Large-scale electrophoresis for protein purification: exploiting isoelectricity |
| |
| Institution: | 1. Janssen R&D, DPDS BTDS Analytical Development, Hochstr. 201, 8200 Schaffhausen, Switzerland;2. Analytical Development, Biogen, Morrisville, NC 27709, USA;3. UCB, Analytical Development Sciences for Biologicals, Chemin du Foriest, 1420 Braine-l''Alleud, Belgium;4. Boehringer Ingelheim Pharma GmbH & Co KG, Innovation Unit, Birkendorfer Str. 65, 88397 Biberach an der Riss, Germany;5. Analytical Research and Development, NBE Analytical R&D, AbbVie Deutschland GmbH& Co. KG, Knollstraße, 67061 Ludwigshafen, Germany;6. GlaxoSmithKline, 1250 S Collegeville Rd, Collegeville, PA 19426, USA;7. Merck Serono SpA, Guidonia Montecelio, Italy, an affiliate of Merck KGaA, Darmstadt, Germany;8. Pfizer, Inc. Biotherapeutics Pharmaceutical Sciences, Analytical Research and Development 875 W. Chesterfield Parkway, Chesterfield, MO 63017, USA;9. Novartis Pharma AG, TRD Biologics & CGT, GDD, 4002 Basel, Switzerland;10. Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2, Penzberg, 82377, Germany;11. Lonza AG, Drug Product Services, Hochbergerstr. 60G, CH-4057 Basel, Switzerland;12. Byondis B.V., Downstream Processing, Nijmegen, the Netherlands;13. Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA;14. Process Development, Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA;15. Bayer AG, Product Supply, Analytical Development and Clinical QC for Biotech Products, Friedrich-Ebert-Str. 217-233, 42117 Wuppertal, Germany;p. Sanofi R&D, Biologics Drug Products Development,13 quai Jules Guesde, 94403 Vitry-sur Seine, France;1. Janssen R&D, DPDS BTDS Analytical Development, Hochstr. 201, 8200 Schaffhausen, Switzerland;2. Analytical Development, Biogen, Morrisville, NC 27709, USA;3. UCB, Analytical Development Sciences for Biologicals, Chemin du Foriest, 1420 Braine-l''Alleud, Belgium;4. Boehringer Ingelheim Pharma GmbH & Co KG, Innovation Unit, Birkendorfer Str. 65, 88397 Biberach an der Riss, Germany;5. Analytical Research and Development, NBE Analytical R&D, AbbVie Deutschland GmbH& Co. KG, Knollstraße, 67061 Ludwigshafen, Germany;6. GlaxoSmithKline, 1250 S Collegeville Rd, Collegeville, PA 19426, USA;7. Merck Serono SpA, Guidonia Montecelio, Italy, an affiliate of Merck KGaA, Darmstadt, Germany;8. Pfizer, Inc. Biotherapeutics Pharmaceutical Sciences, Analytical Research and Development 875 W. Chesterfield Parkway, Chesterfield, MO 63017, USA;9. Novartis Pharma AG, TRD Biologics & CGT, GDD, 4002 Basel, Switzerland;10. Pharma Technical Development Analytics, Roche Diagnostics GmbH, Nonnenwald 2, Penzberg, 82377, Germany;11. Lonza AG, Drug Product Services, Hochbergerstr. 60G, CH-4057 Basel, Switzerland;12. Byondis B.V., Downstream Processing, Nijmegen, the Netherlands;13. Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA;14. Process Development, Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA;15. Bayer AG, Product Supply, Analytical Development and Clinical QC for Biotech Products, Friedrich-Ebert-Str. 217-233, 42117 Wuppertal, Germany;p. Sanofi R&D, Biologics Drug Products Development,13 quai Jules Guesde, 94403 Vitry-sur Seine, France |
| |
| Abstract: | Preparative electrophoresis methods (including isoelectric focusing in immobilized pH gradients) in gel phases are characterized by low loadings barely a few mg protein per ml matrix), low recoveries (rarely exceeding 70%), and heavy contamination from neurotoxic gel materials (the unreacted gel monomers and ungrafted oligomers). These drawbacks can be minimized by a version of isoelectric focusing in which the need for protein of interest to pass the gel is eliminated: only the contaminants traverse the gel. This is achieved by circulating a liquid sample between two gels held at controlled pHs. The method can provide: (1) high rate of sample processing (up to 1 g h?1); (2) high purification (in general to charge homogeneity); and (3) high recoveries (>95%). A large-scale membrane apparatus has been built, with a cross- sectional diameter of 9 cm. Large Pt electrode disks provide even current flow. In this electrolyser, 10 g of Eglin C (produced by recombinant DNA technology) have been purified to homogeneity in around 10 h from 1 l of a partially enriched preparation. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
