Development of novel chemically defined media for CHO cell applications
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Development of novel chemically defined media for CHO cell applications

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novel chemically defined media for CHO cell applications

novel chemically defined media for CHO cell applications

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Development of novel chemically defined media for CHO cell applications Development of novel chemically defined media for CHO cell applications Presentation Transcript

  • Development of novel chemically-defined media for CHO cell applications Dr. Dipl-Ing. Jörg von Hagen Head of Process Development & Launch Management Merck Millipore, Darmstadt, Germany Cell Culture World Congress, Munich 2013
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 2 CHO Cell Media & Feed – Performance evaluation Perspectives 2
  • A Comparison of IgG Production in CVC-200 and Two Competitor Media and Feed Supplements. 3.5 3 80 2.5 60 2 1.5 40 IgG Titer (g/L) Cell Viability (% Viable Cells) 100 1 20 0.5 0 0 0 3 4 5 6 7 8 9 10 11 12 13 14 Elapsed Time (Day) Competitor A Competitor B Cellvento CHO-200 Competitor A Competitor B Cellvento CHO-200 IgG Production in Cellvento CHO-200 was 2-3 fold higher than two competitor products over a 14 day fed batch culture. 3 View slide
  • A Comparison of Cell Growth in CVC-200 and Two Competitor Media and Feed Supplements Cell Growth (VCD) - Competitive Evaluation in Fed Batch 14.00 VCD (10e6 c/ml) 12.00 10.00 8.00 6.00 4.00 2.00 0.00 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Elapsed Time (Day) Competitor A Competitor B Cellvento CHO-200 Cell growth in Cellvento CHO-200 achieved above 1x107 cells /mL in a 14 day fed batch culture. 4 View slide
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 5 CHO Cell Media & Feed – Performance evaluation Perspectives 5
  • Integral Viable Cell Density (10e6 cell day/ml) Comparison of Average Cell Growth in Batch Culture 25.00 20.00 Lot 006_1 15.00 Lot 006_2 Lot 006_3 10.00 5.00 0.00 3 4 5 6 Elapsed Time (Days) Cell growth in Cellvento CHO-200 was consistent in batch culture across three lots of media 6
  • Comparison of Cell Growth in Fed Batch Culture Three Lots of Feed tested 18,0 16,0 Viable Cell Density (10e6 c/ml) 14,0 12,0 Feed Lot 007_1 10,0 Feed Lot 007_2 8,0 Feed Lot 007_3 6,0 4,0 2,0 0,0 0 5 10 15 Elapsed Time (Day) Cell growth in Cellvento CHO-200 was consistent in fed batch culture across three lots of feed 7
  • Comparison of IgG Production in Fed Batch Culture Comparison of Volumetric Titers Across Three Lots of Feed 2.0 1.8 1.6 1.4 IgG (g/L) 1.2 Feed Lot 007_1 1.0 Feed Lot 007_2 0.8 Feed Lot 007_3 0.6 0.4 0.2 0.0 7 10 Sample Day IgG production in Cellvento CHO-200 was consistent in fed batch culture across three lots of feed 8
  • PSD Media 9
  • PSD Feed 10
  • Reproducibility amino acid content Cellvento CHO 200 media Amino acid concentration (in % of the theoretical value) 120 100 80 Batch 1 Batch 2 60 Batch 3 40 20 0 His 11 Asn Ser Arg Presentation title in footer | 00 Month 0000 Gly Asp Glu Thr Ala Pro Lys Tyr Met Val Ile Leu Phe
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 12 CHO Cell Media & Feed – Performance evaluation Perspectives 12
  • A Comparison of Cell Growth in Spin Tubes and 3L Benchtop Bioreactors 16 Viable Cell Density (10e6 c/ml) 14 12 10 3L Cellready Bioreactor 8 50 ml Spin Tubes 6 4 2 0 0 2 4 6 8 10 12 14 Elapsed Time (Day) Similar growth performance was achieved with Cellvento CHO-200 in spin tubes and benchtop bioreactors over a 14 day fed batch culture. 13
  • A Comparison of IgG Production in Spin Tubes and 3L Benchtop Bioreactors 1,8 1,6 1,4 IgG (g/L) 1,2 50 ml Spin Tubes 1 3L Cellready Bioreactor 0,8 0,6 0,4 0,2 0 7 10 Sample Day IgG titers averaged 1.5 g/L after 10 days of fed bath culture using Cellvento CHO-200 in spin tubes and benchtop bioreactors. 14
