Toxicon 151 (2018) 63–73

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Current technology for the industrial manufacture of snake antivenoms T

∗
Guillermo León , Mariángela Vargas, Álvaro Segura, María Herrera, Mauren Villalta,
Andrés Sánchez, Gabriela Solano, Aarón Gómez, Melvin Sánchez, Ricardo Estrada,
José María Gutiérrez
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

A R T I C LE I N FO A B S T R A C T

Keywords: Snake antivenoms are formulations of animal immunoglobulins used in the treatment of snakebite envenoma-
Snake tion. The general scheme for producing snake antivenoms has undergone few changes since its development
Venoms more than a century ago; however, technological innovations have been introduced in the manufacturing pro-
Envenomation cess. These medicines must comply with identity, purity, safety and efficacy profiles, as requested by the current
Antivenom
Good Manufacturing Practices (GMPs) applied to modern biopharmaceutical drugs. Industrial production of
Industrial biotechnology
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.

1. Introduction proteins). Notwithstanding, most of the adverse reactions induced by

Snakebite envenomation is an important public health problem in
most tropical countries around the world (Kasturiratne et al., 2008;
WHO, 2016). Intravenous administration of specific antivenoms is the
only scientifically demonstrated therapy to control snakebite en-
venomations (WHO, 2016). Antivenoms are preparations of im-
munoglobulins (i.e., whole molecules or their F(ab′)2 or Fab fragments)
purified from the plasma of animals immunized with snake venoms
(Gutiérrez et al., 2011).
As pharmaceutical products, antivenoms must comply with identity,
purity, safety and efficacy profiles, according to Good Manufacturing
Practices (GMPs). The identity of antivenoms is determined by the
animal species used as immunoglobulin source and the venoms used as
immunogens (e.g., equine anti-bothropic immunoglobulins; León et al.,
2016). Purity is determined by the combination of the neutralizing
activity of hyperimmune raw plasma and the performance of the
methods used to separate the neutralizing immunoglobulins from other
plasma proteins. Consequently, the potency/total protein ratio is a good
parameter to evaluate antivenom purity (Segura et al., 2012).
On the other hand, safety refers to the assurance that antivenoms
are free of microbial contaminants (e.g., bacteria or their endotoxins,
fungi, virus or prions) and have satisfactory physicochemical char-
acteristics (e.g., low concentration of total protein, low content of im-
munoglobulin aggregates and minimal content of non-immunoglobulin
current antivenoms have been related to the inherent immunochemical
characteristics of heterologous immunoglobulins, such as antic-
omplementary activity and immunogenicity (León et al., 2013).
Pre-clinical efficacy of antivenoms is usually assessed based on the
anti-lethal potency at which they are formulated, i.e. the capacity of
antivenoms to neutralize venom-induced lethality (Gutiérrez et al.,
2017). However, evaluation of the ability of antivenoms to recognize
the venom toxins and neutralize hemorrhagic, defibrinogenating,
myotoxic, necrotizing, edema-forming, and neuromuscular blocking
activities, depending on the venom, offer additional information that
contributes to the prediction of their clinical performance (Gutiérrez
et al., 2013).
Besides the pre-clinical estimation of antivenom potency, the effi-
cacy of antivenoms in the clinical setting is affected by other factors
that do not depend on antivenom formulation, such as: 1) severity of
envenomation, 2) specificity of the antivenom towards the venom of the
snake causing the envenomation, 3) time between snakebite and anti-
venom administration, 4) route by which the antivenom is injected, and
5) dose of antivenom administered. Therefore, the only way to de-
monstrate and guarantee the clinical efficacy and safety of an anti-
venom formulation is through properly conducted clinical studies, such
as dose-finding trials and especially through controlled, double-blind,
randomized studies (WHO, 2016).
Large-scale production of clinically validated antivenoms requires

∗
Corresponding author.
E-mail address: guillermo.leon@ucr.ac.cr (G. León).

https://doi.org/10.1016/j.toxicon.2018.06.084
Received 7 March 2018; Received in revised form 28 May 2018; Accepted 25 June 2018
Available online 28 June 2018
0041-0101/ © 2018 Elsevier Ltd. All rights reserved.
G. León et al.
the implementation of industrial procedures for: 1) production of re-
ference venom pools, 2) production of hyperimmune plasma, 3) pur-
ification of the antivenom immunoglobulins, 4) formulation of the an-
tivenom, 5) stabilization of the formulation, and 6) quality control of
in-process and final products required to verify that all antivenom
batches in the production line meet the product specifications. All these
procedures should be carried out according to GMP principles (WHO,
2016). In this work, a general review of the current technologies used
for the industrial manufacture of snake antivenoms is presented. Since
most antivenom producers usually do not publish their industrial pro-
cedures, the information in this review is mainly based on the methods
tested and used by Instituto Clodomiro Picado.
2. Production of reference venom pools
Despite the fact that snake venom toxins can be produced by re-
combinant technologies (Li et al., 2017) or from cultures of venom
gland cells (Carneiro et al., 2006), virtually all antivenom manu-
facturers still utilize venoms produced by mechanical stimulation of the
venom glands, i.e. ‘milking’, of specimens maintained in captivity
(Chanhome et al., 2001; Chacón et al., 2012; Sasa et al., 2017), or of
wild specimens that, after venom collection, are released to nature (e.g.,
venoms produced by the Irula snake catchers cooperative in India).
Collections maintained in captivity can be composed by animals
collected from nature or/and by ex-situ bred specimens (Corrales et al.,
2014). In both cases, the health of the snakes must be closely monitored
by qualified veterinarians. In addition to clinical examination, de-
termination of normal hematological and plasma-chemistry values is
important to achieve this goal (Gómez et al., 2016; Muliya and Bhat,
2016).
The selection of species used as source of venom for antivenom
manufacture is mainly based on their medical importance (WHO,
2016). Nonetheless, intraspecific variability of venoms (i.e., individual,
ontogenic, wild/captive and regional variability) must be considered to
define the characteristics of the specimens (i.e., minimum number of
specimens, age, sex and geographical origin) used to produce reference
venom pools which relative composition guarantees the representa-
tiveness of venoms of wild snakes in the region (Chippaux et al., 1991;
Gutiérrez et al., 2009, 2010; WHO, 2016).
