Quality of horse F(ab’)2 antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity

Abstract

BackgroundAmong 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.MethodsSamples 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.ResultsHorse 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.ConclusionsAnti-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.

The Preclinical Evaluation of a Second-Generation Antivenom for Treating Snake Envenoming in India

“…The three batches of second-generation antivenom, when subjected to 15% reducing SDS-PAGE, exhibited nearly identical profiles to their conventional antivenom counterparts, suggesting that they contained an F(ab’) 2 formulation. Electrophoresis revealed the presence of two bands between 25 and 37 kDa ( Figure 1 ), which correspond to light and heavy chains of F(ab’) 2 molecules, respectively, consistent with the previously reported profiles of F(ab’) 2 preparations of equine origin [ 15 , 16 , 17 , 18 , 19 , 20 ].…”

Evaluation of the Inhibitory Potential of Synthetic Peptides Homologous to CDR3 Regions of a Monoclonal Antibody against Bothropic Venom Serine Proteases

“…Since the production of the first antivenom by Hawgood and Calmette in 1999, obtained by inoculating rabbits with the venom of the Naja tripudians snake [45], few changes have been made, such as the purification of IgGs by caprylic acid and the use of papain and pepsin to obtain F(ab')2 or F(ab) fragments, respectively [46]. Therefore, it is extremely important to develop new pharmacological tools to support and improve the currently used immunotherapy, given the need to improve the neutralization of toxins present in animal venoms [46]. Likewise, challenges exist in the production of antivenoms, as well as difficulties in distribution, and the requirements for specialized personnel to administer antivenoms, among other factors.…”

A Chimeric Sudan Virus-Like Particle Vaccine Candidate Produced by a Recombinant Baculovirus System Induces Specific Immune Responses in Mice and Horses

“…In addition, we have conducted research on therapeutic antibodies with the goal of preparing antibodies for the post-exposure treatment of SUDV. Horses were selected as immunized animals in our study because horse anti-immunoglobulins have been used in the treatment of various viral infections [37][38][39]. SUDV VLPs can effectively induce humoral immune responses in horses after immunization ( Figure 8A).…”