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High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling

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Abstract

Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.

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Abbreviations

CDR:

complementary determining regions

CQA:

critical quality attributes

FQ:

3-(2-furoyl)quinoline-2-carboxaldehyde

ICH:

International Conference of Harmonization

IAM:

iodoacetamide

LIF:

laser-induced fluorescence

LED-IF:

LED-induced fluorescence

LED:

light-emitting diode

mAb:

monoclonal antibody

mCGE:

multicapillary gel electrophoresis

NGHC:

non-glycosylated heavy chain

PNGase F:

peptide N-glycosidase F

QA:

quality analysis

QC:

quality control

SDS:

sodium dodecyl sulfate

pre-LC:

truncated light chain

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Acknowledgments

This research was supported by the OTKA grant # K-81839 of the Hungarian Research Council. This work was also partially supported by the National Office for Research and Technology of Hungary (TECH-09-A1-2009-0113; mABCHIC). The kind gift of the multi-capillary electrophoresis unit from QIAGEN is also greatly appreciated.

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Authors and Affiliations

  1. Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary, 4032

    Ákos Szekrényes, Márta Kerékgyártó, Andrea Székely & András Guttman

  2. QIAGEN GmbH, 40724, Hilden, Germany

    Udo Roth & Karen Kowalewski

  3. Biosystems International Kft, 4032, Debrecen, Hungary

    István Kurucz

Authors
  1. Ákos Szekrényes
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  2. Udo Roth
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  3. Márta Kerékgyártó
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  4. Andrea Székely
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  5. István Kurucz
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  6. Karen Kowalewski
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  7. András Guttman
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Correspondence to András Guttman.

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Szekrényes, Á., Roth, U., Kerékgyártó, M. et al. High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling. Anal Bioanal Chem 404, 1485–1494 (2012). https://doi.org/10.1007/s00216-012-6213-2

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  • DOI: https://doi.org/10.1007/s00216-012-6213-2

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