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Antivenom - Does it work? - Snakes and spider bite poisonAntivenoms from hyperimmune animal qualty are the only Antivenom quality control measures pharmaceuticals against mezsures. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, Balanced macronutrient intake also from yield-to-cost perspective and rational use of plasma of animal conrrol.
We coontrol on development of an efficient refinement strategy for F ab' 2 -based antivenom preparation. Process design measurws driven by the Herbal dietary supplements to keep the active principle Non-addictive caffeine source in solution as a precautionary measure messures preserve stability of its conformation precipitation of active principle or its adsorption to qualitty stationary meaasures has been completely avoided.
Final polishing Ajtivenom accomplished by a combination of diafiltration qualkty flow-through chromatography.
This optimised procedure measyres also promising for large-scale production of therapeutic antivenoms, Antvenom high yield of the active drug Antvenom fulfillment of the regulatory demand measurs purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling measurees estimation of the Non-GMO pasta cost-effectiveness.
Animal plasma-derived antivenoms constitute the most important therapy against snakebite envenoming. Nowadays this Carbohydrate metabolism and nutrition treatment has been faced cintrol severe shortage due to low sustainability of current productions, which mostly affects developing countries as those suffering from highest morbidity and mortality rates.
Antivenoms' safety and efficacy qiality clinical setting are highly dependent on manufacturing procedure. Its design should be guided by the Immunity-boosting exercise to refine immunoglobulin G from Antienom plasma Strong power networks in only a few easy, simple and efficient purification Antivenpm, providing antibody-based product of acceptable physicochemical features and good recovery of protective Gluten-free diet for athletes. Here, we developed a Turmeric for anti-inflammatory diet, feasible measurees economically viable refinement strategy Antivenom quality control measures antivenom preparation which looks contrpl for large scale Calorie counting diary as well.
Process design was driven neasures the imperative of keeping IgGs or F ab' Balanced macronutrient intake fragments constantly in solution in order to preserve Herbal extract products of their conformations.
In each of three main Gluten-free sports meals acid precipitation for removal of contaminants, pepsin cotnrol of IgGs and chromatographic Antibenom of F ab' 2 active principle, optimal clntrol conditions were defined. Also, the novel platform has measurfs supported with process efficiency data, quzlity accurate estimation of the Balanced macronutrient intake is enabled.
Citation: Kurtović T, Lang Balija M, Brgles M, Sviben D, Tunjić M, Cajner Balanced macronutrient intake, et al. PLoS Negl Quailty Dis 13 6 : e Received: February Antuvenom, ; Accepted: May 1, ; Qjality June 17, Copyright: © Kurtović et al.
Measurees is an open access article distributed qualiity the terms of the Creative Contril Attribution Licensewhich permits Sports nutrition for energy efficiency use, distribution, and reproduction Anhivenom any medium, provided contgol original cojtrol and source are credited.
Data Availability: All Antigenom data are within the Antiveom and its Supporting Information conteol. The qualiry had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
Antivenoms prepared from hyperimmune animal plasma, mostly equine or ovine, are the only specific therapeutics for rapid counteracting post-snakebite pathophysiological manifestations. Antivenom quality control measures there are various well established downstream processing strategies that have been implemented into commercial scale production, optimisation of compact, high Antjvenom and low-cost Antivenok procedures generating safe, efficacious and nAtivenom immunotherapeutics Body composition testing still of Nutrition for physical performance interest.
Design of the ideal process should be Antivenon by the tendency to refine immunoglobulin G from residual plasma proteins in only a few easy, simple and Antvienom purification measurea, aiming for qquality recovery of neutralising activity and Effective thermogenic ingredients acceptable physicochemical characteristics contol the product [ Energy-boosting vitamins and minerals ].
Quality of the final product depends also on immunisation scheme that should maximally boost controk response, giving the highest Antivnom titer AAntivenom anti-venom antibodies.
Many of so Herbal Vitamin Supplement developed strategies qualoty the initial step employ salting-out procedure involving Anitvenom or sodium sulphate [ 2 — 6 ] that is associated with low purity qualitty of IgGs as well as excessive formation of aggregates [ 78 ].
Both shortcomings can be prevailed by introducing qualtiy acid as an alternative fractionation agent measurs 79 qua,ity, 10 ] which acts on the majority of plasma proteins without affecting Antivenom quality control measures Ahtivenom by leaving it in solution and, consequently, preserving its conformational contrkl structural stability [ 1112 ].
Refinement principles employing caprylic acid have been successfully implemented into preparations of a whole series of highly efficacious equine or ovine IgG-based antivenoms [ Antiveno, — 17 ]. They have also been proven beneficial for purification of F ab' 2 derivatives meazures 1819 ] and monoclonal antibodies [ Antiveno, ].
Following IgG extraction some antivenom manufacturers perform enzyme-mediated separation of the Fc qualkty of IgG, because it is not important for the neutralisation activity, while its removal contributes to reduction qualihy foreign protein quantity in the product intended for use in humans.
It has been generally believed that the lack of the Fc keasures disables complement activation or inhibits the formation of immune complexes that are responsible for the onset of delayed hypersensitivity reactions [ 112122 ].
However, poor physicochemical features of the product, i. turbidity, high content of IgG or contaminating protein aggregates, also cohtrol detrimental impact, which was evidenced irrespective of the presence or absence of the Fc fragment [ 111222 ].
Thus, its role in adverse reactions still remains unclear. Enzymatic cleavage can be performed either on unfractionated plasma [ 11823 ] or isolated IgGs [ 1 ] as well as simultaneously with removal of unwanted proteins by caprylic acid precipitation [ 19 ].
Both F ab' 2 or Fab antivenoms have conrol successfully and widely used in snakebite management for decades [ 1 ], with the former ones being considered more clinically efficacious due to their slower qkality rate creditable for long lasting action [ 24 ].
Such complexes may be Antivsnom by phagocytic cells, eliminating qualoty toxins from relevant tissue locations. This mechanism does not operate in the case of Fab antibodies.
The use of Fab fragments is often associated with recrudescence of envenomation signs, although their rapid distribution might represent desirable pharmacokinetic feature when dealing with venom toxins of comparable molecular weight.
Ion-exchange chromatography has been introduced into some refinement strategies as well, proving suitable for separation of F ab' 2 fragments from other plasma proteins under conditions preferring antibody adsorption on cation-exchange stationary phase material [ 18 ].
Additionally, it has been recognised also as a method of choice for controp final polishing where an anion-exchange approach is favourably used [ 25 ].
Other chromatography techniques are also applicable, for example, purification of F ab' 2 fragments by means of qualjty chromatography exclusively [ 26 ]. Our study Antivenmo for integrating the most efficient segments of the existing technological knowledge from the field into a compact, feasible and economically viable purification strategy measuures preparation of equine plasma-derived antivenom based on Quallty ab' 2 fragments.
At the same time, effort was put into the preservation of highest process qualtiy and fulfillment of the regulatory requirements concerning final product purity and aggregate content. The aim was also to precisely quantify the recovery of the active drug in each process step to enable accurate estimation of the cost-effectiveness of the designed procedure.
