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Antivenom production techniques

Antivenom production techniques

Sports performance nutrition must be tailored Ajtivenom combat the venom of a particular Antivenom production techniques. Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent anti-venom used in snakebite envenomation. Manufacturing by-products that affect pepsin purity lacked capability of binding.


Animal Heroes: Animal plasma helps make antivenom - Landline

Antivenom production techniques -

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Antivenoms prepared from hyperimmune animal plasma, mostly equine or ovine, are the only specific therapeutics for rapid counteracting post-snakebite pathophysiological manifestations.

Although there are various well established downstream processing strategies that have been implemented into commercial scale production, optimisation of compact, high yielding and low-cost manufacturing procedures generating safe, efficacious and available immunotherapeutics is still of great interest.

Design of the ideal process should be guided by the tendency to refine immunoglobulin G from residual plasma proteins in only a few easy, simple and efficient purification steps, aiming for good recovery of neutralising activity and regulatory acceptable physicochemical characteristics of the product [ 1 ].

Quality of the final product depends also on immunisation scheme that should maximally boost humoral response, giving the highest possible titer of anti-venom antibodies.

Many of so far developed strategies as the initial step employ salting-out procedure involving ammonium or sodium sulphate [ 2 — 6 ] that is associated with low purity profile of IgGs as well as excessive formation of aggregates [ 7 , 8 ].

Both shortcomings can be prevailed by introducing caprylic acid as an alternative fractionation agent [ 7 , 9 , 10 ] which acts on the majority of plasma proteins without affecting IgG fraction by leaving it in solution and, consequently, preserving its conformational and structural stability [ 11 , 12 ].

Refinement principles employing caprylic acid have been successfully implemented into preparations of a whole series of highly efficacious equine or ovine IgG-based antivenoms [ 13 — 17 ].

They have also been proven beneficial for purification of F ab' 2 derivatives [ 18 , 19 ] and monoclonal antibodies [ 20 ]. Following IgG extraction some antivenom manufacturers perform enzyme-mediated separation of the Fc portion of IgG, because it is not important for the neutralisation activity, while its removal contributes to reduction of foreign protein quantity in the product intended for use in humans.

It has been generally believed that the lack of the Fc fragment disables complement activation or inhibits the formation of immune complexes that are responsible for the onset of delayed hypersensitivity reactions [ 11 , 21 , 22 ].

However, poor physicochemical features of the product, i. turbidity, high content of IgG or contaminating protein aggregates, also exhibit detrimental impact, which was evidenced irrespective of the presence or absence of the Fc fragment [ 11 , 12 , 22 ].

Thus, its role in adverse reactions still remains unclear. Enzymatic cleavage can be performed either on unfractionated plasma [ 1 , 18 , 23 ] or isolated IgGs [ 1 ] as well as simultaneously with removal of unwanted proteins by caprylic acid precipitation [ 19 ]. Both F ab' 2 or Fab antivenoms have been successfully and widely used in snakebite management for decades [ 1 ], with the former ones being considered more clinically efficacious due to their slower elimination rate creditable for long lasting action [ 24 ].

Such complexes may be removed by phagocytic cells, eliminating the toxins from relevant tissue locations. This mechanism does not operate in the case of Fab antibodies.

The use of Fab fragments is often associated with recrudescence of envenomation signs, although their rapid distribution might represent desirable pharmacokinetic feature when dealing with venom toxins of comparable molecular weight. Ion-exchange chromatography has been introduced into some refinement strategies as well, proving suitable for separation of F ab' 2 fragments from other plasma proteins under conditions preferring antibody adsorption on cation-exchange stationary phase material [ 18 ].

Additionally, it has been recognised also as a method of choice for the final polishing where an anion-exchange approach is favourably used [ 25 ]. Other chromatography techniques are also applicable, for example, purification of F ab' 2 fragments by means of affinity chromatography exclusively [ 26 ].

Our study aimed for integrating the most efficient segments of the existing technological knowledge from the field into a compact, feasible and economically viable purification strategy for preparation of equine plasma-derived antivenom based on F ab' 2 fragments.

At the same time, effort was put into the preservation of highest process yield and fulfillment of the regulatory requirements concerning final product purity and aggregate content. The aim was also to precisely quantify the recovery of the active drug in each process step to enable accurate estimation of the cost-effectiveness of the designed procedure.

A standard mouse diet Mucedola srl. Animal monitoring for signs of pain, suffering and distress associated with procedure was performed following severity assessment protocol. Crude venom of V. ammodytes Vaa and two pools of Vaa -specific hyperimmune horse plasma HHP were provided by the Institute of Immunology Inc.

Caprylic acid, o -phenylenediamine dihydrochloride OPD , iodoacetamide IAA , dithiothreitol DTT , bovine serum albumin BSA , Tween 20, thimerosal, 2- N-morpholino ethanesulphonic acid MES monohydrate and Tris base were from Sigma-Aldrich, USA.

