High Throughput Screening Technologies for developing purification processes of proteins Michel Eppink, Synthon BV, Nijmegen
Introduction Overview From small to large processes New protein purification technologies Resin Screening Studies Resin Scouting Studies Case Studies I + II Conclusions
Introduction
Why Purification of Proteins? Development of robust purification processes (DSP) for therapeutic proteins is essential Time to market for DSP development saves money (DSP is a costly process) Reduce the amount of column/filtration steps and increase the overall yield Diminish the amount of impurities such as host cell related proteins
First Recombinant Proteins Insulin (first recombinant product commercialized 1983) Granulocyte Colony Stimulating Factor (GCSF) Gonadotrophins (HCG, FSH) Interferon α/β Interleukins Growth Hormone Erythropoetin (EPO)
Monoclonal Antibodies Large biomolecules (150 kd) Contain both heavy (50 kd) and light (25 kd) chain Expression mainly in eucaryotic cell lines (NS0, PerC6, CHO, HEK or other cell lines) Mainly glycosylation at the C H chains Activity determined by F ab region
Principle of a Chromatographic Step Ligand: -Ionic -Hydrophobic -Affinity Column with Resin Sample Chromatogram mau BKA0023 IMAC003 :10_ UV1_ 280nm BKA0023 IMAC003 :10_ Logbook 40 00 Equilibration Flow through Wash Elution 30 00 20 00 10 00 sample injection Flow through Start w ashing Elution Wash Wash 2 Wash 3 Start elution strip 1M NaOH 0 30 0 40 0 50 0 600 m l
Development DSP processes in early years Batch wise resin screening in small tubes Process Development with in house equipment
From Small to Large Processes
Small Large Experimental space
From small to large Miniaturization Scouting Studies Research Scale Development Scale Large Scale 0.01 0.2 ml 0.05-10 ml 200-300 ml 1-10 % (1-10 liter) 100% (10-500 liter)
New Purification Technologies
Robotic Handling System Small scale purification in microtiterplate and/or minicolumn format Automatic handling of the samples (Fast) assay analysis (EIA, HPLC, UV, Protein, etc.) Parallel testing of different purification conditions (ph, salt, additives, organic solvents, resins, etc.) Resin screening/scouting studies can be performed in a reproducible way
Freedom EVO Protein Chromatography Workstation Freedom EVO 100 LiHa / RoMa Tecan Reader optional, for result analysis Hotels/ carriers sample and buffer preparation Te-Chrom Te-Stack optional, for collection of fractions
Resin Screening Studies
Resin Screening Studies I Essential part of the screening studies is the pipetting robot Pipet chromatographic resin with high reproducibility from a 12-well plate Into a 96-well filter plate (in the near future maybe a 386- well plate would probably be feasible?) Analysis with UV/VIS Reader
Resin Screening Studies II Robot Liquid Handling Batchwise chromatography (96 experiments /day) Titer (ELISA/UPLC) HCP (ELISA) Protein UV CE MS (MALDI/ESI) SDS-PAGE Centrifugation/Vacuum filtration Screening of Resins Low/medium resolution Determination of DBC No flow/bedheight properties map
Case Study I
Case Study I Cell supernatant of a CHO cell line contains a glycoprotein with a molecular mass of approx. 30-40 kd (heterodimer) Development intermediate step with HIC resins (screening/scouting) HTS occurs with robotic system Detection is performed by UV, protein and/or product specific tests
Case Study I Resins screened Elution profile s with different at (NH salt 4 ) concentrations 2 SO 4 concentrations
Resin Scouting Studies
Atoll RoboColumns Pre-packed with the resin of choice per each row Bed heights: 2.5, 5, 10, 30 mm; inner diameter: 5 mm Column volumes: 50, 100, 200, 600 µl
Freedom EVO Protein Chromatography Workstation Sample loading Collection of fractions
Workflow in High Throughput Chromatography Sample preparation/ clarification Preparation of Elution Buffers Column equilibration Sample Loading Elute with Buffer X Elute with Buffer Y Collect Fractions Analysis (Tecan (TECAN Reader) Reader)
Case Study II
Case Study II Comparison of chromatograms Common LC instrumentation vs. RoboColumns processed on Freedom EVO Example study: Separation of 2 proteins Separation of lysozyme and cytochrome c on a cation exchange column, using a step gradient of 0,1 M or 1 M NaCl respectively
Case Study II Chromatograms for the separation of two proteins (lysozyme, cytochrome c (each 1 mg/ml) on cation exchangers packed in 200µl Atoll columns for 8 RoboColumns using 0.1 M NaCl or 1 M NaCl respectively, processed on a common LC instrumentation or Freedom EVO respectively xe+3 OD 280 mau nm [mau] 2 1 Shift due to lower dead volume 0 0 5 10 15 (min) Time [min] Chromatograms from a common LC instrumentation and Freedom EVO are identical Extremely high reproducibility from column to column Data kindly provided by TimSchroeder, Atoll, Germany
Platform Technology for DSP processes Pipetting robot studies Selected resin(s) Robot/Column Scouting studies Selected resin Robot/Column optimization studies ph, buffer, salt, organic solution screening ph, buffer, salt, organic solution screening Parameter screening (Experimental Design) M.H.M. Eppink (2007). Biopharm International, 20, 3, 44-50. M.H.M. Eppink (2009). Biopharm International, Mar 2, Supplement
Conclusions Robot handling equipment important for Downstream Processing (DSP) Fast performance of purification processes Large screening window Small amount of product needed
Acknowledgement TU Delft Marcel Ottens Synthon Guy de Roo Kim Burgers Xiaonan Li Meng Liu TECAN Marc Brus Dennis Bertens Dirk de Regt Atoll Jurgen Friedle Tim Schroeder Kalsruhe Institute of Technology Juergen Hubbuch