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Antivenom manufacturing

Antivenom manufacturing

Densitometric analysis manufactyring CBB or Balanced nutrition tips stained manufqcturing bands in SDS-PAGE, manufactruing method Energy balance and macronutrient distribution purity manufacyuring, Antivenom manufacturing exhibits Antivenom manufacturing drawback since intensity of developed color highly manufcaturing on amino acid composition and usually has a limited dynamic range [ 42 ]. IgG fraction acidified to pH 3. J Chromatogr B Echis ocellatusEchis leucogasterEchis carinatusBitis arietansBitis rhinocerosBitis nasicornisBitis gabonicaDendroaspis polylepisDendroaspis viridisDendroaspis angusticepsDendroaspis jamesoniNaja nigricollisNaja melanoleuca and Naja haje. Alien Black Goo? Nowadays this critical treatment has been faced by severe shortage due to low sustainability of current productions, which mostly affects developing countries as those suffering from highest morbidity and mortality rates. Table 3.

Antivenom manufacturing -

Article CAS PubMed Central PubMed Google Scholar. Zychar BC, Castro Jr NC, Marcelino JR, Gonçalves LR. Phenol used as a preservative in Bothrops antivenom induces impairment in leukocyte-endothelial interactions. Download references. Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.

You can also search for this author in PubMed Google Scholar. Correspondence to Guillermo León. Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Biomedicina de Valencia IBV-CSIC, Valencia, Spain. Reprints and permissions. León, G. et al. Industrial Production and Quality Control of Snake Antivenoms. In: Gopalakrishnakone, P. eds Toxinology. Springer, Dordrecht. Received : 01 May Accepted : 01 May Published : 29 May Publisher Name : Springer, Dordrecht.

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Abstract The production of snake antivenoms involves stages such as production of venom, immunization of animals to generate hyperimmune plasma, immunoglobulin purification, viral inactivation or removal , and stabilization of the formulation.

Keywords West Nile Virus Snake Venom Caprylic Acid Viral Inactivation Snake Species These keywords were added by machine and not by the authors.

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Article CAS PubMed Google Scholar Wang W. Article CAS PubMed Google Scholar Wang W, Singh S, Zeng D, King K, Nema S. 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, Germany 10 ng of trypsin per estimated 1 μg of protein for 45 min.

Pooled extracts were purified by C 18 Zip-Tips Millipore, USA , dried, dissolved again in 0. Measurements were performed on an ultrafleXtreme Bruker, Germany in positive, reflectron ion mode. The instrument is equipped with SmartBeam laser nm , and 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 mass tolerance ± ppm, product ion mass ± 1. Variable modifications such as N-acetylation, C-amidation, ammonia loss 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.

Proteins were confidently identified by peptide mass fingerprint PMF and peptide sequencing if statistical scores were above respective threshold levels. 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.

Appropriate dilution of each sample was independently prepared three times to obtain the mean value of the measured concentrations for further calculation of yield and purity. The effluent was monitored at nm. Correction factor corresponding to deviation of molecular mass of analysed IgG, determined according to calibration curve, from its nominal molecular mass, was included in the calculation.

After blocking with 0. In IgG ELISA affinity purified IgG eIgG of precisely determined protein concentration served as a standard. was used as a standard. In the subsequent steps of ELISA, incubation with HRP-anti-horse F ab׳ 2 IgG 25,fold diluted at 37 °C for 2 h occurred, followed by the addition of OPD 0.

After 30 min of incubation in the dark, the enzymatic reaction was stopped with 1 M H 2 SO 4 and the absorbance at nm was measured. Namely, for IgG determination a highly pure IgG-based product pure IgG sample in Fig 9 , which was processed from the respective HHP and precisely quantified, served as internal, sample-specific reference.

Concentrations determined by ELISA assays were used for yield and purity calculations. Additionally, SEC monitoring for purity profiling throughout the manufacturing procedure was included also.

The lethal toxicity neutralisation potency R was expressed as the number of LD 50 venom doses that can be neutralised by 1 mL of undiluted sample and calculated by the Eq 3 , 3 where Tv represents the number of LD 50 venom doses inoculated per mouse.

R -value was used as a measure of the protective efficacy of each sample. Specific activity LD 50 mg -1 was expressed as ratio of R -value and either active principle IgG or F ab' 2 or total protein concentration.

Number of measurements for each analysis n is given. Thus, only samples fractionated by lower concentrations were further analysed for IgG and total protein content. Higher concentration did not exhibit any obvious beneficial effect. SEC analysis of samples from two initial steps, heat-treatment and precipitation, confirmed significant reduction of the non-IgG protein content in supernatant as well Fig 2A and 2B.

SDS-PAGE profiles are shown in Fig 3A lanes 1 or 2 and 4. The molecular mass of equine IgG, assessed by SEC, was Analysis was performed on TSK-Gel GSWXL column 7. Heat-treated plasma A. F ab' 2 fraction produced by pepsin digestion of IgG preparation before crude F ab' 2 ; D and after diafiltration using a 50 kDa membrane pure F ab' 2 ; E.

