Javier Amayra - Biotechnological Screening in Animal Cell Culture


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Biotechnological Screening in Animal Cell Culture

By Javier Amayra

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Javier Amayra - Biotechnological Screening in Animal Cell Culture

  1. 1. HexaScreen®Biotechnological Screening in Animal Cell Culture Equipment to reduce required time & costs for bioprocess development EXPOQUIMIA 2011 www.telstar-lifesciences.com
  2. 2. The Team •Cell Culture •Manufacturing •Bioreactors •Marketing & Sales + • Measurement Systems •Internationalization + + •Biocompatible materials www.telstar-lifesciences.com
  3. 3. Increasing importance of animal cell culture. www.telstar-lifesciences.com
  4. 4. Animal Cell Culture BioprocessesThe potential of animal cell culture arises from the capability of this type ofcells to carry out complex post-translational modifications providing proteinswith the required biological activity to be used for therapeutical anddiagnostic applicationsCHO (Chineese Hamster Ovary)NSO (mouse mieloma)BHK (Baby Hamster Kidney)HEK (Human Embryo Kidney)PER.C6 (Human Retinal Cells)MDCK (Madin Darby Canine Kidney)Sf9 (insect cells, Spodoptera frugiperda) www.telstar-lifesciences.com
  5. 5. Bioprocess Development: Animal Cell Culture”The optimization of the bioprocess is specially important for animal cell culture” – Less Cellular Concentration than Bacterium and Yeast: • Bacterium and Yeast cultures: [X]f ≈ 109 cells/ml • Animal cell cultures: [X]f ≈ 106 cells/ml If Ps=ct, we have 1000 times more product with bacterium or yeast than with animal cells – More expensive & complex process: • Culture media: – Bacterium and Yeast cell culture: Simple undefined mediums containing only salts (usually NaCl) and carbon & nitrogen sources (usually within yeast extract and tryptone). – Animal cell culture: Rich & complex mediums containing salts, carbon & nitrogen sources, but also complements (AA, vitamins, trace elements…) and serums (FCB, FBS…). • Easier to be contaminated: – Bacteria, yeast, mycoplasma or cross contamination. – Specific challenges for animal cell culture: slow growth and specific production rates, cell sensitivity (shear stress, nutrient limitation, metabolite accumulation..). www.telstar-lifesciences.com
  6. 6. Bioprocess Development: Main Steps Genetic Engineering Culture Conditions Operational Conditions Best Clones: Best Conditions: Optimal Process: Specific Productivity [X] [X] Growth rate Death times www.hexascreen.comfrom F. Wurm. Nat. Biotechnol., vol. 22:1393-1398 (2004)
  7. 7. Multiple aspects need to be optimized to establish optimal and profitable production processes Purification process Culture Fisico-chemical medium parameters PROCESS Clonal OPTIMIZATION Operation selection strategy Cell Process line Scale-up control GMP productionHigh cell concentration, high specific productivity, maintain product quality www.telstar-lifesciences.com
  8. 8. Bioprocess Development: Phases • PHASE I: Initial screening IMPORTANT!!! 1. Cell line selection (bacterium, yeast, animal cell…) 2. Cell modification (genetic engineering) Check the 7/8 3. Clonal activity selection activity of theclones Select which cells are producing the protein target protein in all 4. Protein target characterization: phases Measurement of the protein activity • PHASE II: Advanced screening 1. Clonal growth selection (select which cells grow faster…) 1/2 2. Culture medium composition: serum, glutamine, glucose…clones 3. Initial cell concentration. 4. Effects of stirring, [O2] and other culture conditions 5. Needs of the cell culture adaptation (ex: from adherent to suspension) • PHASE III: Lab scale production 1. Scale-up the advanced screening optimum conditions 2. Purification process 3. Type of Bioreactor (Batch, Fed-Batch, Perfusion) • PHASE IV: Pilot Plant and Industrial scale production 1. Scale-up the lab scale optimum production conditions www.telstar-lifesciences.com
