Design of experiments for micellar electrokinetic chromatography method development for the monitoring of water-soluble vitamins in cell culture medium.

Biopharmaceutical production takes place in complex processes which should be thoroughly understood. Therefore, the iConsensus project focuses on developing a monitoring platform integrating several process analytical technology tools for integrated, automated monitoring of the biopharmaceutical process. Water-soluble vitamin monitoring using (microchip) capillary electrophoresis (CE) is part of this platform. This work comprises the development of conventional CE methods as the first part towards integrated vitamin monitoring. The vitamins were divided based on their physical-chemical properties to develop two robust methods. Previously, a method for the analysis of cationic vitamins (pyridoxine, pyridoxal, pyridoxamine, thiamine and nicotinamide) in cell culture medium was developed. This work focused on the development of a micellar electrokinetic chromatography method for anionic and neutral vitamins (riboflavin, d-calcium pantothenate, biotin, folic acid, cyanocobalamin and ascorbic acid). By employing multivariate design of experiments, the background electrolyte (BGE) could be optimised within one experiment testing only 11 BGEs. The optimised BGE conditions were 200 mM borate with 77 mM sodium dodecyl sulphate at a pH of 8.6. Using this BGE, all above-mentioned cationic, anionic and neutral vitamins could be separated in clean samples. In cell culture medium, most anionic and neutral vitamins could be separated. Combining the two methods allows for analysis of cationic, anionic and neutral vitamins in cell culture medium samples. The next step towards integrated vitamin monitoring includes transfer to microchip CE. Due to the lack of fast and reliable methods for vitamin monitoring, the developed capillary methods could be valuable as stand-alone at-line process analytical technology solutions as well.