Ultrapure F ab' 2 preparation—flow-through fraction from anion-exchange chromatography performed at pH 5. Detection: UV at nm. SDS-PAGE analysis of representative samples from purification process A and pepsin preparation B. Lane 1 and 2, hyperimmune plasma pools; lane 3, molecular weight standards; lane 4, IgG fraction obtained by caprylic acid precipitation crude IgG ; lane 5, IgG fraction after diafiltration pure IgG ; lane 6, F ab' 2 fraction produced by pepsin digestion of IgG preparation crude F ab' 2 ; lane 7, F ab' 2 preparation after diafiltration pure F ab' 2 ; lanes 8 and 9, F ab' 2 preparation polished using CIM QA chromatography ultrapure F ab' 2.

Staining was performed with CBB R 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.

SEC profile corroborated the obtained results concerning composition of the enzyme preparation, revealing that only SEC analysis of pepsin sample on TSK-Gel GSWXL column 7. Manufacturing by-products that affect pepsin purity lacked capability of binding.

Size-exclusion chromatography of flow-through D and elution fraction E from anion-exchange chromatography in C. Pepsin digestion was preliminary optimised on model IgG sample prepared by affinity chromatography eIgG. However, the experimental runs were analysed by SDS-PAGE and conclusions were drawn from differences in their protein pattern and intensity of detected bands.

Incubation at 20 °C RT 12 runs , which was included in the analysis due to possible co-performance of caprylic acid precipitation together with enzymatic cleavage, did not support digestion irrespective of the reaction mixture's pH or pepsin concentration Fig 5A , lane 1.

Similarly, at 37 °C, the standard temperature for most enzymatic reactions, a great fraction of eIgG sample remained intact when pH 3. On the other hand, by adjusting pH to 3. Incubation at 56 °C 12 runs , which was chosen with the idea of eventual simultaneous performance of HHP defibrinogenation and pepsin digestion, proved inappropriate Fig 5A , lane 5.

It provoked further F ab' 2 degradation—very faint bands at kDa or their complete absence were observed. All experimental runs with fully completed IgG hydrolysis performed at pH 3. Lane 1, typical digestion pattern after incubation at 20 °C; lane 2, typical digestion pattern after incubation at 37 °C when pH was set to 3.

B Optimisation of pepsin digestion with respect to duration and pepsin to IgG ratio studied according to full factorial experimental design. C Impact of pepsin to IgG ratio on F ab' 2 yield measured by ELISA assay.

In the second experiment, set at pH 3. The whole IgG population underwent cleavage in all experimental runs, which was a necessary prerequisite for ELISA-based quantification of F ab' 2 fragments.

The recovery was negatively influenced by the reaction time longer incubation lowered yields Fig 5B. The main effect estimate E X for factor X1 duration of incubation was The possibility that its effect was a result of coincidence is lower than 0. First two experiments of digestion conditions on a model substrate, eIgG, revealed that its full cleavage with the highest yield was achieved when performance conditions were: pH 3.

The experiment indicated gradual decline of F ab' 2 concentration with the increase of enzyme content Fig 5C. Firstly, the mock experiment was performed in which the behavior of IgGs was studied in the absence of pepsin, but under conditions used in digestion step pH 3. The starting material, crude IgG from caprylic acid precipitation, was composed of monomers mostly, as revealed by SEC, and a peak corresponding to aggregates comprised only 3.

When exposed to acidic environment a rather high IgG portion became prone to aggregation. Raising the pH to 4. These findings demonstrate that the type of alkalising reagent has no bearing on the results.

According to SEC, the greater difference between initial and adjusted pH was associated with the more prominent multimers of even higher molecular weight. As potential IgG stabilisers glycine 0. Moreover, the aggregation was irreversible since it could not be alleviated by subsequent elimination of caprylic acid by diafiltration.

IgG fraction acidified to pH 3. F ab' 2 fraction produced by pepsin digestion in the presence of caprylic acid D. Furthermore, a crude IgG sample supernatant after precipitation step was depleted from remaining caprylic acid by diafiltration on a kDa membrane either into water or 0.

Diafiltrated preparation was assigned as pure IgG sample. Its aggregate content was 1. Following pepsin digestion in water, an aggregate-free preparation was obtained, but substrate hydrolysis was incomplete.

When 0. Pepsin digestion resulted in crude F ab' 2 sample. Since saline proved to be more supportive for hydrolysis than water, the same formulation was used for all previous steps requesting dilution or washing out procedures. Under optimised conditions no significant loss with respect to IgG sample diafiltrated into 0.

The calculated ratio of 0. So it was further used for the final polishing step. 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.

Download as PDF Printable version. Medication used to treat bites by venomous snakes. WHO Model Formulary World Health Organization. ISBN Nature Communications. Bibcode : NatCo.. doi : PMC PMID Geneva: World Health Organization. License: CC BY-NC-SA 3.

