Abstract
Whole IgG antivenoms are prepared from hyperimmune animal plasma by various refinement strategies. The ones most commonly used at industrial scale are precipitation by sodium or ammonium sulphate (ASP), and caprylic acid precipitation (CAP) of non-immunoglobulin proteins. The additional procedures, which have so far been used for experimental purposes only, are anion-exchange (AEX) and cation-exchange chromatography (CEX), as well as affinity chromatography (AC) using IgG’s Fc-binding ligands. These protocols extract the whole IgG fraction from plasma, which contains both venom-specific and therapeutically irrelevant antibodies. Such preparations represent a complex mixture of various IgG subclasses whose functional and/or structural properties, as well as relative distribution, might be affected differently, depending on employed purification procedure. The aim of this work was to compare the influence of aforementioned refinement strategies on the IgG subclass distribution, venom-specific protective efficacy, thermal stability, aggregate formation and retained impurity profile of the final products. A unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma was used as a starting material, enabling direct comparison of five purification approaches. The highest purity was achieved by CAP and AC (above 90% in a single step), 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 IgG sample from AEX processing. Alpha-1B-glycoprotein was present in CAP IgG fraction, as well as in those from ASP- and AEX-based procedures. 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 preparation as measured in vivo. AC and CEX remarkably affected drug’s venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. Presented data might improve the process of designing and establishing novel downstream processing strategies and give guidance for optimization of the current ones by providing information on potency-protecting and purity-increasing properties of each purification principle.
Highlights
Snakebite envenoming is a medical emergency in most parts of the world, serious in tropical countries where it causes significant socioeconomic problems, for the victims but for their entire families and communities as well [1,2]
immunoglobulin G (IgG) preparations derived from the unique pool of Vipera ammodytes ammodytes (Vaa)-specific hyperimmune horse plasma (HHP)
Products obtained by ammonium sulphate precipitation (ASP1) as well as anion- and cation-exchange chromatography procedures (AEX1 and CEX1) retained noticeable amount of impurities (Figure 1; Table 1)
Summary
Snakebite envenoming is a medical emergency in most parts of the world, serious in tropical countries where it causes significant socioeconomic problems, for the victims but for their entire families and communities as well [1,2]. Antivenom usage is not completely devoid of adverse reactions whose pathogenesis has not yet been fully understood [9] For such effects have been attributed to the properties of the therapeutic itself (total amount of proteins, purity, specific activity, aggregate content, form of active drug, and formulation) and/or to some extent to heterologous nature of animal IgGs compared to humans [9,10]. Still, most of these quality-related features are a result of the production procedure itself
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