engleski

Quality-related properties of the whole IgG snake antivenoms obtained by five different refinement protocols

Aim: Whole IgG antivenoms represent a complex mixtures of both venom-specific and therapeutically irrelevant antibodies of various IgG subclasses. They are extracted from hyperimmune animal plasma by diverse refinement principles that most probably have different impact on their functional and/or structural properties. The aim of this work was to compare the influence of five purification protocols on purity, aggregate content, IgG subclass distribution, venom-specific protective efficacy and thermal stability of the final IgG preparations. Methods: IgG fraction was extracted from unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma by each of the following purification approaches: ammonium sulphate precipitation (ASP), caprylic acid precipitation (CAP), anion- (AEX) and cation-exchange chromatography (CEX) and affinity chromatography (AC). Purity and aggregate content were monitored by size-exclusion-HPLC, impurities were identified by mass spectrometry, venom neutralization potency by ED50 assay in mice, thermal stability by thermal shift assay and IgG subclass distribution by in-house ELISA. Results: The highest purity was achieved by CAP and AC, while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated sample from AEX-based sample. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparations. AC and CEX remarkably affected drug’s venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. AC-derived sample was the only one whose thermal stability was reduced. Main conclusion: Our results showed that employed refinement protocols have noticeable impact on IgG features we have studied which might affect both safety and effectiveness of the final product.

plasma processing ; IgG antivenom ; IgG subclasses ; aggregates ; thermal stability

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