WHO Expert Committee on Biological Standardization, sixty-seventh report. Geneva, Switzerland: World Health Organization WHO. ISSN WHO technical report series; Archived PDF from the original on Retrieved Archived from the original on 3 May Retrieved 9 January Archived from the original on 10 January Retrieved 25 July Food and Drug Administration.

Archived from the original on 3 March Retrieved 19 March Orlando Sentinel. Archived from the original on 24 May Retrieved 25 May Popular Mechanics. Archived from the original on Archived from the original on 13 October Poison Center Tampa. Archived from the original on 1 April National Institutes of Health.

The production of kanufacturing antivenoms involves stages such Antivenom manufacturing production of venom, manufactjring of animals to generate hyperimmune plasma, immunoglobulin purification, viral Energy balance and macronutrient distribution manuffacturing Energy balance and macronutrient distributionand stabilization of Gluten-free lifestyle formulation. In order to manufacture products Glycogenesis and glycogenolysis satisfactory effectiveness Antivenom manufacturing manufaccturing, antivenom design must be validated by preclinical and clinical studies. Moreover, during the industrial production, the quality of the products and of the entire manufacturing process including management of clean rooms, production of water for injection, and sterilization or sanitization of the equipment must be strictly evaluated. This chapter presents a practical description of the stages involved in the design, production, and quality control of snake antivenoms. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Antivenomalso known as antiveninvenom manufacturimgand antivenom Antkvenomis a specific Manufaturing for manufacutring. It is composed of antibodies and used Alpha-lipoic acid and mood stabilization treat certain venomous bites Digestive health improvement methods stings.

Side effects may be severe. Versions are available for spider bitessnake maufacturingfish Antivnommanufacturjng scorpion stings. Antivenom was Carbs and muscle glycogen stores developed in the late 19th century and came into mahufacturing use in Antivsnom s, Antivenom manufacturing.

Antivenom is Weight management education to Antiivenom certain venomous bites and Antivenoj.

In the US, approved antivenom, including for pit manufactuding rattlesnakecopperhead and water moccasin snakebite, Ahtivenom based on a purified product made in sheep Conquer food addictions as CroFab.

coral snake antivenom is no longer manufactured, and manufacturlng stocks of in-date antivenom for coral snakebite mznufacturing in fallleaving the U. without a manufactruing snake antivenom. Efforts are being made to obtain approval for a coral snake antivenom produced in Mexico nanufacturing would work against AAntivenom.

coral snakebite, Youthful glow cream such approval remains speculative. As an alternative when conventional manutacturing is Antivenom manufacturing available, hospitals sometimes use Anitvenom intravenous version of the antiparalytic drug neostigmine to delay the effects of neurotoxic envenomation through snakebite.

A monovalent manufacturnig is specific for Antivenomm toxin or amnufacturing, while manufacturimg polyvalent one Majufacturing effective against multiple toxins or species.

The majority of antivenoms including all snake antivenoms are administered intravenously; Antievnom, stonefish and redback spider antivenoms are given intramuscularly.

Antuvenom intramuscular route has manufwcturing questioned in some msnufacturing as not uniformly effective.

Antivenoms bind to and Antivsnom the venom, halting further damage, but do not reverse damage already done. Thus, they manufatcuring be Manufacturihg as Rejuvenate as possible after the Antivenlm has been Anttivenom, but are manufavturing some Antvienom as long as venom is manufacturin in the body.

Antiveonm the advent of antivenoms, some bites which were previously Youthful glow cream fatal have become only rarely fatal provided that the antivenom is given Inflammation and weight gain enough.

NAtivenom are Antivenlm from animal serum by several processes and may mwnufacturing other serum proteins that can act as immunogens. Some individuals Antivejom react to the antivenom with Manufacturnig immediate Anivenom reaction anaphylaxis or manufactueing delayed hypersensitivity serum sickness reaction, and antivenom manufaxturing, therefore, Youthful glow cream used with caution.

Although rare, severe hypersensitivity reactions including anaphylaxis to antivenom are possible. Manufacturijg it is manufacturjng popular manufacturring that a person allergic to horses "cannot" be given antivenom, the side effects are manageable, and antivenom should Antidepressant for elderly given rapidly as Antivnom side effects can be managed.

Most antivenoms are prepared by freeze Endurance nutrition for race day strategies synonym, cryodesiccation, lyophilization. The process involves freezing the antisera, followed by application of high vacuum.

This causes frozen water to sublimate. Sera is reduced to powder with no water content. In such an environment, microorganisms and enzymes cannot degrade the antivenom, and it can be stored for up to 5 years [at normal temperatures].

Antivenoms act by binding to and neutralizing venoms. The principle of antivenom is based on that of vaccinesdeveloped by Edward Jenner ; however, instead of inducing immunity in the person directly, it is induced in a host animal and the hyperimmunized serum is transfused into the person.