Important proteomic, toxicological and/or antigenic differences
have been demonstrated in venoms of geographically separated popu-
lations of snakes such as the monocled cobra (Naja kaouthia) from
Malaysia, Thailand and Vietnam (Tan et al., 2015a); the Russell's viper
(Daboia russelii) from different regions of India (Kalita et al., 2017); the
Indian krait (Bungarus caeruleus) from India (Oh et al., 2017), Pakistan
and Sri Lanka; and the spectacled cobra (Naja naja) from India, Pakistan
and Sri Lanka (Wong et al., 2018). On the other hand, some evidence
suggests that environmental conditions could also produce variations
between venoms of wild and captive snakes (Modahl et al., 2010; Saad
et al., 2012). However, recent studies show that this is not a general
rule (Freitas-de-Sousa et al., 2015; McCleary et al., 2016).
The relative composition of the reference venom pools (i.e. the
proportion of venoms of a particular number of specimens with dif-
ferent age, sex and geographical origin) must be determined based on
detailed biochemical and toxicological analysis of venom variability.
Only in this way the representativeness of reference venom pools can be
guaranteed (Calvete et al., 2014). In the absence of such studies,
composition of reference venom pools could be arbitrarily defined, i.e.
6–12 specimens of each age/sex group to ensure heterogeneity, as well
as including similar groups from at least the northern, southern, wes-
tern and eastern populations of the species, since these are likely to
have the greatest variation.
After defining the relative composition of the reference venom
pools, they must be produced on an industrial scale. The number of
specimens required for industrial production of antivenoms is directly
related to the ratio between the amount of venom consumed in
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Toxicon 151 (2018) 63–73
immunization and quality control activities and the yield of venom of
each species. For this calculation, it must be also considered that venom
yield decreases throughout life in specimens maintained in captivity
(Bolaños, 1972; our unpublished observations). An example related to
antivenoms for sub-Saharan Africa is the contrast between two medi-
cally-relevant species, Echis ocellatus and Bitis arietans. The former is a
relatively small snake with a low venom yield, whereas the latter gives
a large volume of venom. Therefore, the number of specimens that need
to be kept in captivity for regular venom production is much higher in
the case of E. ocellatus than in B. arietans. A similar scenario can be
found with the species Micrurus nigrocinctus and Bothrops asper, where
M. nigrocinctus is difficult to keep in captivity (Chacón et al., 2012) and
many snakes are needed to provide the venom quota used in antivenom
manufacture. On the other hand, species such as B. asper produce large
quantities of venom; therefore, the number of snakes to be maintained
in captivity is lower.
Venoms are obtained by mechanical massage of venom glands and
collected in vessels, which material must be validated in order to pre-
vent the loss of relevant toxins owing their protein-binding properties
(Goebel-Stengel et al., 2011). During this procedure, snake handling
can be facilitated by short-acting general anesthesia (e.g., CO2 inhala-
tion). However, this practice may result in a reduction of the venom
yield. To protect fresh venoms from auto-degradation (Sousa et al.,
2001) they must be frozen as soon as possible after collection, and
stored at −20 °C until their stabilization.
Reference venom pools are used as immunogens, i.e. to stimulate
the immune system of the animals selected as source of antivenom
antibodies (da Silva and Tambourgi, 2011; León et al., 2011), and to
test the ability of the raised antibodies to neutralize the toxic effects
induced by the venoms (i.e., used for quality control; Gutiérrez et al.,
2017). Therefore, reference venom pools must keep the antigenic and
toxic properties of fresh venoms of wild specimens (Farias et al., 2018)
during long-term storage (Jesupret et al., 2014; Hatakeyama et al.,
2018).
Stabilization of venoms is achieved by desiccation or freeze-drying.
During desiccation, the liquid venom at room temperature is submitted
to negative pressure until all the water has evaporated, which normally
takes several days. In these conditions, the venom proteins experience
high and sustained physical stress that could produce some denatura-
tion and loss of activity and antigenicity. During freeze-drying, the solid
venom, frozen at temperatures as low as – 40 °C, is exposed to negative
pressure during several days. In these conditions, the water is sub-
limated, exposing the venom proteins to very low physical stress.
Depending on the composition of the venom and the performance of the
procedure, the toxic activities of dried venoms can differ from those of
the original fresh venoms (Meier et al., 1991). Therefore, procedures for
venom stabilization must be carefully selected, standardized and vali-
dated.
Frequently, venom freeze-drying is achieved under poorly con-
trolled conditions, and this is an aspect that demands improvement in
venom-producing laboratories. Usually, venoms previously frozen at
−20 °C are cooled down to −70 °C for 18 h, in a laboratory freezer.
Then, venom is placed in a flask of a benchtop manifold freeze-drier,
where sublimation (primary drying) is performed at room temperature
and 100–200 mTorr, a process that lasts for a period that depends on
the volume of venom. Since heat is transferred to the product from the
surrounding environment, the desorption step (the secondary drying)
cannot be properly carried out with this type of equipment. The best
control of the process is accomplished if a shelf freeze dryer is used.
Quality control of reference venom pools must include the toxicological
and biochemical characterization of the venoms (e.g., quantification of
venom activities such as lethal, hemorrhagic, necrotizing, proteolytic,
phospholipase A2, coagulant and fibrinolytic activities, together with
the analysis of SDS-PAGE and reverse phase HPLC profiles; Camey
et al., 2002; WHO, 2016). The characterization of many different bat-
ches of venom is necessary to determine reference values that allow the
G. León et al.
identification of defective batches.
Characterization of reference venom pools must also include their
performance in the determination of the neutralizing potency of anti-
venoms. This is perhaps the most important characteristic that a re-
ference venom pool must achieve. We have unpublished evidence in-
dicating that two different batches of venom with similar toxicity
profiles can yield different results when used in the estimation of po-
tency of a single batch of antivenom. This outcome may be due to
structural changes induced by stress experienced by toxins during
freeze-drying (Heller et al., 1999), but this hypothesis needs to be ex-
perimentally addressed. This example illustrates that the performance
of reference venom pools in the determination of the neutralizing
ability of antivenoms should be controlled by using reference anti-
venoms (Fukuda et al., 2006; Araújo et al., 2008, 2017).
3. Production of hyperimmune plasma
It is likely that, in the future, antivenoms will be formulated in-
cluding chemical inhibitors of toxins, or human antibodies produced by
recombinant technologies (Laustsen et al., 2016). However, the current
industrial production of antivenoms is based on the immunization of
animals and the fractionation of hyperimmune plasma as source of
antivenom immunoglobulins.