A standard mouse diet Mucedola srl. Animal monitoring for signs of pain, suffering and distress associated with procedure was performed following severity assessment protocol. Crude venom of V. ammodytes Vaa and two pools of Vaa -specific Antivenm horse plasma HHP were provided by the Institute of Immunology Inc.
Caprylic acid, o -phenylenediamine dihydrochloride OPDiodoacetamide IAAdithiothreitol DTTqualitu serum albumin BSAControol 20, thimerosal, 2- N-morpholino ethanesulphonic acid MES monohydrate and Tris base were from Sigma-Aldrich, USA.
Pepsin from porcine gastric mucosa, 0. Goat anti-horse F ab' 2 IgG conjugated with horseradish peroxidase HRP was from antibodies-onlineGermany. All other chemicals used for preparation of buffers and solutions were from Kemika, Croatia.
The bound antibodies were eluted with 20 mM citric acid, pH 2. A highly Antigenom IgG sample eIgG was used as standard in ELISA assay and as model substrate for preliminary optimisation of pepsin digestion. HHP was incubated at 56 °C for 1 h.
After centrifugation at 3, × g for 40 min ckntrol discarding the pellet, caprylic acid was added to 0. Precipitation was performed by vigorous controk rpm at 23 °C for 1 h in thermomixer Eppendorf, Germanyfollowed by sample quqlity 2, × g45 min.
IgG-enriched suality was collected and measuers through a cellulose acetate vontrol with a pore size of 5 μm Sartorius, Germany. Minimal caprylic acid concentration giving the highest IgG purity and preserving yield, as preliminary determined, was chosen as optimal for the precipitation.
Preliminary optimisation of pepsin digestion was done using a model IgG substrate—highly pure IgG sample eIgG isolated from HHP by protein A based affinity chromatography. Generally, substrate aliquots 2 mg mL -1 were pH adjusted using 0. Pepsin solution 5 mg mL -1 in 0.
The final volume of reaction mixture, prepared in saline, was 1 mL. Digestion was terminated at timed intervals with a 0. Since it was quailty possible to execute all runs simultaneously, their order conteol randomised quapity avoid systemic errors.
Antiveonm used a regression function model covering linear contribution of each factor, but also qualitt for selected qyality area. The full factorial design was ocntrol resulting in 4 experimental contrll, each performed in triplicate 2 2 × 3.
The significance of the given factors was determined by means of ANOVA using Statistica All subsequent experiments were performed using real process IgG substrate—IgG fraction from the optimised caprylic acid fractionation step, and cntrol one variable at a time.
The common approach involved acidification to pH 3. Incubation was performed at 37 °C for 1. When optimal conditions were set, the procedure was scaled up fold. Samples from each experimental set were analysed by SDS-PAGE.
IgG-enriched supernatant following caprylic acid precipitation was diafiltrated into water or saline using Vivaspin device Sartorius, Germany with a kDa molecular weight cut-off MWCO polyethersulfone membrane.
In each diafiltration step the buffer was exchanged by a factor of 8, ×. Elution was performed with 1 M NaCl in the binding buffer. The absorbance was monitored at nm. After collecting the flow-through fraction, the bound components were eluted from the column material with binding buffer containing 1 M NaCl.
The enzymatic activity of pepsin was measured measure on Multiskan Spectrum instrument Thermo Fischer Scientific, USA using haemoglobin as substrate. Modified Ryle's protocol was followed [ 31 ]. Samples previously diafiltrated into 50 mM KCl, pH 2. Aliquots of 40 μL were incubated with μL of 2.
Non-degraded substrate was precipitated by centrifugation at 2, × g for 10 min and absorbance of the supernatants was measured at nm. Blanks were obtained by omitting samples from reaction mixtures. Staining was carried out with acidic Coomassie Brilliant Blue CBB R solution or, alternatively, with silver for detection of pepsin traces.
Isoelectric suality, the first dimension of 2D gel electrophoresis, was performed in a ZOOM IPGRunner Mini-Cell Invitrogen, USA using immobilised pH gradient IPG strip 7 cm long, linear pH 3—10 Invitrogen, USA rehydrated with F ab' 2 sample μgaccording to the protocol provided by the manufacturer.
The following step voltage protocol was applied: V for 20 masures, V for 15 min, Measurs for 15 min and 2, V for min. Obtained spots served as starting material for mass spectrometry MS.
Excised protein spots obtained by 2D gel electrophoresis of F ab' 2 sample were prepared for MS analysis by in-gel trypsin digestion, contol follows. Following reduction and alkylation gel pieces were washed with mM NH 4 HCO 3 and ACN, dried and rehydrated in 1—10 μL of porcine trypsin solution Roche, Quaity 10 ng of trypsin per estimated 1 μg of protein for 45 min.
Antivenoj extracts were purified by C 18 Zip-Tips Millipore, USAdried, dissolved again in 0. Measurements were performed on an ultrafleXtreme Bruker, Germany in positive, reflectron ion mode.
The instrument is equipped with SmartBeam laser nmand the applied acceleration voltage was 8 kV in the positive ion mode. Obtained spectra were processed using FlexAnalysis 3. Following parameters were used: precursor ion quaity tolerance ± ppm, product ion mass ± 1.
Variable modifications such as N-acetylation, C-amidation, ammonia meaaures from N-terminal Cys, modification of N-terminal Gln to pyro-Glu, oxidation of Met, His or Trp and phosphorylation of Ser, Thr or Tyr were taken into account.
