 Stat 1 Clone 59 DG44-2B 4 mM Clone 7 8 mM C2 Stat 4 Stat 3 Clone 46 Clone 36 Average Ranking Average ranking used to select host cell lines to progress Top 5 cell lines progressed 18
  19. 19. Simultaneous comparison of host cell lines: Summary ► For each screen, cell lines were ranked according to their performance • The average ranking from all experiments gave an overall score for each host cell line ► Top ranked host cell line isolated from cloning of parental C2 host • Clone 11 ► Not all cell lines identified performed better than parental C2 host • 2 cell lines from the chemostat and cloning approaches ranked lower ► Performance of cell lines varied between the screens: • The chemostat derived cell lines ranked towards the top when measuring growth properties in shake-flasks • In contrast, these cell lines performed poorly when assessing growth in semi-solid medium, with only low numbers of colonies being produced • Demonstrates importance of assessing multiple characteristics ► Top 5 host cell lines, Clone-11, Sort-5, Sort-4, 1mM and Clone-27, were chosen to evaluate further in a ‘mini-pool’ assessment (representative of early stage stable cell line generation) 19
  20. 20. Mini-pool evaluation of candidate host cell lines 1. Transfection mAb 1 mAb 2 mAb 3 2. Removal of HT 3. Addition of selective pressure and plating into 96-well plates at 5000 cells/well 5. Progression to 24- well plates 4. Screening of confluent colonies by OctetTM 6. Screening by OctetTM 7. Confirmation of the top host cell line(s) for progression 20 Experiment biased towards mAb 1 as mAb 2 and mAb 3 have previously proven to be “difficult-to-express”
  21. 21. 24-well plate screen titre 30 25 20 15 10 5 0 mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 mAb 1 mAb 2 mAb 3 DG44-2B Clone 11 Clone 27 Sort 4 Sort 5 1 mM (in 1mM GLN) 1 mM (in 8mM GLN) 24wp titre (mg/L) ► Rank results from mini-pool evaluation combined with previous ranking results to select the top 2 host cell lines ► Decision made to ensure cell lines from different directed evolution routes progressed to mitigate risk: • Sort-5 and Clone-27 • DG44-2B included as a control 21
  22. 22. Cell Line Development Run-Through 22
  23. 23. Process map of the CLD run-though 23 Transfection & Selection 96-well plate screen Fed-batch shake-flask screen ClonePix™ screen 24-well plate screen Batch shake-flask screen Stability study ambr15™ & Bioreactor evaluation Titre assessment used a generic antibody standard curve and Octet™ assay Titre assessment used Octet™ or ProA HPLC & a specific antibody standard curve • Clone 27 and Sort 5 assessed in CLD run-through • New FDB expression vector used with the new host cell lines • Original host/vector system included as a comparator up to the RCB stage • Original system also uses different medium/feed system Model mAb 1 Host cell line Research cell bank
  24. 24. 24 CLD run-through: Transfection to recovery Pools from Clone-27 host Transfection recovery Pools from Sort-5 host Pools from DG44-2B host recovery recovery Host cell line Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Pools from the original host cell line (DG44-2B) had the longest recovery period -HT + Selection reagent
  25. 25. CLD run-through: ClonePix™2 screening 25 FITC1000 Exterior Mean Intensity Apollo Alternative Host Cell Line DG44-2B 40000 30000 20000 1 0000 0 FITC1 000 Exterior Mean Intensity Clone-27 Sort-5 D G44-2B Exemplar colony image ► The fluorescence intensity and colony size were lower for the original host/vector system (DG44-2B)
  26. 26. CLD run-through: 96-well plate screen 26 ► The highest titres achieved were from colonies originating from the Clone-27 host ► A greater number of colonies from the original system had expression below the detection level; the max titre was considerably lower >150 top colonies progressed DG44-2B Sort-5 Clone-27 250 200 150 100 50 0 0 100 200 300 400 500 600 700 800 Titre (mg/L) 250 200 150 100 50 0 0 100 200 300 400 500 600 700 800 Titre (mg/L) 250 200 150 100 50 0 0 100 200 300 400 500 600 700 800 Titre (mg/L) Colony Number