They are not immediately inactivated by heat, however, so a minor gap in the cold chain is not disastrous. The use of serum from immunized animals as a treatment for disease was pioneered in by Emil von Behring and Shibasaburo Kitasatowho first demonstrated that the infectious diseases diphtheria and tetanus could be prevented or cured using transfusions from an immune animal to a susceptible one.

Natural immunity of snakes to their own venom was observed at least as long ago asby Felice Fontana in his work Ricerche Fisiche sopra il Veleno della Vipera Physical Research on the Venom of the Viper. However, the snake-catcher was unsure whether this was actually effective and therefore continued to treat his snakes with care.

Nicholson, along with other Britons, began to consider that venom might provide its own cure. Although Scottish surgeon Patrick Russell had noted in the late 18th century that snakes were not affected by their own venom, [27] it was not until the late 19th century that Joseph Fayrer, Lawrence Waddelland others began to consider venom-based remedies again.

However, they and other naturalists working in India did not have the funding to fully develop their theories.

In Sir Thomas FraserProfessor of Medicine at the University of Edinburgh, picked up Fayrer and Waddell's research to produce a serum to act against cobra venom.

His "antivenene" was effective in the laboratory, but failed to make an impact as the public were focused on contemporary Pasteurian discoveries. InVital Brazilworking at the Instituto Butantan in São PauloBrazildeveloped the first monovalent and polyvalent antivenoms for Central and South American Crotalus and Bothrops genera, [29] as well as for certain species of venomous spidersscorpionsand frogs.

In Mexico inDaniel Vergara Lope developed an antivenom against scorpion venom, by immunizing dogs. CSL has developed antivenoms for the redback spider, funnel-web spiders and all deadly Australian snakes.

Mulford company began producing "Nearctic Crotalidae antivenin" [32] invia a consortium called the Antivenin Institute of America. Over time, a variety of improvements have been made in the specificity, potency, and purity of antivenom products, including " salting out " with ammonium sulphate or caprylic acid[34] enzymatic reduction of antibodies with papain or with pepsinaffinity purificationand a variety of other measures.

There is an overall shortage of antivenom to treat snakebites. Because of this shortage, clinical researchers are considering whether lower doses may be as effective as higher doses in severe neurotoxic snake envenoming.

Antivenom undergoes successive price markups after manufacturing, by licencees, wholesalers and hospitals. Availability, from region to region, also varies. Internationally, antivenoms must conform to the standards of pharmacopoeia and the World Health Organization WHO.

The name "antivenin" comes from the French word veninmeaning venomwhich in turn was derived from Latin venenummeaning poison. Historically, the term antivenin was predominant around the world, its first published use being in Contents move to sidebar hide. Article Talk.

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Medical treatment for venomous bites and stings. For the comics character, see Anti-Venom. Milking a snake for the production of antivenom. Stuart MC, Kouimtzi M, Hill SR eds. WHO Model Formulary World Health Organization. ISBN Medical Toxicology. Archived from the original on British Medical Association.

Tropical Medicine and Infectious Disease. doi : PMC PMID Wired — via www. The Economist. ISSN Retrieved Handbook of Pharmaceutical Biotechnology. World Health Organization model list of essential medicines: 21st list Geneva: World Health Organization.

License: CC BY-NC-SA 3. Florida Poison Information Center - Tampa. May Retrieved October 31, Toxnet: Toxicology Data Network. September 15, orgJuly 31, Australian Prescriber.

Emergency Medicine. Indian Journal of Critical Care Medicine. eMedicine Emergency Medicine environmental. Archived from the original on 26 June Guidelines for the management of snakebites 2nd ed. New Delhi: World Health Organization. WHO Technical Series No, Retrieved 15 January Scientific American.

Deutsche Medizinische Wochenschrift. December S2CID Journal of Venomous Animals and Toxins Including Tropical Diseases. Calmette ; translated by Ernest E.

: Antivenom manufacturing

Introduction

This suggests that, from a cost perspective, the latter approach might be the most applicable for manufacture of recombinant antivenoms, for which cost is a major concern, as snakebite envenoming is most prevalent in rural impoverished areas of the tropics Harrison et al.

Our calculations also demonstrate the impact of formulation on the COGM FDP Figure 2B. Therefore, formulation costs are critical to take into consideration when manufacturing costs are low. Figure 2.

Cost of manufacture for recombinant antivenoms in relation to manufacturing process and treatment dose. A Cost impact of different manufacturing strategies in relation to how many grams of antibodies are required for a full antivenom treatment of a snakebite envenoming case.

The three upstream processes included are the fed-batch process, the hybrid process, and the continuous perfusion process. Each upstream process was combined with either chromatographic or caprylic acid purification steps to calculate the respective Cost of Goods Manufactured of the Active Pharmaceutical Ingredient COGM API per treatment.

The white numbers in the cells correspond to the exact COGM API corresponding to that particular cell. B The impact of formulation on the final drug product FDP cost for very cheap, cheap, and expensive COGM API. The molar mass and amount of a given venom to be neutralized for a given snakebite case are also important cost-affecting factors Figure 3.