  9. 9. Screening Bioreactors: Differences – Reproducibility problems when scaling up from usual screening phases into lab or pilot & production plant scales due to: • Homogeneity: Non agitated systems can produce cells or nutrientReproducibility accumulations giving not representative and impossible to repeat problems experiments. • [O2] limitations: Can produce a decrease of the cell culture growth velocity, metabolic differences or stopping the cellular cycle leading to an unreal productivity determination (higher or lower). – Lack of probes: few knowledge of the main cellular growth parameters : cell concentration, pH & pO2. – Lack of automatization: human manipulation is highly needed. – Current screening agitated systems (spinner flasks)Cost and time characteristics include high volumes and post- issues experiment treatments. www.telstar-lifesciences.com
  10. 10. Screening : The HexaScreen Alternative Initial Screening Advanced Screening Lab Scale New automated & controlled screening platform Bioreactors Usual scale-up procedure Spinner flask Multiwell plates T-flask0,1 1 10 100 1000+ Vessel volume (ml) www.hexascreen.com
  11. 11. Bioprocess Development: Phases Bioprocess development for a biotechnology product requires a number of steps Scale-up Methodology: Laboratory to Pilot to Industrial Initial HEXABATCH Screening Advanced Screening Lab scaleSince Biotechnological Processes are notcompletely known, the transition from bench to Pilot Plant Production Plantfinal volume is done step by step. www.telstar-lifesciences.com
  12. 12. Screening: HexaBatch®Design Criteria – Simultaneous multiple experiment capability. – Low minibioreactor volumes (10-15 mL), but not too low to allow cell culture similarities with lab scale bioreactors. – Agitation requirement to allow system homogeneity, but without upsetting cell viability. – Parts in contact with cells must be manufactured with single use biocompatible plastic (no contamination,…). – Cell growth (optical density), pH and dissolved oxygen monitoring via non invasive probes. www.hexascreen.com
  13. 13. HexaScreen®: HexaBatch Features Initial/Advanced Pilot Plant HexaBatch Screening / Industry Stirred & O2 fed Culture Stirred CultureEquipment used & Stationary Culture System to allow Systems:environment Systems: Heterogeneous homogeneity Homogeneous Multiple experiments run simultaneously to Unique cellMethodology Multiple experiments decrease the time culture process required for the screening phase From 2 to 10-15 ml (bench topVessel size Micro liters and milliliters thousands of scale) liters pH, pO2 and OD (cell Discontinuous and ContinuousProcess control concentration) on-line manual and monitored monitoring Disposable bioreactor made of sterileAsepsis Difficult / uncontrolled Necessary biocompatible plastic material www.telstar-lifesciences.com
  14. 14. HexaScreen®: HexaBatch ElementsHexaBatch version consists in two differentiated parts,the 6-minibioreactors’ plate and the workstation with acomputer/software .– Single use Minibioreactors’ Plate. Previously sterilized inside a plastic bag, includes 6 individual vessels equipped with gas filters, one septum for inoculation, miniaturized ports for probe’s allocation and a magnetic actuator for stirring.– Workstation. Contains the chamber where the minibioreactors’s plate remains during its culture, while maintaining optimal agitation & temperature (common), sterility, providing individual aeration and acquiring pH, DO and OD data. WorkStation is controlled via software. www.telstar-lifesciences.com
  15. 15. HexaBatch: Minibioreactor plate characteristics1-. Optical port for OD and pH measurements. 2 3 4 12-. Gas filters (inlet and outlet).3-. Optical port for DO measurements via fluorescence.4-. Septum for cell inoculation.5-. Low shear magnetic pendular agitation (optimal for animal cell).6-. Thermostated general bath. 87-. Vessel liquid volume: 10-15 ml.8-. Biocompatible and disposable plastic, 7 6 5 sterile provided. Possible plasma treatment to promote cell adherence / non- adherence. www.telstar-lifesciences.com
  16. 16. HexaBatch: Minibioreactors Inoculation www.hexascreen.com
  17. 17. HexaBatch: Minibioreactors to Workstation www.hexascreen.com
  18. 18. HexaBatch: Variables, Controls & Monitoring Variable Control Monitoring InformationAeration Time Controlled No NoAgitation Set-point controlled No NoVessel Set-point controlled Monitored System functionalityTemperatureGas & Filters Filters functionality – Set-point controlled MonitoredTemperature Avoids evaporation Monitored and Cell density information,Optical Density Free evolution correlated related to total cellsDissolved Free evolution Oxygen concentration, MonitoredOxygen (constant aeration) related to alive cells Cell activity information,pH Free evolution Monitored related to alive cells www.telstar-lifesciences.com
