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PET for PAT? Process Evaluation Tools for Process Analytical Technologies in Manufacture of Biological Products

PET for PAT? Process Evaluation Tools for Process Analytical Technologies in Manufacture of Biological Products. Charles L. Cooney Department of Chemical Engineering MIT, Cambridge, MA 02139. Advisory Committee for Pharmaceutical Science April 13, 2004. SOME ISSUES.

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PET for PAT? Process Evaluation Tools for Process Analytical Technologies in Manufacture of Biological Products

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  1. PET for PAT?Process Evaluation Tools for Process Analytical Technologies in Manufacture of Biological Products Charles L. Cooney Department of Chemical Engineering MIT, Cambridge, MA 02139 Advisory Committee for Pharmaceutical Science April 13, 2004

  2. SOME ISSUES • What does the pipeline for new biological products look like? • What will be the path for Follow-on Biologics? • How does the biological product respond to physical process change? • Do we have adequate analytics to address the uncertainties in the biological products industry? • How can we assure robustness to design and operation of biological products manufacture?

  3. Where are we going? Multiple processes for the same or similar products • Products • Antibodies and Replacement proteins • Vaccines • Cellular therapies • Gene therapies • Processes • Recombinant protein production • Tissue engineering - tissue repair • Transgenic plants & animals • Challenges • Rapid and cost effective development & scale-up • Continuous improvement & process change • Follow-on biologics • Complex biologicals – cellular therapies and tissue engineering Complex processes for complex products There is tension between the safety and economic agenda; where is the proper balance?

  4. Parameter Control Process for Biological Products Raw Materials Product Environmental Conditions Information Flow How does the biological process respond to physical change?

  5. THE OXYGEN DILEMMA • Required for efficient growth and recombinant protein expression • Potential in vivo or in vitro protein oxidation e.g. Met, Cys • Oxygen induced stress response

  6. 100ml 100 mL 10 L 10,000 L Homogeneous Homogeneous DO Homogeneous 10% 40% O2 Gradients in Large-Scale Fermentors • How do O2 gradients affect cell? • How does cell respond? • Effects on recombinant protein production? Heterogeneous

  7. methionine sulfoxide methionine COOH COOH H2N-C-H H2N-C-H CH2 CH2 CH2 CH2 oxidation S S O CH3 CH3 Model System: a1-Antitrypsin • Elastase inhibitor (44 kDa) 10 met and 1 unpaired cys • Activity lost with oxidation of active site MET358 Oxidation of met358 --> partial loss of neutrophil elastase activity & complete loss of porcine pancreatic elastase • Use in protein replacement therapy • Cytoplasmic expression in E. coli BL21 M358 M351

  8. Observed Problem in Synthesis • Recombinant a1-antitrypsin (soluble at 30C) • Degraded in E. coli • Proteolysis is oxygen-dependent • What is the connection between O2 and proteolysis?

  9. WHERE IS THE PROBLEM?AND SOLUTION?

  10. Protease ClpP • Proteolysis in E. coli • Majority requires ATP • ~70% by Lon and ClpP/AX • Current Strain BL21 is Lon- • ClpP • Protease subunit of ClpP/AX • complex • Heat shock protein • ClpP- strain (SG1146A) • E. coli BL21 ClpP- Figure (Wickner & Maurizi, PNAS 1999)

  11. O2-Enhanced Degradation is Eliminated in ClpP- Strain Wild Type SG1146A (ClpP-) • Some background degradation (~18%) remains • Other protease responsible

  12. Do we have the analytical techniques to probe a cell’s global response to its physical environment?

  13. DNA Microarray Experiments • 3,812 Genes representing 89% of E. coli genome • Multi-Gene Groups • 167 protein complexes • 190 pathways • 333 transcription units

  14. Hyperoxic Stress Responses • Increasing N2→ Air → O2 • Sustained Response • Increasing Air → O2 • Short-Term Response

  15. O2 Dependent Genes • SoxRS Response • soxS, fur, sodA, nfo • Iron Uptake • fur, sodA, fepB • Fe-S Proteins • bioB, ilvD, leuB, mutY, fdx, yfhI • Fe-S Cluster Assembly • b2530 (iscS), b2531, hscA, fdx

  16. What is the right next step?

  17. When we introduce a process to make a biotherapeutic product do we know the “optimum” conditions for quality and quantity? SELF ASSESSMENT During routine manufacture, do we improve the quality and quantity of product?

  18. What is the way forward? • Is there a better way than incremental adjustment to optimize and scale a biological process? • We live with variance; have we taken adequate opportunity to observe it and learn? • Can we explore experimental space more effectively? • How do we embrace risk and manage it? • How do we assure ourselves that we have a robust process?

  19. PROCESS EVALUATION TOOLS • Leverage analytical technology on process and product • Look at the global system response • Explore how biological and parameter variance propagate through the process? • Interrogate the cell at the molecular scale • Multi-scale analysis – scale down to scale up • Understand the interdependencies in experimental space • Understand the connection between molecular processes, process performance & product quality

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