Decomplexation proteomic analysis and purity assessment of a biologic for snakebite envenoming: Philippine Cobra Antivenom.

BACKGROUND: Philippine Cobra Antivenom (PCAV) is the only snake antivenom manufactured in the Philippines. It is used clinically to treat envenoming caused by the Philippine Spitting Cobra (Naja philippinensis). While PCAV is effective pharmacologically, it is crucial to ensure the safety profile of this biologic that is derived from animal plasma. METHODS: This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions. CONCLUSIONS: Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.

Situation of snakebite, antivenom market and access to antivenoms in ASEAN countries.

INTRODUCTION: Snakebite envenoming is a neglected tropical disease posing public health challenges globally. The Association of Southeast Asian Nations (ASEAN) countries are among the tropical regions with disproportionately high incidence of snakebite. Hence, this study aimed to review the situation of snakebite, antivenom market and access to antivenoms in ASEAN. METHODS: This mixed-methods study included comprehensive literature review and in-depth interviews with key informants to assess the situation of management system of snakebite, antivenom market and access to antivenoms in seven ASEAN countries, including Malaysia, Thailand, Indonesia, Philippines, Vietnam, Lao PDR and Myanmar. Data were analysed by a framework method. RESULTS: ASEAN have developed various strategies to improve outcomes of snakebite victims. Five domestic antivenom manufacturers in the region produce up to 288 375 vials of antivenoms annually with the value of US$13 058 053 million which could treat 42 213 snakebite victims. However, there remain challenges to be addressed especially the lack of snakebite-related informatics system, inadequate antivenoms at the healthcare facilities and when the majority of snakebite victims seek traditional healers instead of conventional treatment. CONCLUSION: Improving the situation of snakebite and antivenom is not only about the availability of antivenom, but the whole landscape of surrounding management and supporting system. The assessment of the situation of snakebite and antivenom is crucial for countries or regions where snakebites are prevalent to recognise their current standpoint to inform the development of strategies to achieve the goal set by the WHO of halving the global burden of snakebite by 2030.

Immunoreactivity and neutralization capacity of Philippine cobra antivenom against Naja philippinensis and Naja samarensis venoms.

BACKGROUND: The Philippine cobra (Naja philippinensis) and Samar cobra (Naja samarensis) are two WHO Category 1 medically important venomous snakes in the Philippines. Philippine cobra antivenom (PCAV) is the only antivenom available in the country, but its neutralization capacity against the venoms of N. philippinensis and hetero-specific N. samarensis has not been reported. This knowledge gap greatly hinders the optimization of antivenom use in the region. METHODS: This study examined the immunological binding and neutralization capacity of PCAV against the two cobra venoms using WHO-recommended protocols. RESULTS: In mice, both venoms were highly neurotoxic and lethal with a median lethal dose of 0.18 and 0.20 µg/g, respectively. PCAV exhibited strong and comparable immunoreactivity toward the venoms, indicating conserved venom antigenicity between the two allopatric species. In in vivo assay, PCAV was only moderately effective in neutralizing the toxicity of both venoms. Its potency was even lower against the hetero-specific N. samarensis venom by approximately two-fold compared with its potency against N. philippinensis venom. CONCLUSION: The results indicated that PCAV could be used to treat N. samarensis envenomation but at a higher dose, which might increase the risk of hypersensitivity and worsen the shortage of antivenom supply in the field. Antivenom manufacturing should be improved by developing a low-dose, high-efficacy product against cobra envenomation.