Tandem single-chain variable fragment (scFv) bispecific antibodies (bsAb) are one of the most promising bsAb formats reported thus far. Yet, because of their increased aggregation propensity, high impurity content due to low expression level, smaller size and lack of the Fc region, it is challenging to isolate these products with high yield and purity within a limited number of purification steps in a scalable fashion. A robust purification process that is able to circumvent these issues is therefore of critical importance to allow effective isolation of this group of antibodies. We investigated the addition of sodium chloride (NaCl), calcium chloride (CaCl2), and L-arginine monohydrochloride (Arg·HCl) to the elution buffer of Protein L affinity chromatography, and propose here a novel mechanism for the modification of Protein L binding avidity that can lead to enhanced high molecular weight (HMW)-monomer separation, a preferential strengthening effect of the HMW-Protein L interaction compared to the monomer-Protein L interaction. In particular, we found Arg·HCl to be the most effective salt additive in terms of purity and recovery. The mechanism we propose is different from the widely reported chaotropic effect exerted by salt additives observed in Protein A chromatography. We also demonstrate here that a final eluate containing <1% HMW species and <100 ppm host cell proteins can be obtained within a two-step process with an overall yield of 65%, highlighting the promising suitability of Protein L affinity chromatography for the purification of kappa light chain-containing tandem scFv bsAb.

中文翻译：

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研究蛋白L亲和层析中盐添加剂对串联单链可变片段双特异性抗体的纯化作用。

串联单链可变片段（scFv）双特异性抗体（bsAb）是迄今为止报道的最有希望的bsAb格式之一。然而，由于它们的聚集倾向增加，由于低表达水平，较小的尺寸和缺乏Fc区而导致的高杂质含量，以有限的纯化步骤以可扩展的方式分离具有高产率和纯度的这些产物具有挑战性。 。因此，能够避免这些问题的可靠的纯化方法对于有效分离这组抗体至关重要。我们研究了在蛋白质L亲和色谱的洗脱缓冲液中添加氯化钠（NaCl），氯化钙（CaCl2）和L-精氨酸一盐酸盐（Arg·HCl），并在此提出了一种新的修饰蛋白质L结合亲和力的机制，该机制可导致增强的高分子量（HMW）-单体分离，与单体-蛋白质L相互作用相比，HMW-蛋白质L相互作用的优先增强作用。特别是从纯度和回收率方面，我们发现Arg·HCl是最有效的盐添加剂。我们提出的机制不同于蛋白质A色谱中观察到的盐添加剂所产生的离液效应。我们还在此处证明了可以在两步过程中以65％的总收率获得包含<1％HMW物种和<100 ppm宿主细胞蛋白的最终洗脱液，突出了Protein L亲和层析对纯化的有希望的适用性含κ轻链的串联scFv bsAb。