caa a22 4500 445802642 CHVBK 20180317145146.0 cr unu---uuuuu 170323e20110601xx s 000 0 eng 10.1007/s10337-011-2003-9 doi (NATIONALLICENCE)springer-10.1007/s10337-011-2003-9 CZE with On-line Micellar Sample Stacking for Determination of Protein Concentration of Biopharmaceuticals [Elektronische Daten] [Medhat Al-Ghobashy, Martin Williams, Götz Laible, David Harding] 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. Springer-Verlag, 2011 Capillary zone electrophoresis nationallicence Large-volume sample stacking nationallicence Polyethylene oxide nationallicence Biopharmaceuticals nationallicence Human myelin basic protein nationallicence Al-Ghobashy Medhat Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, 11562, Cairo, Egypt aut Williams Martin Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand aut Laible Götz Agresearch, Ruakura Research Centre, Hamilton, New Zealand aut Harding David Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand aut Chromatographia Springer-Verlag 73/11-12(2011-06-01), 1145-1153 0009-5893 73:11-12<1145 2011 73 10337 https://doi.org/10.1007/s10337-011-2003-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10337-011-2003-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Al-Ghobashy Medhat Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, 11562, Cairo, Egypt aut NATIONALLICENCE 700 1- Williams Martin Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand aut NATIONALLICENCE 700 1- Laible Götz Agresearch, Ruakura Research Centre, Hamilton, New Zealand aut NATIONALLICENCE 700 1- Harding David Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand aut NATIONALLICENCE 773 0- Chromatographia Springer-Verlag 73/11-12(2011-06-01), 1145-1153 0009-5893 73:11-12<1145 2011 73 10337 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer