Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Zagreb, Croatia.
Center of Excellence for Virus Immunology and Vaccines, Zagreb, Croatia.
Front Immunol. 2022 May 17;13:889736. doi: 10.3389/fimmu.2022.889736. eCollection 2022.
During the pre-vaccine era of the COVID-19 pandemic convalescent plasma has once again emerged as a major potential therapeutic form of passive immunization that in specific cases still represents irreplaceable treatment option. There is a growing concern that variable concentration of neutralizing antibodies, present in convalescent plasma which originates from different donors, apparently affects its effectiveness. The drawback can be overcome through the downstream process of immunoglobulin fraction purification into a standardized product of improved safety and efficacy. All modern procedures are quite lengthy processes. They are also based on fractionation of large plasma quantities whose collection is not attainable during an epidemic. When outbreaks of infectious diseases are occurring more frequently, there is a great need for a more sustainable production approach that would be goal-oriented towards assuring easily and readily available immunoglobulin of therapeutic relevance. We propose a refinement strategy for the IgG preparation achieved through simplification and reduction of the processing steps. It was designed as a small but scalable process to offer an immediately available treatment option that would simultaneously be harmonized with an increased availability of convalescent plasma over the viral outbreak time-course. Concerning the ongoing pandemic status of the COVID-19, the proof of concept was demonstrated on anti-SARS-CoV-2 convalescent plasma but is likely applicable to any other type depending on the current needs. It was guided by the idea of persistent keeping of IgG molecules in the solution, so that protection of their native structure could be assured. Our manufacturing procedure provided a high-quality IgG product of above the average recovery whose composition profile was analyzed by mass spectrometry as quality control check. It was proved free from IgA and IgM as mediators of adverse transfusion reactions, as well as of any other residual impurities, since only IgG fragments were identified. The proportion of S protein-specific IgGs remained unchanged relative to the convalescent plasma. Undisturbed IgG subclass composition was accomplished as well. However, the fractionation principle affected the final product's capacity to neutralize wild-type SARS-CoV-2 infectivity, reducing it by half. Decrease in neutralization potency significantly correlated with the amount of IgM in the starting material.
在 COVID-19 大流行的疫苗前时代，恢复期血浆再次成为一种主要的潜在被动免疫治疗形式，在某些特定情况下仍然是不可替代的治疗选择。人们越来越担心，来自不同供体的恢复期血浆中存在的中和抗体浓度不同，显然会影响其效果。通过将免疫球蛋白分离纯化为标准化的、安全性和有效性提高的产品，可以克服这一缺点。所有现代程序都是相当冗长的过程。它们还基于对大量血浆进行分离，而在疫情期间无法获得这种血浆。当传染病爆发更加频繁时，非常需要一种更可持续的生产方法，这种方法的目标是确保随时可以获得具有治疗相关性的免疫球蛋白。我们提出了一种通过简化和减少处理步骤来改进 IgG 制备的策略。该方法设计为一个小型但可扩展的过程，提供一种即时可用的治疗选择，同时与病毒爆发期间恢复期血浆的可用性增加相协调。考虑到 COVID-19 的持续大流行状态，该概念验证是在抗 SARS-CoV-2 恢复期血浆上进行的，但可能适用于任何其他类型，具体取决于当前的需求。该方法的指导思想是保持 IgG 分子在溶液中的持久性，从而确保其天然结构得到保护。我们的制造工艺提供了一种高质量的 IgG 产品，其平均回收率高于平均水平，其组成谱通过质谱分析作为质量控制检查。结果表明，该产品不含作为不良反应输血反应介质的 IgA 和 IgM，也不含任何其他残留杂质，因为只鉴定出 IgG 片段。与恢复期血浆相比，S 蛋白特异性 IgG 的比例保持不变。也完成了未受干扰的 IgG 亚类组成。然而，分馏原理影响了最终产品中和野生型 SARS-CoV-2 感染性的能力，使其降低了一半。中和效力的降低与起始材料中 IgM 的量显著相关。
