Hydroxocobalamin association during cell culture results in pink therapeutic proteins.
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.
Department of Analytical Sciences; Amgen Inc; Seattle, WA USA.
Kenneth M
KM
Prentice
Department of Purification Process Development; Amgen Inc; Seattle, WA USA.
Ronald
R
Gillespie
Department of Analytical Sciences; Amgen Inc; Seattle, WA USA.
Nathan
N
Lewis
Department of Drug Product Development; Amgen Inc; Thousand Oaks, CA USA.
Kiyoshi
K
Fujimori
Department of Cell Sciences and Technology; Amgen Inc; Seattle, WA USA.
Rebecca
R
McCoy
Department of Purification Process Development; Amgen Inc; Seattle, WA USA.
Julia
J
Bach
Department of Purification Process Development; Amgen Inc; Seattle, WA USA.
Lisa
L
Connell-Crowley
Department of Analytical Sciences; Amgen Inc; Seattle, WA USA.
Catherine M
CM
Eakin
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D018929:Cell Culture Techniques; D002623:Chemistry Techniques, Analytical; D003116:Color; D003124:Colorimetry; D003412:Cricetulus; D006879:Hydroxocobalamin / Q000032:analysis* / Q000737:chemistry*; D008027:Light; D015394:Molecular Structure; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D011506:Proteins; D006879:Hydroxocobalamin
cobalamin; color; monoclonal antibody; process control; vitamin B12