  27. 27. CLD run-through: 24-well plate screen 200 150 100 50 200 150 100 50 0 0 50 100 150 200 250 Titre (mg/L) 27 Titre (mg/L) >30 top colonies progressed ► The highest titres achieved were from colonies originating from the Clone-27 host ► Colonies from the original host/vector system had much lower titres DG44-2B Sort-5 Clone-27 0 0 50 100 150 200 250 Titre (mg/L) 200 150 100 50 0 0 50 100 150 200 250 Colony number
  28. 28. 28 CLD run-through: Suspension culture Viable cell density (VCD) and viabilities achieved during a routine subculture of the top 36 producers Clone-27 Sort-5 DG44-2B 4 3 2 1 0 VCD (x1E6) 100 90 80 70 60 50 Viability (%) Viability (%) VCD (x 106 cells/mL) Clone-27 Sort-5 DG44-2B Cell lines from DG44-2B host (original system): ► Difficulty in adaptation to suspension ► Lower VCD during routine subculture ► Several top ten producers in batch screen not suitable for progression to fed-batch screen due to poor growth
  29. 29. 29 CLD run-through: Batch shake-flask screen * Suspension adapted cell lines ► All recombinant cell lines from Clone-27 in the batch screen achieved titres above 300 mg/L ► One cell line from DG44-2B produced >300 mg/L; the majority produced <100 mg/L DG44-2B Sort-5 Clone-27 600 500 400 300 200 100 0 Titre (mg/L) 600 500 400 300 200 100 0 Titre (mg/L) 600 500 400 300 200 100 0 Titre (mg/L) * * * * * * cell growth was not acceptable for progression
  30. 30. CLD run-through: Growth profiles from the fed-batch shake-flask screen 30 Clone-27 Sort-5 DG44-2B 25 20 15 10 5 25 20 15 10 5 25 20 15 10 5 ► Non-optimised process for Clone-27 and Sort-5 ► A greater number of cell lines from Clone-27 host grew to >10 x 106 cells/mL; all cell lines were harvested on day 14 ► Only one cell line from Sort-5 host achieved high VCD; several cell lines were harvested before day 14 ► Cell lines from DG44-2B generally had poor growth profile and some were harvested as early as day 5 Note: different medium/feed system required for DG44-2B 0 0 2 4 6 8 10 12 14 VCD (x 106 cells/mL) Culture time (days) 0 0 2 4 6 8 10 12 14 Culture time (days) 0 0 2 4 6 8 10 12 14 Culture time (days)
  31. 31. ►Lactate: • Clone-27 and Sort-5 typically back-metabolise lactate • Cell lines from DG44-2B generally reached higher lactate concentrations throughout culture ►Ammonium: • Harvest ammonium concentration for all cell lines from Clone-27 and Sort-5 were below 15 mM • Cell lines from DG44-2B generally had higher ammonium concentration throughout the culture and at harvest 31 CLD run-through: Further observations from the fed-batch shake-flask screen
  32. 32. 32 CLD run-through: Product concentration achieved from the fed-batch shake-flask screen ► A higher proportion of cell lines from Clone-27 had product titres ~2 g/L and above ► Most cell lines from Sort-5 produced between 1 and 1.5 g/L ► Productivity of cell lines from DG44-2B were considerably lower at <1 g/L Cell lines DG44-2B Sort-5 Clone-27 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Titre (g/L) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Titre (g/L) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Titre (g/L)
  33. 33. 33 CLD Run-through: Specific productivity from the fed-batch shake-flask screen DG44-2B Sort-5 Clone-27 ► Despite lower titres, cell lines from Sort-5 had higher specific productivity (QP) - due to lower growth and IVC ► With the exception of one cell line, all cell lines from DG44-2B had QP less than 12 pg/cell·day 50 40 30 20 10 0 Qp (pg/cell.day) 50 40 30 20 10 0 Qp (pg/cell.day) 50 40 30 20 10 0 Qp (pg/cell.day)
