Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, HR-10000 Zagreb, Croatia.
Center of Excellence for Virus Immunology and Vaccines, CERVirVac, Rockefellerova 10, HR-10000 Zagreb, Croatia.
Toxins (Basel). 2022 Jul 13;14(7):483. doi: 10.3390/toxins14070483.
Antivenoms contain either pure animal IgGs or their fragments as an active substance, and are the only specific therapeutics against envenomation arising from snakebites. Although they are highly needed, the low sustainability of such preparations' manufacture causes constant global shortages. One reason for this is the stability of the product, which contributes not only to the manufacture sustainability, but the product safety as well. It has been hypothesized that the roughness of conditions to which IgGs are exposed during downstream purification disturbs their conformation, making them prone to aggregation, particularly after exposure to secondary stress. The aim of this research was to investigate how the roughness of the downstream purification conditions influences the stability properties of purified IgGs. For this purpose, equine IgGs were extracted from unique hyperimmune plasma by two mild condition-based operational procedures (anion-exchange chromatography and caprylic acid precipitation) and three rougher ones (ammonium sulphate precipitation, cation-exchange chromatography and protein A affinity chromatography). The stability of the refined preparations was studied under non-optimal storage conditions (37 °C, 42 °C, and a transiently lower pH) by monitoring changes in the aggregate content and thermal stability of the pure IgGs. Mild purification protocols generated IgG samples with a lower aggregate share in comparison to the rougher ones. Their tendency for further aggregation was significantly associated with the initial aggregate share. The thermal stability of IgG molecules and the aggregate content in refined samples were inversely correlated. Since the initial proportion of aggregates in the samples was influenced by the operating conditions, we have shown a strong indication that each of them also indirectly affected the stability of the final preparations. This suggests that mild condition-based refinement protocols indeed generate more stable IgGs.
抗蛇毒血清含有纯动物 IgG 或其片段作为活性物质，是治疗蛇咬伤引起的中毒的唯一特效疗法。尽管它们需求量很大，但由于这些制剂制造的可持续性低，导致全球持续短缺。造成这种情况的一个原因是产品的稳定性，它不仅有助于制造的可持续性，而且对产品的安全性也有影响。有人假设，IgG 在下游纯化过程中所暴露的条件的粗糙度会干扰其构象，使它们容易聚集，尤其是在暴露于二次应激后。本研究的目的是研究下游纯化条件的粗糙度如何影响纯化 IgG 的稳定性特性。为此，通过两种温和的基于条件的操作程序（阴离子交换色谱和辛酸沉淀）和三种更粗糙的程序（硫酸铵沉淀、阳离子交换色谱和蛋白 A 亲和色谱）从独特的高免疫血浆中提取马 IgG。通过监测纯 IgG 的聚集含量和热稳定性的变化，在非最佳储存条件（37°C、42°C 和短暂较低的 pH 值）下研究了精制制剂的稳定性。与较粗糙的程序相比，温和的纯化方案产生的 IgG 样品中聚集物的含量较低。它们进一步聚集的趋势与初始聚集物的含量显著相关。IgG 分子的热稳定性和精制样品中的聚集物含量呈负相关。由于样品中聚集物的初始比例受操作条件的影响，我们已经表明了一个强烈的迹象，即它们中的每一个也间接地影响了最终制剂的稳定性。这表明温和的基于条件的精制方案确实能产生更稳定的 IgG。
