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Comparison of five protocols for the whole IgG snake antivenom purification in terms of stability, purity and immunoglobulin subclass composition

The whole IgG antivenoms are obtained from the hyperimmune animal plasma by various refinement strategies. Besides therapeutic action, the “refined” preparations still commonly cause clinical side effects attributable to contaminating proteins and /or aggregates. There has been a hypothesis in the literature, that some purification procedures (precipitation of IgGs, low pH-mediated elution of IgGs in chromatography), which induce transient conformational changes of IgG molecules, make their structure less stable and more prone to aggregation, in contrast to others that leave them in solution throughout (caprylic acid precipitation of non-IgG plasma proteins). Our goal was to verify this hypothesis by comparing IgGs isolated by five different commonly employed protocols from the same plasma in terms of stability, purity and immunoglobulin subclass composition. The fractionation methods were: ammonium sulfate 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 generated in AC, CEC and ASP, as shown by SDS-PAGE and SEC-HPLC. Some protocols influenced IgG subclass composition, with AC generating the highest loss of IgG(T), as shown by ELISA. Less pure IgG fractions (from ASP, AEC and CEC) 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. However, one month storage of IgGs at 37 °C did not influence either Tm or aggregate content of analysed preparations. Our so far results indicate that different procedures gain IgGs of variable purity and subclass composition which might affect both safety and effectiveness. Still, conformational changes during purification procedures might not be the trigger for increased aggregation.

antivenom, purification, IgG, stability, purity

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