Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms.

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.

Assessment of the viral safety of antivenoms fractionated from equine plasma.

Antivenoms are preparations of intact or fragmented (F(ab')2 or Fab) immunoglobulin G (IgG) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. They are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. Manufacturing processes usually include pepsin digestion at acid pH, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. Most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. Nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. This paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. Analysis of production parameters indicate that acid pH treatments and caprylic acid precipitations, which have been validated for the manufacture of some human IgG products, appear to provide the best potential for viral inactivation of antivenoms. As many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products.

Improved purification and yield of the Egyptian snake Cerastes cerastes antitoxin by the use of caprylic acid.

The aim of this study was to obtain anti-snake antiserum by optimizing the conditions of extraction and purification and test its ability to neutralize local myonecrosis. Extraction and purification was achieved through adjustment of the pH, pepsin concentration, time of digestion, and caprylic acid concentration. Our results indicate that the best conditions to obtain anti-snake antiserum from ammonium sulfate fractionated plasma are pH 3.3, 3.5 g/l pepsin, digestion for 90 min at 37 degrees C, and 0.5% caprylic acid. Antiserum purified using this method has greater neutralizing ability of myonecrosis than ammonium sulfate (ammSO4) fractionated product.

Cross neutralization of dangerous snake venoms from Africa and the Middle East using the VACSERA polyvalent antivenom. Egyptian Organization for Biological Products & Vaccines.

This study was performed to assess the ability of polyvalent snake venom anti-serum, produced by the Egyptian Organization for Biological Products & Vaccines (VACSERA), to neutralize several toxic activities of snake venoms, not only of those included in the antivenom mixture, but also some additional venoms of snakes from Egyptian, African, and Middle Eastern habitats. In general, the results revealed that polyvalent snake venom anti-serum from VACSERA is highly effective in neutralizing Egyptian snake venoms, especially Naja haje, Naja nigricolles, Naja pallida, Cerastes cerastes, Cerastes cerastes cerastes, Cerastes vipera, Pseudocerastes persicus fieldi, and Walterinnisia egyptia. The antivenom was also effective against Naja haje, Walterinnisia egyptia, and Bites aritans from Saudi Arabia. High activity was obtained against venoms from Naja haje, Naja nigricolles, and Naja pallida of Sudan, as well as the African Naja melanoleuca, Naja mossambica, Naja naja oxiana, Bites gabonica, and Vipera lebetina. Only moderate effectiveness was obtained with Echis coloratus and Echis carinatus, and the polyvalent antiserum was ineffective against the venom of Naja nivea.

Development of an improved method for production of antiscorpion F(ab')2 fragment of IgG with high yield and potency.

The main objective of the present work was to obtain a stable and highly purified scorpion antitixon, rich in specific antibodies, free of immunologically irrelevant plasma proteins or gross proteins. Our results indicate that the most potent yielded and purified F(ab')2 antivenom preparation was obtained when the first discarded precipitate was washed with 14% ammonium sulphate saline; then after the second addition of ammonium sulphate, the mixture was stirring overnight followed by precipitation of most non-immunoglobulin proteins with the aid of caprylic acid to produce antivenom rich in specific antibodies with higher yield and potency compared to the method commonly used.