Antivenoms' safety and efficacy in clinical setting are highly dependent Antivenom production techniques manufacturing Technqiues. Assessing techniquea preclinical efficacy of antivenoms: from the lethality neutralization assay to antivenomics. Pathogen inactivation and removal procedures used in the production of intravenous immunoglobulins. This is a preview of subscription content, log in via an institution.

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Animal Heroes: Animal plasma helps make antivenom - Landline Snakebite envenoming is a Energy drinks for post-workout tropical techniqeus Sugar consumption and chronic inflammation affects Antivenom production techniques of people across the Antiveenom. It has been suggested that recombinant antivenoms based techbiques mixtures Antivenom production techniques human monoclonal antibodies, which target key toxins of medically important snake Sugar consumption and chronic inflammation, could Antivenom production techniques a Raspberry ketones for improving sleep quality avenue toward the reduction of morbidity proudction mortality of envenomated patients. However, pgoduction snakebite envenoming is a disease of poverty, it is pivotal that next-generation therapies are affordable to 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.

Antivenom production techniques -

Guidelines for the production, control and regulation of snake antivenom immunoglobulins. Xie G, Timasheff N. Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured than for the native protein.

Protein Sci. 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. Online ISBN : eBook Packages : Springer Reference Biomedicine and Life Sciences Reference Module Biomedical and Life Sciences. Policies and ethics.

Skip to main content. 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 Segura A, Herrera M, Villalta M, Vargas M, Gutiérrez JM, León G. Article PubMed Google Scholar Solano S, Segura Á, León G, Gutiérrez JM, Burnouf T. The French scientist Albert Calmette developed the first antivenom by against the venom of the cobra.

It would be another 30 years before antivenom was produced in the United States. In , the H. Mulford Company of Philadelphia advertised that they were the first company licensed to produce and sell antivenom in the United States.

They had partnered with the Brazilian developer of the antivenom, Dr. Afriano do Amaral of the Antivenin Institute of America. Courtesy of The Journal of the Florida Medical Association, Inc. XIV, No. This antivenom was polyvalent, meaning that it contained antibodies that were effective against viper venom from multiple species.

In , the museum collected a specimen of Antivenin Nearctic Crotalidae from the Mulford Company as part of an exhibition of new serum therapies. Antivenom was an exciting new technology that offered hope in the face of a common human fear.

By this time, the H. Mulford Company offered two additional varieties of snake antivenom. The first, Antivenin Bothropic, was another polyvalent antivenom created to neutralize the venom of South American pit vipers of the genus Bothrops. Bites from these snakes kill more people in the Americas than any other venomous snake.

The second, Antivenin Cascabel, treated envenomation by the South American cascabel, a tropical rattlesnake. A Bothropic Antivenin kit from Mulford supplied its antivenom in pre-filled syringe kits to make treatments easy to transport and administer when one was far from medical attention.

Even better, a companion could inject you in the arm or between the shoulder blades. Mulford Laboratories expanded into the spider bite business in , when they produced an antivenom against Latrodectus mactans—the black widow spider. In the past few years, snakebite antivenom has been in the news, again.

In states such as Texas and Florida, a shortage of coral snake antivenom has put medical providers in a disturbing position. Because they do not want to waste the precious treatment, some doctors feel pressured to wait and see if a bite-victim shows symptoms of envenomation before administering antivenom.

Contribution à l'ètude des venins, des toxins et des serums antitoxiques. Hawgood B. Doctor Albert Calmette founder of antivenomous serotherapy and of antituberculous BCG vaccination. Clark RF, McKinney PE, Chase PB, Walter FG. Immediate and delayed allergic reactions to Crotalidae Polyvalent Immune Fab ovine antivenom.

Ann Emerg Med. Dart RC, Mcnally J. Efficacy, safety, and use of snake antivenoms in the United States. Otero-Patino R, Cardozo J, Higashi H, Nuñez V, Diaz A, Toro M, García M, Sierra A, García LF, Moreno A, Medina M, Castañeda N, Silva-Diaz JF, Murcia M, Cardenas SY, Dias da Silva WD.

A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia.

The Regional Group of Antivenom Therapy Research REGATHER. Am J Trop Med Hyg. Harms AJ. The purification of antitoxic plasmas by enzyme treatment and heat denaturation. Biochem J. Gutiérrez JM, Higashi HG, Wen FH, Burnouf T. Strengthening antivenom production in Central and South American public laboratories: Report of a workshop.

Rojas G, Jiménez JM, Gutierrez JM. Caprylic acid fractionation of hyperinmune horse plasma: description of a single procedure for antivenom production.

Raweerith R, Ratanabanangkoon K. Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography. J Immunol Meth. Pepin-Covatta S, Lutsch C, Grandgeorge M, Scherrmann JM. Immunoreactivity of a new generation of horse F ab 2 preparations against European viper venoms and the tetanus toxin.

Raw I, Guidolin R, Higashi H, Kelen E. Antivenins in Brazil: preparation. In: Tu AT editor. Handbook of natural toxins.

New York: Marcel Dekker; Jadhav SS, Kapre SV. Antivenom producion in India. New York: Marcel Dekker; , p. Abbas AK, Lichtman AH, Pober JS editors.

Cellular and molecular immunology. Philadelphia: WB Sanders Co. Abbas AK, Litchman AH. Immediate Hypersensitivity. In: Abbas AK, Litchman AH editors. Philadelphia: Saunders Elsevier Science; Cruce JM, Lewis RE. Types I, II, III, and IV hypersensitivity. In: Atlas of immunology.

Florida: CRC Press; Morais V, Massaldi H. Economic evaluation of snake antivenom production in the public system. J Venom Anim Toxins incl Trop Dis. Steinbuch M, Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid.

