Quantitative biopharmaceutical applications of capillary electrophoresis

Various modes of capillary electrophoresis offer numerous possibilities for biopharmaceutical analysis. Chapter 2 studied capillary zone electrophoresis (CZE) for characterization of glycoprotein erythropoietin by its isoform distribution. The CZE method for identification of recombinant human erythropoietin described in the European Pharmacopoeia has shown poor reproducibility. In this study, it was found that the root cause of the irreproducibility was due to inadequate capillary conditioning. The method was optimized to make it more robust and suitable for quality control laboratory for analysis of epoetin alfa. Further, a procedure for removal of polysorbate 80 from formulated epoetin alfa was developed, allowing the material to be analyzed using the modified CZE method.;Chapter 3 describes a micellar electrokinetic chromatography (MEKC) method for the determination of residual cell culture media components including folic acid, hypoxanthine, mycophenolic acid, nicotinic acid, riboflavin, and xanthine. Acceptable limits of detection and quantitation were obtained. The MEKC method was compared to the corresponding CZE method using the same running buffer containing no sodium dodecyl sulfate (SDS). The effect of SDS concentration on separation, the pH of the running buffer, and the detection wavelength were studied and optimal MEKC conditions were established.;Chapter 4 describes a capillary SDS gel electrophoresis method for purity and impurity analysis of monoclonal antibodies. The study investigated the effect of sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide, and reduction conditions with 2-mercaptoethanol. It was observed that the sample buffer at slight acidic conditions (pH 5.5-6.5) greatly decreased thermally induced fragmentation of non-reduced CNTO3157. As such, a citrate-phosphate buffer at pH 6.5 was used for sample preparation to replace the commonly used Beckman sample buffer (pH 9.0).;Chapter 5 details an investigation of the effect of sample injection length on efficiency and detection sensitivity enhancement in MEKC using the sweeping technique under conditions of suppressed electroosmotic flow. The relationship between injection length and retention factor was derived and experimentally confirmed. A method for predicting the optimal injection length for a given analyte was developed.