“Current Best Practise in Biomanufacturing and thePlatzhalter Bild Critical Role of Innovation” International Vellore Symposium “Bioprocess Industry-Academia Interaction” July 2011 Dr. Uwe Gottschalk, VP Purification Technologies, Sartorius Stedim Biotech
What are the hot Topics? 7th Annual Survey of Biopharmaceutical Manufacturing. Eric S. Langer, BioPlan Associates Inc.
The USDP/DSP Interface in a World of High Titers Data adapted from: F. Wurm Production of recombinant Protein Therapeutics in Cultivated Mammalian Cells. Nature Biotechnology 22, 1-6 (2004)
DSP is Mass not Volume driven Jim Davis, Lonza Economics of Monoclonal Antibody Production: The relationship between upstream titer and downstream costs; IBC San Diego March 2008
Current Best Practise in DSP High Titer Implications: Increasing biomass and contaminant levels Protein A pool volumes and step cost DNA & HCP levels post Capturing Polishing load volumes and conductivity Pathogen clearance as a moving target
Capture Costs: Why bother? Jim Davis, Lonza Economics of Monoclonal Antibody Production: The relationship between upstream titer and downstream costs; IBC San Diego March 2008
Emerging capture technologies expected to have limited market potential inupcoming years Technology Description Maturity Risk Relevance • CIM, BIA Separations, methacrylate based monoliths Monoliths • Similar to membrane adsorbers • Purification of large biomolecules (viruses, plasmid DNA, conjugates), good resolution Expanded • Upfront/DSM work on single use technology for MABs Capture and intermediate purification bed ad- • Already used in depletion of valuable biomolecules from particle • No significant sorption containing feedstreams at large scale (milk, juice, etc.) market in upcoming years expected – • Membrane adsorber technology Potential for Direct Capture • Own development and IP, depletion of valuable biomolecules from Niches (e.g. MA particle containing feedstreams Vaccines, DNA) • Currently low priority, combines cell harvest and capture chromatography step Ligands • instAction, Prometic (mimetic ligands), BAC, GEHC pipeline, Repligen Protein A Precipitation/ • Used at large scale in plasma fractionation (precipitation) Crystallization/ and APIs (crystallization) • No immediate Extractionn • Not developed for mABs commercialization possible • Potential for Affinity • Polybatics disruptive nano- • Disruptive technology technology particles • Single use alternative to Protein A • Platform character Already used In development Start of development Low risk Moderate risk High riskSource: Sartorius
Alternatives to Protein A Capture• Product precipitation batch/continuous• Impurity precipitation (followed by non-Protein A process)• Alternative Capturing (Protein A Mimetics, Mixed Mode, CEX)Issues: Selectivity, Scale up, Reproducibility, Comparability
addresses: Protein A pool volumes and step costD. Low BioManufacturing Paris 2007
Alternative Protein A Chromatography Formats:Goal: Intensified Use/Volume Reduction• Simulated Moving Bed (SMB) and related: » Tarpon („single use flow path“) » Novasep » Chromacon » Chromatan » ... ______________________________________________________________________• Expanded Bed Chromatography » DSM/Upfront („single use flow path“)Issues: Complexity, Scale up, Reproducibility, Comparability
Alternative Protein A Formats:Goal: Low Cost – Real Single Use 2000: Oleosin Platform 2005: TMV Nanoparticles 2010: Bio Polyester Platform Polyester Synthase Polyester Granule Immunoabsorbent nanoparticles based on a 100-300 nm tobacco mosaic virus displaying protein ALimitation: Oleosin yields < 1kg/ha S. Werner et al. PNAS 103, 17678 - 17683 Grage, K. and Rehm, B.H.A. (2008) Bioconj. Chemistry, 19(1):254-62.
Two Birds – one Stone: Contaminant Precipitation at Pfizer and Medarex addresses: Fig 7a. precipitation based process Fig 7b. TFF based process Contaminant precipitation Protein A pool volumes TFF and step cost DNA & HCP levels post HCP < 1000 ng/mg CEX HCP < 10ng/mg CEX Dilution Capturing Dilution Q Membrane Q Membrane HCP < 1000 ng/mg HCP BDL 2 g/ml 20 g/ml Dilution VF Mix Mode VF Process Scale Precipitation of Precipitation of Process-Derived Impurities in Mammalian Cell Culture Impurities in Non-Protein A Broth; J. Glynn et al. In: Gottschalk U Purification Schemes for MAb; J. Wang (ed) Process-scale Purification of et al. BioPharm Intl. 10/2009, 2-9 Antibodies. Wiley, NY.
Selection Guide Convective Media Q,S Low salt Polishing in flowthrough: STIC viruses, DNA, Host ng is hi cell proteins, Pol High salt endotoxins, HIC aggregates Convective Media Cap Purification: large t ure proteins (Factor Q,S VIII), viruses (vaccines), phages...
Sartobind STIC® - Next Generation of Membrane AdsorbersShares same cellulose base membrane as Q: >3 μm pore size0.5 μm Here is the binding capacity Sartobind Q Grafted Quaternary ammonium Sartobind STIC Direct derivatisation Binding capacity + ligand density is distributed more evenly + pore accessibility Primary amine (Sartobind STIC PA) I. Tatárova, I., R. Fáber, R. Denoyel, M. Polakovic, J. Chromatography A 1216 (2009) 941March 7, 2011 Page 9
Host cell protein removal from up 10 kg mAb per L (pH 8, 500 ppm HCP load, 10MV/minApplication Note Sartorius-Stedim Biotech: 85032-540-18, 05/2011
Sartobind STIC Sartobind Q Sartobind STICBinding Capacity (g/m²)BSA 2 mS/cm @ 0 mM NaCl 9 19BSA 20 mS/cm @ 200 mM NaCl 1 10DNA 7 mS/cm @ 50 mM NaCl 2 6Removal of ΦX174 (LRV)LRV 1.4 mS/cm @ 0 mM NaCl 3,7 5,1LRV 6.7 mS/cm @ 50 mM NaCl 0,1 5,1LRV 16.8 mS/cm @ 150 mM NaCl 0,1 4,8Removal of MVM (LRV) at Wuxi ApptecTrial 1 16.8 mS/cm @ 150 mM NaCl 2.10 3,82Trial 2 16.8 mS/cm @ 150 mM NaCl 1.81 >4,96
Source: 2nd Annual Survey of the Bioprocessing Marketfor Single-Use SolutionsAspen Brook Consulting, 2010
Current Challenge in UF • Process efficiency should be HIGH • Membrane cleaning should be EASY • Final Mab concentration may reach 20% • Maximum system pressure is LIMITED E ECO Pumping Requirements 8 L/m2/min 2 L/m2/min Viscosity High Low MAb Concentration >15% <10%
Select the Right Products Flux vs. Concentration for Mab Sartorius ECO & E cassette Crossflow Rate: 360 L/m2-Hr 100 90 ECO 80 eate FLux (LMH) 70 60 50 40 E Perm 30 20 10 0 10 100 1000 Concentration (g/l)