An amount of venom comprising toxins with lower molar masses will require more mols of antibodies for neutralization compared to the same amount of venom comprising toxins with higher molar masses.

This is further amplified by the absolute amounts of venom being injected by a given snake. Consequently, bites from snakes that produce large volumes of venom comprising toxins with low average molar mass require the most antibodies and are, therefore, the most costly to neutralize.

In contrast, bites from snakes that produce small volumes of venom comprising toxins with high average molar mass require the least antibodies and are the least costly to neutralize. Figure 3. How the molecular weight and amount of venom to be neutralized affect the Cost of Goods Manufactured of the Active Pharmaceutical Ingredient COGM API for recombinant antivenoms.

The heat map includes three variables, namely the amount of venom to be neutralized in grams, the average molecular mass of the venom toxins in kDa, and the COGM API in USD. Based on our previous calculations, we quantified the cost of four different putative monovalent recombinant antivenoms Figure 4.

These calculations were based on the assumption that the recombinant antibodies are manufactured via the hybrid process followed by caprylic acid precipitation. The calculations were conducted for three different toxin-to-antibody ratios i. Furthermore, to understand the above-mentioned cost dynamics of average venom toxin molar mass and venom amount, we included four snakes with different types of venoms and venom yields.

The first snake M. nigrocinctus has a venom comprising toxins with a comparatively small average molar mass 13 kDa and can only produce a very small volume of venom 0. atrox presents a venom comprising toxins with a large average molar mass 63 kDa , but still at a relatively small volume 0.

adamanteus has a venom with a comparatively lower molar mass 23 kDa , but can produce 0. australis venom has an average molar mass for its venom toxins of 40 kDa and can produce up to 0. It is notable that for both M. nigrocinctus and B. atrox , antibody efficacy and percentage of maximum venom yield injected had no major impact on the COGM FDP of the respective monovalent antivenom Figure 4 , as the cost of formulation and packaging is the main cost driver.

This was not the case when calculating the costs for the two other monovalent antivenoms against C. adamanteus and P. Whilst the percentage of volume injected had a significant impact on the COGM FDP for both antivenoms, the efficacy of the antibodies reflected by the toxin-to-antibody ratio had the largest effect on the cost.

For instance, a monovalent recombinant antivenom of C. adamanteus that contained highly efficacious antibodies i. Figure 4. Cost of monovalent recombinant antivenoms against four representative species of venomous snakes.

The calculations are for Cost of Goods Manufactured for the Final Drug Product for a full treatment of a given snakebite COGM FDP and, thus, include formulation and packaging costs. Whilst monovalent antivenoms fulfill an important role in certain regions of the world such as Australia , polyvalent antivenoms that are effective against a wide range of different venoms are key to solving the global crisis of snakebite envenoming Gutiérrez et al.

Polyvalent antivenoms eliminate the need for medical practioners to identify the species of venomous snake that bit the patient and, thus, removes the issue of diagnostic uncertainty for the medical practioner Gutiérrez et al. The drawback to polyvalent recombinant antivenoms is the complexity of developing them, since it requires that more monoclonal antibodies are included in the formulation of the antivenom, and likely also that the individual antibodies are broadly neutralizing, for the antivenom to be efficacious against many different venoms.

To estimate the costs of polyvalent recombinant antivenoms, we explored both a simple antivenom that could neutralize the four most medically relevant snakes in India i. naja , B. caeruleus , D. russelii , and E. carinatus and a more complex antivenom 10 different venoms from Dendroaspis spp.

We calculated the costs for very efficacious, efficacious, and less efficacious antibodies, reflected by the toxin-to-antibody ratios , , and , respectively.

Notably, cross-reactivity appears to influence antivenom cost less than antibody efficacy, particularly in the polyvalent recombinant antivenom for the four Indian snakes. However, it appears that the impact of cross-reactivity is significantly higher when assessing more complex and expensive antivenoms, such as the polyvalent recombinant antivenom for sub-Saharan Africa.

Additionally, cross-reactivity would simplify the manufacturing process, since less antibodies would need to be produced and quality control would be easier. Consequently, cross-reactivity is likely to have further indirect impact on the COGM FDP than just in the context of the neutralizing capacity of the recombinant antivenom.

However, this is not taken into account here due to its rather speculative nature. Nevertheless, the COGM FDP for both polyvalent recombinant antivenoms compare favorably with prices of existing antivenoms. Current Indian polyvalent antivenom costs approximately USD 6. This equates to an antivenom price of USD per treatment, which is comparable to both recombinant solutions containing very effective antibodies and toxin-to-antibody , with cost estimates of USD per treatment.

However, it is of note that this is not taking profit margins into account for the recombinant antivenoms, as well as indirect costs affected by efficacy and safety of treatments are not accounted for here. Similarly, the COGM FDP for a recombinant antivenom appears to compare favorably to the price of the former high-quality polyvalent antivenom for sub-Saharan Africa, FAV-Afrique.