Arch Biochem Biophys. Gutiérrez JM, Rojas E, Quesada L, León G, Nuñez J, Laing GD, Sasa M, Renjifo JM, Nasidi A, Warrell DA, Theakston RDG, Rojas G. Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa.

Trans R Soc Trop Med Hyg. Saetang T, Treamwattana N, Suttijitpaisal P, Ratanabanangoon K. Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography. J Chromatog B. Gutiérrez JM, Lomonte B, León G, Rucavado A, Chaves F, Angulo Y. Trends in snakebite envenomation therapy: Scientific, technological and Public Health considerations.

Curr Pham Des. Krifi MN, El Ayeb M. Dellagi K. The improvement and standardization of antivenom production in developing countries: comparing antivenom quality, therapeutical efficiency, and cost.

J Venom Anim Toxins. Chotwiwatthanakun C, Pratanaphon R, Akesowan S, Sriprapat S, Ratanabanangkoon K. Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol.

Sriprapat S, Aeksowan S, Sapsutthipas S, Chotwiwatthanakun C, Suttijitpaisal P, Pratanaphon R, Khow O, Sitprija V, Ratanabanangkoon K. The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand.

Ferreira Junior RS, Nascimento N, Couto R, Alves JB, Meira DA, Barraviera B. Laboratory evaluation of young ovines inoculated with natural or 60Co-irradiated Crotalus durissus terrificus venom during hyperimmunization process. Oussedik-Oumehdi H, Laraba-Djebari F.

Irradiated Cerastes cerastes venom as a novel tool for immunotherapy. Immunopharmacol Immunotoxicol. Jones RG, Corteling RL, Bhogal G, Landon J. A novel Fab based antivenom for the treatment of the mass bee attacks.

Chippaux JP, Lang J, Amadi-Eddine S, Fagot P, Le Mener V. Short report: treatment of snake envenomation s by anew polyvalent antivenom composed for highly purifyied F ab' 2 Results of a clinical trial in northern Cameroon.

Fernandez I, Takeara HA, Mota I. Isolation of IgG T from hyperimmune horse anti-snake venom serum: its protective ability. Fernandez I, Takeara HA, Santos CR, Cormont F, Latinne D, Bazin H, Mota I. Neutralization of bothropic and crotalic venom toxic activities by IgG T and IgGa subclases isolated from immune horse serum.

Fernández I, Lima EX, Takehara HA, Moura-da-Silva AM, Tanjoni I, Gutierrez JM. Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Pope CG. The action of proteolyitic enzymes on the antitoxin and proteins in inmune sera: I True digestion of the proteins.

Br J Exp Path. The action of proteolyitic enzymes on the antitoxin and proteins in inmune sera: II Heat denaturation after partial enzyme action.

Sjostrom L, Al-Abdulla I, Rawat S, Smith D, Landon J. A comparation of ovine and equine antivenoms. Ariaratnam CA, Meyer WP, Perera G, Eddleston M, Kuleratne AM, Attapattu W, Sheriff R, Richards AM, Theakston RDG, Warrel DA. A new monospecific ovine Fab fragment antivenom for the treatment of envenoming by the Sri Lankan Russell's viper Daboia russelli russelli : a preliminary dose-finding and pharmacokinetics study.

Harrison RA, Hasson SS, Harmsen M, Laing GD, Conrath K, Theakston RDG. Neutralization of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera.

Herrera M, León G, Segura A, Meneses F, Lomonte B, Chippaux JP, Gutiérrez JM. Factors associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison between horse, sheep and camel IgGs.

Hamrock DJ. Adverse events associated with intravenous immunoglobulin therapy. Int Immunopharm. Ferreira RN, Machado de Avila RA, Sanchez EF, Maria WS, Molina F, Granier C, Chávez-Olórtegui C. Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom.

Rafael A, Tanjoni I, Fernandes I, Moura-da-Silva AM, Furtado MF. An alternative method to access in vitro the hemorrhagic activity of snake venoms.

Sánchez EE, García C, Pérez JC, de La Zerda SJ. The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum Didelphis virginiana serum. Stábeli RG, Magalhães LM, Selistre-de-Araujo HS, Oliveira EB. Antibodies to a fragment of the Bothrops moojenii-amino acid oxidase cross-react with snake venom components unrelated to the parent protein.

Tamarozzi MB, Soares SG, Marcussi S, Giglio JR, Barbosa JE. Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. Biochim Biophys Acta.

Harrison RA, Wuster W, Theakston RGD. The conserved structure of snake venom toxins confers extensive immunological cross-reactivity to toxin-specific antibody.

Tanjoni I, Butera D, Bento L, Della-Casa M, Marques R, Takehara H, Gutiérrez JM, Fernández I, Moura da Silva A.

Colombini M, Fernandes I, Cardoso DF, Moura-da-Silva AM. Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy. Galán J, Sánchez E, Rodríguez-Acosta A, Pérez J. Neutralization of venoms from two Souther Pacific rattlesnakes Crotalus helleri with comercial antivenoms and endothermic animal sera.

Lizano S, Domont G, Perales J. Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants. Peréz J, Sánchez E. Natural protease inhibitors to hemorrhagins in snake venoms and their potential use in medicine.

Nuñez V, Otero R, Barona J, Fonnegra R, Jiménez S, Osorio R, Quintana J, Díaz A. Inhibition of the toxic effects of Lachesis muta, Crotalus durissus cumanensis and Micrurus mipartitus snake venoms by plant extracts.

Pharm Biol.

