Refinement strategy for antivenom preparation of high yield and quality

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness.

源自超免疫动物血浆的抗蛇毒血清（antivenoms）是目前唯一针对蛇咬伤的特异性治疗药物。下游加工策略的优化一直备受学界关注，其价值不仅体现在提升纯化纯度表现，还可从产率成本比（yield-to-cost）以及动物源血浆的合理利用维度加以考量。本研究报道了一种针对基于F(ab')2片段的抗蛇毒血清制剂的高效精制工艺开发。本工艺的设计遵循核心准则：将活性成分始终维持于溶液状态，作为保障其构象稳定性的预防性措施，且完全避免了活性成分发生沉淀或吸附至色谱固定相的情况。研究团队采用2%（V/V）辛酸（caprylic acid）从超免疫马血浆中提取免疫球蛋白G（IgG），去除残留沉淀剂后，通过胃蛋白酶（pepsin）进行酶解；通过将胃蛋白酶与底物的质量比调整为4:300（w/w）、将pH值设为3.2、孵育时长设定为1.5小时，实现了免疫球蛋白G组分不完全酶解、F(ab')2片段过度酶解与活性成分保护效力损失三者间的平衡。最终纯化抛光步骤采用渗滤（diafiltration）与穿流色谱联用的方式完成。经尺寸排阻高效液相色谱（size-exclusion HPLC）检测与串联质谱（MS/MS）验证，本工艺制备的F(ab')2制剂纯度达100%且无聚集物产生，总产率可达75%及以上，优于目前已报道的同类工艺。该优化工艺在治疗性抗蛇毒血清的大规模生产中同样具有应用前景，因其不仅实现了活性药物的高收率，还满足了基于纯度要求的监管需求；研究人员在每一步纯化步骤中均精准测定了活性物质的回收率，从而能够准确评估该工艺的成本效益。