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Article

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches

1
University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Rockefellerova 10, HR-10000 Zagreb, Croatia
2
Centre of Excellence for Virus Immunology and Vaccines, CERVirVac, Rockefellerova 10, HR-10000 Zagreb, Croatia
3
Vienna University of Technology, Institute for Chemical Technologies and Analytics, Getreidemarkt 9/164, A-1060 Vienna, Austria
*
Authors to whom correspondence should be addressed.
Received: 25 September 2020 / Revised: 10 December 2020 / Accepted: 11 December 2020 / Published: 14 December 2020
(This article belongs to the Special Issue Novel Strategies for the Diagnosis and Treatment of Snakebites)
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. View Full-Text
Keywords: plasma processing; IgG antivenom; IgG subclasses; aggregates; thermal stability plasma processing; IgG antivenom; IgG subclasses; aggregates; thermal stability
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MDPI and ACS Style

Mateljak Lukačević, S.; Kurtović, T.; Lang Balija, M.; Brgles, M.; Steinberger, S.; Marchetti-Deschmann, M.; Halassy, B. Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches. Toxins 2020, 12, 798. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12120798

AMA Style

Mateljak Lukačević S, Kurtović T, Lang Balija M, Brgles M, Steinberger S, Marchetti-Deschmann M, Halassy B. Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches. Toxins. 2020; 12(12):798. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12120798

Chicago/Turabian Style

Mateljak Lukačević, Sanja, Tihana Kurtović, Maja Lang Balija, Marija Brgles, Stephanie Steinberger, Martina Marchetti-Deschmann, and Beata Halassy. 2020. "Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches" Toxins 12, no. 12: 798. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12120798

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