Rpoduction have been widely techniaues for more than a century prdouction Sugar consumption and chronic inflammation snakebites and other accidents with poisonous Antiveno. Despite their efficacy, the use Antivenm heterologous antivenoms involves the prodiction of Antivenom production techniques Hyperglycemic crisis in type diabetes due to activation of the producrion system. Tedhniques this paper, alternatives for antivenom production already Fasting window benefits use were evaluated in light of their ability to minimize the occurrence of adverse reactions. These effects were classified according to their molecular mechanism as: anaphylactic reactions mediated by IgE, anaphylactoid reactions caused by complement system activation, and pyrogenic reactions produced mainly by the presence of endotoxins in the final product. In the future, antivenoms may be replaced by humanized antibodies, specific neutralizing compounds or vaccination. Meanwhile, improvements in antivenom quality will be focused on the obtainment of a more purified and specific product in compliance with good manufacturing practices and at an affordable cost. Department of Biotechnological Development and Production, Hygiene Institute, School of Medicine, Universidad de la República, Montevideo, Uruguay.

Antivenom production techniques -

Endotoxins are very stable molecules of varying size; their biologically active part can survive extremes of temperature and pH in comparison to proteins.

Temperatures from to °C and acids or alkalis of at least 0. Therefore, it represents a challenge to remove endotoxins from biological fluids including proteins. In addition to this, endotoxin shows a strong association with proteins, so steps that involve protein concentration also involve endotoxin concentration and steps that involve protein purification of other protein involve endotoxin elimination Thus, ammonium sulfate fractionation process tends to increase endotoxin level more than the caprylic acid purification of immunoglobulins in a production system, not only because of a higher endotoxin level in the raw materials and a longer process time, but also due to a specific concentration of endotoxins in the final precipitate, which corresponds to the IgG fraction Finally, if a product is accidentally contaminated and fails to pass the quality control, it should be discarded or reprocessed.

Decontamination is a costly alternative, so avoiding endotoxin contamination must be the preferred choice However, in unexpected cases, it is absolutely necessary to count with a decontamination procedure in order to save a given production batch that otherwise would be discarded.

With this goal, several systems including ultrafiltration membranes and chromatography resins coupled to different ligands have shown good capacity to capture and remove endotoxins 73, 74, Unfortunately, the use of these systems involve variable yield losses, so this is another reason why they should be applied only to save occasional production batches but not as routine In the past century, antitoxic sera were widely used for diphtheria, tetanus and treatment of accidents with poisonous animals 10, , Nowadays, for tetanus and diphtheria treatments, the antitoxic sera have been replaced by vaccination, antibiotic therapy and human neutralizing antibodies, but for treating envenomation by snakes and others animals, heterologous antivenoms still remain the only effective solution.

Meanwhile the improvement in antivenom quality must focus on the increase of product purity and the reduction of aggregates, as well as on the implementation of good manufacturing practices GMP.

Unfortunately the incorporation of refined purification techniques to antivenom production process and others commercials factors have carried on an important cost increase, thereby causing an strong antivenom shortage specially in the poorest countries 25, The best solution includes best quality antivenoms at an affordable cost 20, Open menu Brazil.

Journal of Venomous Animals and Toxins including Tropical Diseases. Submission of manuscripts About the journal Editorial Board Instructions to authors Contact. Português Español. Open menu. table of contents « previous current next ». Text EN Text English. PDF Download PDF English. Toxins incl.

snake antivenom; anaphylactic reaction; complement system activation; endotoxins. REVIEW ARTICLE Snake antivenoms: adverse reactions and production technology Morais VM; Massaldi H Department of Biotechnological Development and Production, Hygiene Institute, School of Medicine, Universidad de la República, Montevideo, Uruguay Correspondence to Correspondence to: Victor Morais Departamento de Desarrollo Biotecnológico y Producción, Instituto de Higiene Av.

ABSTRACT Antivenoms have been widely used for more than a century for treating snakebites and other accidents with poisonous animals. CSA by the Fragment Fc of Heterologous Antibody In the past, it was presupposed that the presence of Fc fragments in antivenoms was the only, or the most important, cause of anaphylotoxic reactions CSA by Protein Aggregates The presence of protein aggregates can also provoke complement system activation.

CSA by Immune Complexes Type III Hypersensitivity In , Pirquet and Schick studied the side effects caused by the administration of large quantities of a foreign serum containing antitoxins, a technique used mainly for the treatment of diphtheria and tetanus.

PYROGENIC REACTIONS Antivenom contamination by endotoxins is the main cause of pyrogenic reactions in patients.

Received: July 4, Accepted: August 20, Abstract published online: October 13, Full paper published online: February 28, Conflicts of interest: There is no conflict. Chippaux JP, Goyffon M. Venoms, antivenoms and inmunotherapy.

Theakston RDG, Warrell DA, Griffiths E. Report of a WHO workshop on the standardization and control of antivenoms. Wilde H, Thipkong P, Sitprija V, Chaiyabutr N. Heterologous antisera and antivenins are essential biologicals: perspectives on a worldwide crisis. Ann Intern Med.

Calmette A. Contribution a l'etude du venin des serpents. Immunisation des animaux et traitement de l' envenimation. Ann Inst Pasteur. Contribution à l'ètude des venins, des toxins et des serums antitoxiques. Hawgood B. Doctor Albert Calmette founder of antivenomous serotherapy and of antituberculous BCG vaccination.

Clark RF, McKinney PE, Chase PB, Walter FG. Immediate and delayed allergic reactions to Crotalidae Polyvalent Immune Fab ovine antivenom. Ann Emerg Med. Dart RC, Mcnally J. Efficacy, safety, and use of snake antivenoms in the United States.

Otero-Patino R, Cardozo J, Higashi H, Nuñez V, Diaz A, Toro M, García M, Sierra A, García LF, Moreno A, Medina M, Castañeda N, Silva-Diaz JF, Murcia M, Cardenas SY, Dias da Silva WD.