  34. 34. No substantial difference in product characteristics between cell lines from different hosts 34 Size exclusion chromatography Capillary electrophoresis-SDS (reduced) •The majority of the product from all recombinant cell lines in Exemplar Overlay monomeric form (≥98%) Exemplar graph Cation-exchange N-linked glycans chromatography electropherogram •Major peaks identified as LC and HC •Very low percentage of non-glycosylated (~0.5%) and other variants. •Low percentage of acidic and basic variants •Lower percentage of main peak and additional basic peaks for DG44-2B* cell lines * Different medium/feed system Basic peaks overlays Cell lines from new hosts Cell lines from DG44-2B • Predominant glycan species did not change between host cell lines • Man5 varied from 2 to 15% between cell lines •Some minor differences with DG44-2B but this had different medium / feed system Clone-27 derived cell line • Adapting all cell lines to this system removed the minor differences •No considerable aggregation detected CONFIDENTIAL
  35. 35. 35 Comparison of top producers in the ambr15TM screen point size: Qp 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 IVC (x 106 cells.h/mL) Clone-27 Sort-5 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Harvest Titre (g/L) ► The highest titre was achieved by a Clone-27 derived cell line • 3 g/L ► Cell lines from Sort-5 generally had higher Qp but lower IVC
  36. 36. 3.0 2.5 2.0 1 .5 1 .0 Product Concentration (g/L) ambr15TM Boxplot of IVC data from Dolly and Phoenix in fed shake-flasks and Ambr 36 Decision on lead host cell line Box plot summary of key data for cell lines derived from Clone-27 and Sort-5 Fed-batch shake-flask ambr15TM Clone-27 Sort-5 Sort-5 Dolly FOG IVC Phoenix FOG IVC Dolly Ambr IVC Phoenix Ambr IVC 200 1 50 1 00 50 0 IVC (x1 E6 cells.day/mL) Boxplot of Qp data from Dolly and Phoenix in fed shake-flasks and Ambr Qp (pcd) Dolly FOG Qp Phoenix FOG Qp Dolly Ambr Qp Phoenix Ambr Qp 45 40 35 30 25 20 1 5 1 0 Qp (pcd) Clone-27 ► Clone-27 selected as lead host cell line: ► Clone-27 cell lines achieved the highest titres and IVCs in both shake-flask and ambr15TM screens ► Difference in QP reduced between Sort-5 and Clone-27 cell lines in ambr15TM Fed-batch shake flask Fed-batch shake flask ambr15TM IVC (x 106 cells.d/mL) Clone-27 Sort-5 Clone-27 Sort-5 Clone-27 Sort-5 Clone-27 Sort-5
  37. 37. 37 New hosts/vector system Original host/vector system Higher Titres Better growth Suitable PQ More undesirable waste metabolites Poor growth & productivity CLD run-through: Summary The new host/vector system performed better than the original system at every screening stage Vector + Host Suitable PQ CLD run-through
  38. 38. Summary ► Four directed evolution approaches used to develop new host cell lines • FACS; cloning; chemostat; subculture regime ► Multiple host cell lines with improved characteristics identified • Growth rate and expression capability • Need to look at multiple characteristics when assessing potential new host cell lines demonstrated ► Top two potential host cell lines assessed in full CLD process • Performance of both superior to original system, which had been included as a comparator ► A final new host cell line was selected • Product concentrations up to 3 g/L observed in ambr15™ using a non-optimised process • Improved growth characteristics • Acceptable product characteristics 38
  39. 39. Summary - continued ►Aim: To develop a mammalian expression platform which rapidly leads to efficient, robust and high quality biomanufacturing processes ►New host cell line ►New vector ►Optimised cell line development process ►Complementary medium and feed platform – under development 39   
  40. 40. Acknowledgements ►Adeline Bayard ►Naz Dadehbeigi ► Clare Lovelady ► Leon Pybus ► Fay Saunders ► Alison Young ► All members of the Mammalian Cell Culture R&D team – UK and USA ►Analytical Development, FDB 40
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