Most antivenoms are produced by techniques developed over 50 years ago, with minor modifications. Herein we revise the core of traditional antivenom production processes aiming to optimize key determinants for both consistent antivenom production and the best balance between F(ab')2 quality and recovery. Factorial design analysis revealed that pepsin digestion of 1:3 saline diluted equine plasma for 60 min under pH: 3.20, 37 °C temperature and a 1:15 pepsin to protein ratio conditions, allowed to achieve maximal IgG to F(ab')2 conversion with minimal protein aggregate formation. Further downstream processing by salting out with ammonium sulfate was also studied by factorial analysis. The influence of ammonium sulfate (AS) concentration, temperature (T) and the albumin to total plasma protein ratio plasma (Alb:P) were assayed, revealing that both AS, T and their interaction have a significant impact in F(ab')2 quality and recovery. Taking into account the existing compromise between F(ab')2 monomer recovery and quality two alternative conditions were selected: 14 g/dl AS at 56 °C and, alternatively 16 g/dl AS at 30 °C. Reasonable yields (42%) and product quality (2.5% of aggregates) without significant changes in production cost of traditional methodologies was achieved under the optimized conditions found.

大多数抗蛇毒血清是由 50 多年前开发的技术生产的，经过微小的修改。在此，我们修订了传统抗蛇毒血清生产流程的核心，旨在优化关键决定因素，以实现一致的抗蛇毒血清生产以及 F(ab')2 质量和回收率之间的最佳平衡。因子设计分析显示，在 pH 值：3.20、37 °C 温度和 1:15 胃蛋白酶与蛋白质比率条件下，胃蛋白酶消化 1:3 生理盐水稀释的马血浆 60 分钟，可实现最大 IgG 对 F(ab')2以最少的蛋白质聚集体形成进行转化。还通过因子分析研究了通过用硫酸铵盐析出的进一步下游加工。测定了硫酸铵 (AS) 浓度、温度 (T) 和白蛋白与总血浆蛋白比率血浆 (Alb:P) 的影响，揭示 AS、T 及其相互作用对 F(ab')2 质量和回收率有显着影响。考虑到 F(ab')2 单体回收率和质量之间的现有折衷，选择了两种替代条件：56 °C 下的 14 g/dl AS 和 30 °C 下的 16 g/dl AS。在发现的优化条件下，在传统方法的生产成本没有显着变化的情况下，实现了合理的产量 (42%) 和产品质量 (骨料的 2.5%)。