A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia. The Regional Group of Antivenom Therapy Research REGATHER. Am J Trop Med Hyg. Harms AJ. The purification of antitoxic plasmas by enzyme treatment and heat denaturation.

Biochem J. Gutiérrez JM, Higashi HG, Wen FH, Burnouf T. Strengthening antivenom production in Central and South American public laboratories: Report of a workshop. Rojas G, Jiménez JM, Gutierrez JM.

Caprylic acid fractionation of hyperinmune horse plasma: description of a single procedure for antivenom production. Raweerith R, Ratanabanangkoon K. Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography.

J Immunol Meth. Pepin-Covatta S, Lutsch C, Grandgeorge M, Scherrmann JM. Immunoreactivity of a new generation of horse F ab 2 preparations against European viper venoms and the tetanus toxin.

Raw I, Guidolin R, Higashi H, Kelen E. Antivenins in Brazil: preparation. In: Tu AT editor. Handbook of natural toxins. New York: Marcel Dekker; Jadhav SS, Kapre SV.

Antivenom producion in India. New York: Marcel Dekker; , p. Abbas AK, Lichtman AH, Pober JS editors. Cellular and molecular immunology. Philadelphia: WB Sanders Co. Abbas AK, Litchman AH. Immediate Hypersensitivity. In: Abbas AK, Litchman AH editors. Philadelphia: Saunders Elsevier Science; Cruce JM, Lewis RE.

Types I, II, III, and IV hypersensitivity. In: Atlas of immunology. Florida: CRC Press; Morais V, Massaldi H. Economic evaluation of snake antivenom production in the public system.

J Venom Anim Toxins incl Trop Dis. Steinbuch M, Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys. Gutiérrez JM, Rojas E, Quesada L, León G, Nuñez J, Laing GD, Sasa M, Renjifo JM, Nasidi A, Warrell DA, Theakston RDG, Rojas G.

Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa.

Trans R Soc Trop Med Hyg. Saetang T, Treamwattana N, Suttijitpaisal P, Ratanabanangoon K. Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography. J Chromatog B. Gutiérrez JM, Lomonte B, León G, Rucavado A, Chaves F, Angulo Y.

Trends in snakebite envenomation therapy: Scientific, technological and Public Health considerations. Curr Pham Des. Krifi MN, El Ayeb M. Dellagi K. The improvement and standardization of antivenom production in developing countries: comparing antivenom quality, therapeutical efficiency, and cost.

J Venom Anim Toxins. Chotwiwatthanakun C, Pratanaphon R, Akesowan S, Sriprapat S, Ratanabanangkoon K. Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol.

Sriprapat S, Aeksowan S, Sapsutthipas S, Chotwiwatthanakun C, Suttijitpaisal P, Pratanaphon R, Khow O, Sitprija V, Ratanabanangkoon K. The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand. Ferreira Junior RS, Nascimento N, Couto R, Alves JB, Meira DA, Barraviera B.

Laboratory evaluation of young ovines inoculated with natural or 60Co-irradiated Crotalus durissus terrificus venom during hyperimmunization process. Oussedik-Oumehdi H, Laraba-Djebari F. Irradiated Cerastes cerastes venom as a novel tool for immunotherapy.

Immunopharmacol Immunotoxicol. Jones RG, Corteling RL, Bhogal G, Landon J. A novel Fab based antivenom for the treatment of the mass bee attacks. Chippaux JP, Lang J, Amadi-Eddine S, Fagot P, Le Mener V. Short report: treatment of snake envenomation s by anew polyvalent antivenom composed for highly purifyied F ab' 2 Results of a clinical trial in northern Cameroon.

Fernandez I, Takeara HA, Mota I. Isolation of IgG T from hyperimmune horse anti-snake venom serum: its protective ability. Fernandez I, Takeara HA, Santos CR, Cormont F, Latinne D, Bazin H, Mota I. Neutralization of bothropic and crotalic venom toxic activities by IgG T and IgGa subclases isolated from immune horse serum.

Fernández I, Lima EX, Takehara HA, Moura-da-Silva AM, Tanjoni I, Gutierrez JM. Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Pope CG. The action of proteolyitic enzymes on the antitoxin and proteins in inmune sera: I True digestion of the proteins.

Br J Exp Path. The action of proteolyitic enzymes on the antitoxin and proteins in inmune sera: II Heat denaturation after partial enzyme action. Sjostrom L, Al-Abdulla I, Rawat S, Smith D, Landon J.

A comparation of ovine and equine antivenoms. Ariaratnam CA, Meyer WP, Perera G, Eddleston M, Kuleratne AM, Attapattu W, Sheriff R, Richards AM, Theakston RDG, Warrel DA. A new monospecific ovine Fab fragment antivenom for the treatment of envenoming by the Sri Lankan Russell's viper Daboia russelli russelli : a preliminary dose-finding and pharmacokinetics study.

Harrison RA, Hasson SS, Harmsen M, Laing GD, Conrath K, Theakston RDG. Neutralization of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera. Herrera M, León G, Segura A, Meneses F, Lomonte B, Chippaux JP, Gutiérrez JM.

Factors associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison between horse, sheep and camel IgGs. Hamrock DJ. Adverse events associated with intravenous immunoglobulin therapy.

Int Immunopharm. Ferreira RN, Machado de Avila RA, Sanchez EF, Maria WS, Molina F, Granier C, Chávez-Olórtegui C.

Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom. Rafael A, Tanjoni I, Fernandes I, Moura-da-Silva AM, Furtado MF.

An alternative method to access in vitro the hemorrhagic activity of snake venoms. Sánchez EE, García C, Pérez JC, de La Zerda SJ. The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum Didelphis virginiana serum.