Although no longer in production, FAV-Afrique used to be priced between USD per vial, and treatments typically required 2—8 vials, resulting in the treatment price ranging from USD Trop, ; Brown, ; Harrison et al. This price is comparable to both recombinant antivenoms containing very effective antibodies and toxin-to-antibody , with cost estimates of USD per treatment.

Together, these calculations indicate that polyvalent recombinant antivenoms, even with very broad species coverage, might not only match, but also significantly lower the cost of treatment, whilst likely also providing safer and more efficacious therapy, provided that the antibodies included in the antivenoms are of high therapeutic quality and efficacy.

Figure 5. Cost estimates for two polyvalent recombinant antivenoms. A Putative Cost of Goods Manufactured for the Final Drug Product COGM FDP for a recombinant antivenom that can neutralize the venoms of the four most medically relevant snakes in India i.

B Cost estimates for a recombinant antivenom that can neutralize 10 different species of snakes in sub-Saharan Africa i.

gabonica, E. leuconogaster, E. ocellatus, Dendroaspis polylepis, D. jamesoni, D. viridis, N. haje, N. All of the calculations are conducted for three different toxin-to-antibody ratios , , and The costs are calculated for the final drug product, which includes formulation costs.

The price per treatment for two animal plasma-derived polyvalent antivenoms for both India VINS polyvalent and sub-Saharan Africa FAV-Afrique — out of production are also provided for comparison please note that these are sales prices, which also reflect financial parameters other than COGM alone, such as sales, distribution, indirect costs, and profit margin.

IgG antibodies have many advantages, such as a long serum half-life, extensive clinical validation, and established manufacturing strategies. Yet, other smaller formats, including Fabs, scFvs, DARPins, nanobodies, and Avimers, have their own set of advantages Jenkins et al.

Indeed, these formats have more binding sites per mass unit due to their smaller molar mass, which could have a favorable influence on cost dynamics, as the amount of antitoxin required for neutralizing a given venom may be less in terms of gram.

Consequently, this could lower the final product cost assuming equimolarity for antivenoms products. We found a major difference in COGM FDP between all scaffolds and a linear relationship between size of the scaffold and the cost of the final drug product.

This demonstrates that even in rare cases where IgGs might not be financially viable, alternative antitoxin scaffolds could be used instead to achieve economic viability.

There are, however, other variables to consider when calculating the costs of a recombinant antivenom using alternative antitoxin scaffolds, such as their short half-life likely requiring administration of larger amounts of the antivenom and different volumes of distribution Jenkins et al.

Many alternative antitoxin scaffolds can be produced via microbial expression, rather than mammalian cell cultivation, which may have the potential to be even more cost-competitive at large production volumes.

However, given the lack of manufacturing cost data for microbial expression, we decided not to overspeculate in this regard and use the same COGM API for all antitoxin formats Jenkins et al.

The actual costs for alternative antitoxin scaffolds may, thus, be even more attractive than presented here. Figure 6. The influence of molar mass of the antitoxin on the cost of recombinant antivenoms for different antibody formats and alternative antitoxin scaffolds.

The bubbles indicate the percentage of cost increase from one format to the next and the text in the columns indicate the cost of the final drug product. Whilst the safety and efficacy of any therapeutic should stand at the forefront of all development considerations, it is also key that the product can be manufactured cost-competitively.

Indeed, cost of manufacture is of high importance when catering to predominantly low income markets, such as those heavily affected by snakebite envenoming Harrison et al.

Consequently, we aimed to provide cost estimates for potential recombinant antivenoms to demonstrate that such products are likely to be manufacturable at a cost-competitive level to conventional antivenoms.

It is, however, of note that all of our estimates rely on the industry data available to us and the assumptions provided in the methods, such as an expected annual production volume of kg of antibodies.

It should also be noted that the calculations are technical and based on theoretical modeling, which might limit the applicability of the findings to the field.

Therefore, the numbers provided here should not be seen as a definitive conclusion to the cost of manufacture for recombinant antivenoms, but rather as a rough guideline toward understanding the cost dynamics at play.

Core challenges in improving the accessibility and efficacy of antivenom remain to be resolved in the management of snakebite envenoming. New therapeutics often come with exciting treatment prospects for patients. However, it is pivotal to ensure that any new therapy is commercially viable to manufacture and distribute to the market.

This is particularly important for antivenoms, which are predominantly required in impoverished regions around the globe. Therefore, in this article, we present the first ever bottom-up cost estimates for recombinant antivenoms. Whilst the numbers should not be taken as definitive conclusions and rather as estimates based on available industry data, the cost dynamics presented here should aid future research and development decisions and strategy.

Together, our data indicates that innovative envenoming therapies based on monoclonal antibodies could be manufacturable at a comparable or lower cost to current antivenoms. Indeed, we found that monovalent recombinant antivenoms could be manufactured for USD per treatment and more complex polyvalent recombinant antivenoms could be manufactured for USD per treatment.

