State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; Department of Chemical Engineering, Beijing Institute of Petro-chemical Technology, Beijing 102617, China.
J Immunol Methods. 2018 Sep;460:45-50. doi: 10.1016/j.jim.2018.06.008. Epub 2018 Jun 15.
Protein A chromatography has been a popular method for purification of therapeutic monoclonal antibodies (mAb). Protein A chromatographic media using alkali-resistant rProtein A ligands from site-directed coupling method have been pursued for both high dynamic capacities and excellent stabilities. However, the mechanism of rProtein A leaking under cleaning-in-place (CIP) conditions is not very clear and difficulties have been commonly encountered when improving the media's chromatographic performance. We investigated the chromatographic performance of site-directed coupled rProtein A chromatographic media during CIP procedure. Trace amount of ligands leaked during the chromatographic media's incubation in 0.5 M NaOH was detected, explaining for the decline of chromatographic media's CIP performance. Decrease of rProtein A's concentration in 0.5 M NaOH was consistent with chromatographic media's binding capacity. A novel rProtein A chromatographic media were prepared by site-directed coupling a newly-constructed alkali-resistant rProtein A to highly cross-linked agarose-based matrix. The media had a dynamic binding capacity of 63.2 mg hIgG/mL higher than 48.1 mg hIgG/mL of the commercial one, and the CIP performance was improved greatly with the remained dynamic binding capacity increased from 86% to 95% of the initial value after 40 CIP cycles.
Protein A 亲和层析一直是用于治疗性单克隆抗体（mAb）纯化的热门方法。使用定向偶联方法的耐碱 rProtein A 配体的 Protein A 亲和层析介质，因其高动态容量和出色的稳定性而备受关注。然而，rProtein A 在就地清洗（CIP）条件下泄漏的机制尚不清楚，在改善介质的色谱性能方面通常会遇到困难。我们研究了定向偶联 rProtein A 亲和层析介质在 CIP 过程中的色谱性能。在 0.5 M NaOH 中孵育层析介质时检测到痕量配体泄漏，这解释了层析介质 CIP 性能下降的原因。0.5 M NaOH 中 rProtein A 浓度的降低与层析介质的结合能力一致。通过定向偶联新构建的耐碱 rProtein A 到高度交联的琼脂糖基质上，制备了一种新型 rProtein A 亲和层析介质。该介质的动态结合容量为 63.2mg hIgG/mL，高于商业介质的 48.1mg hIgG/mL，经过 40 次 CIP 循环后，CIP 性能得到了极大改善，剩余的动态结合容量从初始值的 86%增加到 95%。