Pepsin from porcine gastric mucosa, 0. Goat anti-horse F ab' 2 IgG conjugated with horseradish peroxidase HRP was from antibodies-online , Germany.

All other chemicals used for preparation of buffers and solutions were from Kemika, Croatia. The bound antibodies were eluted with 20 mM citric acid, pH 2. A highly purified IgG sample eIgG was used as standard in ELISA assay and as model substrate for preliminary optimisation of pepsin digestion.

HHP was incubated at 56 °C for 1 h. After centrifugation at 3, × g for 40 min and discarding the pellet, caprylic acid was added to 0. Precipitation was performed by vigorous stirring rpm at 23 °C for 1 h in thermomixer Eppendorf, Germany , followed by sample centrifugation 2, × g , 45 min. IgG-enriched supernatant was collected and filtered through a cellulose acetate filter with a pore size of 5 μm Sartorius, Germany.

Minimal caprylic acid concentration giving the highest IgG purity and preserving yield, as preliminary determined, was chosen as optimal for the precipitation. Preliminary optimisation of pepsin digestion was done using a model IgG substrate—highly pure IgG sample eIgG isolated from HHP by protein A based affinity chromatography.

Generally, substrate aliquots 2 mg mL -1 were pH adjusted using 0. Pepsin solution 5 mg mL -1 in 0. The final volume of reaction mixture, prepared in saline, was 1 mL. Digestion was terminated at timed intervals with a 0.

Since it was not possible to execute all runs simultaneously, their order was randomised to avoid systemic errors. We used a regression function model covering linear contribution of each factor, but also non-linear for selected experimental area. The full factorial design was employed resulting in 4 experimental runs, each performed in triplicate 2 2 × 3.

The significance of the given factors was determined by means of ANOVA using Statistica All subsequent experiments were performed using real process IgG substrate—IgG fraction from the optimised caprylic acid fractionation step, and examining one variable at a time. The common approach involved acidification to pH 3.

Incubation was performed at 37 °C for 1. When optimal conditions were set, the procedure was scaled up fold. Samples from each experimental set were analysed by SDS-PAGE. IgG-enriched supernatant following caprylic acid precipitation was diafiltrated into water or saline using Vivaspin device Sartorius, Germany with a kDa molecular weight cut-off MWCO polyethersulfone membrane.

In each diafiltration step the buffer was exchanged by a factor of 8, ×. Elution was performed with 1 M NaCl in the binding buffer. The absorbance was monitored at nm.

After collecting the flow-through fraction, the bound components were eluted from the column material with binding buffer containing 1 M NaCl. The enzymatic activity of pepsin was measured spectrophotometrically on Multiskan Spectrum instrument Thermo Fischer Scientific, USA using haemoglobin as substrate.

Modified Ryle's protocol was followed [ 31 ]. Samples previously diafiltrated into 50 mM KCl, pH 2. Aliquots of 40 μL were incubated with μL of 2. Non-degraded substrate was precipitated by centrifugation at 2, × g for 10 min and absorbance of the supernatants was measured at nm.

Blanks were obtained by omitting samples from reaction mixtures. Staining was carried out with acidic Coomassie Brilliant Blue CBB R solution or, alternatively, with silver for detection of pepsin traces.

Isoelectric focusing, the first dimension of 2D gel electrophoresis, was performed in a ZOOM IPGRunner Mini-Cell Invitrogen, USA using immobilised pH gradient IPG strip 7 cm long, linear pH 3—10 Invitrogen, USA rehydrated with F ab' 2 sample μg , according to the protocol provided by the manufacturer.

The following step voltage protocol was applied: V for 20 min, V for 15 min, V for 15 min and 2, V for min. Obtained spots served as starting material for mass spectrometry MS.

Excised protein spots obtained by 2D gel electrophoresis of F ab' 2 sample were prepared for MS analysis by in-gel trypsin digestion, as follows. Following reduction and alkylation gel pieces were washed with mM NH 4 HCO 3 and ACN, dried and rehydrated in 1—10 μL of porcine trypsin solution Roche, 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. 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, org , July 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. Wellcome Collection. Smithsonian Institution. Ricerche fisiche sopra il veleno della vipera. Wellcome Library. In Lucca : Nella stamperia di Jacopo Giusti. Indian Journal of History of Science.

PLOS Neglected Tropical Diseases. Power House Museum. Archived from the original on 7 August Retrieved 24 February Bulletin of the Antiven Institute of America. US: Antivenin Institute of America. Bulletin of the World Health Organization. Emergency Medicine Journal. Retrieved 9 January Clinical Toxicology.

September The Dangerous Snakes of Africa. Ralph Curtis Books. Dubai: Oriental Press. com Dictionary. Progress in the characterization of venoms and standardization of antivenoms.

Geneva: WHO Offset Publications. 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. 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.

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