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Speeding up media design in cell culture - a novel high throughput approach for rapid cell culture media development
 

Speeding up media design in cell culture - a novel high throughput approach for rapid cell culture media development

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Speeding up media design in cell culture

Speeding up media design in cell culture

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    Speeding up media design in cell culture - a novel high throughput approach for rapid cell culture media development Speeding up media design in cell culture - a novel high throughput approach for rapid cell culture media development Presentation Transcript

    • Speeding up media design: a novel high throughput approach for rapid cell culture media development Cell Culture World Congress 2013, Munich, 26 February 2013 Authors: Arnaud Périlleux, Yolande Rouiller, Martin Jordan and Matthieu Stettler Biotech Process Sciences, Upstream Development Group Merck Serono S.A. Vevey, Switzerland
    • Merck Serono at a glance  Merck Serono SA is the largest division of Merck KGaA – Established in January 2007,17’000 employees, EUR 5.9 billion in 2011, headquarter in Darmstadt, Germany – In the United States and Canada, Merck Serono operates under the name of EMD Serono (a separately incorporated affiliate of Merck Serono)  Merck Serono SA process development and production site in Vevey, Switzerland – One of the largest and most technologically advanced biotech centers in the world (4 x 5K and 8 x 15K production capacity) – Production of Rebif® (INF beta-1a) since 1999 – Production of Erbitux® (Cetuximab) since 2011 Merck Serono’s headquarter in Darmstadt (top) and development and production site in Vevey (bottom) 2 Cell Culture World Congress | 26 February 2013
    • Presentation scope  Overview of Merck Serono’s high throughput cell culture methods  Case study #1: High throughput media blending – How to obtain a new high performance medium in one single experiment?  Case study #2: Product quality optimization – How are quality analyses possible with micro-scale culture systems?  Perspectives – Strategies to quickly develop processes with strong quality requirements 3 Cell Culture World Congress | 26 February 2013
    • Merck Serono’s HT cell culture methods Overview Integrated development approach Cell line evaluation Predictive, scalable and comprehensive set of tools High throughput cell culture methods for fed-batch processes assessing 24 to 400+ cultures in parallel Medium development High performance cell lines Media Blending Feed Blending DoE Feed development High performance processes CFD Process development Process validation Quality by Design 4 Cell Culture World Congress | 26 February 2013 Deliver the right product quality
    • Merck Serono’s HT cell culture methods High throughput evaluation of cell lines in fed-batch Transfection and selection A B C D Cloning in static 384 well plate Adaptation in shaking 96DWP A Fed-batch in shaking 96DWP Up to 500 clonal cell lines 0.5 mL 0.5 mL 10 mL Scale up to shake tubes B Fed-batch in shake tubes C 5 Fed-batch in lab-scale bioreactors Cell Culture World Congress | 26 February 2013 30 mL 15 mL Fed-batch in micro-scale bioreactors D 12 cell lines 4 candidate cell lines 3 000 mL
    • Case study #1: High throughput media blending A high-throughput media design approach for high performance mammalian fed-batch cultures Authors: Yolande Rouiller, Arnaud Périlleux, Natacha Collet, Martin Jordan, Matthieu Stettler, Hervé Broly Manuscript accepted, to be published in mAbs
    • Case study #1: High throughput media blending Workflow 47 components 16 media formulations varying 43 out of 47 components Blends automatically mixed in 96DWP Predicted vs. Actual 3 passages prior a 14-days fed-batch Titer (mg/L) Data analysis 376 media blends tested 3000 2000 1000 0 0 2 4 6 8 101214 Elapsed time (days) 7 Cell Culture World Congress | 26 February 2013 Data acquisition
