Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1340, Issue -, Pages 68-78Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2014.03.010
Keywords
Chromatin; IgG purification; Protein A; Capacity; Turbidity; Aggregates
Funding
- Exploit Technologies Pte. Ltd.
- Validated Biosystems
- Biomedical Research Council of the Singapore Agency for Science and Technology Research
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Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA + histone proteins), nonhistone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A. Formation of turbidity in eluted IgG during pH titration is caused by neutral-pH insolubility of chromatin hetero-aggregates. NaOH is required at 500 mM to remove accumulated chromatin. A chromatin-directed clarification method removed 99% of histones, 90% of non-histone proteins, achieved a 6 log reduction of DNA, 4 log reduction of lipid-enveloped virus, and 5 log reduction of non-enveloped retrovirus, while conserving 98% of the native IgG. This suspended most of performance compromises imposed on protein A. IgG binding capacity increased 20%. Host protein contamination was reduced about 100-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Aggregates were reduced to less than 0.05%. Turbidity of eluted IgG upon pH neutralization was nearly eliminated. Column cleaning was facilitated by minimizing the accumulation of chromatin. (C) 2014 Elsevier B.V. All rights reserved.
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