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

Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562 Egypt; Biotechnology Centre, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Chromatographia (Impact Factor: 1.44). 73(11):1145-1153. DOI: 10.1007/s10337-011-2003-9

ABSTRACT 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 0.26μgmL−1 (3.64 nM BSA). A linear relationship between protein concentration and integrated peak area was obtained over a wide concentration
range (8.49–135.87μgmL−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.

KeywordsCapillary zone electrophoresis–Large-volume sample stacking–Polyethylene oxide–Biopharmaceuticals–Human myelin basic protein

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