engleski

Comparative study of different whole IgG snake antivenom refinement strategies and their impact on the purified IgG features

The main treatment for snake bite envenoming is antivenom consisting of antibodies obtained from hyperimmunised animal plasma. Still, the “refined” preparations commonly cause clinical side effects attributable to contaminating proteins and/or aggregates. According to the literature, certain purification methods induce conformational changes of IgG molecules making them more prone to aggregation while others, leaving them in solution throughout, don’t have such an impact. The aim of this study was to verify this hypothesis by comparing IgGs isolated by five different protocols with regard to stability, purity and immunoglobulin subclass composition. The fractionation methods were: ammonium sulphate precipitation (ASP), anion (AEC) and cation (CEC) exchange chromatography, affinity chromatography (AC) and caprylic acid precipitation (CAP).The highest purity was achieved by CAP and AC, while the highest aggregates content was observed in samples from AC, CEC and ASP. Contaminating proteins in IgG preparations were identified by mass spectrometry (MS) analysis. The most prominent impact on the subclass composition had AC, causing the highest loss of IgG(T). Less pure IgG fractions were additionally purified by CAP step prior stability study. Pure IgGs had different melting temperatures (Tm) in thermal shift assay, which might be the consequence of diverse subclass composition. One-month storage of IgGs at 37 °C didn’t influence either Tm or aggregates content of analysed preparations. The results indicate that different procedures gain IgGs of variable purity and subclass composition which might affect both safety and effectiveness. However, conformational changes during purification procedures might not be the trigger for increased aggregation.

antivenom, purification, IgG, stability, purity

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