    • Case study #1: High throughput media blending  Protocol – 3 passages prior fed-batch inoculation, an antibody expressing cell line is diluted in each of the 376 blends • Guaranty to obtain a medium suitable for both expansion and fed-batch process Viable Cell Density (x106 cells/mL) Evaluation of 376 media blends – Performance assessment 8 Cell Culture World Congress | 26 February 2013 20 15 10 Titer (mg/L) 2 4 6 8 10 12 14 2 4 6 8 10 12 14 2500 2000 1500 1000 500 • Cell count, Cell viability with a Guava Easycyte • Titer quantification with an Octet Fed-batch process 0 -6 3500 -8Ctrl 1-4 -2 0 3000 Ctrl 2 • Standardized and controlled experimental setup • Guaranty to obtain a medium adapted to the feed system Cell expansion 3 passages 5 – Each of the 376 cultures is diluted individually targeting a specific cell density – On day 2, 4, 7 and 10, a reference proprietary feed is added 25 KQe 0 -8 -6 -4 -2 0 Time (days)
    • Case study #1: High throughput media blending Data analysis opportunities Data analysis using three approaches 1st approach: Excel spreadsheet Ranking of various tested conditions Identification of key media formulations Identification of key components Selection of promising formulations 9 2nd approach: DoE (Design Expert) Design of predicted best formulation List of key components to be further optimized Cell Culture World Congress | 26 February 2013 3rd approach: MVA (Simca P++)
    • Case study #1: High throughput media blending Data analysis with Design of Experiment (DoE) Titer at Day 14 Predicted values  DoE analysis allows – To identify the key formulations (out of the 16) – To design new media formulations  DoE analysis does not allow – To understand why some media are better than others 3000 2000 1000 R2 = 0.88 Adj. R2 = 0.84 Pred. R2 = 0.76 0 0 10 Prediction 1 Prediction 4 Prediction 2 Prediction 5 Predicted Values Prediction 3 Average Prediction PDL F16 F15 F14 F13 F12 F11 F10 F9 F8 F7 F6 F5 10.47 238 3933 10.42 226 3908 10.45 230 3902 5th F4 3960 4th F3 10.43 226 3rd F2 Titer 2nd Cell Culture World Congress | 26 February 2013 IVC 1st 35% 30% 25% 20% 15% 10% 5% 0% F1 % of each formulation in the predicted medium Compositions of best predicted media based on the 16 formulations 1000 2000 3000 Observed values 10.39 224 3885 Average 10.43 228 3910
    • Case study #1: High throughput media blending Data analysis with MultiVariate Analysis (MVA)  MVA analysis allows – To rank the 43 components in terms of influence on performance – To identify the key components that could be interesting for further optimization (in orange and yellow)  MVA analysis does not allow – To have a strong confidence in the conclusions due to the relatively low number of conditions tested compared to the high number of factors evaluated Components with strong influence in MVA Components that correlate by design with key components 11 Cell Culture World Congress | 26 February 2013 Titer Day 14 (mg/L) 0.4 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 L-Serine D-Biotin L-Arginine Thymidine L-Aspartic acid L-Leucine L-Glutamic acid Hypoxanthine Zinc Sulfate myo-Inositol NaH2PO4 L-Histidine Sodium Selenite Putrescine L-Tyrosine Riboflavin Choline Chloride Pyridoxine L-Lysine x HCl L-Phenylalanine L-Isoleucine Calcium Chloride Folic acid Vitamin B12 Thiamine Pluronic Ethanolamine L-Aspargine Cupric sulfate L-Cysteine Niacinamide (B3) Glycine L-Threonine L-Tryptophan L-Proline Magnesium Sulfate L-Alanine L-Methionine Sodium pyruvate Potassium Chloride L-Valine D-Pantothenic acid x 1/2Ca Ferric ammonium citrate Coefficients scaled & centered (components 1 to 3 of PLS model) Influence of increased levels of the tested components in the PLS regression of titers at day 14 Ferric Ammonium Citrate (mg/L)
    • Case study #1: High throughput media blending Scale-up, confirmation and conclusions Reference New medium medium Cell line 1 Cell line 2 Cell line 3  8 media from the ranking approach and 1 from the DoE approach were scaled up in shake tubes  1 medium was selected for scale up in bioreactor and evaluated on several cell lines – The 3 cell lines showed from 30 to 60% titer improvement Titer (mg/L) – data not shown 6000 5000 4000 3000 2000 1000 0  Conclusions 0 2 4 6 8 10 12 14 16 18 Time (days) – Media blending allows in one single experiment to identify new media formulations with high potential for performance improvement – Same approach can be made with feeds – An approach combining medium and feed blending can be designed 12 Cell Culture World Congress | 26 February 2013