: Antivenom quality control measures| Advancing antivenom | University of Hawaii Cancer Center, Honolulu, Hawaii, USA. Reprints and permissions. Chippaux, J. Antivenom Safety and Tolerance for the Strategy of Snake Envenomation Management. In: Gopalakrishnakone, P. eds Snake Venoms. Springer, Dordrecht. Received : 09 October Accepted : 09 February Published : 04 March Publisher Name : Springer, Dordrecht. Online ISBN : eBook Packages : Springer Reference Biomedicine and Life Sciences Reference Module Biomedical and Life Sciences. Policies and ethics. Skip to main content. Abstract Discovered years ago, passive immunotherapy remains the sole etiological treatment of envenomations, particularly those caused by snakes and scorpions. Keywords Envenomation Antivenom Adverse reactions Treatment Immunoglobulin snake Scorpion. References Abroug F, Ouanes-Besbes L, Ouanes I, Dachraoui F, Hassen MF, Haguiga H, Elatrous S, Brun-Buisson C. 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Article PubMed Google Scholar Visser LE, Kyei-Faried S, Belcher DW, Geelhoed DW, van Leeuwen JS, van Roosmalen J. Article CAS PubMed Google Scholar Warrell DA. Article PubMed Google Scholar WHO. Chippaux Faculté de Pharmacie, University Paris Descartes, Sorbonne Paris Cité, Cotonou, France J. Chippaux Center for the Study and Research of Malaria Associated with Pregnancy and Childhood Cerpage , Cotonou, Benin J. Massougbodji Institute of Biotechnology, National Autonomous University of Mexico IBt-UNAM , Cuernavaca, Mexico R. Stock Authors J. Chippaux View author publications. View author publications. Editor information Editors and Affiliations Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore P. Gopalakrishnakone Biomedical Research Institute National Institute of Advanced Industria, Tsukuba, Ibaraki, Japan Hidetoshi Inagaki Department of Molecular Biology and Biot, Tezpur University, Tezpur, Assam, India Ashis K. Mukherjee Egyptian Society of Natural Toxins and Z, Faculty of Science Suez Canal University, Ismailia, Egypt Tarek R. Rahmy University of Hawaii Cancer Center, Honolulu, Hawaii, USA Carl-Wilhelm Vogel. As a negative control, BSA was used. The use of a specified amount of preservative m -cresol in PAV preparations for their long-term storage was approved by the WHO in The m -cresol content was determined by reversed-phase ultra-high performance liquid chromatography RP-UHPLC of SL PAV on an Acclaim C 18 RP-UHPLC column 2. The flow rate was 0. The isocratic programme for the mobile phase was optimized for 18 min The detection of m -cresol was observed at nm The percentage of m -cresol was determined against a standard curve of m -cresol run in UHPLC under identical experimental conditions. Thereafter, cell viability was determined by the MTT-based method 91 and the result was expressed as PAV-induced cell death in percentage , if any, with respect to control PBS-treated cells H 2 O 2 was used as a cytotoxic agent positive control. The immunological cross-reactivity of SL snake venoms against SL PAV and Indian PAV raised against venoms of the Big Four snakes of India was determined by ELISA and Western blot analysis 19 , 43 , 44 , Briefly, for ELISA ng of venom was coated for overnight at 4 °C in microtiter ELISA plate. After washing the wells by using washing buffer phosphate buffer saline containing 0. For negative control, the venom samples were treated with naïve horse IgG and developed in parallel. After incubation with primary antibody, the excess antibodies were washed using washing buffer and incubated with anti-horse IgG HRP-conjugated secondary antibody produced in rabbit for 2 h at room temperature dilutions. The reaction was stopped immediately by 2 M H 2 SO 4 and the absorbance was read at nm. For presenting the data, the absorbance values of PAV against venom samples was deducted from the absorbance of negative control. Immunoblotting experiments were performed as described previously by resolving the venom proteins in The SDS-PAGE protein bands were transferred to PVDF membrane in a semi-dry gel transfer system Amersham Bioscience, UK at 1. The transfer efficiency was checked by Ponceau S staining of the membranes. The excess unbound antibodies were washed with TBS-T and incubated with anti-horse IgG ALP-conjugated secondary rabbit anti-horse antibodies for 1 h at room temperature. Venom samples treated with horse naïve IgG served as negative control. Densitometry analysis of the blots was done using ImageQuant TL software 8. Briefly, venom 10 µg was pre-incubated with PAV µg in a predetermined ratio , protein: protein for 30 min at 37 °C followed by assaying the mixture for enzymatic activities and in vitro pharmacological properties of venom 44 , 45 , 51 , In vivo neutralization of lethality and other pharmacological effects of snake venom hemorrhagic activity, necrotizing activity, pro-coagulant activity, defibrinogenating activity, and myotoxicity of SL snake venoms by PAV raised against these snakes were evaluated in laboratory inbred Swiss albino mice males and females weighing between 18 and 20 g, age 3 to 4 weeks, following the WHO guidelines Ltd, Pune. Dry food pellets Nutritive Life Sciences, Pune and purified filtered water were provided ad libitum. To determine the LD 50 , graded concentrations of venom from each species of snake in 5 mL of normal saline were injected intravenously into a group of five mice. Animals were observed for 48 h and deaths during this period, if any, were recorded. The LD 50 was calculated by the Reed and Muench method by using the following formula. The mixtures were incubated for 30 min at 37 °C, and then aliquots of a precise volume maximum 0. After injection, deaths were recorded at 48 h intravenous test and the results were analyzed using Reed and Muench method. The ED 50 results were expressed as mg of venom neutralized by per mL of PAV and value was calculated using following formula After 3 h post injection, mice were euthanized using a carbon dioxide asphyxiation method. The area of the injected skin was removed and the size of the hemorrhagic lesion was measured using calipers in two directions. The mean diameter of the hemorrhagic lesion for each venom dose was calculated and the mean lesion diameter was plotted against each venom dose to determine the minimum hemorrhagic dose MHD. One unit of MHD is defined as the dose of venom that produces 10 mm diameter of skin hemorrhage. To determine the venom necrotizing activity, the above procedure was followed and the size of the dermonecrotic lesion was measured. The mean diameter of the dermonecrotic lesion for each venom dose was calculated and mean lesion diameter was plotted against venom dose to determine the minimum necrotic dose MND , which is defined as the venom dose that produces skin necrosis with a diameter of 5 mm. To determine the in vivo defibrinogenating activity, graded concentrations of venom in 0. injected in a group of five mice. After 1 h following the injection, blood was withdrawn by cardiac puncture from the anesthetized mice and transferred to glass tubes. Clot formation was observed visually by the tilting of the tubes. The minimum defibrinogenating dose MDD is defined as the amount of injected venom that does not show in vitro blood clot formation. Control animals received an injection of the same volume of normal saline. Blood was withdrawn from the tail tip of the anesthetized mice 3 h post injection and serum creatine phosphokinase CPK activity was determined using a commercial diagnostic kit Tulip Diagnostic, India 93 , For the neutralization assay, a challenge dose of venom was incubated with different concentrations of PAV at 37 °C for 30 min and the venom-antivenom mixture was injected into a group of five mice to determine the neutralization of the above mentioned pharmacological activities of venom 14 , The significance of difference for more than one set of data was analyzed by a one way ANOVA. Authors confirm that all methods were carried out in accordance with relevant guidelines and regulations and informed consent was obtained from all subjects. All the animal experimental protocols were approved by Premium Serum and Vaccines Pvt. Kasturiratne, A. et al. The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 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Article ADS PubMed PubMed Central CAS Google Scholar. Senji Laxme, R. Faisal, T. Proteomics , 1—13 Quantitative proteomic analysis of venom from Southern India common krait Bungarus caeruleus and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom. Expert Rev. Proteomics 16 , — Chanda, A. Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom. Proteomics 15 , — Proteomic analysis and antivenomics study of Western India Naja naja venom: Correlation between venom composition and clinical manifestations of cobra bite in this region. Deka, A. Proteomics of Naja kaouthia venom from North East India and assessment of Indian polyvalent antivenom by third generation antivenomics. Proteomics , Proteomic investigation of Sri Lankan hump-nosed pit viper Hypnale hypnale venom. Toxicon 93 , — Quantitative proteomics to reveal the composition of Southern India spectacled cobra Naja naja venom and its immunological cross-reactivity towards commercial antivenom. Toxicon 38 , — Gnanathasan, A. Saw-scaled viper bites in Sri Lanka: Is it a different subspecies? Clinical evidence from an authenticated case series. The Am. Katkar, G. NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction. Acta , — Rucavado, A. Characterization of aspercetin, a platelet aggregating component from the venom of the snake Bothrops asper which induces thrombocytopenia and potentiates metalloproteinase-induced hemorrhage. Thakur, R. Elucidation of procoagulant mechanism and pathophysiological significance of a new prothrombin activating metalloprotease purified from Daboia russelii russelii venom. Toxicon , 1—12 Book Google Scholar. Liu, C. Snake venom proteome of Protobothrops mucrosquamatus in Taiwan: Delaying venom-induced lethality in a rodent model by inhibition of phospholipase A2 activity with varespladib. Bhat, M. Isolation and characterization of a lethal phospholipase A2 NN-IVb1-PLA2 from the Indian cobra Naja naja naja venom. Preclinical evaluation of the efficacy of antivenoms for snakebite envenoming: State-of-the-art and challenges ahead. Toxins 9 , Article PubMed Central CAS Google Scholar. Syndromic approach to treatment of snake bite in Sri Lanka based on results of a prospective national hospital-based survey of patients envenomed by identified snakes. Karunanayake, R. A study of snake bite among children presenting to a paediatric ward in the main Teaching Hospital of North Central Province of Sri Lanka. BMC Res. Notes 7 , Ibrahim, N. Categorization of venoms according to bonding properties: An immunological overview. Shaikh, I. Development of dot-ELISA for the detection of venoms of major Indian venomous snakes. Toxicon , 66—73 Whitaker, R. Venom, antivenom production and the medically important snakes of India. Tumbare, S. Venom yield of major Indian poisonous Snakes in captivity. Bombay Vet. Daniel, J. The Book of Indian Reptiles and Amphibians — Bombay Natural History Society India, Englemann, W. Snakes: Biology, behavior and relationship to man. Croom-Helm Publishing Colondon , 51 Broad, A. Namely, each of the investigated pH values 4. The presence of 0. Although the highest F ab' 2 yield was achieved under conditions employing pH 4. A Detection of pepsin in Unosphere Q fractions by "negative" silver staining method. Molecular mass markers are at right side. Staining was performed with AgNO 3. Optimal conditions for pepsin removal from F ab' 2 preparation determined in a batch mode, i. those that allow adsorption of pepsin traces and other residual acidic impurities exclusively, at the same time enabling passing of the active principle through anion-exchange resin without binding, were transferred to column chromatography in the flow-through mode. Concerning the loading of the sample, two different approaches were investigated. Alternatively, the digestion product was first diafiltrated into binding buffer using a 50 kDa membrane pure F ab' 2 Figs 2E and 3A and then submitted to final polishing which produced completely pure F ab' 2 -based preparation ultrapure F ab' 2 sample without any loss Figs 2F and 3A. The recovery for the CIM QA chromatography step was ± 2. The final product was completely aggregate-free Fig 2F and depleted from pepsin, as confirmed by SDS-PAGE and absence of a ''negative'' band at position corresponding to its molecular weight following silver staining Fig 7B. The chromatographic behavior of pepsin was studied under conditions employed for the final F ab' 2 polishing by applying the enzyme preparation in quantities 10 times higher than that present in the digestion reaction mixture Fig 4C. Thus, the potential of CIM QA disk for pepsin binding and removal was verified. Manufacturing by-products that affect pepsin purity did not bind Fig 4D. No enzymatic activity could be detected in the flow-through fraction. In comparison to the sample applied to column 8. In 2D gel electrophoresis sample was intentionally overloaded so that even minor contaminants could be revealed. Predominantly segments of F ab' 2 fragments—different isoforms of heavy chain constant regions and light chain variable regions, were detected and identified Fig 8 , S1 Table. Of the rest discrete protein spots only transthyretin was confirmed while others remained unidentified. In the first dimension F ab' 2 μg was focused using IPG strip under denaturing conditions linear pH 3— Molecular mass markers are at left side. According to the lethal toxicity neutralisation assay in mice, each molecule of both active principle types exhibited comparable activity, proving that Fc portion is not relevant for neutralisation of venom toxins. Namely, 1 mg of IgGs from pure IgG sample is able to neutralise 1. Purification factors of 2. Purification factors obtained through manufacturing procedure are indicated. The production of immunotherapeutics has always been a struggle of finding balance between retaining the potency of the product and reducing the appearance of its side effect-inducing properties. From the standpoint of antivenom manufacturing, consistent quality, safety and clinical efficacy are usually ensured through removal of the immunogenic Fc part of the IgGs previously fractionated from other plasma proteins and purification of the F ab' 2 -based preparation from residual contaminants. Although deprived from innovative technological breakthroughs, our refining scheme Fig 9 provides a finely tuned approach through which high yield and fulfillment of regulatory demands in the most straightforward way were achieved. Also, since the process efficiency has been supported with quantitative data, economic feasibility can be easily evaluated. The development of the processing platform was demonstrated on HHP pool raised against V. ammodytes venom S2 Fig. The emphasis was put on the active principle handling by preserving it in solution throughout the manufacturing procedure. Unwanted precipitation of IgGs was avoided by employing caprylic acid-mediated fractionation as a method introduced by Steinbuch and Audran [ 9 ]. This method is generally considered to represent a mild treatment. Preferential use of caprylic acid in the range of 1. Higher concentrations are usually associated with excessive tubidity and slower filtration rates [ 18 , 36 , 38 , 39 ]. Fernandes et al. Since caprylic acid precipitation or pepsin digestion do not change IgG subclass distribution, ELISA with a sample-specific correction of results [ 33 ] was found suitable for precise quantification of active principle. For instance, the resolution of SEC does not allow detection of potential impurities such as IgA, IgE and ceruloplasmin, which overlap with major peaks corresponding to IgG and albumin, as already emphasised [ 41 ]. Densitometric analysis of CBB or silver stained protein bands in SDS-PAGE, another method of purity profiling, also exhibits major drawback since intensity of developed color highly depends on amino acid composition and usually has a limited dynamic range [ 42 ]. The majority of loss occurred during the heat treatment step, probably due to entrapment of portion of IgG molecules into the denatured fibrinogen network. The literature in general lacks supportive data concerning recovery. For instance, Rojas et al. Optimal conditions for pepsin digestion of equine IgGs, established on highly pure preparation from protein A chromatography, proved inadequate when crude IgG sample from precipitation step was employed as substrate because in the acidic environment residual caprylic acid provoked aggregation Fig 6. Therefore, diafiltration as an intermediate step was introduced, also contributing to purity increase Table 1 , which was comparable to that reported by other groups [ 19 , 37 , 41 ]. ELISA-based yield assessment was additionally supported by in vivo assay, revealing purification factor of about 2-fold Table 3. This is in line with literature as antivenoms derived from the whole IgGs purified by caprylic acid fractionation usually display low degree of aggregation [ 8 , 12 , 43 ]. In addition, protective efficacy of IgG preparation against V. ammodytes venom was congruent to that of the respective plasma pool Table 3 , indicating preservation of IgG subclasses and invariance