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. 2020 Nov 30:1159:122400.
doi: 10.1016/j.jchromb.2020.122400. Epub 2020 Oct 13.

An improved protocol for large scale production of high purity 'Fc' fragment of human immunoglobulin G (IgG-Fc)

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An improved protocol for large scale production of high purity 'Fc' fragment of human immunoglobulin G (IgG-Fc)

Jatin B Tandale et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

Abstract

We describe a simplified approach for purification and characterization of human 'IgG-Fc' fragment used widely as immunochemical tool and for therapeutic purposes. The 'Fc' fragment was purified from human IgG in a 3-stage column chromatography. The purified 'Fc' fragment appeared as a dimer glycoprotein with an apparent molecular mass of 52,981 Dalton (Ultraflex MALDI TOF/TOF). The Size-exclusion HPLC profile of the purified 'Fc' fragment of human IgG matched that of a commercially procured reference 'Fc' fragment material. The purity of the 'Fc' fragments was >99% by SDS-PAGE and size-exclusion HPLC. The results of Western blotting, immunoelectrophoresis, and mass spectrometry analysis indicate a high purity of the 'Fc' fragment. Peptide mass fingerprint analysis of the purified 'Fc' protein yielded peptides that partially match the known database sequences of FCG3B_HUMAN (Uniprot ID: O75015). This method of purification of the 'Fc' fragment is suitable for achieving high purity level of 'Fc' fragment protein. With this purification approach, the cost of the purified 'Fc' fragment of human IgG is significantly reduced as compared with the current market price of IgG-Fc fragment protein in international market. The purified 'IgG-Fc' fragment protein was found to be negative for major viral markers.

Keywords: Human IgG; Human proteins; Papain digestion; Purification; ‘Fab’ fragment; ‘Fc’ fragment.

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