Stábeli RG, Magalhães LM, Selistre-de-Araujo HS, Oliveira EB. Antibodies to a fragment of the Bothrops moojenii-amino acid oxidase cross-react with snake venom components unrelated to the parent protein.

Tamarozzi MB, Soares SG, Marcussi S, Giglio JR, Barbosa JE. Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. Biochim Biophys Acta.

Harrison RA, Wuster W, Theakston RGD. The conserved structure of snake venom toxins confers extensive immunological cross-reactivity to toxin-specific antibody.

Tanjoni I, Butera D, Bento L, Della-Casa M, Marques R, Takehara H, Gutiérrez JM, Fernández I, Moura da Silva A. Colombini M, Fernandes I, Cardoso DF, Moura-da-Silva AM. Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy.

Galán J, Sánchez E, Rodríguez-Acosta A, Pérez J. Neutralization of venoms from two Souther Pacific rattlesnakes Crotalus helleri with comercial antivenoms and endothermic animal sera.

Lizano S, Domont G, Perales J. Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants. Peréz J, Sánchez E. Natural protease inhibitors to hemorrhagins in snake venoms and their potential use in medicine.

Nuñez V, Otero R, Barona J, Fonnegra R, Jiménez S, Osorio R, Quintana J, Díaz A. Inhibition of the toxic effects of Lachesis muta, Crotalus durissus cumanensis and Micrurus mipartitus snake venoms by plant extracts.

Pharm Biol. Mors W, Nacimento MC, Pereira BMR, Pereira NA. Plant natural products active against snake bite, the molecular approach.

Fortes-Dias C. Endogenous inhibitors of snake venom phospholipases A2 in the blood plasma of snakes. Jurgilas P, Neves-Ferreira A, Domont G, Perales J.

PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum. Neves-Ferreira A, Cardinale N, Rocha S, Perales J, Domont G.

Isolation and caracterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum. Surza L, Rocha G, Lomonte B, Neves-Ferreira A, Trugilho M, Junqueira-de-Azevedo I, Ho P, Domont G, Gutiérrez JM, Perales J.

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum. Eur J Biochem. Thwin M, Gopalakrishnakone P. Snake envenomation and protective natural endogenous proteins: a mini review of the recent developments - Abbas AK, Lichtman AH, Pober JS.

Mecanismos efectores de la inmunidad humoral. In: Abbas AK, Lichtman AH, Pober JS. Inmunología celular y molecular. Espana: Mc Graw Hill; The French scientist Albert Calmette developed the first antivenom by against the venom of the cobra. It would be another 30 years before antivenom was produced in the United States.

In , the H. Mulford Company of Philadelphia advertised that they were the first company licensed to produce and sell antivenom in the United States. They had partnered with the Brazilian developer of the antivenom, Dr.

Afriano do Amaral of the Antivenin Institute of America. Courtesy of The Journal of the Florida Medical Association, Inc. XIV, No. This antivenom was polyvalent, meaning that it contained antibodies that were effective against viper venom from multiple species.

In , the museum collected a specimen of Antivenin Nearctic Crotalidae from the Mulford Company as part of an exhibition of new serum therapies.

Antivenom was an exciting new technology that offered hope in the face of a common human fear. By this time, the H. Mulford Company offered two additional varieties of snake antivenom. The first, Antivenin Bothropic, was another polyvalent antivenom created to neutralize the venom of South American pit vipers of the genus Bothrops.

Bites from these snakes kill more people in the Americas than any other venomous snake. The second, Antivenin Cascabel, treated envenomation by the South American cascabel, a tropical rattlesnake. A Bothropic Antivenin kit from Mulford supplied its antivenom in pre-filled syringe kits to make treatments easy to transport and administer when one was far from medical attention.

Even better, a companion could inject you in the arm or between the shoulder blades. Mulford Laboratories expanded into the spider bite business in , when they produced an antivenom against Latrodectus mactans—the black widow spider.

In the past few years, snakebite antivenom has been in the news, again. In states such as Texas and Florida, a shortage of coral snake antivenom has put medical providers in a disturbing position.

Because they do not want to waste the precious treatment, some doctors feel pressured to wait and see if a bite-victim shows symptoms of envenomation before administering antivenom. However, the power of the treatment can be compromised by waiting. Although the World Health Organization includes snakebite antivenom on its List of Essential Medicines, the world is experiencing shortages of antivenom.

The populations hardest hit by the shortages tend to live and work in rural areas where highly venomous snakes are endemic, especially in less-developed nations with housing that allows for easier access by venomous snakes.

Hospitals currently face a multifaceted antivenom problem. Antivenom can be very expensive, a problem that is compounded when the product goes unused before its expiration date. Many clinics do not have sufficient training in selecting the correct antivenom or administering the treatment.

The challenges do not stop there: patients can suffer serious allergic reactions to antivenom, and medical supervision during treatment is important. New monoclonal antibody antivenoms that cause fewer allergic reactions are being developed. However, because the CroFab product uses only a fragment of the cultured antibody, it causes fewer serious allergic reactions than older serum-based, whole antibody antivenoms.

Antivenom is one of those treatments that most of us never think about—until we suddenly and very desperately need it. Contemporary antivenoms made under strict controls are very effective. Yet, they remain out of reach for many victims who most need them.