of venom-specific antibody content through manufacturing process. As such, the pure IgG sample, although firstly regarded only as input material for subsequent antivenom manufacturing, later was recognised as possible production exit point as well due to compliance with regulatory frameworks concerning neutralisation potency and physicochemical profile [ 1 ]. Efficient pepsin cleavage aims for total IgG fraction breakdown and circumvention of over-digestion. The reaction should progress to the degree of Fc part removal only. In our protocol, complete removal yet limited hydrolysis was dependent on recognising 0. We considered the avoidance of any kind of burden for the manufacturing process by introducing purity-compromising reagents such as pepsin in unrationaly high amounts. Namely, even shorter reaction time can be used but apparently seeks for higher enzyme concentrations to achieve comparable recovery levels [ 26 ]. When diafiltration on 50 kDa membrane was employed for purity enhancement, basically only F ab' 2 product was detected Figs 2E and 3A. Since no evidence of high molecular weight material was observed, dissociation of aggregates due to buffer exchange and washing out of released protein segments is very likely. Physicochemical profile of the pure F ab' 2 sample was equally good or better from that of some other final F ab' 2 products generated by various methodologies from hyperimmune plasma or serum on laboratory scale [ 18 , 19 , 26 , 38 , 41 ]. As already noticed by Jones and Landon [ 25 ], diafiltration was only partially efficient at removing pepsin Fig 4A and 4B. Therefore, a third and final purification step was introduced. Polishing was performed by means of anion-exchange chromatography at pH 5. The methodology has already been successfully demonstrated on digestion product of ovine serum-derived IgG fraction [ 23 , 25 ]. SEC profile indicated that completely pure and aggregate-free F ab' 2 -based preparation was achieved Figs 2F and 3A. In order to get a deeper insight on its purity or contaminant profile, as additional insurance of the final product quality, 2D gel electrophoresis and MS analysis were performed. Among traces of impurities only transthyretin was identified Fig 8 , S1 Table. Other low-abundance protein spots did not contain sufficient material for successful MS analysis and their identification failed. Exceptionally high purity of the final product is creditable to supplementing action of diafiltration and anion-exchange chromatography. ELISA-based calculation has been supported by the result of a lethal toxicity neutralisation assay in mice Table 3. Functionality of the final product in terms of protective efficacy was comparable to that of the respective plasma pool and thus fully preserved. Thus, apart from quantity loss, reduction of neutralisation potency of F ab' 2 fragments due to denaturation induced by acidic conditions during pepsin digestion step can be excluded also, meaning that a good balance between pH level and reaction time was achieved. Others reported that performance of digestion at pH of 2. In addition, congruent protective efficacy of the final product and starting material points that no substantial IgG subclass loss and, consequently, redistribution of the venom-specific antibody content occurred. In conclusion, fractionation of the venom-specific plasma was efficiently performed on laboratory scale by sequence of optimised purification steps—precipitation of unwanted proteins by caprylic acid, removal of precipitating agent from IgG-enriched fraction, pepsin digestion, diafiltration of the obtained F ab' 2 preparation and its final polishing by flow-through chromatography. During the whole process IgGs or F ab' 2 fragments were kept in solution, ensuring quality and, therefore, safety of the final product. Manufacturing protocol has been performed independently several times on two plasma pools of slightly different protective efficacy. Also, two analysts were involved. Refining scheme resulted in the completely pure, aggregate- and pepsin-free active principle with overall yield advantageously comparable to others so far reported. Suitability for larger scale production, as well as estimation of its cost-effectivenes, should be determined through additional study, together with stability, pre-clinical and clinical efficacy of the final product prepared according to optimised procedure. List of identified proteins is given in S1 Table. Proteins are denoted by numbers as in S1 Fig. Other protein spots were assigned based on PMF spectra overlapping or remained unidentified. Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. Author summary Animal plasma-derived antivenoms constitute the most important therapy against snakebite envenoming. Introduction Antivenoms prepared from hyperimmune animal plasma, mostly equine or ovine, are the only specific therapeutics for rapid counteracting post-snakebite pathophysiological manifestations. Snake venom, plasma pools, animals and reagents Crude venom of V. Optimisation of IgG purification by caprylic acid precipitation HHP was incubated at 56 °C for 1 h. Pepsin digestion optimisation Preliminary optimisation of pepsin digestion was done using a model IgG substrate—highly pure IgG sample eIgG isolated from HHP by protein A based affinity chromatography. Diafiltration steps IgG-enriched supernatant following caprylic acid precipitation was diafiltrated into water or saline using Vivaspin device Sartorius, Germany with a kDa molecular weight cut-off MWCO polyethersulfone membrane. Pepsin activity The enzymatic activity of pepsin was measured spectrophotometrically on Multiskan Spectrum instrument Thermo Fischer Scientific, USA using haemoglobin as substrate. MALDI-MS analysis Excised protein spots obtained by 2D gel electrophoresis of F ab' 2 sample were prepared for MS analysis by in-gel trypsin digestion, as follows. Protein concentration determination Throughout the isolation procedure total protein concentration was estimated spectrophotometrically by use of the Eq 2 [ 32 ], 2 where Ehresmann's factor " f " for equine IgG of 0. Production process yields and sample purity calculation Concentrations determined by ELISA assays were used for yield and purity calculations. Download: PPT. Fig 1. Preliminary determination of optimal caprylic acid concentration for precipitation step of the purification protocol. Fig 2. The assessment of purification steps by size-exclusion chromatography. Table 1. Purities and yields of the intermediates and the final product obtained by developed downstream processing protocol. Pepsin characterisation Commercial pepsin preparation involved in the manufacturing procedure had 7 times lower total protein concentration in comparison to the one derived from the weighted mass. Fig 4. Verification of pepsin removal by the final polishing procedure. Optimisation of pepsin digestion Preliminary screening of digestion conditions. Pepsin digestion on IgG obtained by caprylic acid precipitation. Fig 6. Fig 7. SDS-PAGE analysis of F ab' 2 preparation after flow-through chromatography under different pH conditions and detection of pepsin traces. Table 2. Characterisation of the unbound fractions following incubation of F ab 2 preparation containing pepsin with UNOsphere Q stationary phase under variuos pH conditions. Final polishing step. Efficacy of flow-through chromatographic final polishing in pepsin removal. Protective efficacies of IgG and F ab' 2 preparations. Table 3. Discussion The production of immunotherapeutics has always been a struggle of finding balance between retaining the potency of the product and reducing the appearance of its side effect-inducing properties. Fig 9. Flow sheet of downstream processing steps with corresponding samples and performance rationales. Supporting information. S1 Fig. s TIF. S2 Fig. |
| References | Among traces of impurities only transthyretin was identified Fig 8 , S1 Table. Modified Ryle's protocol was followed [ 31 ]. WHO guidelines for the production, control and regulation of snake antivenom immunoglobulins. Google Google Scholar. Fernández I, Lima EX, Takehara HA, Moura-da-Silva AM, Tanjoni I, Gutierrez JM. Calmette's groundbreaking work with equine antiserum resulted in the first, unrefined antivenom in Sci Rep 11 , |
