Low pH formulation of whole IgG antivenom: Impact on quality, safety, neutralizing potency and viral inactivation (original) (raw)
Related papers
2009
We have evaluated for the first time the impact of a solvent/detergent (S/D) treatment on the quality and in vivo neutralization potency of horse-derived whole IgG antivenom used in the treatment of viperid snake bite envenoming in Central America. The S/D treatment by 1% tri (nbutyl) phosphate (TnBP) e 1% Triton X-45 at 22e25 C was applied either on starting plasma or on purified immunoglobulins. The S/D agents were removed from both fractions by extractions with oil. S/D-treated plasma was subjected to caprylic acid precipitation to purify the immunoglobulins. Products were formulated, sterile-filtered, and filled into 10-mL vials, stored at 5 AE 3 C, and subjected to routine quality controls, SDS-PAGE, determination of anti-Bothrops asper venom antibody titre by ELISA, in vivo B. asper venom-neutralization potency tests, and safety test, comparatively with an antivenom manufactured by caprylic acid fractionation without S/D treatment. Results indicate that these conditions of S/D treatment on purified immunoglobulin yielded an antivenom of high turbidity that induced weight loss in animals. In contrast, antivenom fractionated from the S/D-treated plasma had physico-chemical and biological characteristics indistinguishable from those of the non-S/D-treated antivenom. S/D treatment of horse plasma may be considered to increase the viral safety of antivenoms.
Biotechnology Progress, 2013
Viral safety remains a challenge when processing a plasma-derived product. A variety of pathogens might be present in the starting material, which requires a downstream process capable of broad viral reduction. In this article, we used a wide panel of viruses to assess viral removal/inactivation of our downstream process for Snake Antivenom Immunoglobulin (SAI). First, we screened and excluded equine plasma that cross-reacted with any model virus, a procedure not published before for antivenoms. In addition, we evaluated for the first time the virucidal capacity of phenol applied to SAI products. Among the steps analyzed in the process, phenol addition was the most effective one, followed by heat, caprylic acid, and pepsin. All viruses were fully inactivated only by phenol treatment; heat, the second most effective step, did not inactivate the rotavirus and the adenovirus used. We therefore present a SAI downstream method that is cost-effective and eliminates viruses to the extent required by WHO for a safe product.
Biologicals, 2015
There is a need to introduce innovations in the manufacture of snake antivenoms to increase the supply of these products worldwide. In this work, the fractionation of equine hyperimmune plasma with a new methodology that includes an aqueous two phase system (ATPS) as a primary purification step was compared with the traditional method of caprylic acid precipitation. Hyperimmune plasma from horses immunized with the venoms of three snakes from sub-Saharan Africa was used as starting material for the production of both formulations. After being adjusted to the same lethal neutralizing activity, both antivenoms were compared in terms of their immunoreactivity, neutralization of in vitro venom activities, physicochemical characteristics, and stability. Their performance in terms of yield and purity was also assessed. The neutralization profile of in vitro enzymatic activities and the immunoreactivity, analyzed by ELISA and antivenomic approaches, were very similar for both preparations. Likewise, they behaved similarly in stability studies. However, ATPS-fractionated antivenom showed improved physicochemical profile and immunochemical purity and yield, mainly owing to its lower protein content. Additionally, this methodology allowed the recovery of albumin as a byproduct. ATPS purification constitutes a promising technology for antivenom production and should be further evaluated at preclinical and clinical levels.
Biologicals, 2002
Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4°C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.
Assessment of the viral safety of antivenoms fractionated from equine plasma
Biologicals, 2004
Antivenoms are preparations of intact or fragmented (F(ab#) 2 or Fab) immunoglobulin G (IgG) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. They are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. Manufacturing processes usually include pepsin digestion at acid pH, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. Most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. Nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. This paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. Analysis of production parameters indicate that acid pH treatments and caprylic acid precipitations, which have been validated for the manufacture of some human IgG products, appear to provide the best potential for viral inactivation of antivenoms. As many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products.
International Journal of Molecular Biology: Open Access, 2020
The treatment of choice for the snake bite is the administration IgG antibodies that are raised in an animal body thorough immunization of the animals. To avoid potential adverse reactions that arises from introduction of animal antiserum in human body, the antibody needs to be purified from the other non-specific proteins like albumin. Optimal conditions for the fractionation of IgG's were studied; ammonium sulfate was added to plasma up to 40% concentration to separate non-specific proteins, iso electric separation was carried out at pH 5.4, and the final IgG's fractionation by the addition of ammonium sulfate up to 50% concentration. A highly purified and concentrated mixture of IgG's was obtained after removal of ammonium sulfate through dialysis. The resulting anti venom was of good quality in terms of purity and was found to be highly potent against venoms of all four types of snakes. Owing to the high purity and greater potency of the anti venom the method has potential to be used for lab and commercial scale production of anti venom.
Toxicon, 2009
Liquid formulations of antivenom require a cold chain for their distribution and storage, especially in tropical countries characterized by high temperature and humidity (climatic zone IV). Since cold chain is often deficient in many regions, there is a need to develop novel formulations of liquid antivenoms of higher stability at room temperatures. The effect of addition of the polyols mannitol and sorbitol on the thermal stability of caprylic acid-fractionated equine whole IgG antivenoms was assessed in preparations having different concentrations of protein and phenol. Results evidenced that: (1) turbidity increases proportionally to phenol and protein concentration.
Biologicals, 2010
Administration of antivenoms to treat snakebite envenomings has the potential risk of inducing early adverse reactions. The mechanisms involved in these reactions are unclear. In this study, polyspecific antivenom consisting of whole IgG purified from equine plasma by caprylic acid precipitation was administered intravenously to non-envenomed horses (n ¼ 47) and cows (n ¼ 20) at a dose of 0.4 mL/kg. It has been reported that, in humans, this formulation (administered at a dose of 0.4 mL/kg) induces mild noticeable early adverse reactions, such as fever, vomiting, diarrhea, urticaria, generalized rash, tachypnea or tachycardia, in about 15e20% of the patients. Unexpectedly, none of the animals receiving antivenom in our study showed any evidence of early adverse reaction. Moreover, no late adverse reactions, i.e. serum sickness, were observed during 40 days after antivenom administration. Unlike studies performed in envenomed humans, our present results were obtained in a group of non-envenomed individuals. It is concluded that, in addition to the physicochemical characteristics of the formulation, other unknown factors must determine the occurrence of adverse reactions in snakebite envenomed humans treated with equine-derived antivenoms.
Toxicon, 2005
Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by b-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein concentration. Antivenoms fractionated by using b-propiolactone or filtration through quaternary ammonium membrane had a significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that (a) b-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and experimental studies, since it may not adequately predict in vivo complement activation by antivenoms. q
Human Antibodies
The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.