    • Case study #2: Product quality optimization using high-throughput cell culture methods
    • Case study #2: Product quality optimization Are quality analyses possible with micro-scale cultures?  Context – Cell culture process development required to optimize a large number of critical quality attributes (CQAs) • Isoforms (deamidation, oxydation, …), glycoforms, (galactosylation, mannosylation, fucosylation, sialylation), product integrity, process impurities – Most of the CQAs are linked to the recombinant cell line, the medium and feeds, and the process (physical parameters) – To ensure a successful process development (e.g. next generation process), it is important that the new process provides a product with the same quality 14 Cell Culture World Congress | 26 February 2013
    • Case study #2: Product quality optimization Are quality analyses possible with micro-scale cultures?  Challenges – High throughput cell culture methods (i.e. 96DWP) provide small amounts of product Fed batch process in 96DWP 400-500 µL 225-1500 µg – Analytical lab should be able to analyze 400+ samples with 5+ analytical methods • Capture, glycan analysis, charge profile, integrity, …  Achievements – A combination of media blending and DoE generated 400 samples of about 400 µL Capture on Phytips® Charge profile iCE280 Glycan analysis CGE-LIF – Samples were captured using Phytips® – Samples were analyzed in 2.5 weeks on 5 analytical methods – 8800 results were generated 15 Cell Culture World Congress | 26 February 2013 Integrity SE-HPLC Caliper NR
    • Case study #2: Product quality optimization Experiment design Factors tested in DoE  Combine media blending and DoE design Zn N-acetylcystein Cu pH  HCl Fe NaCl Se NaButyrate Mn Hydrocortisone Galactose Spermine Ascorbic acid + Retinol + 4-aminobenzoic acid + a-tocopherol Lipids – 5 new Basal Media (BM) mixed to obtain 11 media – 17 factors tested in DoE by addition on day 5 – Standard platform feeding strategy – Analytics performed on day 14 • Biacore • CGE-LIF, iCE280 • Caliper NR, SE HPLC Fucose Uridine ManNAc Additional factors 2ndary feed Main Feed Glucose 0 16 Cell Culture World Congress | 26 February 2013 3 5 7 10 14
    • Case study #2: Product quality optimization Results – After optimization, the confirmation and scale up of a new manufacturing process version was performed 17 Cell Culture World Congress | 26 February 2013 Model: R2 = 74.9% / R2 adj. = 71.2% BM4 • to select 4 factors and 2 media for further optimization 32 DoE conditions Mix1 • to fix the levels of 4 factors 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 Spermine – Models allow Std. Coef. (Cluster 4) – For most of the CQAs, at least some conditions were able to match the TPP Control (n=4) BM3  Conclusions Mix5 10 Mix4 – Lists of key media and factors were established from models 15 NaCl – Models were defined 20 All data BM1 BM2 BM3 BM4 BM5 Mix1 Mix2 Mix3 Mix4 Mix5 Mix6 BM1 BM2 BM3 BM4 BM5 – Distribution of results was compared to Target Product Profile (TPP) 25 Cu  For each CQA Charge Profile Cluster 4 (%) 30
    • HT cell culture – Speeding up media design Conclusions  High throughput tools are now available for cell culture, but also for purification and analytics  Designing the right experiments allows to get the maximum of these tools and to reduce development timelines  Their use has been mainly directed to media design, but their application to feed design could even provide more interesting results 18 Cell Culture World Congress | 26 February 2013
    • Acknowledgements  Biotech Process Sciences (BPS), Merck Serono SA Vevey, Switzerland – Hervé Broly - Head of BPS – Upstream Development Group – Flavie Robert - Head of Analytical Group 19 Cell Culture World Congress | 26 February 2013
    • Questions 20 Cell Culture World Congress | 26 February 2013