Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids

Michael W. Handlogten, Jared F. Stefanick, Nathan Alves, Basar Bilgicer

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

Original languageEnglish (US)
Pages (from-to)5271-5278
Number of pages8
JournalAnalytical Chemistry
Volume85
Issue number10
DOIs
StatePublished - May 21 2013
Externally publishedYes

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Purification
Fluids
Antibodies
Pharmaceutical Preparations
Haptens
Affinity chromatography
Ammonium Sulfate
Conditioned Culture Medium
Immunoglobulins
Proteins
Salts
Monoclonal Antibodies
Cells
Impurities
Membranes

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids. / Handlogten, Michael W.; Stefanick, Jared F.; Alves, Nathan; Bilgicer, Basar.

In: Analytical Chemistry, Vol. 85, No. 10, 21.05.2013, p. 5271-5278.

Research output: Contribution to journalArticle

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