BIOMAN 2011 CHO- tPA Production System Upstream Processing

1. BIOMAN 2011CHO-tPA Production SystemUpstream Processing Mike Fino MiraCosta College

2. Program Concentrations

3. Program Coursework100-level courses • Basic Techniques in Biotechnology (4-units) • Introductory experience working in a technical environment • Business and Regulatory Practices in Biotechnology (3-units) • Fully online • Overview of the industry from discovery to GMPs • Biostatistics (4-units) • Lecture and lab • UC/CSU transferrable

4. Fully online Courses Program Coursework200-level courses Hybrid Lab courses Advanced Cell Culture Isolation and Purification of DNA Techniques in DNA Amplification Recombinant DNA Principles of Separation and HPLC Techniques in Immunochemistry and ELISA Qualification and Validation in Biotechnology Bioprocessing: Cell Culture and Scale-up Bioprocessing: Large Scale Purifications Biofuels Production and Analysis Data Analysis with Excel Technical Writing for Regulated Environments Career Experience • Internship • Work Co-op

5. Program Certificates Biopharmaceuticals or Biofuels Career & Professional Development Transfer / Long-Range

6. Biopharmaceutical Production • Upstream • Product formation • Downstream • Product purification • Quality Control • Product safety and efficacy

7. A Time of Transition and Translation • The lead candidate coming out of discovery research will now be subjected to a development process that sees it change through a prism of • Scale • Process Control • Compliance

8. From Art to ScienceScale-up • Development is the confluence of business, engineering, manufacturing, quality assurance, quality control, and regulatory affairs • The end goal is process understanding so that a commercial process and product are in a state of control

9. Process Control & Compliance • Say what you do • Document it • Do what you say

10. The Upstream Process • The confluence of inter-related efforts in: • Cell line development • Media development • Bioreactor process design • Bioreactor process control

11. Cell Line Engineering

12. Expression Systems Bacteria Yeast Mammalian Insect Transgenic Plants Transgenic Animals

13. End game of cell line engineering? PCD = picograms per cell per day

15. cont’d

16. Back of the envelope calcs • If we have a cell line that produces our product at 100 pg/cell-day and we want to produce material for a Phase I clinical trial in a single batch then… • Our product is dosed at 1-mg/kg • Our process takes about 1 week • Typical cell densities are 1e6 cells/mL • Assume 20 people in the trial • Assume average person is 75-kg • …how big does my culture need to be?

17. Large scale mammalian cell culture • We use the CHO-tPA system as a model culture system for several reasons • Seeding densities as a way to show why a step-wise • Aseptic technique and processing to underscore the slower growth rates vs microbes • Currently the main choice for any glycosylated protein

18. Inherent Variability: Erythropoietin • Contains 40% carbohydrate, only 2 disulfide bonds • 3, N-linked ASN (24, 38, 83), 1 O-link (SER126) glycosylation sites • Glycosylation at these sites may be responsible for resistance to denaturing conditions • O-linked site not essential for in-vitro or in-vivo activity • Sialic acid residues (avg 10 moles/mole EPO) responsible for preserving phamacokinetic behaviour • Muteins lacking 2 or 3 N-linked sites are poorly secreted • N-linked glycosylation and sialylation is critical to optimal secretion, structure, in-vivo potency

19. The Upstream Process • The confluence of inter-related efforts in: • Cell line development • Media development • Bioreactor process design • Bioreactor process control

22. The Upstream Process • The confluence of inter-related efforts in: • Cell line development • Media development • Bioreactor process design • Bioreactor process control

29. Reactor Selection: Many factors to consider

30. Developing higher yields • In 1986, the industry standard from stable CHO-derived cell lines was a specific productivity of 10 pg/cell/day, 50 mg/L titers, 2e6 cells/mL and processes lasted 7 days • Today, we are seeing 90 pg/cell/day, 10 g/L, 10e6 cells/mL, and processes lasting up to 3 weeks • Generation of recombinant cell lines with high specific productivities • Formulation of media to support high density cell cultivation • Understanding of bioprocess conditions for cell cultivation • Sustained viability of cell lines in high-density batch and fed-batch cultures

31. Stirred Vessel Bioreactor • Process control of the physiologic environment • Disssolved oxygen • pH • Temperature • Medium components (feed: glucose, glutamine; hormones, growth factors) • Waste products (CO2, ammonium, lactate)

32. Batching Types (Process Modes) Single Batch Fed Batch Continuous Batch

33. Single Batch 7-14 days Run is over when there are no more nutrients Batching Types

34. Fed-Batch Addition of concentrated nutrients => higher product concentration 2-3 weeks Intermittent common for viral vaccine rProtein production is largely fedbatch Batching Types

35. Continuous Continuous addition of feed and collection of harvest with cell retention device => higher product concentration Especially for process-labile products Batching Types

36. Parameters measured or controlled in bioreactors

42. The Upstream Process • The confluence of inter-related efforts in: • Cell line development • Media development • Bioreactor process design • Bioreactor process control

43. Essentials to Control a Process • Process Variables • Identify which variables are important to what you’re doing • Bioreactors: pH, DO, Temperature, cell count, nutrient levels, waste levels, CO2 levels • Process Probes • You need some instrument that can convert the chemical or physical phenomenon into an electrical one • Controller • The basic function of a controller is to make a comparison between the current reading of a Process Variable and the desired Setpoint • Based on that comparison, the controller produces an output • Process Manipulation • Based on the Output, you then attempt to manipulate the Process Variable to bring it closer to the Setpoint

44. Bioreactor Control System PV Controller Output SP Depending on the level of the output, something will be changed (e.g. valve opened/closed) to bring the PV closer to the SP Yellow number is the Process Variable, PV White number is the Set Point, SP Blue number is the Controller Output

45. A Control System A simple error calculation will be made: SP - PV = Error Depending on the nature of the Error an Output is calculated Magnitude History Current slope The Output will do something to bring the PV closer to the SP

47. Our BioNet Systems Bioreactor Probes Heating blanket Agitator and motor Control tower Collects, stores, and processes information Controls gases and pump additions Primary interface

48. Bioreactor Control System • MANual mode • Controller not utilized, full operator instruction • Example is agitation – we set it and forget it • AUTO mode • Controller will try to bring the PV in line with the SP • The Output of the AUTO mode will be looking to do something, based on what’s defined in the Setup Page • CAScade mode • These are the devices that are looking for direction from the AUTO output • This is for all the instruments (pumps, valves, MFCs) that will be controlled in order to maintain the SP • Their response is defined in the Setup Page

50. Priming the Base