3.1. Immunization of animals
Horses and sheep are the species preferred as source of antivenom
immunoglobulins for the industrial production of antivenoms, and most
manufacturers use horses. In the past, it was suggested that ovine an-
tivenoms are safer than equine antivenoms (Landon and Smith, 2003),
but clinical evidence has not shown major differences in the incidence
of adverse reactions induced by both types of antivenoms (Abubakar
et al., 2010). Some experimental antivenoms have been developed by
using other species such as camelids and chickens (León et al., 2011),
but they have not been used in clinical trials yet.
Immunization can be done by using venoms of one or several spe-
cies, to produce monospecific or polyspecific antivenoms, respectively
(Gutiérrez et al., 2011). Use of monospecific antivenoms is suitable only
when the identification of the species causing the envenomation is
possible based on the clinical characteristics (Gutiérrez et al., 2011) or
the availability of kits for venom detection (Trevett et al., 1995).
However, when the identification of the offending snake is not feasible,
the use of polyspecific antivenoms is usually preferred (Gutiérrez et al.,
2009; WHO, 2016).
The selection of venoms used as immunogens is based on the
medical importance of the snakes (WHO, 2016). However, raised an-
tibodies can neutralize not only the venoms used during immunization
(homologous venoms), but also the venoms from other species (het-
erologous venoms), which have antigenic similarity with the homo-
logous venoms. Cross-neutralization studies are required to determine
the neutralization scope of antivenoms and to identify venoms or
venom fractions that must be included in the immunizing mixture to
guarantee the efficacy of the antivenom in the geographical region
where it is going to be used (Tan et al., 2015b; Ratanabanangkoon
et al., 2016; Wong et al., 2016; Sánchez et al., 2017; Solano et al.,
2018).
During immunization, repeated injections of venoms are performed
to the animals, generally by the subcutaneous route, to induce the
production of neutralizing antibodies (da Silva and Tambourgi, 2011;
León et al., 2011). DNA immunization has been assessed as an alter-
native immunization strategy for antivenom production (Wagstaff
et al., 2006; Arce-Estrada et al., 2009). However, the titer of neu-
tralizing antibodies obtained by this method did not compete with that
obtained by directly injecting the venom.
Production of antivenom antibodies is potentiated by mixing the
venoms with immunological adjuvants, such as some simple or multiple
65
Toxicon 151 (2018) 63–73
emulsions (e.g., complete and incomplete Freund's adjuvants; Valverde
et al., 2017) and mineral salts (e.g. aluminum hydroxide, calcium
phosphate; Olmedo et al., 2013). Freund's adjuvants are the most ef-
fective for enhancing antibody response towards snake venoms (da
Silva and Tambourgi, 2011; León et al., 2011). However, these ad-
juvants induce adverse effects such as local tissue damage, granulomas,
abscesses and general reactions such as arthritis and fever, and there-
fore their use is regulated by international guidelines (Fukanoki et al.,
2001; Jansen et al., 2007), and should be limited to the first injections
of the immunization schedule. The use of Freund's adjuvants to ad-
minister routine boosters could affect the general health of the animals
and produce unnecessary suffering that is unlike to result in better
neutralizing responses.
After priming immunization, routine boosters can be administered
by using safer adjuvants such as aluminum salts and bentonite colloids,
which adjuvant activity is attributed to its ability to form depots from
which venoms are slowly released, producing a sustained stimulation to
the immune system (León et al., 2011). However, since animals im-
munized with snake venoms coupled to aluminum salts or bentonite
develop low titers of antivenom antibodies (Sadahiro et al., 1981;
Sunthornandh and Ratanabanangkoon, 1994), these adjuvants are
rarely used. In contrast, many producers immunize their animals using
no adjuvant at all, i.e. with venom dissolved in plain saline solution.
Some new adjuvants whose use is likely to increase in the next years
are liposomes (León et al., 2011) and the IMS 3012 nanoparticle ad-
juvant which have shown the ability to induce high titres of neu-
tralizing antibodies towards venoms of Naja naja, Vipera ruselli, Bun-
garus caeruleus and Ecuis carinatus from India with minimal tissue
damage (Waghmare et al., 2009).
Owing to their toxicity, snake venoms can produce tissue damage at
the injection site. Inactivation of venom by chemical or physical
treatments is not recommended because these procedures might affect
the antigenicity of the toxins and consequently reduce the efficacy of
the antivenom towards the native venom (Theakston et al., 2003).
Thus, the use of native venoms is usually preferred for immunization.
Traditionally, immunization with several venoms has been per-
formed by injecting a mixture of the venoms in a single anatomical site.
However, Chotwiwatthanakun et al. (2001) have obtained higher
neutralizing responses by distributing the venom injections in multiple
anatomical sites (i.e., the low dose, low volume, multi-site immuniza-
tion protocol). This method has the additional advantage of reducing
tissue damage induced by venoms and adjuvants.
Using a variation of the low dose, low volume, multi-site im-
munization protocol, and replacing crude venoms by ultrafiltration-
fractions composed by toxins with low molecular weight,
Ratanabanangkoon et al. (2016) eliminated antigens with low toxicity
in some venoms and were able to increase the variety of venoms in the
immunogen mixture that they used. In this way, they induced in horses
an antibody response with wide paraspecificity against major medically
important elapids in Asia. This strategy could be readily adapted to
produce antivenoms against other elapids.
On the other hand, venoms can influence the antibody response
towards other venoms used as co-immunogens. Thus, removal of the
immunosuppressive components of the venoms, or injection of venoms
at different time intervals, are strategies that can be used to improve the
antibody response obtained by the traditional method (Stephano et al.,
2005; Arroyo et al., 2015, 2017). It is therefore relevant that manu-
facturers assess the immunological interactions between the venoms
used for immunization to design immunization protocols that minimize
negative immunomodulatory effects.
Independently of the immunization strategy used, the rise in neu-
tralizing potency in plasma due to the increment of specific antivenom
antibodies has an upper limit value, which is not possible to overcome,
and depends on the physiological restrictions of the animal. However,
since this upper limit is unknown for each animal, the search for better
immunization strategies is a permanent task. At the end of the
G. León et al.
immunization schedule, neutralizing potencies reached by individual
animals usually show a normal distribution. While most animals de-
velop intermediate potencies around an average value, some animals
develop lower or higher potencies (our unpublished observations).
