Study on the hole and knob related impurities in the purification of bispecific antibodies

IF 1.8 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2026-02-01 Epub Date: 2025-10-18 DOI:10.1016/j.pep.2025.106836

Huanlan Chen, Naiqi Ji, Maozhi Zhou, Yingyue Bu

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引用次数: 0

Abstract

The Knob-into-Hole (KiH) design is an effective strategy to promote the heterodimerization of heavy chains. In this research, through the integration of advanced analytical techniques, we made a critical finding regarding heterodimer formation. When mixing materials derived from different clones containing hole-hole homodimers and knob-knob homodimers, the resulting structures initially appear to form the desired knob-into-hole (KiH) configuration. However, detailed characterization by SDS-Caliper NR analysis revealed these were not genuine KiH heterodimers. While this phenomenon presents an optimal apparent outcome, it has significant impact on the optimization of process in heterodimer production. Additionally, a comprehensive assessment was carried out on the efficacy of cation exchange chromatography (CEX) in removing homodimers. This study presents a case for the optimization of the CEX process, utilizing bispecific - antibody materials sourced from different clonal bioreactors. By adjusting the operating pH of CEX, the knob-related impurities and hole-related impurities were reduced to below the detection limit, as shown by iCIEF analysis. Meanwhile, SEC-UPLC analysis indicated that purity increased to 99 %, and SDS-Caliper NR results showed that the percentage of functional KiH improved from 85 % in the input to 97 % after elution. These findings demonstrate that the CEX process contributes to an increase in the proportion of functional KiH.

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双特异性抗体纯化中孔洞和球形杂质的研究。

旋钮入孔设计是促进重链异二聚化的有效策略。在这项研究中，通过整合先进的分析技术，我们在异源二聚体的形成方面取得了重要的发现。当混合来自含有孔-孔同型二聚体和旋钮-旋钮同型二聚体的不同克隆的材料时，所得到的结构最初似乎形成了所需的旋钮-孔（KiH）结构。然而，SDS-Caliper NR分析的详细表征显示这些不是真正的KiH异源二聚体。虽然这一现象呈现出最优的表观结果，但它对异二聚体生产过程的优化有重要影响。此外，还对阳离子交换色谱法（CEX）去除同型二聚体的效果进行了综合评价。本研究利用来自不同克隆生物反应器的双特异性抗体材料，对CEX工艺进行了优化。通过调整CEX的工作pH值，可以将旋钮相关杂质和孔洞相关杂质降低到检测限以下，如iCIEF分析所示。同时，SEC-UPLC分析表明，纯度提高到99%，SDS-Caliper NR结果表明，洗脱后的功能性KiH百分比从输入的85%提高到97%。这些发现表明CEX过程有助于功能性KiH比例的增加。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.