| SYSTEMATIC REVIEW article | This cycle has also contributed to conditions that have allowed lesser quality products and inappropriate marketing to emerge. The arrival of new manufacturers and the presence of spare capacity within some current facilities provide hope, but uncertain market conditions and inadequate financial support will continue to restrict growth of trustworthy antivenoms. Inadequate financial support for antivenom production and variable quality have catalysed the collapse of the antivenom market, which is now characterised by deficient supply, deficient quality control, rising prices and poor profitability. This cycle is a variation on that proposed by Stock et al in [9] , and demonstrates the importance of future financial stimulus in reinvigorating competition and viability of the antivenom market. Inadequate financing within the antivenom industry is the major factor underpinning its decline over the last 40 years, and strategies to solve this crisis must recognise and unwind the economic and commercial drivers on both sides of the supply and demand equation. It is unrealistic to expect that pharmaceutical companies will commit to long-term production of antivenom for an inconsistent and unreliable market that is starved of investment. Even if greater volumes of appropriate antivenom could be produced, without adequate subsidisation it will be priced out of range for most snakebite victims living in underprivileged rural and remote areas. Similarly, corporate executives and regulatory bodies must also accept that there exists a moral imperative for them to contribute their expertise and capabilities, and that existing business models and production frameworks may be inappropriate for the supply of humanitarian products to developing countries. Encouragingly, there has been a small increase in financial support for the development and procurement of new African antivenoms between and Better utilisation of spare production capacity and improved economies of scale will produce greater yields, reduce costs, increase revenues and further enhance the commercial viability of antivenoms. The second major problem eroding the antivenom market is the lack of accountability in quality standards. Possessing the capacity to produce vast amounts of antivenom for sub-Saharan African communities is meaningless if the products are poorly made and ineffective against the snakes in those regions. A current lack of interest, insufficient investment and poor competition are allowing unscrupulous behaviours within the marketplace to go unchecked. Given the ongoing severe shortage of antivenom and the continuing high incidence of envenoming, it is not surprising that opportunistic manufacturers seek to fill the void. The advent of seemingly inexpensive, but low quality or inappropriate antivenoms with poor neutralising ability, not only compromises the reputation of antivenoms in general but also drains important financial resources away from proven snakebite treatment programs and products. Some manufacturers have cited this uneven playing field as a key impediment to future innovation and productivity. Nevertheless, the very high volume output by some manufacturers of alleged inappropriate products still make them key players in the antivenom industry, and potentially integral to future strategies for increasing output of higher quality products. Improving standards and maximising efficiencies ought to be the common goal for all manufacturers. The three groups with emerging new African antivenoms provide hope for the future [41] — [44] , however ensuring that these products, as well as existing antivenoms, are of sufficient quality to be incorporated into a properly funded and sustainable market is paramount [8]. The final quality control checkpoint for all antivenoms entering a country should be the national regulatory authorities. It is essential that NRAs are adequately resourced and transparent to ensure the integrity and robustness of their mechanisms are above reproach. Linking funds for antivenom procurement to improved quality control and assurance measures would enhance the crucial role of local regulatory bodies and incentivise the maintenance of minimum standards. Antivenom's usually rapid and curative effects make it a highly cost-effective intervention [40] , and together with snakebite's surpassing morbidity and mortality [6] , ought to attract attention from global health funding bodies. Leadership and support from groups such as the Global Snakebite Initiative and the World Health Organisation may help to secure essential funds from donors and provide important coordination, transparency and accountability. It will also help to recruit and reform manufacturers capable of contributing a greater supply of effective and appropriate antivenoms. The declining availability of high quality antivenom in sub-Saharan Africa is a real and unnecessary tragedy, and constitutes a major neglected global health concern. The amount of suitable antivenom marketed in these countries has fallen to crisis levels, representing only a fraction of the amount required. Although recent output of antivenom for Africa has increased, and the number of manufacturers able to boost production is growing, inadequate financial support and market uncertainty continue to suppress growth and compromise quality standards. The provision of sufficient funds to identify satisfactory antivenoms, maintain quality control, maximise efficiencies and increase procurement is desperately needed to break the vicious cycle that currently constrains the antivenom industry. The mechanisms to achieve this are realistic and available; science, business and government must collaborate to secure a brighter future for snakebite victims in developing countries. Only then will the goal of providing effective, safe and affordable antivenoms to all who need them, be realised. The author would like to acknowledge the support and feedback received from Drs Linda Allan and Ken Seamon from the University of Cambridge Institute of Biotechnology; David Williams and Ken Winkel from the Australian Venom Research Unit; Profs John Landon and David Warrell; Bridget and Abigail Brown; and The Hawker Scholarship Foundation. Conceived and designed the experiments: NIB. Performed the experiments: NIB. Analyzed the data: NIB. Wrote the paper: NIB. Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Background The worldwide neglect of immunotherapeutic products for the treatment of snakebite has resulted in a critical paucity of effective, safe and affordable therapy in many Third World countries, particularly in Africa. Methods A global survey of snake antivenom products was undertaken in , involving 46 current and former antivenom manufacturers. Conclusion Financial stimulus is urgently needed to identify and develop dependable sources of high-grade antivenoms, support current and emerging manufacturers, and capitalise on existing unutilised production capacity. Author Summary Antivenom is the only specific treatment for systemic envenoming from snakebite, but remains unavailable to thousands of snakebite victims around the world. Lalloo, Liverpool School of Tropical Medicine, United Kingdom Received: June 7, ; Accepted: April 19, ; Published: June 5, Copyright: © Brown. Introduction Snakebite is a significant social and economic problem in many developing countries, however its victims rank among those most neglected by global health campaigns. The rise and fall of antivenom Since Edward Jenner's controversial inoculation of James Phipps with cowpox in , immunotherapy has developed into a diverse industry [4]. Methods Data for this paper was collected from primary and secondary sources, including interviews, surveys, product inserts and literature searches. Companies responded with information regarding the following: quantity and type of antivenom produced, including number of unsold vials; average number of snake antivenom vials required to successfully treat a moderately severe snake envenomation; countries where antivenom is marketed; wholesale cost of antivenom; existing spare production capacity; quality control and regulatory standards; profitability of antivenom products; and attitudes about the use, availability and sustainability of antivenom. Results 1. Epidemiological estimates of antivenom requirement The global incidence of clinically significant snakebite has been calculated to be between , and 2. Current African antivenom market a. Manufacturers table 1. Download: PPT. Table 1. Recent and current sub-Saharan African antivenom manufacturer. Antivenom output and capacity. Table 3. Antivenom quality. Antivenom cost. Antivenom formulation. Global antivenom market a. Antivenom quality and formulation. Attitudes to future antivenom production All companies currently producing antivenom for sub-Saharn Africa indicated a willingness to increase output should market demand improve. Whilst not all manufacturers listed the same reasons, there was some concordance and the responses below have been listed in descending order of frequency: Lack of consistent market demand for antivenom products; Inconsistencies