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Objectives of our project

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Objectives of our project

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  1. Cold ethanol precipitation and calcium-phosphate flocculation of recombinant antibodies University of Natural Resources and Life Sciences Vienna, AustriaDepartment of BiotechnologyNikolaus Hammerschmidt, Ralf Sommer, Anne Tscheliessnig, Henk Schulz, Bernhard Helk, Alois JungbauerIntegrated Continuous BiomanufacturingBarcelona, 21.10.2013

  2. Objectives of our project • Development of different precipitation methods for proteins, with an emphesis on recombinant antibodies • Replacement of chromatography based process by a series of selective precipitation steps • Implementation of the process in continuous mode

  3. Status quo - Commercial mAb processes SynagisTM HerceptinTM Rituxan MabCampathTM RemicadeTM Cell removal Cell removal Cell removal Cell removal Cell removal AC AC AEX AC AC Virus inactivation Virus inactivation CEX Virus inactivation Virus inactivation AEX CEX Virus inactivation CEX CEX SEC Virus clearance CEX AEX Virus clearance HIC Virus clearance Virus clearance AEX AEX Sterile filtration Sterile filtration AEX Virus clearance Sterile filtration SEC Sterile filtration Sterile filtration S. Sommerfeld, J. Strube, Chem. Eng. Proc. 44 (2005) 1123–1137

  4. Status quo - Commercial mAb processes SynagisTM HerceptinTM Rituxan MabCampathTM RemicadeTM Cell removal Cell removal Cell removal Cell removal Cell removal AC AC AEX AC AC Virus inactivation Virus inactivation CEX Virus inactivation Virus inactivation AEX CEX Virus inactivation CEX CEX SEC Virus clearance CEX AEX Virus clearance HIC Virus clearance Virus clearance AEX AEX Sterile filtration Sterile filtration AEX Virus clearance Sterile filtration SEC Sterile filtration Sterile filtration S. Sommerfeld, J. Strube, Chem. Eng. Proc. 44 (2005) 1123–1137

  5. Design by solubility curve 1 Solubility curves • mAb→ blue line • Impurities→ red line • Below solubility curve: protein in solution • Above solubility curve: protein precipitates logS = logS0 – βω (1) (1) Juckes I.R.M.: Fractionation of proteins and viruses with polyethylene glycol. Biochim. Biophys. Acta 229: 535-546 (1971)

  6. Design by solubility curve 2 Solubility curves • Region 1: Impurities and mAb precipitate • Region 2: impurities precipitate, mAb in solution • Region 3: mAb precipitates • Region 4: mAb and impurities in solution

  7. Ethanol – effect on antibody Excess enthalpy of water-ethanol mixtures [1] V.P.M. Belousov, I.L.Vestn. St.-Peterb. Univ. Ser. 4 Fiz. Khim., Vestn. St.-Peterb. Univ. Ser. 4 Fiz. Khim., 22 22 (1970) 101.

  8. Precipitation - effect on secondary structure ATR FT-IR spectra Dissolvedprecipitatevsdrugsubstance Dissolvedprecipitatevs 4 monthstorageat -10°C

  9. Cold ethanol precipitation platform process Clarified supernatant • 4-step process • Advantages of ethanol: • Low toxicity • Miscible with water • No explosive gaseous mixtures under normal working conditions • Highly volatile • Chemically inert • Cheap and easily available • FDA: Ethanol is class 3 solvent (Solvents with Low Toxic Potential) 1st CaCl2 precipitation ~4 mM phosphate, pH 8.5, 250 mM CaCl2, 20°C 1st ethanol precipitation pH 6.5, -10°C, 25%(v/v) EtOH 2nd CaCl2 precipitation ~4 mM phosphate, pH 8.5, 250 mM CaCl2, 20°C 2nd ethanol precipitation pH 6.5, -10°C, 25%(v/v) EtOH

  10. Purity data: mAb1

  11. Purity data: mAb2

  12. Purity data: mAb3

  13. Cold ethanol precipitation platform process Clarified supernatant • Currently 5-step process • Advantages of ethanol: • Low toxicity • Miscible with water • No explosive gaseous mixtures under normal working conditions • Highly volatile • Chemically inert • Cheap and easily available • FDA: Ethanol is class 3 solvent (Solvents with Low Toxic Potential) 1st CaCl2 precipitation ~4 mM phosphate, pH 8.5, 250 mM CaCl2, 20°C 1st ethanol precipitation pH 6.5, -10°C, 25%(v/v) EtOH 2nd CaCl2 precipitation ~4 mM phosphate, pH 8.5, 250 mM CaCl2, 20°C 2nd ethanol precipitation pH 6.5, -10°C, 25%(v/v) EtOH IEX monolith

  14. Polishing by IEX flowthrough [1] A. Jungbauer, R. Hahn, Journal of Chromatography A 1184 (2008) 62. From: http://www.biaseparations.com/pr/1702/cimmultus-qa-8-advanced-composite-column • Negative purification • High pI of therapeutic mAbs exploited • Impurities bound (DNA, HCPs), product in flow through • Monolith – masstransferbyconvection

  15. Purity data: mAb1

  16. Continuous reactor – Scale up and throughput Reactordiameterdoubled throughputinceases 4x atconstant linear velocity

  17. Economic evaluation (CoGs) – Gantt charts Classicalprocess: Fed-batch + chromatography Hybrid: Fed-batch + continuous precipitation Processing constraint: 5 days Fully continuous: Perfusion + continuous precipitation

  18. Economic evaluation – 3 scenarios

  19. Economic evaluation – Increasing titer Diameter ofpAcolumn: > 2 m Precipitationscaleswithprocessedvolume, not titer!

  20. Advantages Challenges Advantages and challenges of new process • Suitable for high titer processes • Disposable format possible • Reduction of footprint • Platform process • Can be run in batch AND continuous mode • Automatisation • GMP facilities already exist (blood plasma industry) • Rapid mixing and cooling • Adaptation to continuous mode • New to the field

  21. Acknowledgments • Alois Jungbauer • Anne Tscheließnig • Ralf Sommer • Novartis AG – Bernhard Helk • Novartis AG – Henk Schulz

  22. Questions??? Thank you!

  23. Stirred tank reactor – Tubular reactor Batch Continuous from Mettler Toledo using built-in probes Self-construction

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