开发一种创新的盐介导 pH 梯度阳离子交换色谱方法，用于治疗性抗体的表征。
Development of an innovative salt-mediated pH gradient cation exchange chromatography method for the characterization of therapeutic antibodies.
机构信息
School of Pharmaceutical Sciences, University of Geneva, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 1;1160:122379. doi: 10.1016/j.jchromb.2020.122379. Epub 2020 Sep 7.
The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.
单克隆抗体（mAb）在肿瘤学和自身免疫性疾病中的成功应用为开发具有更广泛结构和物理化学性质的治疗性抗体铺平了道路。开发了一种 pH 梯度，该梯度结合了 2-（N-吗啉基）乙磺酸（MES）和 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲液，并通过氯化钾介导，足以在阳离子交换层析（CEX）中保留酸性 mAb（pI<7），同时保持对碱性 mAb（pI>7）的合适分离性能。首先，通过应用盐梯度来单独评估 MES 和 HEPES 缓冲液在其有用 pH 范围内的性能。然后将 MES 和 HEPES 缓冲液结合的盐介导 pH 梯度的性能与商业 pH 梯度试剂盒进行比较。发现开发的条件优于盐梯度方法，并为商业 pH 梯度试剂盒提供了有用的替代方案。在这项研究中，开发的条件被应用于从其两个亲本 mAb 中分离双特异性抗体（BsAb）。
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