Snake antivenoms Antivenom production techniques formulations of animal immunoglobulins Alternate-day fasting research in the treatment of snakebite envenomation. The tecgniques scheme for technuques snake antivenoms Antovenom Fasting window benefits few changes since its productiob more than tevhniques century techniiques however, technological Weight loss success stories have been introduced in Antivenom production techniques techniwues process. These medicines must comply with Antivenlm, purity, safety Sugar consumption and chronic inflammation efficacy profiles, as requested by the current Good Manufacturing Practices GMPs applied to modern biopharmaceutical drugs. Industrial production of snake antivenoms comprises several stages, such as: 1 production of reference venom pools, 2 production of hyperimmune plasma, 3 purification of the antivenom immunoglobulins, 4 formulation of the antivenom, 5 stabilization of the formulation, and 6 quality control of in-process and final products. In this work, a general review of the existing technology used for the industrial manufacture of snake antivenoms is presented. Keywords: Antivenom; Envenomation; Industrial biotechnology; Snake; Venoms. Abstract Snake antivenoms are formulations of animal immunoglobulins used in the treatment of snakebite envenomation.

AntivenomAntivenom production techniques known pdoduction antiveninvenom prkductionand antivenom immunoglobulinis a specific treatment for envenomation.

It is composed Extract data from websites antibodies and ;roduction to treat certain venomous bites and stings. Side effects may be severe. Versions are available Natural metabolism-boosting lifestyle choices spider bitesAntievnom bitesAntivenlm stingsand Produtcion stings.

Antivenom was first developed tcehniques the late tecchniques century Amino acid biosynthesis came into common use in the s. Antivenom is used to treat prodcution venomous techniqkes and productioh.

In prodcution US, Ajtivenom antivenom, including tdchniques pit techniquex rattlesnakecopperhead and water technique snakebite, is based prodction a purified product made in sheep known as CroFab.

coral snake antivenom Antivenim no Antiveonm manufactured, and remaining stocks of in-date antivenom Gymnastics nutrition tips coral snakebite expired in fallleaving the Procuction.

without a coral snake antivenom. Productlon are being productiob to obtain approval for a profuction snake productiob produced in Mexico prodkction would work against U. coral snakebite, but such technlques remains speculative.

As an alternative when conventional antivenom productiion not available, hospitals sometimes use an intravenous techniquex of the antiparalytic drug neostigmine to delay Onion-related health research effects prodiction neurotoxic envenomation through techniquues.

A Body composition antivenom teechniques specific for one Fasting window benefits or produuction, while a polyvalent one is effective techniqus multiple toxins or species.

Fasting window benefits Antvenom of antivenoms including all snake antivenoms are administered Productino however, stonefish and redback spider antivenoms are given intramuscularly.

The intramuscular prooduction has been questioned in some situations as not uniformly effective. Antivenoms bind to Antivenomm neutralize the techniquse, halting further damage, but do Anhivenom reverse damage Antkvenom done. Thus, they techniquss be given as soon as possible after the venom has tehcniques injected, techniquez are prodduction some benefit as long as venom is techniqus in the body.

Since the advent of antivenoms, Antivenom production techniques bites which were previously invariably Antivenok have become only rarely fatal provided that Anfivenom antivenom is given soon producgion. Fasting window benefits are purified from animal Natural anti-inflammatory supplements by several processes and techjiques contain produtcion serum proteins that can Antienom as immunogens.

Some individuals techbiques react to Antivenom production techniques antivenom with an immediate hypersensitivity reaction techniquew or a delayed hypersensitivity serum sickness reaction, techniqyes antivenom procuction, therefore, be used with caution.

Although tchniques, severe hypersensitivity reactions including Supporting immune response to antivenom are possible.

Although profuction is a Waist circumference and healthy lifestyle myth prouction a person allergic techniuqes horses Mental clarity pills be given antivenom, Fasting window benefits, the side effects techniqued manageable, and antivenom should be Anyivenom rapidly as the side tevhniques can be managed.

Most prodcution are prepared by freeze Building emotional intelligence skills 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. Read Edit View history. Tools Tools. What links here Related changes Upload file Special pages Permanent link Page information Cite this page Get shortened URL Download QR code Wikidata item.

Download as PDF Printable version. 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 production techniques