The determination of the minimum plasma potency required to
establish a sustainable production of effective, safe and low-cost anti-
venoms requires a careful analysis that must consider: 1) plasma po-
tencies that are feasible to reach with the immunization strategy used,
2) possibility of concentrating the antivenom immunoglobulins during
the formulation process, and 3) maximal dose (i.e., volume) of anti-
venom that could be administered in patients to ensure a therapeutic
effect.
Some researchers have estimated the therapeutic dose of anti-
venoms from the relationship between the amount of venom that a
snake can inject and the potency of the antivenom determined in a
murine model (Simpson and Norris, 2007). However, this approach is
likely to yield inaccurate results, because it does not consider the re-
lationship between the toxicokinetics of venom toxins and the phar-
macokinetics of antivenom immunoglobulins (Gutiérrez et al., 2003),
nor that the potency of an antivenom varies depending on the venom
challenge dose used in the assay (Solano et al., 2010). The antivenom
dosage to be used in therapy can only be defined based on dose-finding
clinical trials.
Determination of the minimum plasma potency used for industrial
production of antivenoms has also economic implications that must be
considered: if only high-potency plasmas are used, less vials of more
potent but more expensive antivenom will be produced, than if all the
plasmas (including those of lower potency) are used. Once the specifi-
cation of plasma potency has been established, animals that regularly
do not comply with this standard must be separated from the group and
replaced by new ones.
3.2. Bleeding of animals
After immunization, antivenom antibodies-enriched blood is col-
lected. Animals subjected to bleeding must be in good physical condi-
tions, as previously determined by auscultation of cardiorespiratory and
digestive systems, conjunctival and gingival mucosal examination, ca-
pillary filling test, overall clinical evaluation, and determination of
hemoglobin and hematocrit values.
Normal ranges of hematocrit and hemoglobin change from one
breed of animal to another. Very often, these values are lower in horses
subjected to snake venom immunization when compared to normal
horses of the same breed (Angulo et al., 1997). In general terms, horses
with hematocrit and hemoglobin values lower than 30% and 10 g/dL,
respectively should not be considered for large-scale bleeding. Con-
versely, animals with hematocrit higher than 45% may be suffering
from dehydration and should not be subjected to bleeding either.
Industrial bleeding/self-transfusion of horses can be performed
following a variety of protocols. For example, in the protocol described
by Angulo et al. (1997), horses weighing between 450 and 550 kg were
subjected to bleeding/self-transfusion during four consecutive days.
During the first three days, volumes of blood corresponding to 1.5%,
1.5% and 0.8% of body weight were harvested, and no blood was
collected in the fourth day. In the second, third and fourth days, self-
transfusion of red blood cells collected the preceding day was per-
formed to prevent animals from developing anemia. It has been de-
monstrated that equine erythrocytes can be stored for at least 35 days
before transfusion without apparent damage (Niinistö et al., 2007). The
last step of the procedure was the hydration of animals by the in-
travenous administration of 19 L of Ringer's lactate solution. After a 2
months rest period, animals were completely recovered and ready to
undergo a new immunization/bleeding cycle. Using this protocol, it is
possible to obtain yields of 10–12 L of plasma/horse, without producing
adverse acute or chronic physiological alterations in the animals
(Angulo et al., 1997). Other strategies of bleeding/self-transfusion are
66
Toxicon 151 (2018) 63–73
used by other laboratories.
Throughout bleeding, a strict aseptic technique is mandatory to
prevent microbial contamination of the blood. Before jugular veni-
puncture, the skin of the horse must be shaved, washed and disinfected.
The hands of the operator must be also washed and disinfected.
Protection of plasma by the addition of thimerosal, phenol or cresol,
and storage at 2–8 °C contribute to preserve the microbiological quality
of the plasma. In the case that plasma must be stored during several
months before process, freezing storage could be an option to prevent
microbial growth.
During decades, antivenom producers used autoclaved glass bottles
with an anticoagulant such as Acid Citrate Dextrose (ACD) to collect the
blood (Levine and Broderick, 1970). Plasma can be separated, after
sedimentation of red blood cells, by aspiration with a mechanical
pump, in a clean environment. Alternatively, closed systems of sterile
plastic bags containing an anticoagulant can be used. With this system,
plasma is protected from microbial contamination, with minimal en-
vironmental control. The rate of contaminated bags can be as low as
0.1% (our unpublished observations).
Automated plasmapheresis has been suggested as a cleaner and
more controlled method to harvest equine plasma (Ziska et al., 2012)
with a lower extent of cellular contamination (Feige et al., 2003).
However, the yield and microbiological quality of plasma obtained by
this method are not necessarily better than those obtained by using
autoclaved bottles or plastic bags. Prospective comparison of manual
and automated plasmapheresis methods regarding cost-effectiveness,
safety and animal welfare risk-benefit, is a pending task.
The processing of plasmas obtained by using a poor aseptic tech-
nique is the principal cause of in-process and final product con-
tamination with bacterial endotoxins. Therefore, the microbiologic
quality of plasma should be carefully controlled. Specifications of en-
dotoxin content in plasma should be at least as strict as those for en-
dotoxin content in the final product (see below).
4. Purification of antivenom immunoglobulins
Few years after starting the clinical use of antivenoms, which in-
itially consisted of crude hyperimmune serum (Calmette, 1894), it was
suggested that purification of the active substance of antivenoms was
relevant to concentrate the neutralizing potency of the product and to
reduce the high incidence of adverse reactions induced by crude serum
or poorly-purified immunoglobulin preparations. Then, several techni-
ques based on differential precipitations of proteins (Atkinson, 1900;
Gibson, 1906; Steinbuch and Audran, 1969) and enzyme digestion
(Parfentjev, 1936; Pope, 1939) were gradually introduced in the pro-
tocols of antivenom manufacture.
Currently, there is a diversity of methods used to purify im-
munoglobulins from hyperimmune plasma at the industrial scale (e.g.,
pepsin digestion, salting out, caprylic acid precipitation, anionic ex-
change chromatography and ultrafiltration). Indeed, there is not a
standard protocol to purify antivenoms, and manufacturers use dif-
ferent methodologies (WHO, 2016). Examples of protocols used to
produce antivenoms based on whole Igs or F(ab′)2 fragments are shown
in Figs. 1 and 2, respectively. As a rule, the method that renders the
highest purification with the fewest number of steps should be pre-
ferred. However, each laboratory selects the techniques included in its
production protocols, establishes the sequence in which they are per-
formed, and defines the conditions for each procedure, according to its
own criteria. Consequently, many producers have unique protocols to
manufacture their products, and therefore there is great variation be-
tween products in terms of quality and production costs.
