BioDrugs 1997 May; 7 (5): 366-375 BIOPHARMACEUTICALS 1173-8804/97/0005-0366/$05.00/0 © Adis International Umited. All rights reserved. Antivenoms A Review of Current Status and Future Developments R. David G. Theakston 1 and Damon C. Smith2 1 World Health Organization Collaborating Centre for the Control of Antivenoms, Liverpool School of Tropical Medicine, Liverpool, England 2 Medical College of Sf Bartholomew's Hospital, London, England Contents Summary . 366 1. Standard Methods of Antivenom Production 367 2. Storage of Antivenoms . 368 3. Labelling of Antivenoms . 368 4. Different Types of Antivenoms . 369 5. Recent Developments in Immunotherapy 369 5.1 Choice of Host Animal . 369 5.2 Affinity Purification . 370 5.3 Use of Fab Fragments . 370 5.4 Future Developments in Immunotherapy 370 6. Antivenom Reactions . 371 7. Assessment of Antivenom Quality 372 7.1 Conventional Methods 372 7.2 Clinical Trials. 372 8. Conclusions . 373 Summary The first advance on crude filtered immune serum or plasma for treating sys­ temically envenomed bite victims was the use of IgG (predominantly equine) precipitated by salt treatment. This was followed by the development of F(ab')z anti venoms, prepared using pepsin digestion to remove both the highly reactive and nonspecific Fc part of the IgG molecule and also other non-antibody proteins from the material. Within normal limitations, both mono specific (raised against the venom of a single species) and poly specific (raised against a mixture of venoms of different species) F(ab')z anti venoms have proved to be generally effective. Many prepa­ rations are poor for a wide range of reasons often not related to the method of preparation. The main problem associated with them is the high reaction rate in patients caused by complement-mediated anaphylactic reactions. The most significant recent development in immunotherapy has been the pro­ duction of ovine Fab anti venoms prepared by replacing pepsin digestion with papain digestion. Theoretically, these possess advantages over F(ab')2 anti­ venoms, such as a greater volume of distribution and more rapid kinetics. A major disadvantage of Fab anti venoms is, however, their shorter clearance time, which Recent and Future Developments of Antivenoms 367 may result in inadequate blood anti venom concentrations for neutral ising venom entering the system late from a venom depot at the bite site. Affinity purification of both Fab and F(ab')2 anti venoms also results in a major increase in specific activity when compared with the original serum source, but this adds greatly to the cost of production, rendering its use prohibitive in the rural tropics where bites and stings are a major problem. The use of sheep as opposed to horses for immunisation also results in a cheaper product, due to the lower cost of animal maintenance; theoretically, sheep preparations should also be safer, causing fewer sensitivity reactions, due to the nature of ovine as opposed to equine protein. Additionally, it is a great advantage in the rural tropics to have a Iyophilised, as opposed to a liquid, anti venom because of the longer shelf-life of the former at ambient tropical temperatures. However, Iyophilisation does add considerably to the cost of production, involves additional testing and has also in the past been reported to result in some decrease in neutral ising potency. There may well be a case for the development of a combined ovine Fab/F(ab')2 anti venom which combines the advantages of each product. Alternatives to con­ ventional anti venoms should also be explored in the future. 1. Standard Methods of curs.!4,5] Unfortunately, horses do not tolerate such Antivenom Production adjuvants well,f6] Other additives, such as benton­ ite,!7] aluminium hydroxide gel and sodium algin­ Because of biochemical and immunological dif­ atel8] also have a similar overall effect. ferences between snake and other animal venoms Although it is remarkable that even today a few from different areas of the world, anti venoms are producers supply crude filtered horse serum alone produced commercially by about 70 different lab­ with all its additional attendant problems,!2] most oratories in 37 countries. I 1,2) Antivenoms are usu­ antivenoms usually comprise an ammonium sul­ ally made by immunising large animals with an phate-precipitated, pepsin-digested, concentrated individual venom or a range of venoms from large F(ab'h fraction of the IgG molecule (fig. I) sepa­ venom pools, which take account of seasonal, ge­ rated from the serum or plasma of hyperimmunised ographical, genetic and sex-related variation in horses.l7,9.11] The heat-labile fraction of the IgG venoms within the same species. Ideally, an anti­ molecule, the Fc fragment, produces no direct anti­ venom should be capable of neutralising all the venom effect and is responsible for some of the toxic components in a venom, and to achieve this early reactions induced by inadequately purified it is generally considered best to use unmodified anti venoms. This fragment is therefore removed by venom for immunisation, rather than to rely on de­ pepsin digestion; the ammonium sulphate used for toxification techniques which may result in thc rc­ precipitation of the IgG is eliminated by dialysis duction or complete loss of important antigens. 