Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris |
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Authors: | Jiang Youwei Li Fang Zha Dongxing Potgieter Thomas I Mitchell Teresa Moore Renée Cukan Michael Houston-Cummings Nga Rewa Nylen Adam Drummond James E McKelvey Troy W d'Anjou Marc Stadheim Terrance A Sethuraman Natarajan Li Huijuan |
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Institution: | GlycoFi Inc., A Wholly-Owned Subsidiary of Merck & Co. Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA. |
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Abstract: | A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity. |
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