  19. 19. HexaBatch: Software Specifications HexaScreen®’s control & acquisition program runs on a Windows platform and will guide the user, as a wizard, through the processes of workstation’s configuration, calibration and data acquisition.General specifications• Automatic processes: – Workstation’s configuration – Thermal stabilization – Oxygen calibration – Optical calibration – Data acquisition (minibioreactors’ behaviour monitoring graphs)• Additional tasks: – Create new or edit already existing experiment set-ups – Create user defined graphs – Create reports – Export reports to EXCEL, PDF and HTML files www.hexascreen.com
  20. 20. HexaBatch: Advantages & Benefits Features Advantages Benefits - Stirring, gas exchange and oxygen supply are - Focused.Specially designed for animal cell specially designed for handling animal cell - Suitable for both suspension andculture. culture. adherent cultures. - Reducing working volume means lessBenchtop Scale: development costs in medium, cell culture, - Lower operation cost.Small Volumes from 10 to 15 ml”. enzymes…Parallel bioreactor system: - Less time required to achieve final results - Faster time to market.“6 multiple parallel experiments - Reproducible results: statistic data can be - Save time.for device”. obtained from replicated experiments. - Faster product development cycles. - Precision & control over all - Real-time kinetic information: experimentation.Automated on-line cell concentration (OD), DO & pH. - Lower labour and time-consuming inmeasurements. - No off-line control processes required. order to invest in other valuable - No maintenance required during experiment. tasks. - No post-experiments treatments. - Save money & time.Single use. - Avoid possible cross-contaminations. - Easier experiment validations.Easy to use. - No expert personal required. - Less effort required.Convenient. - Easier scale up to lab-size reactors. - Optimized culture parameters. - Save automatically the data acquired from all - Control on all acquisitionPC controlled. the experiments done giving a comprehensive experiment data. documentation. - Possibility to do final reports easier. www.hexascreen.com
  21. 21. HexaBatch: Applications & Cell Lines Cultured Applications Examples Cell Lines Cultured - Adherence and suspension lines. Suspension:Cellular screening. - Clone selection. - Hybridoma. - Growth parameters measurements (cellCellular characterization. - Genetically modified hybridoma. density and cell activity). - CHO cells (adapted).Cellular adaptation. - Adherent to suspension. - HEK (adapted) Adherent:Medium definition and - Commercial medium comparison.optimization. - Medium’s components definition. - Vero cells. - New drugs tests. - Ovine Mesenchymal Stem Cells.Cellular tests. - Toxicity tests. - Apoptosis tests. - CHO. - HEK. - Initial cellular concentration, cultureProcess optimization. conditions… www.hexascreen.com
  22. 22. Advantages of single-use technology Fast setup. No need for Cleaning. No risk of cross-contamination. Minimizes utility requirements. Minimizes validation. Minimizes space floor. Minimizes labor. Minimizes engineering design. Reduces COGS. Minimizes maintenance. Environmentally friendly:  Use for generate electrical power by incineration.  Estimated savings in WFI at over 80%.  Estimated 72% saving in electricity compared to conventional manufacturing facility. www.hexascreen.com
  23. 23. Advantages of single-use technology www.hexascreen.com
  24. 24. Systems comparison for screening• Systems to evaluate: • HexaBatch On-line measurements • 1L glass Bioreactor • T-flasks for suspension cell cultures Off-line measurements • T-flasks for adherent cell cultures www.telstar-lifesciences.com
  25. 25. HexaBatch Case Study: Conditions• Case Study parameters: • Number of conditions: 2 • Number of repetitions: 3 • Total number of cultures: 6 • Batch culture time: 3 days • Cell line: CHO • Culture media price (CDCHO): 103,52 €/L • Qualified personnel costs: 30 €/hour• It is assumed that there is only a one-liter bioreactor, so steps of the experiment are performed sequentially.• For off-line measurements (T-Flasks), only one sample is taken each day, with a total of 3 cell concentration measurements during culture. (no metabolites concentration measured) www.hexascreen.com
