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Fed-batch or perfusion for the production of biopharmaceuticals by animal cell cultivation? Veronique Chotteau, PhD., Cell Technology group, KTH, School of Biotechnology. [email protected] Workshop on Industrial scale cultivation of cells in pharmaceutical or antibody production systems April 23 2009, Royal Institute of Technology, Stockholm, Sweden Mode of operations Batch bioreactor medium & additives addition during the cultivation O2 tension Fed-batch pH From 10 to 21 days temperature Typically 5 days Batch-refeed = easy option Perfusion continuous medium renewal during the cultivation cell broth cell-free supernatant cells separation device harvest collection tank/bag From 3 weeks to 6 months System with internal separation device Perfusion devices in processes using stirred tank reactors - Filtration fresh medium Spin filter cell-free supernatant LEVEL CONTROL QUI CK CONNECT FLUI D I NLET ADDI TI ON PUMP Alternating tangential flow (ATF) VALVE FI LTRATE PUMP FI LTRATE HF MODULE OR SCREEN MODULE HOUSI NG LI QUI D LEVEL CONTROLLER EXHAU PROCESS VESSEL DI APHRAGM I ON OFF ATF PUMP FI LTER STAND Hollow fibre etc,… Sources: Refine, Microgon Perfusion devices in processes using stirred tank reactors - Centrifugation Centritech centrifuge Cell-free supernatant (to harvest tank) Feed = cell broth (from bioreactor) Concentrated cells (to bioreactor) Cell-free supernatant (to harvest tank) Feed = cell broth (from bioreactor) Concentrated cells (to bioreactor) Westfalia centrifuge Sources: Kendro, Westfalia Perfusion devices in processes using stirred tank reactors - Sedimentation fresh medium cell-free supernatant cell sedimentation Inclined settler Sedimentation by gravity Sources: Applisens Acoustic settler Forced sedimentation by acoustic wave Comparison of several perfusion systems Properties Spin filter ATF Hollow fibre Centritech Acoustic settler Inclined settler Fouling (or poor operation) -- + -- + + - Simplicity of operation + ++ ++ - + + ++ ++ - - -- ++ ++ + ++ + + - Easiness to optimise the operation parameters Easiness to obtain and maintain sterility ++ Scalability ++ Possibility for re-sterilisation yes if external yes yes yes yes if external yes if external Residence time of cells in separator and connection tubing 0 if internal 1–2 min 10 sec. 2 – 9 min 3 – 14 min 10 – 20 min (?) Running cost + - - - + + Purchase cost + + + - + + Characteristics of fed-batch and perfusion principles Fed-batch Perfusion • Continuous medium addition • • Addition of selected components • Continuous medium addition and removal of used medium Addition of complete medium energy source (e.g. glucose, glutamine), amino acids, vitamins, salts, metal trace, growth factors • Removal of the by-products, etc. – e.g. toxic lactate, ammonia, CO2 – protease • Dilution of the by-products, etc. e.g. toxic lactate, ammonia • Changing environment for the cells • Constant environment for the cells • Environment of lower stability of the product of interest • Environment of higher stability of the product of interest • Alkali addition • Less alkali addition for pH control Implications of the characteristics of fed-batch and perfusion at early clinical phase development Fed-batch Perfusion • Continuous medium addition • • Addition of selected components • Need to develop the feed strategy, e.g. feed medium, feed rate, … Continuous medium addition and removal of used medium Addition of complete medium Require only the selection of a medium ‘good’ enough • Dilution of the by-products, etc. presence of by-products • Removal of the by-products, etc. • Changing environment for the cells • Constant environment for the cells • Environment of lower stability of the product of interest • Environment of higher stability of the product of interest • Alkali addition • Less alkali addition for pH control Consequences of fed-batch and perfusion techniques Fed-batch Perfusion • Accumulation of the product of interest • Continuous dilution of the product of interest lower concentration • • Single harvest Smaller harvest volume • • Multiple harvests variation? In total larger volume of harvest increase work load of down-stream • Larger bioreactor (up to 20000 L) less available, less ‘convenient’ • Smaller bioreactor (up to 500 or 1000 L) more available, even disposable • • Technically less complex Technically more complex – higher risk for failure – higher risk for contamination – requires perfusion device knowhow Perfusion in 2 L bioreactor with ATF (Karin Tördahl, Véronique Chotteau) - The first two runs Medium sponsorin g IrvineScie ntific, US, CA Collaborati on & cartridge sponsorin g, GE Healthcare Selection of perfusion or fed-batch in biopharmaceutical industry Obvious selection of perfusion if • Instable protein – production of proteases by the cells, e.g. serine protease, metalloprotease – physical instability at cultivation pH and temperature • Product of interest toxic for the cells Selection of perfusion or fed-batch in biopharmaceutical industry • Preference for fed-batch – High titer and high cell density – Simplicity (technical) of fed-batch process – Lower contamination & failure risk – Dominance of antibodies stable proteins – Usage of technical platforms with fed-batch process – Large companies availability of large bioreactors 3 perfusion, 6 batch/fed-batch, 4 unknown process in ‘Table 2: Numerical summary of BLA product generated in mammalian cell-culture systems 1996-2000’, Chu & D.K. Robinson, Curr. Opinion in Biotechn. 2001, 12:180. • Selection of perfusion when – Know-how present in company – Previous experience of product – Equipment adapted for perfusion – Desire to use smaller bioreactors Simulation of a comparison of fed-batch and perfusion – case of cell specific productivity = 5 pg/cell/day Simulation of a comparison of fed-batch and perfusion – case of cell specific productivity = 5 pg/cell/day Simulation of a comparison of fed-batch and perfusion – case of cell specific productivity = 5 pg/cell/day Simulation of the product mass in a 1 L bioreactor in fed-batch or perfusion as a function of the cell specific productivity Notice: Konstantinov (2006) cell specific productivity 20 to 60 pg/cell /day (Bayer), Wurm (2004) cell specific productivity 20 pg/cell /day High viability & constant environment favorable for protein Example of fed-batch (Wurm 2004 cited ‘courtesy of Lonza’) Example of perfusion (Konstantinov 2006 perfsuion rate from 6 RV/day 2 RV/day) Basic development for simple fed-batch or perfusion processes • Assumptions: small company, cell producing 5 pg/cell/day, ‘simple’ protein • Purpose: process ‘good enough’ for production of phase I material (up-scale at 10 L bioreactor) • Rough estimate of USP & DSP development cost • Conclusions • Perfusion – best knowledge within big/middle biotech/big pharma – very attractive due to smaller bioreactor volumes and disposable bioreactors (up to 1000 L) • Perfusion can be an interesting alternative for small companies – not higher cost – favorable for proteins • Requires perfusion device – simple – robust – scalable – affordable cost ATF Questions?