Implementation of a Micro Bioreactor System for Platform Cell Culture Process Development at Cobra Biologics Kristina Lae, Scientist, Cell Culture Cobra Biologics, Södertälje, Sweden
Cobra Biologics and Cobra Biologics Södertälje
Cobra Biologics Sites Keele: Virus and Small Scale Microbial Södertälje: Mammalian Cell Culture (Monoclonal antibody, recombinant proteins) Now: Modern facility, with non- GMP and GMP 250L SUB Comprehensive analytics and cell line development capability Future: 1,000L disposable SUB for commercial capacity Now: 14 year track record of developing processes and GMP manufacturing for pioneering medicines Future: Commercial virus capacity 250L disposable SUB Matfors: Fill Finish and Large Scale Microbial Now: 20 year track record for clinical and commercial supply with 600L microbial GMP capacity. New aseptic fill/ finish facility including lyophilisation Future: Expansion space for fill/finish or bio-production
Product Areas Proteins Bacteria Plasmids Bacteriophages Whole cells Animal cells Viruses Proteins Novel proteins Monoclonal antibodies
Goals and Drivers Our goals to meet customer requirements Increase flexibility for different molecules and different technologies Decrease time lines and start up times Increase the GMP capacity Potential drivers for early stage clinical development programmes Productivity & cost of goods Rapid generation of clinical materials & obtaining clinical data Development costs Product quality Regulatory expectations
Platform Strategy for Customer Projects Customer project Cell line development Process development Supply of Material (tox) GMP Production Cello Plates Shake flasks Culti flasks Ambr Micro Bioreactors Shake flasks Culti flasks 5L BR 25L Wave 50L SUB 250L SUB 25L Wave 250L SUB 100L SS Media, feed and buffer preparation using single use bags Utilization of singles use materials increases flexibility and reliability of the processes used as part of the cell culture platform strategy
Cell Line Development and Small Scale Process Development Transfection and clone selection High throughput colony detection, titre determination and scale up in static culture using Cello Robotic System Second titre screen in shake flask culture Small scale cultivation Shake flasks Up to 1L Culti flasks Small volumes (8-12 ml) Easy handling Results comparable to shake flasks Micro Bioreactors Ambr Micro Bioreactor system
Cello Robotic System for Cell Line Development Plate handling robot (Stäubli) Up to three incubators and a cold storage unit Two pipetting robots High resolution microscope Software, managing the complete process from clone screening to expansion to 6 well plates Database with full traceability for each clone Cello Robotic System manufactured by TAP Biosystems
Material Supply at Different Scales Delivery of a requested amount of material for purification or analytical development or toxicological studies Delivery of GMP-material for pre-clinical supply Material from clonal cell lines or cell pools Fed-batch, batch or perfusion Supply of material from a certain scale Shake flask 2x Wave (5-25L) 2x 50L SUB (25-50L) 250L SUB (125-250L) Supply of material in 250L scale Scale up in Shake flask Seed train using Wave Production in 250L SUB
Single Use Bioreactors Less cleaning and no cleaning verification needed Time is saved between batches, quicker re-start and shorter lead times Flexible systems Less complex Easy to train personnel Reduced risk for contamination Easy to clean, less sanitation agent Compatible with excising clarification equipment Seamless transfer from lab to GMP production Similar equipment
GMP Supply Time Line 1 w 2.5 w 2 w Set-up and cleaning Scale up and Production in 100L SS Cleaning and cleaning verification One slot is 5.5 w 1 day 3 days 2 w 1 day Scale up Wave Set up Production in 250 L SUB Cleaning One slot is 3 w Since less cleaning and no cleaning verifications are needed a new project can be started directly if the scale up is done in parallel.
