Cold ethanol precipitation and calcium-phosphate flocculation of recombinant antibodies University of Natural Resources and Life Sciences Vienna, Austria Department of Biotechnology Nikolaus Hammerschmidt, Ralf Sommer, Anne Tscheliessnig, Henk Schulz, Bernhard Helk, Alois Jungbauer Integrated Continuous Biomanufacturing Barcelona, 21.10.2013

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 08.10.2013

Status quo - Commercial mAb processes HerceptinTM Rituxan MabCampathTM SynagisTM RemicadeTM Cell removal Cell removal Cell removal Cell removal Cell removal AC AC AC AEX AC Virus inactivation Virus inactivation Virus inactivation CEX Virus inactivation CEX AEX CEX Virus inactivation CEX AEX CEX SEC Virus clearance Virus clearance HIC Virus clearance Virus clearance AEX AEX Sterile filtration Sterile filtration Sterile filtration Virus clearance AEX SEC Sterile filtration Sterile filtration S. Sommerfeld, J. Strube, Chem. Eng. Proc. 44 (2005) 1123–1137 08.10.2013

Status quo - Commercial mAb processes HerceptinTM Rituxan MabCampathTM SynagisTM RemicadeTM Cell removal Cell removal Cell removal Cell removal Cell removal AC AC AC AEX AC Virus inactivation Virus inactivation Virus inactivation CEX Virus inactivation CEX AEX CEX Virus inactivation CEX AEX CEX SEC Virus clearance Virus clearance HIC Virus clearance Virus clearance AEX AEX Sterile filtration Sterile filtration Sterile filtration Virus clearance AEX SEC Sterile filtration Sterile filtration S. Sommerfeld, J. Strube, Chem. Eng. Proc. 44 (2005) 1123–1137 08.10.2013

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) 08.10.2013

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 08.10.2013

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. 08.10.2013

Precipitation - effect on secondary structure ATR FT-IR spectra Dissolved precipitate vs drug substance Dissolved precipitate vs 4 month storage at -10°C 08.10.2013

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 08.10.2013

Purity data: mAb1 Dilution factor IgG IgG yield Monomer HCP HCP Reduction [µg/ml] step overall [ppm] Supernatant 2563.4 109230 1st CaCl2 precipitation 1.06 2379.4 98% 66462 1.6 1st EtOH precipitation - SN 1.35 38.8 2% 1st EtOH precipitation - PP 1.00 2172.4 91% 89% 15224 4.4 7.0 2nd CaCl2 precipitation 1.07 1991.7 88% 3863 3.9 28.3 2nd EtOH precipitation - SN 22.5 2nd EtOH precipitation - PP 1816.4 80% 99.9% 1201.6 3.2 90.9 08.10.2013

Purity data: mAb2 Dilution factor IgG IgG yield Monomer HCP HCP Reduction [µg/ml] step overall [ppm] Supernatant 1.00 1952.9 0% 180099 1st CaCl2 precipitation 1.07 1807.6 99% 66462 2.7 1st EtOH precipitation - SN 1.35 34.8 3% 1st EtOH precipitation - PP 1649.0 89% 88% 31648 2.1 5.7 2nd CaCl2 precipitation 1487.3 100% 13760 2.3 13.1 2nd EtOH precipitation - SN 19.2 2% 2nd EtOH precipitation - PP 1390.9 94% 83% 90% 8276 1.7 21.8 08.10.2013

Purity data: mAb3 Dilution factor IgG IgG yield Monomer HCP HCP Reduction [µg/ml] step overall [ppm] Supernatant 0.00 3322.2 0% 81752 1st CaCl2 precipitation 1.09 2825.6 92% 64212 1.2 1st EtOH precipitation - SN 1.35 7.3 1st EtOH precipitation - PP 1.00 n.a. 2nd CaCl2 precipitation 1.08 2336.6 89% 82% 3863 2.3 17.2 2nd EtOH precipitation - SN 3.3 2nd EtOH precipitation - PP 2162.4 93% 76% 99% 3701 22 48.7 08.10.2013

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 08.10.2013

Polishing by IEX flowthrough Negative purification High pI of therapeutic mAbs exploited Impurities bound (DNA, HCPs), product in flow through Monolith – mass transfer by convection [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 08.10.2013

Purity data: mAb1 mAb1 IgG Yield IgG monomer DNA HCP HCP Reduction   [µg/ml] step overall  [ppm] [ppm] Supernatant 2509 ± 0 2583 ± 0 136424 ± 0 1st CaCl2 2272 ± 28 96% ± 1% 96% ± 1% 30 ± 1 107010 ± 3387 1.3 ± 0.0 1st CEP 2161 ± 41 95% ± 2% 91% ± 2% 166 ± 58 28350 ± 2559 3.8 ± 0.4 4.8 ± 0.5 2nd CaCl2 1845 ± 27 83% ± 1% <LLOQ 6406 ± 801 4.5 ± 0.5 21.5 ± 2.5 2nd CEP 1743 ± 3 79% ± 2% 99.92% ± 0.02% 136 ± 37 1254 ± 182 5.1 ± 0.3 110.3 ± 15.4 DEAE AEX 1715 ± 49 99% ± 1% 78% ± 2% 99.95% ± 0.01% 121 ± 21 80 ± 14 15.7 ± 1.1 1739.2 ± 326.7 08.10.2013

Continuous reactor – Scale up and throughput Diameter [cm] Throughput L/min L/24h kg/24h 1 0.1 136 0.44 2 0.4 543 1.74 5 2.4 3393 10.86 10 9.6 13572 43.43 Assumption: Linear flow rate: 2 cm/s; titer: 4 g/L; yield: 80% Reactor diameter doubled  throughput inceases 4x at constant linear velocity 08.10.2013

Economic evaluation (CoGs) – Gantt charts Classical process: Fed-batch + chromatography Hybrid: Fed-batch + continuous precipitation Processing constraint: 5 days Fully continuous: Perfusion + continuous precipitation 08.10.2013

Economic evaluation – 3 scenarios Assumptions Phase I, II Phase III Very large commercial 4 g/L, 20% batch-failure rate 70% DSP yield 10 kg Resins discarded Multi-product plant 4 g/L, 20% batch failure rate 3 batches at comm. scale 4 g/L, 5% batch failure rate Target production: 500 kg/a 08.10.2013

Economic evaluation – Increasing titer Diameter of pA column: > 2 m Precipitation scales with processed volume, not titer! 08.10.2013

Advantages and challenges of new process Advantages Challenges 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 08.10.2013

Acknowledgments Alois Jungbauer Anne Tscheließnig Ralf Sommer Novartis AG – Bernhard Helk Novartis AG – Henk Schulz 08.10.2013

Questions??? Thank you! 08.10.2013

Stirred tank reactor – Tubular reactor Batch Continuous from Mettler Toledo using built-in probes Self-construction 08.10.2013