High Throughput Bioreactor Mimetic in Early and Late Stage Process Development
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The document discusses the evaluation of the ambrTM system as a high-throughput bioreactor mimetic for accelerating biopharmaceutical development. It highlights case studies demonstrating the system's capability in reproducing key observations from classical bioreactors and emphasizes the importance of process control and scalability in cell culture development. Ultimately, the findings suggest that ambrTM can significantly reduce process development time for investigational biopharmaceuticals, thus expediting their path to clinical trials.
High Throughput Bioreactor Mimetic in Early and Late Stage Process Development
- 1. High throughput bioreactor mimetic in l d l t t d l tearly and late stage process development American Chemical Society – Biochemical Technology (BIOT) Division 245th – ACS National Meeting, New Orleans, LA Shahid Rameez, Ph.D.Shahid Rameez, Ph.D. Scientist I, Process Development KBI Biopharma Inc, Durham, NCp
- 2. Overview Ambr System Bioreactor Mimetic In process Mimetic KBI Evaluation p Activities & Design Space Evaluation Experimental Design in Early and Late C t l St t i y Stage Process Development Control Strategies
- 3. A key bottleneck in biopharmaceutical development has been they p p rapid development of robust and scalable manufacturing processes that can permit accelerated progress of products into clinical trials. Innovations in this area can have a very significant impact on the overall economics of biopharmaceutical drug development by d i h i i k h h li idecreasing the time it takes to reach the clinic. Mammalian cell culture processes typically have the longestp yp y g experimental duration with 2-3 weeks being a typical duration for the production bioreactor step with additional time spent on the seed culturescultures.
- 4. Shake flasks provide the capability to perform high throughput experiments, but with an inability to control process parameters likeexperiments, but with an inability to control process parameters like agitation rate, dissolved oxygen (DO) and pH. Th t l h l h d l i b t ffi i t These parameters play a huge role when developing a robust, efficient cell culture process which dictates the product quality and yield. Moreover, reproducibility and scalability of process and culture performance is a pre-requisite for successful and efficient process development at a scale down level.development at a scale down level. At KBI we did case studies evaluating the ambr™ system (an a tomated micro scale bioreactor s stem) to establish the roleautomated micro-scale bioreactor system) to establish the role this system could play in accelerating biotech drug development.
- 5. Automated Miniaturized Bioreactors 1mL tips ambrTM Technology 1mL or Liquid Handler 4mL tips Handler Used Tips Discard Culture stations; each holding 12 bioreactors
- 6. ambrTM Deck Layout Culture stations 1ml tips 4ml tips4ml tips 24 deep-well plate Tip box lid Plate lid
- 7. The Vessel Vessel Cap: In-line filter on gas supply DO sensor on gas supply Impeller pH sensorp
- 8. Culture Station Nomenclature Culture station 1 (CS1) Culture station 2 (CS2) CS1-1 CS1-2 CS1-3 CS1-4 CS1-5 CS1-6 Culture station 1 (CS1) CS2-1 CS2-2 CS2-3 CS2-4 CS2-5 CS2-6 Culture station 2 (CS2) 7 8 9 10 11 12 7 8 9 10 11 12 CS1-7 CS1-8 CS1-9 CS1-1 CS1-1 CS1-1 CS2-7 CS2-8 CS2-9 CS2-1 CS2-1 CS2-1 Id l ki l i 13mL W ki l i 11 15mLIdeal working volume is 13mL; Working volume range is 11 – 15mL Things to consider: – Feed strategy and Sampling strategyFeed strategy and Sampling strategy – Low volumes – gas entrainment / vortexing – High volumes – Limits kLa.
- 9. PART 1: Reproducibility for Results and Key Observations during Cell Culture Process Development The processes evaluated in ambrTM were previously developed from a rigorous cell culture process development performed in classical bioreactors of various scales. The process were successfully carried in bioreactors across various scales: 2L, 10L d 200L10L and 200L. This study aimed at studying the reproducibility of the key observations of the processes in ambrTM. Thus a reverse engineering approach was adopted, where we were cognizant about the outcomes from most of the experiments as far as inducing process variations was concerned. The reproducibility of key historical results in ambrTM would corroborate towards its capability as a high throughput bioreactor mimetic in cell culture process development.
- 10. K Ob i f Hi i l D Case Study 1: Reproducibility evaluation for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary (CHO) cell line. Key Observations from Historical Data: • Temperature Shift during the cell culture process was found to be the most important process factor to regulate the productivity of the antibody titer. • The CHO cell line performed better at lower pH set point of 6.85 as compared to pH set point of 7.00. • Feeding intermittently had shown to regulate growth and productivity in the process. Intermittent feeding had showed better results than just Day 0 additions for feed. • A highly basic and a critical feed, referred here as FDX, had to be added without pre- neutralization with acids to avoid osmolality increase in cultures. Thus, a better control had to be established in the bioreactors to control the pH drift with addition of FDX. h h d b b h d hThis was achieved in classical bioreactors by tuning the PID controllers and with regulation in cascade feedback gassing of CO2 and Air.
- 11. Results: Both lower pH set-points and Temperature Shift showed higher cell growth, better cell viabilities. DO as suspected at negligible effect on cell growth and viability. Time courses for viable cell growth and viability for recombinant CHO cell line with changing (A) Process pH (B) Temperature (C) Dissolved Oxygen (DO) levels and (D) Feeding Strategies. The experimental data shows an average of 2-3 vessels in the ambr 24. The error bars show the standard deviationdeviation.
- 12. Results: Both lower pH set-points and Temperature Shift showed higher cell titers. As observed historically for this process, Temperature shift was found to be the most important process factor to regulate the productivity of the antibody titer. The ambrTM system can be used as a high-throughput platform to make key process decisions during the early process development phase of bi h i l d lbiopharmaceutical development.