In general, following the purification steps, the immunoglobulin-
rich fractions are recovered by microfiltration or centrifugation (Rojas
et al., 1994; Mpandi et al., 2007), dialyzed or diafiltered for removing
the low molecular mass components added (Herrera et al., 2009), and
concentrated by tangential flow ultrafiltration to reach the neutralizing
G. León et al.
potency specification (Rosenberg et al., 2009). In some cases, buffer
exchange is required as preparation for the next purification step.
4.1. Enzyme digestion
Digestion of equine plasma with pepsin is performed to eliminate
non-immunoglobulin proteins (e.g., fibrinogen and albumin) and to
remove the Fc fragment of immunoglobulins to obtain F(ab’)2 frag-
ments. Previously, it was assumed that the presence of Fc fragment was
67
Toxicon 151 (2018) 63–73
Fig. 1. Scheme of a general process to produce
antivenoms composed of whole Igs purified by
caprylic acid precipitation. Green boxes at the
periphery depict quality control assays that
need to be done at various points in the pro-
duction line. (For interpretation of the refer-
ences to colour in this figure legend, the reader
is referred to the Web version of this article.)
responsible of the early adverse reactions induced by the intravenous
administration of equine immunoglobulins, and that F(ab’)2-based an-
tivenoms were safer than IgG-based antivenoms. However, it has been
shown that the incidence and severity of early adverse reactions in-
duced by antivenoms do not depend on whether the product is com-
posed of whole IgGs or F(ab’)2 fragments and, instead, are likely due to
the physicochemical quality of the products (León et al., 2013).
Nevertheless, the majority of antivenom manufacturers use pepsin di-
gestion to generate F(ab′)2-based products.
Fig. 2. Scheme of a general process to produce
antivenoms composed by (F(ab′)2 fragments
purified by salting out with ammonium sulphate.
Green boxes at the periphery depict quality
control assays that need to be done at various
points in the production line. (For interpretation
of the references to colour in this figure legend,
the reader is referred to the Web version of this
article.)
G. León et al.
Traditionally, pepsin digestion is performed by using whole plasma
as substrate. However, digestion can be also carried out on purified
immunoglobulins (Pépin-Covatta et al., 1997). Typical digestion with
pepsin is performed by adjusting the pH and temperature of plasma at
3.0–3.3 and 30–37 °C, respectively, adding the enzyme to reach a
concentration of 1 g/L, and incubating the mixture under slow stirring
for 40 min. Digestion is then halted by adjusting the pH to 7.0–7.5
(Pope, 1939).
During pepsin digestion, the neutralizing potency of plasma de-
creases along with the pH at which the digestion is achieved. When the
digestion is performed at pH of 2.8–3.2, ELISA titer of antivenom an-
tibodies is reduced to 35–70% of the initial titer (Morais and Massaldi,
2005), and plasma potency assessed in mice is reduced by half (our
unpublished results). When digestion is performed at pH higher than
3.2, potency loss is reduced, with the inconvenience that there is an
incomplete removal of Fc fragments. At pH values lower than 2.8, the
loss of potency is even higher. Since pepsin digestion reduces the yield
of antivenom, it increases the production costs. Therefore, inclusion of
this step in the antivenom manufacture protocol should be justified by
the demonstration of the improvement of antivenom quality.
On the other hand, enzymatic digestion with papain is used to
produce sheep Fab fragment-based antivenoms. Since equine IgGs are
resistant to papain digestion (our unpublished results), studies of
equine derived Fab-antivenoms are not available. Papain digestion is
performed at room temperature by adjusting the pH of undiluted
plasma to 7.0–7.2, adding 20 g/L of the enzyme powder and 1 g/L cy-
steine, and incubating the mixture under slow stirring for 120 min. In
contrast to pepsin digestion, papain digestion does not reduce the po-
tency of plasma (León et al., 2000).
Owing to their pharmacokinetic properties, Fab fragments are ra-
pidly eliminated from the body by renal filtration after intravenous
administration (Gutiérrez et al., 2003). Consequently, recurrence of
antigenemia and envenomation is frequent in patients treated with this
type of antivenom (Seifert and Boyer, 2001; Bush et al., 2015). Under
these circumstances, additional doses of antivenom are needed.
Therefore, few laboratories have adopted the production of Fab-based
antivenoms for commercial purposes.
4.2. Thermocoagulation
To denature the non-neutralizing degraded proteins, thermo-
coagulation is often incorporated after pepsin digestion of plasma
(Pépin-Covatta et al., 1997). Thermocoagulation is performed by
adding 12–15% ammonium sulfate to the digested plasma, adjusting
the pH of the mixture at 4.0–4.5, and incubating at 56 °C for 60 min. At
the end of the step, the mixture is cooled down and the coagulated
proteins are separated by filtration or centrifugation. This procedure is
used in the preparation of F(ab′)2 antivenoms, but not when producing
whole IgG antivenoms.
4.3. Salting-out
Salting-out occurs when the surface tension of water in a protein
solution is enhanced by the addition of a salt, resulting in an increase of
hydrophobic interactions between proteins and water. Therefore, pro-
teins are forced to reduce their surface area by folding and self-asso-
ciating, which leads them to precipitate (Atkinson, 1900).
Usually, differential precipitation of plasma proteins by salting-out
is performed with ammonium sulfate, although sodium sulfate could
also be used. The general procedure may vary, depending on whether
the molecule to be purified is IgG or F(ab′)2. Nonetheless, in both cases
co-precipitation of impurities along with the active ingredient, and
aggregate formation, are frequent problems associated to the salting-
out procedure (Rojas et al., 1994).
Purification of IgG from equine plasma is performed at room tem-
perature and neutral pH. First, ammonium sulfate is added at a
68
Toxicon 151 (2018) 63–73
concentration of 14–16% (w/v) and the mixture is stirred for 1 h. Then
the mixture is microfiltered or centrifuged to remove the precipitate,
which contains mainly fibrinogen. Afterwards, ammonium sulfate
concentration in the resulting liquid fraction is adjusted to 22–24% (w/
v). Again, the mixture is stirred for 1 h and microfiltered to recover the
precipitated immunoglobulins which are dissolved in water for injec-
tion (WFI) or saline solution (0.15 M NaCl) at pH 7.4 (Gutiérrez et al.,
2011). Salts are then eliminated by dialysis or diafiltration. At the end,
40–50% of the initial IgG is recovered with a purity of around 70%.
