Quality of horse F(ab′)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Autor(es)

Carla Cristina Squaiella-Baptistão ^[1] ; Fábio Carlos Magnoli ^[2] ; José Roberto Marcelino ^[3] ; Osvaldo Augusto SantAnna ^[4] ; Denise V. Tambourgi ^[5]

Afiliação do(s) autor(es)

^[1] Instituto Butantan. Laboratório de Imunoquímica - Brasil

^[2] Instituto Butantan. Laboratório de Imunoquímica - Brasil

^[3] Instituto Butantan - Brasil

^[4] Instituto Butantan. Laboratório de Imunoquímica - Brasil

^[5] Instituto Butantan. Laboratório de Imunoquímica - Brasil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Venomous Animals and Toxins including Tropical Diseases; v. 24, 2018-07-16.

Resumo

Abstract Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab′)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results Horse F(ab′)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab′)2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab′)2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients. (AU)

• Processo FAPESP: 11/51869-1 - Atividade anticomplementar de antissoros equinos produzidos contra venenos,toxinas bacterianas e virais.
• Beneficiário: Denise Vilarinho Tambourgi
• Modalidade de apoio: Auxílio à Pesquisa - Regular