Hydroxocobalamin association during cell culture results in pink therapeutic proteins |
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| Authors: | Kenneth M Prentice Ronald Gillespie Nathan Lewis Kiyoshi Fujimori Rebecca McCoy Julia Bach Lisa Connell-Crowley Catherine M Eakin |
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| Institution: | 1.Department of Analytical Sciences; Amgen Inc; Seattle, WA USA;2.Department of Purification Process Development; Amgen Inc; Seattle, WA USA;3.Department of Drug Product Development; Amgen Inc; Thousand Oaks, CA USA;4.Department of Cell Sciences and Technology; Amgen Inc; Seattle, WA USA |
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| Abstract: | Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing. |
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| Keywords: | vitamin B12 cobalamin color monoclonal antibody process control |
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