Azevedo A M, Rosa P A J, Ferreira I F, Aires-Barros M R
IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisbon, Portugal.
J Chromatogr A. 2008 Dec 12;1213(2):154-61. doi: 10.1016/j.chroma.2008.09.115. Epub 2008 Oct 14.
We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.
我们评估了一种结合水相两相萃取、疏水相互作用色谱（HIC）和尺寸排阻色谱（SEC）的工艺，用于从中国仓鼠卵巢（CHO）细胞上清液中纯化人免疫球蛋白G（IgG）。选择这些单元操作不仅是为了去除目标杂质，还为了便于整合不同的工艺单元，而无需任何预处理步骤。在由聚乙二醇（PEG）和柠檬酸钠组成的水相两相系统（ATPS）中进行萃取，可使抗体在富含柠檬酸盐的相中浓缩，并在富含PEG的相中去除最疏水的化合物。由10%（w/w）PEG 3350和12%（w/w）柠檬酸盐组成的ATPS，在pH 6条件下，IgG的回收率为97%，HPLC纯度为41%，蛋白质纯度为72%。然后将此下层相直接加载到苯基琼脂糖HIC柱上。这个中间纯化步骤允许使用柠檬酸盐流动相捕获抗体，99%的抗体在洗脱级分中回收，HPLC纯度为86%，蛋白质纯度为91%。最后，SEC通过去除IgG聚集体实现最终精制。HIC洗脱级分直接注入Superose 6尺寸排阻柱，得到产率为90%的100%纯IgG溶液。