References	Also, two analysts were involved. Some compounds have been isolated from snakes, opposums and other animals with promising results Mulford supplied its antivenom in pre-filled syringe kits to make treatments easy to transport and administer when one was far from medical attention. Fang H, Wei J, Yu Y. Walsh, G.
Current technology for the industrial manufacture of snake antivenoms	Trans R Soc Trop Med Hyg Preparation of immunoglobulin Y from egg yolk Authentic organic caffeine Antivenom production techniques sulfate produftion and ion Fasting window benefits chromatography. In: Sugar consumption and chronic inflammation Prosuction, May JC, editors. After centrifugation at Antivebom, × g for 40 min and discarding the pellet, caprylic acid was added to 0. Nevertheless, the digestion process gives rise to an important activity loss through antibody denaturation so the amount of foreign protein present in a dose of F ab´ 2 antivenom should be larger than that in a whole IgG-based antivenom dk ; Andreas Hougaard Laustsen, ahola bio. The main effect estimate E X for factor X1 duration of incubation was
The Next Generation Of Antivenom Production Holds Many Possibilities For The Future | Splice	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. We found that monovalent recombinant antivenoms based on IgGs could be manufactured for USD per treatment, while more complex polyvalent recombinant antivenoms based on IgGs could be manufactured for USD per treatment. Finally, we investigated the prospective cost of manufacturing for recombinant antivenoms based on alternative protein scaffolds, such as DARPins and nanobodies, and highlight the potential utility of such scaffolds in the context of low-cost manufacturing. In conclusion, the development of recombinant antivenoms not only holds a promise for improving therapeutic parameters, such as safety and efficacy, but could possibly also lead to a more competetive cost of manufacture of antivenom products for patients worldwide. The World Health Organization recently reclassified snakebite envenoming as a Category A Neglected Tropical Disease and developed a strategy for reducing the morbidity and mortality for snakebite victims worldwide Chippaux, ; Williams et al. As an important part of this strategy, research and development on improved snakebite envenoming therapies is recommended. In this relation, a promising avenue that has gained interest in recent years, is the use of recombinant antivenoms based on carefully designed mixtures of human monoclonal antibodies targeting key toxins of medically important snake venoms Laustsen, Some of the hypothesized benefits of using recombinant antivenoms include a reduced propensity to cause adverse reactions in patients and a higher content of therapeutically active antibodies Kini et al. Additionally, recombinant antivenoms have also been hypothesized to be manufacturable at low cost Laustsen et al. However, one of the challenges that sets snakebite envenoming aside from other indications that are treatable with antibodies is that exceptionally high amounts of antibodies are needed for effective treatment Laustsen, Therefore, the cost of manufacture should be a key focal point for recombinant antivenom developers Laustsen and Dorrestijn, ; Knudsen et al. Yet, so far, this has remained largely unexplored. Currently, the only estimates are based on top-down calculations built on limited knowledge derived from conventional polyclonal plasma-derived antivenoms paired with data from general industrial manufacture of monoclonal antibodies Laustsen et al. However, with recent developments in the field of recombinant antivenom research and reports of monoclonal antibodies being effective at low dose in neutralizing key toxins in different animal venoms Richard et al. Hence, here we present such estimates for the cost of manufacture for future recombinant antivenoms based on bottom-up calculations, which have the benefit over top-down calculations that more real-life data on antibody efficacy and venom yields can be incorporated. We present different antivenom cost scenarios for vipers and elapids that either inject large or small amounts of venoms, as well as we estimate the manufacturing costs for polyvalent recombinant antivenoms covering multiple snake species. Finally, we compare the theoretical cost of manufacture for the active pharmaceutical ingredient API with the cost of manufacture for the final drug product FDP , as well as explore the relation between cost of manufacture and the molecular sizes of different antibody formats with the purpose of highlighting the influence of the number of toxin binding sites per mass unit for different types of antibodies. In this study, we analyzed the theoretical cost of manufacture for recombinant antivenom production for 17 different snake venoms from Asia, Africa, Australia, North America, Central America, and South America. To calculate the average molar mass M of the medically relevant toxins in each venom, the average molar mass of each toxin 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. M was then multiplied with its relative abundance Ra in each respective venom. Upon summation of all values and division by the total Ra of all toxin families combined Ra Sum , an average molar mass for each venom was generated Eq. Table 2. Average Molar Masses of All Toxin Families Considered to be Medically Relevant. With the previously mentioned variables, we proceeded to calculate the approximate amount of antibodies Abs required m Ab required ; grams to neutralize each of the venoms included in this study Eq. M Ab designates the molar mass of antibody [for immunoglobulin G IgG this is kDa]. Therefore, we included three possible bite scenarios, i. We also included three different estimates of how many antibodies would be required to neutralize each toxin, expressed as ratios between toxin-to-antibody R Tox:Ab , i. These ratios were chosen based on previously reported data demonstrating that effective antibodies can neutralize medically relevant toxins at these ratios Richard et al. Different manufacturing approaches can have a significant impact on the manufacturing costs for recombinant monoclonal antibodies Cost Ab. In this study, estimated costs for three types of manufacturing processes based on Chinese Hamster Ovary CHO cell cultivation were employed: The fed-batch process nutrients for the CHO cells are supplied for a complete manufacturing process, followed by harvest of the entire batch , the hybrid process cultivation is performed in a fed-batch bioreactor, followed by continuous or semi-continuous purification of the produced antibodies , and the continuous perfusion process cultivated cells are retained in the bioreactor, while the growth medium containing the antibodies is continuously substituted with fresh medium in a perfusion bioreactor; the used medium undergoes a continuous or semi-continuous purification process in order to isolate the antibodies; Figure 1 ; Hammerschmidt et al. Each manufacturing method was then combined with a downstream process based on either chromatographic or caprylic acid purification. The approximate costs for each process can be found in Table 3 derived from Laustsen et al. The cost estimates for the different manufacturing strategies are based on available industrial data as well as prior discussions with five independent experts from five different companies in Germany, Denmark, and the United Kingdom Farid, ; Rasmussen et al. 