Dept. of Chemical Engineering and Chemical Technology, Imperial College London, Exhibition Road, London SW72AZ, UK.
Biotechnol Prog. 2010 Sep-Oct;26(5):1352-60. doi: 10.1002/btpr.453.
Purification schemes for antibody production based on affinity chromatography are trying to keep pace with increases in cell culture expression levels and many current research initiatives are focused on finding alternatives to chromatography for the purification of Monoclonal antibodies (MAbs). In this article, we have investigated an alternative separation technique based on liquid-liquid extraction called the reverse micellar extraction. We extracted MAb (IgG1) using reverse micelles of an anionic surfactant, sodium bis 2-ethyl-hexyl sulfosuccinate (AOT) and a combination of anionic (AOT) and nonionic surfactants (Brij-30, Tween-85, Span-85) using isooctane as the solvent system. The extraction efficiency of IgG1 was studied by varying parameters, such as pH of the aqueous phase, cation concentration, and type and surfactant concentration. Using the AOT/Isooctane reverse micellar system, we could achieve good overall extraction of IgG1 (between 80 and 90%), but only 30% of the bioactivity of IgG1 could be recovered at the end of the extraction by using its binding to affinity chromatography columns as a surrogate measure of activity. As anionic surfactants were suspected as being one of the reasons for the reduced activity, we decided to combine a nonionic surfactant with an anionic surfactant and then study its effect on the extraction efficiency and bioactivity. The best results were obtained using an AOT/Brij-30/Isooctane reverse micellar system, which gave an overall extraction above 90 and 59% overall activity recovery. An AOT/Tween-85/Isooctane reverse micellar system gave an overall extraction of between 75 and 80% and overall activity recovery of around 40-45%. The results showed that the activity recovery of IgG1 can be significantly enhanced using different surfactant combination systems, and if the recovery of IgG1 can be further enhanced, the technique shows considerable promise for the downstream purification of MAbs.
基于亲和层析的抗体生产纯化方案正试图跟上细胞培养表达水平的提高，许多当前的研究计划都集中在寻找替代色谱法来纯化单克隆抗体 (MAbs)。在本文中，我们研究了一种基于液 - 液萃取的替代分离技术，称为反胶束萃取。我们使用阴离子表面活性剂双(2-乙基己基)磺基琥珀酸钠 (AOT) 的反胶束和阴离子 (AOT) 和非离子表面活性剂 (Brij-30、Tween-85、Span-85) 的混合物从异丙烷中提取 MAb (IgG1) 作为溶剂系统。通过改变水相 pH 值、阳离子浓度以及表面活性剂类型和浓度等参数来研究 IgG1 的萃取效率。使用 AOT/异辛烷反胶束体系，可以实现 IgG1 的良好整体萃取（80%到 90%之间），但通过使用其与亲和层析柱的结合作为活性的替代测量，只能在萃取结束时回收 IgG1 的 30%的生物活性。由于阴离子表面活性剂被怀疑是降低活性的原因之一，我们决定将非离子表面活性剂与阴离子表面活性剂结合使用，然后研究其对萃取效率和生物活性的影响。使用 AOT/Brij-30/异辛烷反胶束体系可获得最佳结果，整体萃取率超过 90%，总活性回收率超过 59%。AOT/Tween-85/异辛烷反胶束体系的整体萃取率在 75%到 80%之间，总活性回收率约为 40%到 45%。结果表明，使用不同的表面活性剂组合系统可以显著提高 IgG1 的活性回收率，如果能够进一步提高 IgG1 的回收率，该技术在单克隆抗体的下游纯化方面具有很大的应用潜力。