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 15 CHO Cell Media & Feed – Performance evaluation Perspectives 15
  • NIR - Near Infrared Spectroscopy A fingerprint method for media performance prediction medium performance high performance low performance PCA-Possibility for clustering good and poor performing media batches Measurement conditions Integrating sphere, PbS-detector, diffuse reflexion Instrument: BrukerVector 22/N Number of scans: 16, Resolution: 8 cm-1, Measurement Range: 12000 – 4000 cm-1
  • Principle component analysis (PCA) Correlation of cellular performance and NIR analysis Supplier C-1 Supplier S Supplier C-2 Supplier B Supplier C-3 EMD Millipore Supplier A PCA of DMEM-F12. Analysis by NIR-Spectroscopy (Bruker, Vektor MPA)
  • Quality Testing of Media … …depends on the appropriate test system 25 cumulative PDL 20 Mill Tech 1-1 15 Mill Tech 1-2 Mill Tech 1-3 Mill Tech 2-1 10 Mill Tech 2-2 Mill Tech 2-3 5 0 0 days 3 days 5 days 7 days 10 days 12 days 14 days 17 days Impact of the milling technology on CHO-S cells grown in chemically defined media. No significant differences are observed in the basic cell culture assay analyzing the cumulative population doubling levels (PDL) using different produced mammalian cell culture media.
  • Sensitive Cell Culture Assays … underline differences in cell culture media performance 70 60 plating efficacy (%) 50 40 30 20 10 0 Supplier A Mill Tech 1-1 Mill Tech 1-2 Mill Tech 1-3 Mill Tech 2-1 Mill Tech 2-2 Mill Tech 2-3 Impact of the milling technology on CHO-S plating efficacy in chemically defined media. CHO-S cells were diluted in the corresponding media and analyzed after 2 weeks on plating efficacy.
  • Impact of Processing Technologies Impact of the milling technology on mAb titer 140 130 Titer ( % to internal reference) 120 110 100 90 80 70 60 50 40 30 20 10 0 Supplier A Mill Tech 2 The media were tested in the production of a monoclonal antibody. Titer was measured by Surface Plasmon Resonance Spectroscopy. Red line indicates the minimum achievable titer according to the specifications.
  • Milling Process Impact on final NBE Attribute Impact of the milling technology on critical antibody attributes 35 Critical Peptides (%) 30 25 20 15 10 5 0 Supplier A Mill Tech 2 Final molecules after capture were analyzed by HPLC to determine the amount (%) of critical peptides that are known to be correlated with lower half life of the drug substance in patient serum samples. The amounts are directly linked with the raw material quality and the production process. Red line indicates the max. value given by the authorities. Dotted red line represents the internal specification limit. Range between dotted blue lines show the target space for good media performance.
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 22 CHO Cell Media & Feed – Performance evaluation Perspectives 22
  • Humidity and impact on powder flowability CDM II DMEM-F12 CDM I After opening the package the first time the CDM II powder is clumpy. The DMEMF12 and CDM I media are opened several times and are free flowing powders.
  • CDM II: particle size distribution CDM II particle size analysis is technically not feasible by air-jet sieving. After 3 min all material gets like chewing gum, whereas for CDM I and DMEM-F12 particle size distribution analysis went without clumping.
  • CDM composition comparison The CDM I and II composition have the following critical hygroscopic ingredients: – Calcium Chloride 2H2O – Choline Chloride – Ferric Nitrate 9 H2O – Ferrous Sulfate 7 H2O – Sodium phosphate, monobasic, monohydrate  the above listed ingredients are present with a concentration in sum of 1.5% in the CDM I and 9.7% in the CDM II formulation.  6.3-fold enrichment of hygroscopic compounds resulted in a final H 2O titer of 4.05% measured by Karl Fischer Titration in the CDM II powder (compared to 1.05 to 1.2 in DMEM-F12 and CDM I.)