Albumin, fibrinogen, fibronectin and α2-macroglobulin are the most
abundant non-immunoglobulin contaminants in this type of formula-
tions (Tan et al., 2016).
On the other hand, purification of F(ab’2) fragments does not in-
clude the first salting-out at 14–16% ammonium sulfate, since fi-
brinogen is digested by pepsin, and usually its fragments are removed
by thermocoagulation. Thus, for the purification of F(ab’)2 fragments
salting out is directly achieved by precipitation with 22–24% (w/v)
ammonium sulfate at pH of 7.0–7.4. Then, the resulting precipitate,
mainly consisting of F(ab’)2 fragments, is recovered by microfiltration
or centrifugation, dissolved in saline solution and diafiltered or dia-
lyzed to eliminate the residual ammonium sulfate (Raw et al., 1991).
4.4. Caprylic acid precipitation
The addition of caprylic acid to plasma causes the irreversible
precipitation of most of the non-immunoglobulin proteins, leaving most
of the immunoglobulins (or their F(ab′)2 or Fab fragments) and low
amounts of some protein contaminants (e.g., ceruloplasmin and trans-
ferrin) in solution (Segura et al., 2012). The mechanism by which
caprylic acid precipitation works is not fully understood. Morais and
Massaldi (2012) proposed that caprylic acid binds to specific sites of the
plasma proteins, thus increasing the hydrophobicity of the interfacial
protein surface, which in turn increases protein-protein interactions
and cause precipitation.
Caprylic acid precipitation is performed at room temperature. The
pH of undiluted plasma (digested with enzymes or not) must be be-
tween 5.5 and 7.5. Then, caprylic acid is slowly added to attain a
concentration of 5–7% (v/v), while the plasma is vigorously stirred.
Regardless of the initial pH of the plasma, at the end of the caprylic acid
addition, the final pH of the mixture is around of 5.4. Precipitation is
complete after 45 min of stirring. Then the precipitated material is re-
moved by microfiltration or centrifugation (dos Santos et al., 1989;
Rojas et al., 1994; Raweerith and Ratanabanangkoon, 2003; Mpandi
et al., 2007). Whole IgGs can be recovered with higher than 90% purity
and 60–65% yield (Rojas et al., 1994). Similar protocols optimized with
different approaches at variable experimental conditions have been
described (Nudel et al., 2012).
4.5. Aqueous two-phase systems (ATPS)
Recently, the development of a method for the purification of an-
tivenom immunoglobulins by differential partition in an aqueous two-
phase system (ATPS), conformed by polyethyleneglycol (PEG) 3350/
potassium phosphate, was described (Vargas et al., 2015). In this
method, NaCl (15% w/v), potassium phosphate (20% w/v) and PEG
3350 (9% w/v) are sequentially added to the plasma until complete
dissolution. The IgGs precipitated in the polymeric phase (i.e., the
upper phase) are recovered by microfiltration, and then dissolved in
water. To increase the purity of the product, the IgGs can be submitted
to an additional purification step with caprylic acid.
Antivenoms produced by ATPS separation showed higher purity and
yield than antivenoms produced by direct caprylic acid precipitation
(Vargas et al., 2015). In addition, this method allows the recovery of
albumin from the saline phase (i.e., the bottom phase). Simultaneous
production of antivenom and albumin from the same plasma batch may
reduce the production cost of antivenoms, and consequently the price at
G. León et al.
which these products are offered.
Until now, the ATPS method has been used only to produce pilot
batches (i.e. 50 kg plasma) for preclinical evaluation. The industrial
scaling up of this method, as well as its evaluation for safety and effi-
cacy at the clinical setting are still pending tasks.
4.6. Chromatographic methods
Ion exchange chromatography is used by some manufacturers to
increase the purity of whole IgGs or F(ab′)2 fragments after primary
purification steps, such as enzyme digestion, salting out or caprylic acid
precipitation (Jones and Landon, 2002; Raweerith and
Ratanabanangkoon, 2003). After primary purification, antibodies con-
stitute more than 90% of the total protein of the preparation. Therefore,
anion exchange chromatography (e.g., DEAE or quaternary ammonium
gels, at pH 7.0–8.0; Gutiérrez et al., 2011), is a frequently used meth-
odology, since contaminants bind to the gel, whereas immunoglobulins
are eluted in the break-through fraction. In contrast, cationic exchange
chromatography (e.g., using exchangers such as carboxymethyl or
sulfonic acid and pH of 4.0–5.0) bind the immunoglobulins, which are
in higher concentration, thus being a less pragmatic methodology.
An additional advantage of anion exchange chromatography is the
removal of endotoxins (Lee et al., 2003). Endotoxin contamination
comprises a potential risk in antivenom manufacture during bleeding,
plasma separation, or immunoglobulin purification, and may remain in
the preparation after the primary purification step. However, the fea-
sibility of reducing endotoxin levels by ion-exchange chromatography
during fractionation does not substitute the need to use non-con-
taminated plasma, i.e. plasma with a content of endotoxin lower than
5.0 EU/mL as starting material for antivenom production.
On the other hand, affinity chromatography has been suggested as
an alternative to purify antivenom antibodies, as it eliminates im-
munoglobulins that do not bind to venom components (Dart et al.,
1997). In this method, an open chromatography column is packed with
snake venom immobilized on an activated Sepharose matrix or a similar
affinity gel. Then, diluted and microfiltered hyperimmune plasma (di-
gested or not), or a solution of purified IgG or IgG-fragments, is passed
through the column. Antivenom antibodies bind to the venom and the
rest of the material is eluted in the unbound fraction. Afterwards, an-
tivenom immunoglobulins are eluted by using low pH or changes in
ionic strength of the loading buffer.
Immunoglobulins obtained by affinity chromatography are re-
covered with purity and yield over 97%. This is the only current
method by which antivenom immunoglobulins can be separated from
non-antivenom immunoglobulins, which are the main contaminant in
current antivenoms (Segura et al., 2012). Nonetheless, application of
affinity chromatography is largely unpractical because it is expensive
and high amounts of venom are required.
4.7. Steps for removing or inactivating viruses
Biotechnology products purified from animal plasma have potential
risk of virus transmission to the patients (EMEA, 1996). Although there
are no reports of equine virus transmission to human patients by par-
enteral administration of antivenoms, protocols of antivenom manu-
facture must include validated steps effective in preventing this risk
(i.e., three steps able to reduce the viral infectivity by at least 4 log10 in
the TCID50 test).
