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

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

8 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sample stacking is of vital importance for analytical CE since it may bring the required sensitivity of analyses. A lot of new relevant papers are published every year and regular surveys seem to be very helpful for experts and practitioners. The contribution presented here is a continuation of a series of regularly published reviews on the topic and covers the last two years. It brings a survey of related literature organized, in accord with the main principle used in the procedure published, in the following mainstream sections: Kohlrausch adjustment of concentrations, pH step, micellar systems and combined techniques. Each part covers literature sorted according to the field of application as, e.g. clinical, pharmaceutical, food, environmental, etc.
    Electrophoresis 01/2011; 32(1):116-26. DOI:10.1002/elps.201000327 · 3.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Capillary electrophoresis has been alive for over 2 decades now, yet its sensitivity is still regarded as being inferior to that of more traditional methods of separation such as high performance liquid chromatography. As such, it is unsurprising that overcoming this issue still generates much scientific interest. This review continues to update this series of reviews, first published in Electrophoresis in 2007, with updates published in 2009 and 2011 and covers material published through to June 2012. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.
    Electrophoresis 01/2013; 34(1). DOI:10.1002/elps.201200396 · 3.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An orthogonal testing protocol was developed and validated to assess the quality of Filgrastim biosimilars. Results were compared to those obtained from the innovator product. Initial screening was carried out using reducing and non-reducing gel electrophoresis. RP-LC was employed for the determination of Filgrastim in the presence of its oxidative degradation products. SEC and CIEF were used under non-denaturing conditions to reveal high molecular weight and charged impurities, respectively. RP-LC assay was found accurate (99.78±0.89) and precise over a linear concentration range of 9.38-300.00μg/ml with a LOD of 8.26μg/ml (0.44mM). SEC was carried out over a molecular weight range of 5.0-150.0kDa. CIEF was optimized using neutrally coated capillaries over a wide-range pH gradient (pH 3.0-10.0). Differences between the studied products were revealed using all these techniques. Impurities above the acceptable limits were detected in both biosimilar products. CIEF revealed heterogeneity in the active ingredient that has not been investigated by the manufacturers. Correlation of the obtained results indicated the presence of not only product-related impurities, but also process-related impurities. Results confirmed the need for in-house validated orthogonal testing protocols to be developed by local regulatory authorities. This should prevent access of substandard biosimilars to price-sensitive markets.
    Journal of pharmaceutical and biomedical analysis 04/2014; 97C:72-80. DOI:10.1016/j.jpba.2014.04.019 · 2.98 Impact Factor
Show more