Ishihara Takashi, Kadoya Toshihiko
CMC R&D Laboratories, Production Division, Kirin Pharma Company Limited, Takasaki, Gunma 370-0013, Japan.
J Chromatogr A. 2007 Dec 28;1176(1-2):149-56. doi: 10.1016/j.chroma.2007.10.104. Epub 2007 Nov 7.
For accelerating the purification process development of human monoclonal antibodies (hmAbs) for pharmaceutical drugs, we designed a standardized method for setting the conditions of the purification process, which could be applied to hmAbs for the early phase of pharmaceutical development. The process includes three sequential chromatography steps: Protein A affinity chromatography (AFC), anion-exchange chromatography (AIEC) and cation-exchange chromatography (CIEC), and also includes a low pH virus inactivation step after the AFC step. We predicted the elution pH in the AFC and elution salt concentration in the CIEC from the amino acid sequences of hmAbs, as described in our previous paper. The mobile phase pH in AIEC and the pH for virus inactivation were also predicted based on the amino acid sequence of hmAb. As a case study, six hmAbs (two of IgG(1), two of IgG(2) and two of IgG(4)) were purified with the standardized method. The recovery, purity and clearance of impurities (DNA, host cell proteins (HCP), and Protein A) were examined. All the six hmAbs were purified with high recovery and high clearance of the impurities. Factors affecting the impurities level in the purified products are also discussed.
为加速用于药物的人单克隆抗体（hmAbs）纯化工艺的开发，我们设计了一种标准化方法来设定纯化工艺条件，该方法可应用于药物开发早期阶段的hmAbs。该工艺包括三个连续的色谱步骤：蛋白A亲和色谱（AFC）、阴离子交换色谱（AIEC）和阳离子交换色谱（CIEC），并且在AFC步骤之后还包括一个低pH病毒灭活步骤。正如我们之前的论文所述，我们根据hmAbs的氨基酸序列预测了AFC中的洗脱pH和CIEC中的洗脱盐浓度。还基于hmAb的氨基酸序列预测了AIEC中的流动相pH和病毒灭活的pH。作为案例研究，使用该标准化方法纯化了六种hmAbs（两种IgG(1)、两种IgG(2)和两种IgG(4)）。检测了回收率、纯度以及杂质（DNA、宿主细胞蛋白（HCP）和蛋白A）的清除率。所有六种hmAbs均以高回收率和高杂质清除率进行了纯化。还讨论了影响纯化产物中杂质水平的因素。