13 ) and lipids are removed by aluminium hydroxide With highly toxic venoms, however, such as those gel agglutination. Filtration is carried out by mo­ of some elapids and sea snakes, the use of formol lecular filtration using gels such as Sephadex®. The toxoids (prepared under acid conditions, as alkalin­ final product is subsequently tested for pyrogenic ity can lead to loss of both toxicity and antigenic­ activity (usually using rabbits), bacterial sterility, ity) was, in the past, considered unavoidable. Ven­ immunological specificity using electrophoresis or oms used in the preparation of anti venoms are best immunodiffusion, and potency before the final pro­ presented combined with an adjuvant, such as cedure of ampouling under sterile conditions to Freund's Complete, which results in a decrease in produce either a lyophilised preparation or a toxici ty because onl y gradual release of venom oc- straightforward fluid anti venom. © Adis International Limited. All rights reserved. BioDrugs 1997 May 7 (5) 368 Theakston & Smith F (ab'h (divalent) Fab (monovalent) World Health Organization Collaborating Centre for the Control of Antivenoms (WHO CCCA) maintained ampoules of I batch (number 2070(6) of Behringwerke North and West Africa anti venom L___l whose expiry date was June 1980 at 22 to 24°C for over 15 years with no loss of neutralising potency; other batches of the same anti venom deteriorate within the expiry period. Even at ambient temper­ P",i" ",,"" ,jO,' P"",;;; ..,,,, ,," atures, most liquid anti venoms maintain clitrity and I activity up to and over the expiry date printed on Fe the label. Facilities for storage of anti venoms are inadequate in many developing countries, both Fig. 1. Schematic representation of the IgG molecule showing F(ab')2' Fab and Fc fragments. Sites of papain and pepsin di­ during transport and at the final destination (hospi­ gestion are also indicated (arrows). tal or dispensary). In the rural tropics, it is possibly preferable to maintain such anti venoms at ambient temperatures rather than to store them in a refrig­ Antivenoms for use against the venoms of ani­ erator with fluctuating electricity supply and con­ mals other than snakes (e.g. medically important sequent fluctuating temperature. r I] scorpions, spiders, ticks, jellyfish and fish) are also Although lyophilised preparations are more ex­ produced by a few companies throughout the world pensive to produce than liquid anti venoms, they are 121 using the same basic system as described above. generally more satisfactory due to the longer shelf­ There are some exceptions to this, one being an life at ambient tropical temperatures in areas where anti venom produced by CSL Ltd. (Australia) cold storage facilities are unreliable or unavailable. against the venom of the funnel web spider (Atrax robustus). This is a protein A-purified intact rabbit IgG.[2 1 It has been reported that over 100 patients 3. Labelling of Antivenoms have received this anti venom with no recorded ad­ verse reactions.112! Simple labelling requirements have been laid down by the WHO Expert Committee on Biologi­ cal Standardisation.IIS ] The label should include in­ 2. Storage of Antivenoms formation on: • potency estimation using a standardised recog- Although there are few published data on the nised method (not yet satisfactory) stability of anti venoms, it is well established that • the nature of the preparation liquid antivenoms stored at different temperatures suffer different rates of activity loss.II.13,14J As a • the method of reconstitution general rule, the higher the ambient temperature, • restrictions, if any, on its use in a particular the more rapid the loss of neutralising potency; it country or area has been concluded that the potency of an anti­ • the identity of each reference venom against venom held in the liquid state is maintained for at which the potency is expressed least I year when stored at 30°C,113] although it has • a list of snake species for which cross-protection also been reported that antivenom stored at 37°C may be expected.ll ] did lose potency at the rate of25% per year and that The instructions contained in the package leanet opacity precedes loss of activity)13! There are ex­ should be clear and printed in letters large enough ceptions to this rule: liquid anti venoms may be to be read under restricted light conditions, and the highly variable both from batch to batch and from ampoule should be screen-printed as paper labels manufacturer to manufacturer. For example, the are susceptible to deterioration in humid condi- ([) Adis Internotional Limited.