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? 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 Multiple processes for the same or similar products Complex processes for complex products There is tension between the safety and economic agenda; where is the proper balance?

4. Process for Biological Products Raw Materials Environmental Conditions Product Parameter Control 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. O 2 Gradients in Large-Scale Fermentors 10,000 L DO 10% 40% 100 mL Homogeneous 10 L Homogeneous How do O 2 gradients affect cell ? How does cell respond? Effects on recombinant protein production? 100  l Homogeneous Heterogeneous

7. Model System:  1 -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 methionine sulfoxide H 2 N-C-H COOH CH 2 CH 3 S H 2 N-C-H COOH CH 2 CH 3 SO oxidation M358 M351

8. Recombinant  1 -antitrypsin (soluble at 30C) –Degraded in E. coli –Proteolysis is oxygen-dependent What is the connection between O 2 and proteolysis? Observed Problem in Synthesis

9. WHERE IS THE PROBLEM? AND SOLUTION?

10. 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) Protease ClpP

11. Wild TypeSG1146A (ClpP-) Some background degradation (~18%) remains Other protease responsible O 2 -Enhanced Degradation is Eliminated in ClpP- Strain

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 N 2 → Air → O 2 Sustained Response Increasing Air → O 2 Short-Term Response

15. O 2 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? During routine manufacture, do we improve the quality and quantity of product? SELF ASSESSMENT

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