Implementation of Ambr Micro Bioreactor System
Need for an Automatic System for Process Development Why did we need an automatic system? Keep time lines short and speeding up the development work More information from one round of process development Reduce costs by reduced work Improve quality of process development Bioreactor process parameters evaluated early in a project Comparable to bioreactor results Limitations in comparability between shake flasks and bioreactors
Screen of market for Micro Bioreactor Systems Requirements Measure and control ph (CO2 and Base) Measure and control DO (Air/O2) Add and take out liquid Simple to handle, not to complex Sufficient culture volume Experiment capacity Process economy Only a few systems on the market Complexity/simplicity Expensiveness Volumetric challenges System capacity
Ambr Micro Bioreactor System 24 single use bioreactors 24 experiments in parallell Individually controlled ph and DO Stirring and temperature control Automated sampling and additions Screening of process parameters Media, nutrition feeds, clones Process parameter optimization ph, DO, temperature etc Ambr Micro Bioreactor System Manufactured by TAP Biosystems
Ambr Micro Bioreactor System Single use bioreactors Optical DO and ph measurements 10-15 ml culture volume Stirrer Dip tube for gassing (CO 2, N 2, O 2 ) Benefits Suitable for design of experiment setups Large amount of data achieved Quick and easy to set up No cleaning Small footprint in the lab Low volumes of media and feed required Single use bioreactor
Implementation of Ambr Micro Bioreactor System Timeline Purchase in May 2011 First run through in May 2011 Implemented in internal projects in June 2011 Successfully used in first customer project in July 2011 Results Comparable to previous results in 5L scale bioreactors Large amount of data for evaluation Quick and easy to set up ph and DO regulation sufficient
Titer (g/l) Case study; Process Development 2,5 2 Media screening Un-optimized batch Feed Screen using Ambr 1 st Round of Feed Optimization using Ambr 1,5 1 0,5 Media screening Performed in culti flasks (12 ml) Feed Screen Performed in Ambr using one base media 1 st round of feed optimisation DoE set-up for selected feed using Ambr 0 Un-optimized Batch Feed Screen Ambr Feed Optimization Ambr Commercially available chemically defined media and feeds 2 g/l IgG achieved after 1 round of feed optimization In total 6 weeks of process development after clone selection
Viable Cell Density [MVC/mL] Viability [%] Cell density [MVC/mL] Viability [%] Results using Ambr Micro Bioreactor system Growth in Ambr and 5L glass bioreactor are comparable Growth in 5L glass bioreactor and 250L SUB are comparable 250 L Ambr 5L BR 5 L Culture time [days] Culture time [days]
Data Data Clean Clean Clean Clean Clean Clean 2v Shortened Process Development Time Line Parameter optimization in BR 18v 2v 24 SH 4x 5L 4x 5L 4x 5L 4x 5L 4x 5L 4x 5L 2x 5L Clone selection 24x MBR 24x MBR 2x 5L Process verification in BR 2v 2v 2v Clone selection can be performed in micro bioreactors More valid results, comparable to bioreactor Parameter optimization in 24 set-ups Evaluation in 2 weeks using Ambr micro bioreactors No cleaning between batches, quick start-up Evaluation in 18 weeks using 5L glass bioreactors 4 BR in parallel using 1 FTE Process verification in small scale bioreactor (2-5L)
Data Data Data Data Data Data Increased Process Development Capacity 2v 5-6 weeks of process development in BR 2v 24 SH 4x 5L 4x 5L 2x 5L Clone selection Process verification in BR 24x MBR 24x MBR 24x MBR 2x 5L 2v 2v 2v 2v Evaluation of different media, feeds, feeding strategies and process parameters Increased number of set-ups increases the ability to find a suitable process 5-6 weeks of process development with 1 FTE Ability to test 8 different set-ups in 5L bioreactor scale Ability to test 48 different set-ups using Ambr
Challenges Culture volume should be kept within 10-15 ml Processes are designed to fit the volume Feed volume restrictions Base and glucose additions predicted in advance to estimate culture volume Sampling volumes must be kept small Daily sampling for metabolites, ph, pco2 and cell count Small volumes are used for all analyses Samples for titer and spent media analysis Analysis methods for titer and amino acid concentrations set up in 96-well format Addition of antifoam necessary Foaming occurs if no anti-foam is added ph control A tight deadband might cause high usage of tips and high addition of base Optimization of data handling during and after run Great amount of data in excel sheets ph and DO curves
Future Improvements using Ambr Compare results from Ambr and 250L SUB in order to evaluate the possibility to further reduce time lines Include Ambr even earlier in cell line development platform to select clones based on bioreactor performance earlier
Summary The Ambr Micro Bioreactor system was successfully implemented during 2011 Ambr is included in our process development platform for customer projects Ambr results are comparable to results from 5L bioreactor scale