REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
J Chromatogr A. 2014 Apr 25;1339:59-64. doi: 10.1016/j.chroma.2014.02.069. Epub 2014 Mar 3.
The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions. The binding capacity of gum arabic coated particles modified with aminophenyl boronic acid (GA-APBA-MP) was were found to be excellent in combination with the ATPS system, yielding high yields of antibody recovery (92%) and purity (98%) from cell culture supernatants. The presence of MPs in the ATPS was found to speed up phase separation (from 40 to 25min), to consume a lower amount of MPs (half of the amount needed in magnetic fishing) and to increase the yield and purity of a mAb purified from a cell culture supernatant, when compared with ATPE or magnetic fishing processes alone.
本研究旨在开发一种从细胞培养上清液中选择性地非色谱法纯化抗体的方法，考察了水相双相萃取（ATPE）与磁分离相结合的潜力。在上清液中，由聚乙二醇（PEG）和葡聚糖组成的双水相体系（ATPS）在不同盐浓度下添加了几种表面修饰的磁性颗粒（MPs）。研究了纯人 IgG 在上下相中的分配以及在 MPs 表面吸附的量，结果表明，用葡聚糖和阿拉伯胶修饰的 MPs 建立了最低的非特异性相互作用。发现用氨苯基硼酸（GA-APBA-MP）修饰的阿拉伯胶涂层颗粒具有优异的结合能力，与 ATPS 系统相结合，可从细胞培养上清液中获得高抗体回收率（92%）和高纯度（98%）。与 ATPE 或磁捕集过程相比，在 ATPS 中添加 MPs 可加速相分离（从 40 分钟缩短至 25 分钟），减少 MPs 的用量（减少一半），并提高从细胞培养上清液中纯化单克隆抗体的产量和纯度。
