Characterization and analysis of scFv-IgG bispecific antibody size variants.
Characterization and analysis of scFv-IgG bispecific antibody size variants.
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DOI：
10.1080/19420862.2018.1505398
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发表时间：
2018-11
期刊：
影响因子：
5.3
通讯作者：
Liu D
中科院分区：
文献类型：
--
作者：
Cao M;Wang C;Chung WK;Motabar D;Wang J;Christian E;Lin S;Hunter A;Wang X;Liu D
双特异性抗体是一类新兴的生物制品，其对于治疗应用越来越感兴趣。在一种双特异性抗体形式中，单链可变片段（scFv）连接至或插入完整免疫球蛋白G（IgG）分子的不同位置以赋予双表位结合。为了提高生物化学稳定性，通常在scFv的重链和轻链区域上改造半胱氨酸残基以形成链内二硫键。虽然这种二硫键通常可以提高稳定性，但它也可能给生产或开发带来意想不到的挑战。我们报告了对于附加的scFv-IgG双特异性抗体观察到的大小变体。结构表征研究表明，大小变体由scFv上的工程化二硫键产生，由此发现工程化二硫键是开放的或由于半胱氨酸的半胱氨酸化或谷胱甘肽化而不能形成链内二硫键。此外，scFv工程化的半胱氨酸还形成分子间二硫键，导致形成高度稳定的二聚体和聚集体。由于单体变体和二聚体均显示出较低的生物活性，因此认为它们是必须监测和控制的产品相关杂质。为此，我们开发和优化了一个强大的，精确的，准确的高分辨率尺寸排阻色谱方法，使用统计实验设计方法。
Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.
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DOI：
10.1093/protein/9.7.617
发表时间：
1996-07-01
期刊：
PROTEIN ENGINEERING
影响因子：
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作者：
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影响因子：
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DOI：
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发表时间：
2000-10-01
期刊：
PROTEIN ENGINEERING
影响因子：
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作者：
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通讯作者：
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影响因子：
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影响因子：
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