Rosa P A J, Azevedo A M, Ferreira I F, de Vries J, Korporaal R, Verhoef H J, Visser T J, Aires-Barros M R
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
J Chromatogr A. 2007 Aug 24;1162(1):103-13. doi: 10.1016/j.chroma.2007.03.067. Epub 2007 Mar 21.
The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.
研究了在几种功能化聚乙二醇（PEG）存在下，人免疫球蛋白（IgG）在聚合物-聚合物和聚合物-盐双水相体系（ATPS）中的分配情况。作为第一步，使用纯IgG进行分配研究，所测试的非功能化体系中目标蛋白保留在下层相时，采用该体系进行研究。研究了功能化PEG浓度增加的影响以及配体类型。之后，首先使用含有纯蛋白和人工蛋白混合物的体系，对最成功的配体进行选择性研究，随后使用含有中国仓鼠卵巢（CHO）细胞上清液的体系进行研究。PEG/磷酸盐ATPS不适用于IgG的亲和分配。在PEG/葡聚糖ATPS中，二戊酸功能化的PEG（PEG-COOH）对IgG表现出很大的亲和力，当使用20%（w/w）的PEG 150-COOH和40%（w/w）的PEG 3350-COOH时，所有IgG都可以在上层相中回收。使用人工蛋白混合物评估这些功能化PEG的选择性，在存在人血清白蛋白和肌红蛋白等典型血清蛋白的情况下，PEG 3350-COOH对IgG没有亲和力，而在含有PEG 150-COOH的体系中，IgG的回收率可达91%。然后在PEG/葡聚糖ATPS中，在PEG 150-COOH存在的情况下，从CHO细胞上清液中对IgG进行了最佳纯化，回收率为93%，纯化因子为1.9，对IgG的选择性为11。当将这种功能化PEG添加到ATPS中时，与非功能化体系相比，选择性提高了60倍。