  • Humidity effects solubility of DMEM F12 2 – 8 °C 4 fold accelerated (5min real) 6 h / 28 °C / 90 % rH.
  • 17° increase in avalanche angle after DMEM F12 exposure to 90% rH DMEM F12 before and after flowability measurement (3h/25°C at 90% rH)
  • DMEM-F12 compacted R = Rosa = pinkish uncompressed 20KN 30KN 40KN 50KN 60KN
  • DMEM-F12 compacted uncompressed 20KN 30KN 1 mm 1 mm 1 mm 1 mm 1 mm 1 mm 40KN 50KN 60KN 1-3 mm particle sizes
  • Solubility of DMEM F12 powder vs. granules
  • Filterability of Powder vs. Granules of DMEM F12 Compaction Filter Medium (kN) Family Filter Name Filter Pore Size (µm) Vmax (L/m2) Amin (m2) 0 0 SHR 0,1 9 305 0,28 Express SHR-2L 0.5/0.1 20 555 0,29 30 30 Express SHR 0.1 11 221 0,29 Express SHR-2L 0.5/0.1 37 911 0,31 60 Express SHR 0.1 10 596 0,30 60 DMEM F-12 Express Express SHR-2L 0.5/0.1 44 219 0,31 The Amin represents the minimal surface of membrane needed to process 500L within the defined time.
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 32 CHO Cell Media & Feed – Performance evaluation Perspectives 32
  • Tyrosine solubility in feed with >110 g/L amino acid & vitamin load L-Tyr 1 L-Tyr 2Na 1 mod L-Tyr I mod L-Tyr II 50 g/L @ pH 7.0 +/- 0.2 70
  • 4,5mM 2Na-Tyr CHO-S growth profile comparing L-Tyrosine Disodium salt vs. modified Tyrosine 4,5mM 2Na-P-Tyr Medium 200 180 160 Lebenszellzahl [vc/ml] x10^5 VCD (x105 vc/mL) 0 1 1 1 1 1 1 1 1 2 3 4 5 6 7 1 29,6333333 33,2 11,5333333 24,15 24,6666667 19 16,4666667 4 753,033333 681,6 61,0666667 528,6 462,9 85,2666667 116,266667 5 1264,53333 1218,13333 73,0666667 1318,4 1197,26667 157,4 135,9 6 1541,13333 1470,4 64,8999992 1409,82503 1441,11333 266,206664 117,206667 7 8 988,936665 333,58 1108,24002 548,75 46,4400007 63,1033333 1393,7 1324,422 1578,69001 1502,80333 375,935004 415,776667 78,376667 71,34 0 1,92 2,07 2,49 4,5mM 2Na-Tyr 0,29546573 140 4.5 mM L-Tyr 2Na 4,5mM 2Na-Tyr 120 100 4,5mM 2Na-P-Tyr 4.5 mM mod L-Tyr 4,5mM 2Na-P-Tyr II 80 60 9.0 mM mod L-Tyr 9mM 2Na-P-Tyr II 40 18,33 0,31 20 9mM 2Na-P-Tyr 0 1 3 9 5 7 time (days) time 11 9 11
  • Presentation Outline 1 2 Lot to Lot Consistency 3 Platform Consistency 4 Media Performance Prediction 5 Powder handling improvements 6 Solubility Toolbox for high performing media 7 35 CHO Cell Media & Feed – Performance evaluation Perspectives 35
  • Perspectives in media preparation • The raw material quality is key for optimal media performance on cellular level • Raw material selection • Raw material pre-processing • Novel raw material development • Supply chain • The production process is directly linked with batch to batch consistency and cellular performance • Milling process • Mixing process • Packaging • The combination of raw materials and the production process is mandatory for excellent dry powder media performance Raw material quality Production process Cellular performance