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）在肿瘤和自身免疫性疾病中的成功应用为开发具有更广泛的结构和物理化学特性的治疗性抗体铺平了道路。开发了结合了2-（N-吗啉代）乙烷磺酸（MES）和4-（2-羟乙基）-1-哌嗪乙烷磺酸（HEPES）缓冲液并用氯化钾介导的pH梯度以充分保留酸性mAb（pI <7）在阳离子交换色谱（CEX）中进行分离，同时保持适合碱性mAb的分离性能（pI> 7）。首先，通过应用盐梯度分别评估MES和HEPES缓冲液在其有用的pH范围内。然后将结合了MES和HEPES缓冲液的盐介导的pH梯度的性能与商用pH梯度试剂盒进行了比较。发现开发的条件优于盐梯度方法，并提供了商业pH梯度试剂盒的有用替代方法。在这项研究中，已开发的条件用于将双特异性抗体（BsAb）从其两个亲本mAb中分离出来。