Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance
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- 作者：Pete Gagnon ; pete_gagnon@mac.com" class="auth_mail ; pete@validated.com" class="auth_mail ; Rui Nian ; Jeremy Lee ; Lihan Tan ; Sarah Maria Abdul Latiff ; Chiew Ling Lim ; Cindy Chuah ; Xuezhi Bi ; Yuansheng Yang ; Wei Zhang ; Hui Theng Gan
- 关键词：Chromatin ; IgG purification ; Protein A ; Capacity ; Turbidity ; Aggregates
- 刊名：Journal of Chromatography A
- 出版年：2 May, 2014
- 年：2014
- 卷：1340
- 期：Complete
- 页码：68-78
- 全文大小：1578 K
文摘
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), non-histone 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 鈭?0%. 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.