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

Content Provider	Springer Nature Link
Author	Al Ghobashy, Medhat A. Williams, Martin A. K. Laible, Götz Harding, David R. K.
Copyright Year	2011
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 0.26 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.
Starting Page	1145
Ending Page	1153
Page Count	9
File Format	PDF
ISSN	00095893
Journal	Chromatographia
Volume Number	73
Issue Number	11-12
e-ISSN	16121112
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-03-22
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Capillary zone electrophoresis Large-volume sample stacking Polyethylene oxide Biopharmaceuticals Human myelin basic protein Analytical Chemistry Biochemistry Plant Sciences Proteomics Pharmacy Organic Chemistry
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Analytical Chemistry Biochemistry Clinical Biochemistry

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