Fortunately, several steps commonly used to produce antivenoms
have been shown to remove or inactivate viruses. Steps such as caprylic
acid precipitation (Dichtelmüller et al., 2002; Burnouf et al., 2007;
Caricati et al., 2013); low pH treatment (Burnouf et al., 2004; Solano
et al., 2012); pepsin digestion (Lazar et al., 2002; Burnouf et al., 2007;
Caricati et al., 2013); and solvent/detergent treatment (Segura et al.,
2009a), are effective for inactivating enveloped virus, but not against
non-enveloped viruses. Other steps like thermal treatment (Burnouf
69
Toxicon 151 (2018) 63–73
et al., 2004) and formulation with phenol (Caricati et al., 2013) in-
activate both enveloped and non-enveloped virus.
5. Formulation
After purification, immunoglobulins must be concentrated or di-
luted, to comply with potency specification. Concentration or dilution
of the final formulation depends on the relationship between the neu-
tralizing ability of the starting plasma and the potency required to
control venom toxicity with a dose of antivenom, a parameter that must
be validated in properly conducted clinical trials.
After adjustment of the total protein concentration, the im-
munoglobulin solution must be formulated. Sodium chloride at
0.7–1.0% (w/v) is added to confer the ionic strength necessary to keep
the immunoglobulins in solution, and pH is adjusted to physiological
values (7.2–7.4). Osmolytes such as sorbitol, mannitol or glycine can be
added to protect immunoglobulins from chemical and thermal stress
(Wang, 1999; Segura et al., 2009b). In the case of freeze-dried anti-
venoms, formulation with lyoprotectant components such as 5.0% su-
crose (m/v) is frequent (Herrera et al., 2017a, 2017b).
Although antivenoms are not presented as multi-dose medicines,
most of the current products are formulated with preservatives such as
phenol 0.15–0.25% (w/v) or cresol 0.30–0.35% (w/v). However, some
preservatives favor the formation of immunoglobulin aggregates, in-
duce turbidity (García et al., 2002; Segura et al., 2009b) and cause
transitory hypotension in rats when administered as bolus injections
(García et al., 2002). Thus, the lowest concentration with proven bac-
teriostatic activity based on antimicrobial efficacy tests (USP, 2014a)
should be used (Abd-Elsalam et al., 2011). Ideally, antivenoms should
not contain preservatives. However, to attain this goal, aseptic condi-
tions must be guaranteed during the aseptic filling of the product.
6. Freeze-drying stabilization
Freeze-drying or lyophilization is the best method for antivenom
stabilization in the presence of thermal stress (Herrera et al., 2014,
2017a). Therefore, freeze-dried antivenoms are the best alternative to
supply remote regions where the cold chain required for the storage of
liquid formulations is not guaranteed. When properly processed, effi-
cacy and safety of freeze antivenoms are not different from that shown
by liquid antivenoms (Mendonça-da-Silva et al., 2017). However, the
physical and chemical stress induced during the freeze-drying proce-
dure may affect the solubility and the neutralizing ability of im-
munoglobulins (Sarciaux et al., 1999; Maury et al., 2005); conse-
quently, the standardization of the freeze-drying procedure must be
carefully addressed.
The freeze-drying process consists of three stages: freezing, primary
drying or sublimation and secondary drying or desorption. During an-
tivenom freezing, the water present in the antivenom forms ice crystals,
leaving free spaces in which immunoglobulins are concentrated until
the formation of a solid amorphous system (Pikal, 2004). For anti-
venoms, the optimal freezing rate is around 1 °C/min until reaching
−40 °C. Annealing is an alternative step to homogenize and maximize
water crystallization.
The following step is the drying of the frozen solid under vacuum.
During this step, the temperature of the product should not exceed the
glass transition temperature (i.e., the temperature at which the anti-
venom is transformed into a solid amorphous system; Schersch et al.,
2010), which, in the case of an equine IgG formulated without ex-
cipients, is around −13 °C (Herrera et al., 2014).
During the first phase of the drying process (primary drying or
sublimation), frozen water is sublimated at a rate that depends on the
programmed shelf temperature (e.g. −15 °C). This phase ends when the
temperature of the product equals that of the shelf. Since this is the
most prolonged step of the freeze-dying process, its optimization has a
large impact on the process economy. The second drying phase (the
G. León et al.
secondary drying or desorption) is performed by exposing the dried
product to temperatures around 30 °C during 3–4 h, to reduce the
content of residual humidity of the product to less than 3%.
The stability of freeze-dried antivenoms depends on the formula-
tion, the freeze-drying process, and the storage conditions. Stability of
antivenoms must be demonstrated for each formulation, under worst-
case storage conditions (WHO, 2016). Results obtained are useful for
real conditions of product storage and transportation at high tempera-
tures (EMEA, 2002). Usually, the shelf-life of freeze-dried antivenoms,
stored at temperatures lower than their glass transition temperatures,
can reach five years (WHO, 2016). Instability of freeze-dried anti-
venoms is evidenced by increases in the time of reconstitution, the
turbidity of the reconstituted product, and eventually on the reduction
of the neutralizing ability of the antivenom.
An analysis of several products distributed in different regions of the
world showed that most of them were able to reconstitute in less than
5 min (Herrera et al., 2017b). This is particularly important in the case
of antivenoms, because efficacy of this therapy depends on the time
lapse between envenomation and antivenom administration, and long
reconstitution times (i.e., 40 min or longer) introduce an inconvenient
delay in the start of the therapy (Hill et al., 2001; Gerring et al., 2013).
7. Quality control of in-process and final products
Quality control is not a separate stage of the antivenom manu-
facture, but an integral part of the whole production process. It begins
with the monitoring of: 1) clean rooms, 2) the production of water for
injection, 3) the sanitization/sterilization of the equipment line, and 4)
the quality of raw materials. However, it also includes the evaluation of
reference venom pools, hyperimmune plasma, and in-process/final
antivenom.
As previously mentioned, quality control of venoms includes the
determination of toxic and enzymatic activities (Camey et al., 2002),
the ability of the venom to estimate the potency of reference anti-
venoms (Fukuda et al., 2006; Araújo et al., 2008, 2017), and the ver-
ification that these activities are conserved during long-term storage
(Jesupret et al., 2014; Hatakeyama et al., 2018).
