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Application of HTP microfluidic culture systems to media and process optimisation

Application of HTP microfluidic culture systems to media and process optimisation. Steven C. Peppers, Ph.D., MBA Principal Scientist, R&D Reg Joseph, B.Sc., MBA Business Area Manager, BioProduction BioProduction Systems and Services Invitrogen Corporation. Metabolic pathways chart from

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Application of HTP microfluidic culture systems to media and process optimisation

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  1. Application of HTP microfluidic culture systems to media and process optimisation Steven C. Peppers, Ph.D., MBA Principal Scientist, R&D Reg Joseph, B.Sc., MBA Business Area Manager, BioProduction BioProduction Systems and Services Invitrogen Corporation

  2. Metabolic pathways chart from Boehrringer-Mannheim Complexity of Cell’s Needs

  3. Complexity of culture systems Interactions within: Uptake of nutrients Receptor signaling Metabolic pathways Physicochemical qualities Dynamic states No tools to fully probe this complexity Build up of components at inappropriate concentrations Special technologies for managing complexity: 1. Statistical designs and strategy 2. Scaled down HTP tools 3. Expertise in cell requirements and media components DOE (Design of Experiment): Statistically sound means of planning and analyzing efficient experiments: Examples: 2-level factorials and fractional factorials, central composite designs, minimum run designs, mixtures, steepest ascent, Box-Behnken, D-optimal

  4. Multi-factor central composite designs Run chart showing coded levels +1 (8) Component B (Glutamine, nM) 0 (6) Component C (Osmolality) -1 (4) 0 (3) +1 (4) -1 (2) Component A (Glucose, g/L) Half-factorial central composite for 3 factors

  5. What’s The Right Tool For The Right Job? • Effective bioprocess development tool: • High throughput—100’s of different conditions • Scalable—Predict performance in ST bioreactors • Reliable—High precision, accuracy and reproducibility • Efficient—Process in time and labor, cost effective

  6. Fluidic Module Sampling Module Incubation Modules Central Robot Optical Sensing Module Loading Cell BioProcessors SimCell System Micro-Bioreactor Array (MBA) 6 Chambers 600 uL Working Vol. Independent Loading Stirring by Bubble Invitrogen working with BioProcessors for 2 yr

  7. Scalability of SimCell Results SimCell at Day 6 pH 6.9 4.45 x106 TC/mL pH 7.2 3.07 x106 TC/mL Difference/Average = 36.7% ST Bioreactors at Day 6 pH 6.9 4.57 x106 TC/mL pH 7.2 3.13 x106 TC/mL Difference/Average = 37.4% Ratio of “Dif/Avg” values = 0.98

  8. Coded Level Complex Factorials in SimCell Goal: Confirm SimCell capability in complex factorials Fractional Central Composite Design, N=192

  9. Response Surface from Analysis

  10. Interaction in Late Log Phase Growth

  11. Interaction in Final tPA Productivity

  12. Optimizing both Cell Density and Productivity Selected OutcomeSet toWeight Cells/mL, days 5-6.5 Maximum 3 Cells/mL, days 6.5-8 Maximum 3 Prod’n of tPA, day8 Maximum 5

  13. SimCell™ at Invitrogen Purchased model with 4 incubators 1008 chambers possible 24-factor 2-Level factorials possible Recently installed at Grand Island (GIBCO) site Currently in OQ phase PQ and early implementation scheduled for 3rd and 4th quarter

  14. General Workflow Plans Hamilton STARplus Compose 100’s of variations of a medium Database and Firewall DOE design SimCell™ System Next Optimization Cycle HTP Assays and Data analysis Occasional scaled-up verification

  15. Improved economics for services *Not a consumable cost; calculated by dividing the full project cost by number of data points

  16. ½ • Benefits • Reduce COGS • # of weighs/product • raw material mgmt, incoming QC • Decrease variability & formulation errors • Eliminate redundancies & counter effects Reduce components Preserve or increase performance 80-100 components – many replicates at varying conc. ¼ ¼ • Let’s get smarter around media components: • Multiple Salt forms* – calcium nitrate + calcium chloride • Hydration levels - L-Histidine vs L-Histidine HCl H2O • Differing forms of the same amino acid - cysteine vs cystine • *May have opposite effects Traditional Mixtures Experiment Improving media design & manufacturing X

  17. Conclusions • SimCell system installed at Invitrogen • Currently going through validation • Beta programs with key cell lines underway • Formal service offering in 2007 • Demonstrated ability to perform complex factorial designs • Developed and validated a fed-batch model using SimCell • Optimize media, process, and feed simultaneously • Cost models enables high value services at reasonable prices

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