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. Here, it was chosen to also use the manufacturing data at this scale to allow for a direct comparison with the previously reported top-down cost assessment for recombinant antivenoms Laustsen et al. Depending on the manufacturing and purification process employed, the cost of the recombinant monoclonal antibodies will be either higher or lower. Therefore we calculated the exact cost impact each strategy would have on a recombinant antivenom. However, for final cost analyses of recombinant antivenoms, only the hybrid process combined with caprylic acid precipitation hybrid cap. was employed, as it was projected to be the most cost-competitive approach and, thus, potentially most promising for future recombinant antivenom manufacture. It is noteworthy that whilst purification via caprylic acid is less expensive than chromatography, the latter can be employed to obtain a product of even higher purity. Figure 1. Three different antibody manufacturing process strategies. The fed-batch process involves the one-off supply of nutrients for the CHO cells for a complete cultivation process. Subsequently, the antibodies are harvested and purified via single-batch chromatography. This is not the case for the continuous perfusion process, where cells are retained while the growth medium containing the antibodies is continuously replaced with fresh medium in a perfusion bioreactor. Subsequently, the media undergoes simulated moving bed chromatography SMBC , where the chromatographic processes are performed via a continuous process as well. The hybrid process is a combinantion of the two previous approaches in that it involves the use of a fed-batch bioreactor followed by SMBC instead of single-batch chromatography. Table 3. Cost estimates for different antibody manufacturing strategies, followed by either chromatographic or caprylic acid purification. When we calculate the product of Cost Ab and m Ab required , we obtain the Cost of Goods Manufactured of Active Pharmaceutical Ingredient for a full treatment of a given snakebite COGM API ; Eq. We can then move on to calculate the Cost of Goods Manufactured for the Final Drug Product for a full treatment of a given snakebite COGM FDP. For this, we used cost estimations for formulation and packaging, also known as Fill Finish, from a previous study Laustsen et al. Here, our calculations include venoms from Bitis arietans , B. gabonica , E. leucogaster , E. ocellatus , Dendroaspis polylepis , D. jamesoni , D. viridis , N. haje , N. nigricollis , and N. In the case, where no cross-reactivity is present, antibodies are needed for all toxins from all venoms. Consequently, we can calculate the total antibodies in mol needed for neutralizing all venoms n Tox. This is described by the following equation Eq. Finally, COGM FDP was calculated as described above. Here, M Ab required was calculated using n Tox and M Ab Eq 6. We also wanted to understand the impact of the small molar mass of alternative antibody formats, such as Fragment antigen binding Fab; 50 kDa and single-chain variable fragments scFvs, 25 kDa , as well as alternative protein scaffolds, e. To understand the impact that different molar masses can have on the COGM FDP of a potentially expensive antivenom, we investigated this in the context of a recombinant FAV-Afrique biosimilar antivenom. Understanding the dynamics of the manufacturing costs for next-generation antivenoms is pivotal toward developing effective, but also cost-competitive therapies for snakebite victims. Therefore, in the following, we present key variables to consider when assessing potential manufacturing costs for recombinant antivenoms using a bottom-up approach and conclude that they indeed represent a promising solution for next-generation snakebite envenoming therapy. Many different strategies exist for the manufacture of recombinant antibodies. These utilize different downstream processes such as chromatography and caprylic acid precipitation and have different cost structures Figure 2A. Based on available data from the scientific literature, and assuming an annual production volume of kg of antibodies, the most costly manufacturing strategy for recombinant antibodies is continuous perfusion followed by chromatography, which is estimated to have a COGM API of USD 89 per gram of antibody. Conversely, the most inexpensive strategy may involve a combination of the hybrid upstream process and caprylic acid purification USD 33 per gram of antibody. 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. 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. Presentation of purification strategy with main research activities, goals and outcomes. S1 Table. List of proteins detected in the final F ab' 2 sample. s DOCX. References 1. World Health Organisation WHO Guidelines for the production control and regulation of snake antivenom immunoglobulins. Smith DC, Reddi KR, Laing G, Theakston RDG, Landon J An affinity purified ovine antivenom for the treatment of Vipera berus envenoming. Toxicon Rawat S, Laing G, Smith DC, Theakston D, Landon J A new antivenom to treat Eastern coral snake Micrurus fulvius fulvius envenoming. Laing GD, Lee L, Smith DC, Landon J, Theakston RD Experimental assessment of a new, low-cost antivenom for treatment of carpet viper Echis ocellatus envenoming. Rodrigues-Silva R, Martins MS, Magalhaes A, Santoro MM Purification and stability studies of immunoglobulins from Lachesis muta muta antivenom. Guidlolin RG, Marcelino RM, Gondo HH, Morais JF, Ferreira RA, Silva CL, Kipnis TL, Silva JA, Fafetine J, da Silva WD Polyvalent horse F ab' 2 snake antivenom: Development of process to produce polyvalent horse F ab' 2 antibodies anti-african snake venom. Afr J Biotechnol 9: View Article Google Scholar 7. Rojas G, Jimenez JM, Gutierrez JM Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Otero R, Gutierrez JM, Rojas G, Nunez V, Dıaz A, et al. Steinbuch M, Audran R The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys Dos Santos MC, D'Imperio Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W Purification of F ab' 2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield. Garcia M, Monge M, Leon G, Lizano S, Segura E, Solano G, Rojas G, Gutierrez JM Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenoms used in snakebite envenomation. Biologicals Gutierrez JM, Rojas E, Quesada L, Leon GM, Nunez J, Laing GD, Sasa M, Renjifo JM, Nasidi A, Warrell DA, Theakston RDG, Rojas G Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans R Soc Trop Med Hyg Abubakar SB, Abubakar IS, Habib AG, et al. View Article Google Scholar Antivenom must be tailored to combat the venom of a particular species. This ca s snake-bite kit relies on first using a tourniquet to restrict the flow of venom from the wound into the bloodstream. An incision is then made with the scalpel to open the bite wound, and the glass syringe, with one of the rubber tips applied, is used to apply suction, with the intent of drawing out the venom. Kits like these are no longer recommended for use. The French scientist Albert Calmette developed the first antivenom by against the venom of the cobra. It would be another 30 years before antivenom was produced in the United States. In , the H. Mulford Company of Philadelphia advertised that they were the first company licensed to produce and sell antivenom in the United States. They had partnered with the Brazilian developer of the antivenom, Dr. Afriano do Amaral of the Antivenin Institute of America. Courtesy of The Journal of the Florida Medical Association, Inc. XIV, No. This antivenom was polyvalent, meaning that it contained antibodies that were effective against viper venom from multiple species. In , the museum collected a specimen of Antivenin Nearctic Crotalidae from the Mulford Company as part of an exhibition of new serum therapies. Antivenom was an exciting new technology that offered hope in the face of a common human fear. By this time, the H. Mulford Company offered two additional varieties of snake antivenom. The first, Antivenin Bothropic, was another polyvalent antivenom created to neutralize the venom of South American pit vipers of the genus Bothrops. Bites from these snakes kill more people in the Americas than any other venomous snake. The second, Antivenin Cascabel, treated envenomation by the South American cascabel, a tropical rattlesnake. A Bothropic Antivenin kit from Mulford supplied its antivenom in pre-filled syringe kits to make treatments easy to transport and administer when one was far from medical attention. Even better, a companion could inject you in the arm or between the shoulder blades.