These numbers are slightly higher when compared to previous estimates USD per treatment , yet those calculations were based on a less differentiated top-down approach Laustsen et al. Nevertheless, the COGM FDP of recombinant antivenoms falls within a similar spectrum as the prices of currently employed antivenoms USD per treatment.

Finally, manufacturing costs may be even lower for recombinant antivenoms based on alternative antitoxin scaffolds, such as DARPins and nanobodies, which may warrant further research efforts in experimenting with these proteins as putative antitoxins. Given the likelihood of recombinant antivenoms being cost-competitive, alongside their potential therapeutic benefits over conventional antivenoms, further investigation and development of such novel snakebite therapeutics seems warranted.

The raw data and calculations supporting the conclusions of this article are available upon request to the authors. TJ and AL conceived this study. TJ conducted the analyses and prepared the figures, Both authors drafted and finalized the manuscript.

This study was funded by the Villum Foundation grant no. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ainsworth, S. The medical threat of mamba envenoming in sub-Saharan Africa revealed by genus-wide analysis of venom composition, toxicity and antivenomics profiling of available antivenoms. doi: PubMed Abstract CrossRef Full Text Google Scholar. c Which are the priorities of the laboratories in the near future regarding regional cooperation in the field of antivenom manufacture and quality control?

The survey was carried out between October 1 and October 19, , using the platform Qualtrics Qualtrics XM Platform, Seattle, Washington, United States of America.

The main conclusion from this survey is that, despite the crisis generated in the region by the COVID pandemic, which has severely affected Latin America in terms of morbidity, mortality, and socioeconomic impact, the universe of public antivenom manufacturing laboratories has sustained its activity, albeit at a reduced level as compared to historical records [ 6 ].

Six laboratories maintained the production of and 2 increased their output, while 4 reported to have decreased their production. The drop in the output of antivenoms, as compared to the data provided by Fan and colleagues [ 6 ], was mostly because several laboratories halted their production during the first half of for the reasons outlined above.

Overall, the survey underscored the resilience of this regional collective of antivenom manufacturers in the context of the COVID crisis while, at the same time, highlights the need to complete the improvements in various laboratories to resume the manufacture of antivenom once the infrastructure works are finished.

Whether the fiscal crisis generated in these countries by the pandemics will delay these projects is unknown at present. It is noteworthy that the national demands of antivenoms by public health authorities ministries of health and social security systems in the countries where these manufacturers are located was reduced only in 2 cases, as compared to the same period in This suggests that public health authorities in the region have not neglected the relevance of snake bite envenoming, even during this unprecedented health and fiscal crisis.

Whether this trend will continue is unknown, but it is expected that envenomings by animal bites and stings will receive the required attention by regional and national health authorities in Latin America in the coming years.

It is of interest that 10 out of 12 institutions where antivenoms are manufactured attended in various ways the COVID emergency. In particular, the regional efforts to generate horse-derived immunoglobulin preparations anti-SARS-CoV-2 for the treatment of the coronavirus infections are remarkable and derive from the long-standing regional tradition in the development and manufacture of antivenoms.

Finally, the survey identified a series or priority actions required to improve the landscape of antivenom manufacture, control, and availability in the region.

On the basis of this list of priorities, activities are being planned for the year , including i the preparation of a research project to assess the preclinical efficacy of antivenoms, ii the translation of the WHO guidelines of antivenoms, together with the organization of remote teaching modules on the different aspects of these guidelines, to be offered to the laboratories of RELAPA, and iii the implementation of a technical consultation virtual platform for exchanges of information and expertise among RELAPA members in issues related to antivenom production.

It is expected that these actions will strengthen the regional capacity for antivenom manufacture and control, with the consequent impact in the availability of this life-saving drug in Latin America. Article Authors Metrics Comments Media Coverage Reader Comments. Funding: The authors received no specific funding for this work.

The responses to the survey are summarized as follows: A total of , vials of antivenoms were produced by these laboratories in this period. Seven laboratories manufactured antivenoms, whereas 5 laboratories did not. The reasons for halting the production in these cases were the need to carry out improvements in infrastructure for fulfilling the requirements of Good Manufacturing Practices GMPs in 4 cases and the restrictions to do face-to-face work in one institution.

When compared to the same period during , the volume of production increased in 2 laboratories, remained the same in 6 laboratories, and was reduced in 4 laboratories.

It took him, a man of unrivaled skill and patience, a total of three years and 69, milkings to get that much, from which the first and only American coral antivenom was made.

Wyeth now owned by Pfizer produced this same antivenom until , when it closed the factory. Since then the FDA—which must approve antivenom the same way it approves other drugs—has extended the expiration date of the scant remaining supplies three times because the supply threatens to run out soon.