with manufacturers receiving payment. Corruption within some global markets and government agencies; Threats from black market re-sale of antivenom products; Lack of appropriate venom for immunogen preparation, A lack of certainty regarding appropriate distribution of their products; Inappropriate clinical use of antivenom products; Lack of adequate animals for raising antisera; and High costs of maintaining livestock for antivenom production;. Discussion This survey of antivenom manufacturers highlights the paucity of antivenom products for sub-Saharan Africa and the unhelpful variability that exists within the current industry. Figure 2. The self-perpetuating cycle responsible for the decline in antivenom production in sub-Saharan Africa. Acknowledgments The author would like to acknowledge the support and feedback received from Drs Linda Allan and Ken Seamon from the University of Cambridge Institute of Biotechnology; David Williams and Ken Winkel from the Australian Venom Research Unit; Profs John Landon and David Warrell; Bridget and Abigail Brown; and The Hawker Scholarship Foundation. Author Contributions Conceived and designed the experiments: NIB. References 1. Winkel K, Mirtschin P, Pearn J Twentieth century toxinology and antivenom development in Australia. Toxicon — View Article Google Scholar 2. Abubakar IS, Abubakar SB, Habib AG, Nasidi A, Durfa N, et al. PLoS Negl Trop Dis 4: e View Article Google Scholar 3. Gold SG, Dart RC, Barish RA Bites of venomous snakes. N Engl J Med — View Article Google Scholar 4. Gutierrez JM Animal Antisera Production; Quality, safety and efficacy problems, Instituto Clodomiro Picado, Universidad de Costa Rica. Presentation to WHO. Available at: www. Accessed Nov Harrison RA, Hargreaves A, Wagstaff SC Snake Envenoming: A disease of poverty. PLoS Negl Trop Dis 3: e View Article Google Scholar 6. Williams DJ, Gutierrez JM, Harrison R, et al. Lancet 89— View Article Google Scholar 7. Theakston D, Warrell D Crisis in snake antivenom supply for Africa. The Lancet View Article Google Scholar 8. Williams D, Gutierrez JM, Calvette JJ, et al. Journal of Proteomics — View Article Google Scholar 9. Chippaux JP The development and use of immunotherapy in Africa. View Article Google Scholar Stock RP, Massougbodji A, Alagon A, Chippaux JP Bringing antivenoms to sub-Saharan Africa, Nature Biotechnology — WHO Snake Antivenoms. Fact Sheet no. Warrell DA Unscrupulous marketing of snakebite antivenoms in Africa: what's in a name? Trans R Soc Trop Med and Hygiene — Chippaux JP Evaluation de la situation épidemiologique et des capacités de prise en charge des envenimations ophidiennes en Afrique subsaharienne francophone, Bull Soc Path Exo Chippaux JP Perspectives in Molecular Toxinology. Ménez A, editor. WHO Rabies and envenomings: a neglected public health issue. Accessed , April Kasturiratne A, Wickremasinghe AR, de Silva N, et al. PLoS Med 5: e Chippaux JP Snake-bites: appraisal of the global situation. Bull World Health Organ — Chippaux JP, Diof A, Stock RP, et al. Toxicon —9. Swaroop S, Grabb B Snakebite mortality in the world. Bull World Health Organ 35— WHO WHO guidelines for the production, control and regulation of antivenom immunoglobulins. Released Chippaux JP, Ramos-Cerrillo B, Stock RP Study of the efficacy of the black stone on envenomation by snake bite in the murine model. Chippaux JP Estimate of the burden of snakebite in sub-Saharan Africa: a meta-analytic approach. Snow RW, Bronzan R, Rogest T, Niamawi C, Murthy S, et al. Ann Trop Med Parasitol This study reflects that there is a general lack of knowledge transfer amongst various actors: most producers, health authorities, and experts expect increased and improved communication and guidance from leading international bodies. Due to the low response rates observed in this study, conclusions drawn herein are not representative of the global situation; yet provide an exploratory insight on the difficulties facing antivenom management. Full Text. Additional File 1 Download Additional File 2 Download Additional File 3 Download. Chippaux J. Snakebites: appraisal of the global situation. Bull World Health Organ ; Kasturiratne A, Wickremasinghe AR, de Silva N, Gunawardena NK, Pathmeswaran A, Premaratna R, et al. The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med ; 5: e Rabies and Envenomings - A neglected public health issue. Geneva: WHO; Harrison RA, Hargreaves A, Wagstaff SC, Faragher B, Lalloo DG. Snake envenoming: a disease of poverty. PLoS Negl Trop Dis ; 3: e Snakebite mortality in India: a nationally representative mortality survey. PLoS Negl Trop Dis ; 5: e Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med ; 3: e Omics meets biology: application to the design and preclinical assessment of antivenoms. Toxins Basel ; 6: Crisis in snake antivenom supply for Africa. Lancet ; Estimating the global burden of snakebite can help to improve management. Snakebite envenoming from a global perspective: Towards an integrated approach. Toxicon ; A multicomponent strategy to improve the availability of antivenom for treating snakebite envenoming. Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. J Proteomics ; The need for full integration of snakebite envenoming within a global strategy to combat the neglected tropical diseases: the way forward. PLoS Negl Trop Dis ; 7: e Antivenoms for the treatment of snakebite envenomings: the road ahead. Biologicals ; Snake Antivenoms: Adverse Reactions and Production Technology. J Venom Anim Toxins incl Trop Dis ; 3. Snake venoms and antivenoms: critical supply issues. J Assoc Physicians India ; Brown NI. Consequences of neglect: analysis of the sub-Saharan African snake antivenom market and the global context. PLoS Negl Trop Dis ; 6: e Guidelines for the production control and regulation of snake antivenom immunoglobulins. |
| Industrial Production and Quality Control of Snake Antivenoms | SpringerLink | However inconsistent market demand, unpredictable financial investment and inadequate quality control discourage further production and threaten the viability of the antivenom industry. Financial stimulus is urgently needed to identify and develop dependable sources of high-grade antivenoms, support current and emerging manufacturers, and capitalise on existing unutilised production capacity. Investing to ensure a consistent and sustainable marketplace for efficacious antivenom products will drive improvements in quality, output and availability, and save thousands of lives each year. Antivenom is the only specific treatment for systemic envenoming from snakebite, but remains unavailable to thousands of snakebite victims around the world. A cycle of inconsistent and low market demand, sub-optimal utilisation, rising costs and reduced output of antivenoms have resulted from long term under-investment in procurement and quality regulatory programs. This study provides a contemporary overview of the African antivenom market within the context of the global market. Globally, 35 companies sold at least 4 million vials of antivenom in Combined unutilised production capacity far exceeds the total projected antivenom needs for Africa. Citation: Brown NI Consequences of Neglect: Analysis of the Sub-Saharan African Snake Antivenom Market and the Global Context. PLoS Negl Trop Dis 6 6 : e Received: June 7, ; Accepted: April 19, ; Published: June 5, Copyright: © Brown. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was made possible because of a scholarship by the Hawker Foundation, awarded to assist research at The University of Cambridge. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The author has read the journal's policy and has the following conflicts: A consultancy with MicroPharm, one of the companies mentioned in this research. However, the majority of this research was undertaken prior to the author's association with MicroPharm. No financial incentive was received for or as a result of this research. Snakebite is a significant social and economic problem in many developing countries, however its victims rank among those most neglected by global health campaigns. Snakebite was recognised by the WHO as a Neglected Tropical Diseases in , and antivenom — the only specific treatment for systemic envenoming - remains largely inaccessible to hundreds of thousands of snakebite victims around the world. Since its introduction and continued refinement throughout the twentieth century, antivenoms have saved countless lives [1]. Whilst good quality products do exist in some developing countries its procurement is often inadequate, leaving snakebite victims without access to proper treatment. Quantifying the gap between what is currently available and what is needed is a critical step towards developing effective solutions to this problem. This study provides a contemporary overview of global antivenom production, focusing particularly on the antivenom market in sub-Saharan Africa. Since Edward Jenner's controversial inoculation of James Phipps with cowpox in , immunotherapy has developed into a diverse industry [4]. Calmette's groundbreaking work with equine antiserum resulted in the first, unrefined antivenom in Pope's improvements to antivenom refinement in the s were another major step forward in safety and potency of antivenom. Unfortunately, further advances since then have been limited. Despite snakebite being over-represented in morbidity and mortality tables [5] , investment in this type of immunotherapy has not been characterised by the same level of publicity or resolve that has characterised vaccine production or monoclonal antibody research. This under-recognition of bites and stings as major medical and social problems, and snakebite's association with poverty, have contributed to the current antivenom crisis [6]. The introduction of antivenom to Africa in the s heralded a decline in morbidity and mortality from snakebites that led to its widespread use and production. Sadly, over the last 30 years, production of this life-saving medication has been neglected by governments and non-government organisations, and abandoned by some manufacturers [7]. The s and s were characterised by a decline in the sale of antivenom in Africa due to growing neglect and prohibitive costs [8]. The WHO has estimated that antivenom supply failure in Africa is imminent [11] , which is further compounded by the presence of non-specific or fake products, inappropriate clinical use and poor community awareness of the benefits of antivenom [12] — [14]. Data for this paper was collected from primary and secondary sources, including interviews, surveys, product inserts and literature searches. Market research surveys were sent to representatives from 46 known antivenom manufacturers in Previous, current and future producers for sub-Saharan African markets were again contacted in and Companies responded with information regarding the following:. Independent testing of potency and proteomic analysis to validate the species of origin was outside the scope of this study, although verification was sought through literature reviews. The global incidence of clinically significant snakebite has been calculated to be between , and 2. Inadequate record keeping and limited primary epidemiological studies makes accurate assessment difficult, and most authors concur that estimates of snakebite incidence under-represent the problem. However a recent metaanalytical study of reported data concluded that probably , snakebites occur in Africa annually [22]. At least 20, deaths each year are attributed to snakebite in Africa [17] , although this is also considered conservative. Other debilitating morbidities result from the neurotoxic, coagulopathic or necrotic components of different venoms, with clinical effects ranging from chronic ulceration, osteomyelitis, chronic renal failure, endocrine disorders, paralysis, stroke and blindness. Three other institutions are developing antivenom against African snake species that have either recently been licensed or are in the final stages of development. Data on the planned output of antivenoms for Africa from these organisations is either not yet available or for experimental purposes only. A further three groups based in Egypt, Saudia Arabia and Iran produce antivenom against snake species found in West Asia and the Arabian peninsula, which may have efficacy against some North African snake species. Another organisation, based in Colombia, appears to have suspended development of a pan-African antivenom after conducting preclinical work in By comparison, , vials of sub-Saharan African antivenom were marketed to African countries in , providing just over 54, average treatments table 3. In , manufacturers reported a combined excess supply of more than 26, vials of unsold African antivenom. By no manufactured antivenom was unsold, however significant unutilised production capacity was reported by 5 of the 8 current producers, including two with manufacturing facilities and quality control procedures regulated by the European Medical Agency EMEA. If utilised, this combined capacity could produce enough antivenom to treat , patients and save thousands of lives. It is evident from product inserts and literature reviews that the potency of antivenom sold in sub-Saharan Africa varies widely. The average number of antivenom vials required to achieve effective neutralisation of a moderate envenoming, based on manufacturers' recommended doses, is 4. Doses for severe envenomings can be several times greater. The actual number of effective antivenom treatments available in Africa, therefore, is potentially only a fraction of the 83, stated above, and may cover as little as 2. Of the 8 current and pending producers of sub-Saharan African antivenoms, 6 manufacture solely polyspecific products, one produces only monospecific, and one produces both polyspecific and monospecific antivenoms. One company utilises ovine antisera instead of equine, and 6 offer lyophilised products. In , 46 one-time antivenom manufacturers across 28 countries were surveyed and 35 reported current production of at least one type of snake antivenom for commercial, government or research purposes. Eleven organisations listed in various media as antivenom manufacturers either no longer produce snake antivenom or did not respond to the survey. Twenty-four of the 35 organisations producing antivenom operate on a commercial basis; 6 were purely government facilities manufacturing non-commercial antivenom for domestic purposes; and 5 companies did not provide financial data. Total global snake antivenom output by surveyed companies exceeded 4 million vials, although this equated to fewer than , effective treatments. This is well below the WHO's worldwide estimated requirement of at least 2 million treatments per year. Globally, twelve manufacturers reported having capacity to increase volume, which if realised could potentially double the current output. There is a clear relationship between wholesale cost of antivenom and throughput Figure 1 , which has important implications for strategies seeking to increase the amount of antivenom produced globally. It was estimated by one company that costs could be reduced 5-fold from an 8-fold increase in output. However, the retail price of antivenom is also heavily influenced by the market's ability to pay for it. On a per vial basis, antivenom developed for use in high-income countries is disproportionately more expensive, represented by the two out-lying plot points in Figure 1. Economies of scale mean that the cost per ampoule decreases as throughput increases. All companies currently producing antivenom for sub-Saharn Africa indicated a willingness to increase output should market demand improve. Manufacturers identified factors that prevented them from raising production, despite a willingness to do so. Whilst not all manufacturers listed the same reasons, there was some concordance and the responses below have been listed in descending order of frequency:. This survey of antivenom manufacturers highlights the paucity of antivenom products for sub-Saharan Africa and the unhelpful variability that exists within the current industry. It also illustrates that despite the exodus of manufacturers in the s and s, willing producers do exist and they possess substantial unutilised production capacity. Unfortunately, inadequate government and non-government funding for procurement and regulatory oversight restrains production of commercial antivenom. This lack of investment is not only the reason for the current crisis in antivenom availability, but also represents the greatest challenge to future improvements in quantity and quality. Although inexpensive and efficacious antivenoms do exist, and compelling moral and legal arguments advocate increased purchase and distribution [39] , [40] , a lack of funding for antivenom acquisition and regulation of quality standards has catalysed the vicious cycle responsible for the decline in production and use over the last 30 years figure 2. This cycle has also contributed to conditions that have allowed lesser quality products and inappropriate marketing to emerge. The arrival of new manufacturers and the presence of spare capacity within some current facilities provide hope, but uncertain market conditions and inadequate financial support will continue to restrict growth of trustworthy antivenoms. 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