CZE with On-line Micellar Sample Stacking for Determination of Protein Concentration of Biopharmaceuticals

Biotechnology Centre, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Chromatographia (Impact Factor: 1.41). 06/2011; 73(11):1145-1153. DOI: 10.1007/s10337-011-2003-9


A capillary zone electrophoresis total protein assay was developed and validated in polyethylene oxide (PEO) dynamically coated capillaries. On-line large-volume sample stacking was employed. Protein samples were denatured using SDS and then injected into PEO-filled capillaries. Such treatment enabled injection of a sample volume of ≈8% of the total capillary volume and stacking of protein-SDS molecules at the interface between the sample plug and the PEO plug. Results showed that SDS enhanced the sensitivity not only by protein denaturation but also by forming micelles, in which protein-SDS partitioned. Sensitivity of the method was further enhanced through using capillaries with (tenfold) extended detection pathlength. Such strategies resulted in a limit of detection of 0.26 μg mL−1 (3.64 nM BSA). A linear relationship between protein concentration and integrated peak area was obtained over a wide concentration range (8.49–135.87 μg mL−1—R
2 = 0.995). The method is particularly useful for determination of total protein concentration in chromatography fractions. It overcomes low UV absorptivity of proteins, presence of UV absorbing additives and high salt content. Contrary to conventional methods for determination of protein concentration, this method does not involve an interaction with a dye. Thus, variations due to differences in surface properties among proteins or due to differences in posttranslational modifications of the same protein are eliminated. The protocol was successfully applied for the determination of the concentration of a biopharmaceutical protein rhMBP in chromatography fractions. This protein has been previously produced in milk of transgenic cows and several charge isoforms were detected.

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