The safety and effectiveness of the immunization process is assessed
by the clinical evaluation of hyperimmune animals used as im-
munoglobulin source (Angulo et al., 1997) and by the potency assay,
respectively. Usually, the potency assay is performed by mixing chal-
lenge dose of venom and variable dilutions of antivenom, to achieve
different venom/antivenom ratios. After an incubation period, aliquots
of the mixtures are injected in animals and the number of deaths is
recorded and used to calculate a mathematical expression of the anti-
venom potency (Araújo et al., 2008, 2017; Solano et al., 2010). This
potency assay is also used to determine the preclinical efficacy of in-
process/final antivenoms.
The quality of the bleeding process is determined by the clinical
status of bleeding animals (Angulo et al., 1997) and the microbiological
profile of the hyperimmune plasma. Maximum endotoxin content al-
lowed in plasma should be calculated as the product of the endotoxin
limit for the final antivenom (see below) and the potency of the final
product, divided by the potency of the crude plasma.
Immunological quality of in-process/final antivenoms is normally
limited to the evaluation of the ability of the antivenom to neutralize
the lethality of homologous venoms, as explained above (Gutiérrez
et al., 2017). Nevertheless, additional information can be obtained from
the assessment of the ability of the antivenom to neutralize other toxic
effects induced by the venoms, such as hemorrhage, defibrinogenation,
myotoxicity, necrosis, edema, and neuromuscular blockade (Gutiérrez
et al., 2013).
The microbiological quality of in-process/final antivenoms is as-
sessed by the sterility test (USP, 2014b) and the pyrogen test, which
evaluates the rise in the rectal temperature of rabbits injected in the
marginal vein with a dose of antivenom, in a period of 3 h (USP,
70
Toxicon 151 (2018) 63–73
2014c). Alternatively, to reduce the use of animals, the pyrogen test can
be substituted by the Limulus amoebocyte lysate assay (LAL; Solano
et al., 2015). In this case, endotoxin limit for final antivenom can be
calculated as the ratio K/M, where K is the threshold pyrogenic dose of
endotoxin per kilogram of body weight (i.e., 5 EU/kg), and M is the
maximum total dose of antivenom administered to a 70 kg patient per
1 h (USP, 2014d; Solano et al., 2015).
Physicochemical quality of in-process/final antivenoms include the
determination of pH, total protein (Gornall et al., 1949) preservative
concentration (Lacoste et al., 1959), chloride concentration (USP,
2014e), turbidity (Segura et al., 2009b), electrophoretic pattern
(Laemmli, 1970; Tan et al., 2016), presence of aggregates (e.g. by FPLC;
García et al., 2002; Tan et al., 2016), residual moisture (e.g. by the Karl
Fisher titration; Herrera et al., 2014), and residual caprylic acid (e.g. by
HPLC; Herrera et al., 2009) and other substances used during fractio-
nation, such as ammonium sulfate.
Neutralizing immunoglobulins or immunoglobulin fragments (i.e.
IgG, F(ab′)2 or Fab) are the active substance of antivenoms. Then, small
amounts of non-immunoglobulin proteins (i.e. albumin, fibrinogen,
transferrin and ceruloplasmin) and high amounts of non-neutralizing
immunoglobulins are the most important protein contaminants in
current antivenoms (Segura et al., 2012; Tan et al., 2016). While non-
immunoglobulin contaminants can be determined by methods such as
FPLC and SDS-PAGE (Segura et al., 2012; Tan et al., 2016), the content
of non-neutralizing immunoglobulins only can be determined by in-
direct methods, such as the potency/total protein ratio (Segura et al.,
2012).
The establishment of specifications for the minimum required con-
tent of active molecules, or the acceptable level of non-immunoglobulin
contaminants in the antivenom is a controversial issue that can be il-
lustrated by the following example. Let's suppose that there are two
antivenoms formulated at the same potency towards the same venom:
“Antivenom A”, composed of 100% immunoglobulins and formulated
at total protein of 10 g/dL, and “Antivenom B”, composed by 80%
immunoglobulins and 20% albumin, and formulated at total protein of
5 g/dL. Which one would have the better safety profile? If it is assumed
that albumin is the most dangerous contaminant in antivenoms, then
“Antivenom A” would be safer, despite that it exposes the patient to a
higher amount of heterologous protein. In contrast, if it is assumed that
the total amount of heterologous protein administered is the most im-
portant factor in the manifestation of adverse reactions, then
“Antivenom B” would be safer.
Theoretically, all batches of antivenom produced by the same
master process should have the same microbiological, physicochemical
and immunological quality. Then, the analysis of many batches of an-
tivenom is required to evaluate the quality of the master process.
Standardization of the master process is required to guarantee that all
batches of antivenom meet the specifications that cannot be tested
every batch, such as stability.
8. Conclusions
Snakebite envenomation is an important health problem worldwide,
and its solution requires improvements in: 1) the understanding of
snakes biology and venom composition and toxicity, 2) the technology
for antivenom production, 3) the strategies for antivenom distribution,
4) the access of patients to medical attention, 5) the training of health
staff in the management of envenomed patients, and 6) the strength-
ening of regulatory authorities to ensure that only effective and safety
antivenoms will be available in the region under their control.
The industrial production of safe and effective antivenoms is an
essential part of this multicomponent strategy. Antivenom manufacture
is a complex process that involves different activities that must be
carefully designed and coordinately performed. These activities in-
clude: 1) production of reference venom pools, 2) production of hy-
perimmune plasma, 3) purification of the antivenom immunoglobulins,
G. León et al.
4) formulation of the antivenom, 5) stabilization of the formulation,
and 6) quality control of in-process and final products. The conforma-
tion of multidisciplinary professional teams to perform these activities
is essential to improve the technology of antivenom production, an
unfinished task encompassing diverse challenges.
Conflicts of interest
All the authors are employed at Instituto Clodomiro Picado. None of
the authors of this paper has a financial or personal relationship with
other people or organizations that could inappropriately influence or
bias the content of the paper. Sponsors were not involved in the study
design; in the collection, analysis and interpretation of data; in the
writing of the review; or in the decision to submit the manuscript for
publication.
Acknowledgements
This study was supported by Vicerrectoría de Investigación,
Universidad de Costa Rica (project 741-B7-108). The authors thank
Andrés Hernández and our colleagues at Instituto Clodomiro Picado for
their collaboration.
Transparency document
Transparency document related to this article can be found online at
http://dx.doi.org/10.1016/j.toxicon.2018.06.084
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