Elsevier

Biologicals

Volume 49, September 2017, Pages 1-5
Biologicals

Interlaboratory study for the establishment of Brazilian Bothrops Reference Venom and Antivenom for potency evaluation of Bothrops antivenom

https://doi.org/10.1016/j.biologicals.2017.08.006Get rights and content

Abstract

A collaborative study was performed for the establishment of the 5th lot of Brazilian Bothrops Reference Venom and the 1st lot of Brazilian Bothrops Reference Antivenom. All Brazilian manufacturers of Antibothrops Immunoglobulins and the National Control Laboratory participated of the study. The declared potency of the 5th lot of the Bothrops Reference Venom is 40.29 μg/0.5 ml, and the potency of the 1st lot of Bothrops Reference Antivenom is 6.51 mg/ml. For the potency evaluation of Bothrops Reference Venom the inter assay precision (gCV) was 3.25% in lab 01; 3.51% in INCQS; 4.71% in lab 03 and 25.11% in lab 02, and the inter laboratory precision was 13.76%. The intra assay precision of Bothrops Reference Antivenom determinations was 4.38% in INCQS; 8.47% in lab 02; 10.51% in lab 03 and 20.05% in lab 01. The inter assay precision was 3.51% in INCQS; 9.65% in lab 02; 18.03% in lab 01 and 20.23% in lab 03. The inter laboratory precision was 15.85%. Despite the high number of invalid results (55.6% for the pharmacopoeial method and 69.4% for the proposed method) the parallel line assay, have better inter laboratorial precision (gCV = 16.62%) than the pharmacopoeial potency assay (gCV = 38.28%).

Introduction

Snake bite envenoming is an important public health problem in Brazil with 24,467 cases notified in 2015, representing an incidence of 13.3 cases per 100,000 inhabitants per year, with a lethality of 0.44%. The most prevalent species associated with snakebite envenoming belong to the genus Bothrops with 73.5% of notification, followed by Crotalus with 7.5%, Lachesis with 3.0% and Micrurus with 0.7%. The remaining 15.3% were due to unidentified snakes and sub notified or partially notified cases [1].

Anti-snake venom production in Brazil started in 1901 at Instituto Butantan [2]. Currently, antivenoms are liquid preparations containing purified equine Fab’2 IgG, manufactured by four public laboratories: Fundação Ezequiel Dias - FUNED, Instituto Butantan - IB, Instituto Vital Brazil IVB and Centro de Produção e Pesquisa de Imunobiológicos – CPPI and are distributed free of charge to public hospitals all over Brazil by the Ministry of Health National Immunization Program.

The Bothropic Antivenom is a pentavalent formulation containing Fab’2 IgG against: Bothrops jararaca 50%, B. jararacussu, B. alternatus, B. moojeni and B. neuwiedi, 12.5% each. The potency requirement is that this formulation must contain immunoglobulins sufficient to neutralize at least 5.0 mg of Bothrops Reference Venom, per milliliter.

Since 1987, the National Quality Control Institute (INCQS) and producer laboratories use a National Reference Venom to perform the potency assays for lot release. The reference venom contains only B. jararaca venom and is described by Brazilian Pharmacopoeia as “a homogeneous mixture of venoms, representing the geographic distribution of the species Bothrops jararaca”. This reference venom is used in a venom-neutralizing potency test. The potency of antivenoms is assessed after the 50% Effective Dose (ED50) determination [3], [4], [5].

So far, the Bothrops National Reference Venom has been manufactured by Instituto Butantan and his potency is established only by INCQS.

In this work, INCQS and three antivenom producers established the potency of the Brazilian Bothrops Reference Venom lot 05 and Brazilian Bothrops Reference Antivenom lot 01 through an inter laboratory study (CPPI does not took part in this study because at that time it was not producing Bothrops Antivenom). The reference venom was used in the assays for determining the potency of intermediate products and lot release. The reference antivenom was used for evaluating a parallel line assay for potency determination of Bothrops Antivenom.

The main object of this study was to establish the potency of the reference venom and antivenom. Despite the study design did not aim the assay validation, the intra assay, inter assay and inter laboratory precision were evaluated. It was also evaluated the invalid assays' rate and the lethality of the challenge dose.

Section snippets

Reference venom

The lot 05 of the Brazilian Bothrops Reference Venom is a freeze dried preparation, a total of 4368 vials were produced with 30 mg of Bothrops jararaca venom obtained through 4430 venoms extractions of Bothrops jararaca captured in different geographical areas in Brazil. It contains 907,60 ± 40,30 μg of protein/mg of venom and an electrophoretic profile with 12 protein bands (5 majors, with molecular weight of 14,4; 18; 28; 32 and 62 kDa) on SDS-PAGE in a 7.5 to 17,5% gradient (Fig. 1).

Reference antivenom

The

Potency determination of Bothrops Reference Venom Lot 05

The declared potency of Bothrops Reference Venom was 40.29 μg/0.5 ml with an upper confidence limit (UCL) of 41.46 μg/0.5 ml and a lower confidence limit (LCL) of 39.46 μg/0.5 ml (n = 11). The Inter laboratory gCV was 14.39% (Table 1).

As the study protocol doesn't required assays in duplicate it was not possible to calculate the intra assay precision. The inter assay precision was determined through gCV. At INCQS the gCV was 3.51% (n = 3), in laboratory 01 it was 3.25% (n = 2), in laboratory 02

Discussion

According to WHO, biological reference standards comprise materials of complex composition that require biological or immunological assays for appropriate characterization. The biological or immunological assays used are usually comparative rather than absolute, and the reference standard is critical to define the qualitative nature and the relative magnitude of the biological or immunological response. Thus, availability of biological reference standards (international, regional or national)

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References (12)

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In memoriam.

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