The quality of antivenom is governed by its safety and efficacy profiles. These quality characteristics are much influenced by the purity of antivenom content. Rigorous assessment and meticulous monitoring of antivenom purity at the preclinical setting is hence crucial. This study aimed to explore an integrative proteomic method to assess the physicochemical purity of four commercially available antivenoms in the region. The antivenoms were subjected to Superdex 200 HR 10/30 size-exclusion fast-protein liquid chromatography (SE-FPLC). The proteins in each fraction were trypsin-digested and analyzed by nano-ESI-liquid chromatography-tandem mass spectrometry (LC-MS/MS). SE-FPLC resolved the antivenom proteins into three major protein components of very high (>200 kDa), high (100–120 kDa) and medium (<60 kDa) molecular weights. The major components (80–95% of total proteins) in the antivenoms were proteins of 100–120 kDa consisting of mainly the light and partially digested heavy immunoglobulin chains, consistent with F(ab’)₂ as the active principle of the antivenoms. However, LC-MS/MS also detected substantial quantity of large proteins (e.g. alpha-2-macroglobulins), immunoglobulin aggregates and impurities e.g. albumins in some products. The method is practical and able to unveil the quantitative and qualitative aspects of antivenom protein compositions. It is therefore a potentially useful preclinical assessment tool of antivenom purity.

抗蛇毒血清的质量取决于其安全性和有效性。这些质量特征很大程度上受抗蛇毒血清成分纯度的影响。因此，在临床前环境中对抗蛇毒血清纯度的严格评估和细致监测至关重要。本研究旨在探索一种综合蛋白质组学方法，以评估该地区四种市售抗蛇毒血清的理化纯度。将抗蛇毒血清进行 Superdex 200 HR 10/30 大小排阻快速蛋白质液相色谱 (SE-FPLC)。每个级分中的蛋白质被胰蛋白酶消化并通过纳米 ESI-液相色谱-串联质谱 (LC-MS/MS) 进行分析。SE-FPLC 将抗蛇毒血清蛋白分解为非常高 (>200 kDa)、高 (100–120 kDa) 和中等 (<60 kDa) 分子量的三种主要蛋白质组分。₂作为抗蛇毒血清的活性成分。然而，LC-MS/MS 也检测到大量的大蛋白（例如 α-2-巨球蛋白）、免疫球蛋白聚集体和杂质，例如某些产品中的白蛋白。该方法是实用的，能够揭示抗蛇毒蛋白成分的定量和定性方面。因此，它是一种潜在有用的抗蛇毒血清纯度的临床前评估工具。