In situ PEGylation of recombinant hirudin on an anion exchange chromatography column |
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| Affiliation: | 1. School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China;2. Department of Biomedical Engineering, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China;3. College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian, 116044, PR China;4. State Key Laboratory Cultivating Base for Long-acting Bio-medical Research of Jiangsu Province, Jiangsu Hansoh Pharmaceutical Group CO., LTD, Lianyunguang, 222000, PR China;1. School of Chemistry and Molecular Bioscience, University of Wollongong, Northfields Avenue, Wollongong 2522, Australia;2. Research School of Chemistry, Australian National University, Canberra 2600, Australia;3. Mark Wainwright Analytical Centre, University of New South Wales, Kensington 2052, Australia;1. Product Development, Apobiologix, 4100 Weston Road, Toronto, Ontario M9L 2Y6, Canada;2. Medical Affairs US, Apobiologix, 2400 N. Commerce Parkway, Suite 400, Weston, FL 33326, USA;1. Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran;2. AryaTinaGene Biopharmaceutical Company, Gorgan, Iran |
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| Abstract: | In this study, an integrated process was developed for successive solid-phase PEGylation of recombinant hirudin variant-2 (HV2) and separation of PEGylated HV2 species on an anion exchange chromatography column (so-called in situ PEGylation). The effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were investigated. The results showed that in situ PEGylation efficiently integrates the reaction, separation and purification into a single-unit operation using the same column. In situ PEGylation could improve the selectivity of PEGylation reactions by significantly reducing the formation of multi-PEG-HV2. The pore sizes and internal surface structures of different resins had a significant impact on the yield of mono-PEG-HV2. In contrast to liquid-phase PEGylation, the yield of mono-PEG-HV2 decreased as PEG size increased during the in situ PEGylation process, indicating that in situ PEGylation is a pore diffusion-controlled process. The in vitro and in vivo anticoagulant activities of mono-PEG-HV2 derived from in situ PEGylation were higher than those from liquid-phase PEGylation, indicating that in situ PEGylation could enhance the bioactivity retention of mono-PEG-HV2. The results of this study demonstrated that in situ PEGylation can be used as an effective approach for the development of PEGylated protein drugs. |
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| Keywords: | Recombinant hirudin PEGylation Ion exchange chromatography Solid-phase PEGylation On-column PEGylation |
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