John Bonham-Carter Director Magellan Instruments Ltd

1. ESACT-UK Annual Meeting 2005Developments in cell culture perfusion processes and their scale-up to manufacturing John Bonham-Carter Director Magellan Instruments Ltd

2. Agenda • Introduction to Magellan Instruments • Overview of BioSep perfusion system • Overview of ATF filtration and perfusion system • Example process data • Conclusions

3. O2 CO2 What Magellan does: • Distribute in the EU the • ATF perfusion & filtration system • Manufacture and sell world-wide the Tandem Gas Analyser

4. Applisens BioSep • A nice “science” solution and improvement to replace spin filters • Relatively easy to use and inexpensive at small scale However • Uses a shear-inducing peristaltic pump (D) • The take-off (B) requires filtration / clarification

5. Applisens BioSep • Large systems are expensive • 1000L scale being developed • Early problems with sterility now addressed • Separation efficiency much reduced at larger scales to ~85% • Separation efficiency can be improved by double pinch valve method, but media losses rise to ~15%

6. Perfusion: Hollow Fibres & TFF • Traditional TFF - tangential flow filtration (TFF), flow is in tangent to the filter surface and the flow sweeps the surface and prevents rapid plugging of the filtration device • Hollow Fibres- one of the types of configurations used in TFF (cross sectional view of one fiber shown to the right)

7. Temp. Gas inlet into headspace or liquid D.O. Level Control pH Agit. Feed Pump Hollow Fiber Unit Permeate Pump Water Jacket Inlet Outlet Circulation Pump Traditional Recirculation Perfusion

8. ATF Setup • Only one vessel connection • Easily adapts to all bioreactors and process vessels

9. Low Shear System • Recirculation pump is replaced by a low shear diaphragm pump • Energy is added to the surface of the liquid, generating a low shear laminar flow • No peristaltic pump required PressurisationExhaust video

10. Unique Action • Simple movement creates self-cleansing effect: • washes away aggregates • cleans filter pores • reduces biofilm build-up Media filtered out Diaphragm moves down Diaphragm moves up Some media backflushes in Media & cells flow out Media & cells flow in

11. Versatile Filter Sizes • Can be used with hollow fiber modules with different pore sizes • Can be used with a screen module for microcarrier-based cultures • Stainless steel, polyester, and other

12. Microcarrier Screen Module • Perfusion of Microcarrier Based Cultures • Rapid Medium Exchange • Some features of the ATF System: • Readily scalable from research to production • System is located external to the vessel • Requires only a single vessel connection • Can be easily steamed in place with the vessel • The Screen Module can be quickly disassembled for cleaning (and the screen itself disposed of)

13. ATF Controller Vacuum Diaphragm Mesh Screen Module Air Inlet Filtrate Pump D.O. Temp. Level Control pH Agit. Gas inlet into reactor headspace Feed Pump Water Jacket Inlet Outlet Bioreactor Microcarrier Module Setup

14. System Approx. Filter Surface Area (m2) Approx. Filtration Capacity* (L/day) ATF 2 0.045 5 ATF 4 0.42 40 ATF 6 2.1 180 ATF 8 4.2 320 ATF 10 9.6 600 ATF 12 16.7 1200 * these numbers are just a guideline, actual capacity and vessel size depend upon process conditions Scale up- from 1 to >1000L

15. Scale Up

17. Process overview Monoclonal Antibody

18. DSP - Potential Cost Savings Cell Culture 44% Capital 100% Use of the ATF may eliminate up to 10% of the downstream costs and save processing time Clarification ~6% Purification 56% Example costs for a recombinant protein production process

19. NIH Microcarrier Process The glucose was controlled above ~1g/L and lactate below ~2g/L. The HeLa cells were seeded at 1.5x10E5 cells/ml onto 5 g/L Cytodex 3 Microcarriers in DMEM+10% FBS with antibiotics. (Error bars are from duplicate cell counting samples from each run.)

20. CHO Batch & Continuous Culture

21. 22.5L Perfusion Run - Avid Biosciences • Process ran for 48 days • The cell line grew to a density of 20 to 40 x 10^6 cells/ml and a final antibody concentration of 75-100mg/L

22. 22.5L Perfusion Results

23. 300L Perfusion Run - Avid Biosciences • Ran process for 38 days • The cell line grew to a density of 17 to 25 x 10^6 cells/ml and a final antibody concentration of 75-100mg/L

24. 300L Results

25. Crucell Process Development

26. Crucell Process Development X tot ATF Perfusion was 22m cells/ml X tot Fed-Batch was 12m cells/ml Xtot in Batch was 5m cells/ml

27. Benefits • Filtered product eliminating or reducing subsequent process steps • Potential to lower capital and labour costs , esp. at scale • Already used at 1000L scale in final manufacturing • Capability to sustain cultures at high cell concentration (>40m) and productivity • Removes recirculating peristaltic pump, reduces shear • Choice of filters allows different process options e.g. viral work

28. Acknowledgements • Esther de Graaf-Groenendijk, Thomas Irish; Crucell BV • Nicole Bleckwenn, Joseph Shiloach; NIH • William Bentley; Department of Chemical Engineering, UMCP, MD • Avid Biosciences • Andrew Sinclair; Biopharm Services • Bill Thompson; Rotherwood Associates • Refine Technology