Azevedo Ana M, Rosa Paula A J, Ferreira I Filipa, de Vries J, Visser T J, Aires-Barros M Raquel
IBB-Institute for Biotechnology and Bioengineering, Centre for Chemical and Biological Engineering, Instituto Superior Técnico, Lisbon, Portugal.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jan 1;877(1-2):50-8. doi: 10.1016/j.jchromb.2008.11.014. Epub 2008 Nov 14.
In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.
在本文中，我们探索了一种从中国仓鼠卵巢（CHO）细胞上清液中纯化人源抗体的替代方法，该方法包括配体增强萃取捕获步骤和阳离子交换色谱法（CEX）。人源抗体的萃取在由葡聚糖和聚乙二醇（PEG）组成的双水相系统（ATPS）中进行，其中PEG分子的末端羟基用氨基酸模拟配体进行修饰，以增强抗体在富含PEG相中的分配。使用实验设计对该捕获步骤进行了优化，中心复合设计能够确定就系统组成而言有利于抗体分配到含有聚乙二醇二酸（PEG-GA）聚合物相中的条件。因此，对于较高浓度的PEG-GA和较低浓度的葡聚糖，可获得更高的回收率。实验观察到，由5.17%（w/w）葡聚糖和8%（w/w）PEG-GA组成的ATPS获得了最高产率。然而，对于两种聚合物的较高浓度，预测纯度更高。使用5%葡聚糖和10%PEG-GA实现了产率和纯度之间的折衷，通过尺寸排阻色谱法测定，其可回收82%的抗体，蛋白质纯度为96%，总纯度为63%。ATPS的上相通过阳离子交换色谱法进一步纯化，观察到最合适的阳离子交换配体是羧甲基，因为磺丙基配体诱导形成多聚体或变性形式。该柱可洗脱上相中89% 的抗体，蛋白质纯度为100%，总纯度为91%。包含配体增强萃取步骤和阳离子交换色谱步骤的整个过程的总产率为73%。