- 13. PART B: Scalability Assessment in Cell Culture Process Development Case Study 2: Comparison across scales for the production of a monoclonal antibody in a recombinant CHO cell line. Ambr (n = 3). 2L (n = 1). 10 ( 4)10L (n = 4). 200L (n = 1). • Harvest Titers within 1 5 1 7 g/L• Harvest Titers within 1.5 -1.7 g/L across all scales. Comparison of time courses for viable cell growth and viability for recombinant CHO cell line in ambrTM and other scales bioreactors: 2, 10L Glass bioreactors and 200L disposable bioreactor.p
- 14. Case Study 3: Comparison across scales for the production of a protein molecule in a recombinant Chinese Hamster Ovary (CHO) cell line. Results: The cell growth, cell viability and cell viabilities were comparable between ambrTM,10 and 200L Bioreactors200L Bioreactors.
- 15. Case Study 3: This protein molecule had two Isodimers (A and B). The levels of Isodimers A and B were a product quality attribute. Results: Ratio of Isodimers A and B were similar (± 5% of mean values) across ambrTM , 10 and 200L Bioreactors. The process decisions and results from ambrTM were reproducible to the results in other scales bioreactors. Both the case studies (with antibody and a non antibody) demonstrate the utility of the ambr™ system as a high throughput system for cell culture process development.p p
- 16. PART C: Control for process pH and DO during Cell Culture Process Development pH control in ambrTM is established using the automated liquid handler based base additions when pH drops below the pH set pointwhen pH drops below the pH set point. When the pH exceeds the pH set point, the CO2 flow rate increases to establish control on the pH drift. CO2: 0 - 1.24 mL/min. Delivered on demand to control pH. O2 : 0 - 1.24 mL/min. Delivered on demand to control dissolved oxygen. N : 0 1 24 mL/min Flow rate is constantN2 : 0 - 1.24 mL/min. Flow rate is constant.
- 17. Online profiles for process pH (top Online profiles for process pH (top figure) and DO (bottom figure) levels during the culture duration for CHO cell line expressing a recombinant antibody in ambrTM. The spikes in the DO profiles corresponded to bioreactor samplingcorresponded to bioreactor sampling, Liquid additions and Sampling. All these disturb the headspace and l h ki l Th i falter the working volume. The time for the DO traces to equilibrate to setpoint after such manipulations would depend on the controller setup.p
- 18. Case Study 4: Artificial perturbations in pH and DO (by adding a basic feed and changing DO set points respectively) during production of an antibody molecule in a recombinant CHO cell process. Through adjustments to the PID control loop and gas flow rates the capability of b ™ t l t dambr™ system was evaluated. Results: Tuning the gas flow limits and proportional gains in the PID loop of ambr™ system. By changing the proportional gain by eight folds and CO2 gas limits by 1.25 and > 2folds as opposed to default manufacturer values , the pH drifts were reduced by 23 and 47 % of initial value, respectively.value, respectively.
- 19. Results: The DO set points were changed to 80% from 20 and 40%, respectively and changed back to original values The level for DO was maintained at 80% for duration of 6changed back to original values. The level for DO was maintained at 80% for duration of 6 hours and returned to original set points 20 and 40% in ≈ 90 and 120 mins, respectively. Th p bilit f ind in d i ti n n h lp in d i nin r t The capability of inducing deviations can help in designing worst-case experiments. It enables to test operating limits with respect to particular key operational parameters (DO, pH) in a process.
- 20. The combination of pH and DO control and an automated liquid handling system in ambrTM system overcomes major limitations of conventional small-scale cultures vessels Conclusions ambr system overcomes major limitations of conventional small scale cultures vessels especially shake flasks. The single-use, pre-calibrated, and instrumented vessels used in ambrTM system provides a platform for high-throughput in cell culture process development while mimicking a stirred-tank bioreactor environment. The reproducibility of key observations observed in historical process developmentT e ep od c b ty o ey obse vat o s obse ved sto ca p ocess deve op e t demonstrated that ambr™ is capable of providing predictive results under bioreactor relevant process conditions. R d ibili l bili d h bili f h d b i h Reproducibility, scalability and the ability of the system to respond to perturbations show ambrTM to be adequate to consider this system for early and late stages of cell culture process development. The studies at KBI aimed to demonstrate the utility of the ambr™ system as a high- throughput bioreactors that can offer the realistic possibility of decreasing the process development time for investigational biopharmaceuticals to reach the clinic.
- 21. Process Characterization Commercial ProcessProcessCell LineDiscovery Manufacturing Biopharmaceutical Development Process. Characterization and Validation DevelopmentDevelopmentDevelopment y Stage RAPID PRODUCT DEVELOPMENT AT KBI Manufacturing ambrTM Process Development (Design Space & Optimization) Cell Line DevelopmentDiscovery Stage Manufacturing • Platform Downstream Processes • High-throughput Resin Screening • Single - Use Technology TM The combination of methodologies such as ambrTM, Platform Downstream Processes, High-throughput Resin Screening and use of Single-use technology can significantly shorten the window for process development and f t imanufacturing.
- 22. Acknowledgements • Joe McMahon President and CEO • Abhinav Shukla, Ph.D. VP, Process Development and Manufacturing • Sigma Mostafa Ph D Director Process DevelopmentSigma Mostafa, Ph.D. Director, Process Development • Haiou Yang, Ph.D. Scientist II, Process Development • Christopher Miller Scientist II, Process Development • Anushya Mani Scientist I Process Development• Anushya Mani Scientist I, Process Development • Joe Jirka Product Specialist, TAP Biosystems Process Development Team at KBI Process Development Team at KBI