  26. 26. HexaBatch Case Study: Time Analysis Fins a Fin 5040 699 Total personnel cost (€) Inoculum Scale-up time 19 totals Bioreactor set-up time operation cost (€) tot 45 43 days Total sterilization, CIP, calibration, post-exp. cleaning) (Set-up, bioreactor 40 Total time Culture culture medium cost (€)Experimental time (days) 35 18 30 25 36 days 20 18 15 10 5 days 5 4 days 4 days 7 3 3 3 1 1 2 0 1 HexaBatch 1L Bioreactor T-flasks T-flasks (adherent cell) www.hexascreen.com
  27. 27. HexaBatch Case Study: Costs Analysis Fins a Fins a a Fins5040 5040 699 Total personnel cost (€) Fins a Fins a a Fins1960 1960 699 totals 5040 699 Total personnel cost (€) Total personnel cost (€) 1960totals totals Total bioreactor operation cost (€) totals Fins a totals Total bioreactor operation cost (€) Total bioreactor operation cost (€) Total culture medium cost (€) totals Fi 2.520 € Total culture medium cost (€) Total culture medium cost (€) 5040 2500699 Total personnel cost (€) Personnel costs 1 (staff hours x costs/hour) totals Lab material costs to Total bioreactor operation cost (€) (Single-use plate costs + T-flask costs for scale-up)Coste de un experimento (€) Total experimental cost (€) ExperimentTotal experimental cost (€) Total experimental cost (€) Total experimental cost (€) Culture medium costs Total culture medium cost (€) 2000 183 € costs (€) 22,5 1800 161 € 22,5 22,5 1500 507 € 1000 151 150 151 151 150 150 21 12 500 978 12 12 9 4 7 21 99 699 44 77 21 21 21 12 0 1 HexaBatch 1L Bioreactor T-flasks T-flasks (adherent cell) www.hexascreen.com
  28. 28. HexaBatch Case Study: Manipulation TimesQualified Personnel Manipulation Time (hours) 60 hours 60 50 40 30 20 33 hours 10 5 hours 45 min 0 1 HexaBatch 1L Bioreactor T-flasks T-flasks (adherent cell) www.hexascreen.com
  29. 29. HexaBatch Results: On-line Optical Density - Hexabatch gives one on-line OD measurement every five minutes vs just one/two per day in the case of T-flasks.Total cells Final cell concentration Off-line Measurements (T-FLASK) , tdup www.hexascreen.com
  30. 30. HexaBatch Results: On-line pH MeasurementsGivesinformation , tdup (indirect)about cellactivity Faster response than OD (no death cells interaction) www.hexascreen.com
  31. 31. HexaBatch Results: On-line Dissolved Oxygen Optimal Range www.hexascreen.com
  32. 32. Conclusion: HexaBatch system HexaBatch System = 1L bioreactor specifications + T-Flasks cheap and fast experimentation • Technological advantages from a bioreactor • System’s homogeneity: stirring and aeration • On-line process monitoring (pH, DO, OD) • Easy maintenance of asepsis • T-flask experiment price, speed and flexibility • Working volume at ml scale • Multiple experiment capability • Minimal needs on qualified personnel www.telstar-lifesciences.com
  33. 33. HexaScreen Applications: Index 1. Pharmacokinetic Tests. 2. Clone Comparison. 3. Media Comparison. 4. Inoculum Concentration. 5. Adaption to Suspension Cultures. www.hexascreen.com
  34. 34. Drug Functional Tests: Pharmacokinetics Antibiotic effect in cell cultures Functional Tests Activity profilesMore advantages to perform functional testsin cell cultures than in animals: - Faster results - Ethical issues www.hexascreen.com
  35. 35. Advanced Screening: Clone ComparisonChose the best clone in terms of:- Cell growth rates.- Cell activity.- Productivity at different sample times activity Growth rates activity www.hexascreen.com
  36. 36. Advanced Screening: Media Comparison Medium component• Best growth medium depletion• Time of action: - Fed-Batch start point. - Infection. - Product recovery. www.hexascreen.com
  37. 37. Advanced Screening: Inoculum Concentration Cell concentration profiles obtained from pH profiles (related to cell activity) www.hexascreen.com
  38. 38. Advanced Screening: Suspension AdaptionCell concentration control along passages Control over adaption process www.hexascreen.com
  39. 39. GRACIAS POR SU ATENCIÓN:JAVIER AMAYRA: jamayra@hexascreen.com General ManagerHexaScreen Culture Technologies S.L.Edifici Eureka, P1M1.2Parc de Recerca de la Universidad Autónoma de Barcelona (UAB)08193 Cerdanyola del Vallès (Barcelona) www.hexascreen.com