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J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Aug 15;965:72-8. doi: 10.1016/j.jchromb.2014.06.011. Epub 2014 Jun 19.

Protein A affinity precipitation of human immunoglobulin G.

Author information

1
Bioseparation Engineering Group, Technische Universität München, 85748 Garching, Germany.
2
Bioseparation Engineering Group, Technische Universität München, 85748 Garching, Germany. Electronic address: s.berensmeier@tum.de.

Abstract

The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit(®) S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1°C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes.

KEYWORDS:

Affinity precipitation; Antibody purification; Eudragit; Immunoglobulin G; protein A

PMID:
24999247
DOI:
10.1016/j.jchromb.2014.06.011
[Indexed for MEDLINE]

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