Evaluation of the Molecular Size Distribution of Rabies Immunoglobulin Using High-Performance Liquid Chromatography

Rabies immunoglobulin is one of the most commonly used antiviral serum drugs. In the Russian Federation, more than 400,000 people are at risk of contracting rabies every year. In order to improve the drug safety when using heterologous immunoglobulin, it is advisable to introduce such a quality indicator as molecular size distribution. This indicator reflects the distribution of single immunoglobulin molecules, their dimers, aggregates, and fragments in the drug. The study of the content of these fractions in the drug using high-performance liquid chromatography is determined by the requirements of the XIV edition of the State Pharmacopoeia of the Russian Federation. The aim of the present study was to establish the conditions for high-performance liquid chromatography when determining the molecular size distribution of heterologous rabies immunoglobulin. A method for determining the indicator of molecular size distribution using a BioSep-SEC-s3000 (“Phenomenex Inc.”, USA) column and a Biologic Duoflow (“BioRad”, USA) chromatographic system is proposed. Commercial and experimental samples of rabies immunoglobulin obtained from horse blood serum were used. When studying chromatographic parameters (resolution between peaks, retention time, peak symmetry, etc.), analysis conditions in accordance with the pharmacopoeial requirements were selected. The resolution values were: between the peaks of monomers and dimers — 1.42; between the peaks of aggregates and monomers – 3.9, between the peaks of monomers and fragments – at least 4.46. The experiment showed the specificity and repeatability of the technique. Additionally, a high degree of purification of the studied commercial samples of rabies immunoglobulin was shown, which is characterized by the absence of fractions of aggregates and fragments. The results obtained allow us to conclude that the proposed method can be used using a BioSep-SEC-s3000 column and a Biologic Duoflow chromatographic system to control the quality of heterologous rabies immunoglobulin in terms of molecular size distribution.