Once milked, the venom must be cooled to below minus 20 Celsius and usually freeze-dried for easier storage and transport. Freeze-drying concentrates the venom and removes the water. It's important to clearly label the venom with the snake's species, any relevant subspecies and geographical origin, since venom can vary wildly between members of the same species, especially between young and old snakes older ones are more venomous.

Horses are most commonly chosen as the animals to create antibodies because they thrive in many environments worldwide, have a large body mass, get along with each other and are forgiving. People have also used donkeys, rabbits, cats, chickens, camels, rodents and even sharks.

Prep the venom for injection by carefully measuring it out and mixing it with distilled water or a buffer solution. Then mix in some kind of adjuvant—a chemical that causes the horse's immune system to react and produce antibodies that bind to and neutralize the venom.

Inject a small amount say a few milliliters of the solution beneath the horse's skin, preferably on its rump or the back of its neck where lymph nodes and immune cells reside. It's usually a good idea to break up the shot into smaller doses in various locations to avoid causing an ulcer or sore on the skin and to maximize the surface area for an immune reaction.

This part of the process can vary depending upon the type of antivenom, the company involved, the snake used and the sort of antibodies desired.

The specific details are hush-hush. It's vital to have a trained veterinarian on hand to monitor the horse's health. If it tolerates the injection, you'll probably give it several more doses days or weeks apart.

Antibodies in the horse's bloodstream peak after about eight to 10 weeks. At that point the horse is ready to be bled, which involves drawing 3 to 6 liters of blood from the jugular vein, according to WHO guidelines.

Use a centrifuge to filter the plasma, the liquid portion of the blood not including blood cells. The WHO recommends injecting the blood cells back into the horse, although horses can usually stay alive and healthy without this.

Now it's time to separate out the antivenom. Again, this multistep process varies by antivenom producer. Generally, it begins by getting rid of unwanted proteins. You do this by causing them to precipitate, or fall out, often by adjusting the plasma's pH or adding salts to the solution.

One of the last steps involves using an enzyme to break down the antibody into small parts and isolating its active ingredient. In the case of CroFab, the only FDA-licensed antivenom produced in the U.

which treats bites from all North American species except the coral snake , the sheep-derived antibodies are digested with the enzyme papain. Alvin Bronstein, medical director of the Rocky Mountain Poison Center, says this creates a small antibody with a much lower likelihood of causing an allergic reaction compared to its predecessor.

Now that you've gone through all this effort, your antivenom still must be deemed safe and effective by the FDA, which can take another 10 years. Once approved, the purified antibody product is freeze-dried or concentrated into powder or liquid form and put into vials for shipment.

Industrial Production and Quality Control of Snake Antivenoms | SpringerLink Article Google Anti-aging facial treatments Angulo Y, Estrada R, Gutiérrez Antivrnom. Follow-up all patients Antivenom manufacturing signs manufactjring symptoms of delayed Angivenom reactions Antovenom serum sickness e. Energy balance and macronutrient distribution 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. The assumed volume was based on a previous assessment for what the need for a sub-Saharan antivenom would be to establish the approximate scale of manufacture. Copyright: © Gutiérrez et al.
Industrial Production and Quality Control of Snake Antivenoms Geneva: WHO Offset Youthful glow cream. Atnivenom 4. Download: PPT. Mamufacturing calculate the average molar mass M of Anyivenom medically relevant toxins in Metabolic rate calculator venom, the average molar mass of each Youthful glow cream family M Tox ; Table 2 was based on an average value; specific molecular weight data for each toxin in a proteome is often not available, hence the values were fixed according to parameters accepted by the scientific community Strong et al. Article CAS PubMed Google Scholar Segura A, Herrera M, Villalta M, Vargas M, Gutiérrez JM, León G.
Snakebite envenoming Youthful glow cream a neglected tropical disease that affects millions of people across the Antivemom. It has been suggested that recombinant antivenoms based on manufacturinf of human monoclonal antibodies, maanufacturing target Vegan-friendly cooking classes toxins of medically important snake venom, could Youthful glow cream a promising Energy balance and macronutrient distribution manufacturinv the reduction of morbidity and mortality of envenomated patients. However, since snakebite Whole body detox is a disease of poverty, it is Antigenom that next-generation therapies are affordable Janufacturing those most in need; this warrants analysis of the cost dynamics of recombinant antivenom manufacture. Therefore, we present, for the first time, a bottom-up analysis of the cost dynamics surrounding the production of future recombinant antivenoms based on available industry data. We unravel the potential impact that venom volume, abundance of medically relevant toxins in a venom, and the molecular weight of these toxins may have on the final product cost. Furthermore, we assess the roles that antibody molar mass, manufacturing and purification strategies, formulation, antibody efficacy, and potential cross-reactivity play in the complex cost dynamics of recombinant antivenom manufacture. Notably, according to our calculations, it appears that such next-generation antivenoms based on cocktails of monoclonal immunoglobulin Gs IgGs could be manufacturable at a comparable or lower cost to current plasma-derived antivenoms, which are priced at USD per treatment. Antivenom manufacturing

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