Abstract
Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %. Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease. Specific activity of these products is largely determined by their fractional composition, and, in particular, by molecular mass distribution (MMD). The size-exclusion-high-performance liquid chromatography (SEC-HPLC) has traditionally been used for determination of the MMD of the target protein in human immunoglobulin-based products. The use of this method for evaluation of molecular parameters of heterologous immunoglobulin requires confirmation of its specificity, accuracy and precision, and establishment of the chromatographic system suitability criteria in the context of a new test object.The aimof the study was to test the applicability of the SEC-HPLC method to the assessment of molecular parameters of anti-Ebola immunoglobulin derived from horse serum.Materials and methods: three batches of purified equine anti-Ebola immunoglobulin were used in the study. Normal equine and human immunoglobulins of the IgG isotype were used as reference standards. The HPLC test procedures described in the European Pharmacopoeia 9.6 and State Pharmacopoeia of the Russian Federation, 14th ed., were used for determination of monomers and other immunoglobulin fractions. An Agilent 1260 Infinity (Agilent, USA) HPLC system with a diode array detector and an Agilent Bio SEC-3 HPLC column were used for quality evaluation of the tested products.Results: the resolution factor between IgG monomer and dimer peaks (1.69 and 2.10), and the chromatographic column efficiency (>2000) make it possible to use the SEC-HPLC system for evaluation of molecular parameters of heterologous immunoglobulin. The study demonstrated reproducibility of the test procedure.Conclusions: the study confirmed the applicability of the SEC-HPLC procedure for evaluation of molecular parameters of anti-Ebola immunoglobulin derived from horse serum. It demonstrated the compliance of the purified immunoglobulin to the national and international quality requirements in terms of «Molecular parameters».
Highlights
Апробация методики эксклюзионной хроматографии для оценки молекулярных параметров иммуноглобулина против лихорадки Эбола из сыворотки крови лошадей
Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %
Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease
Summary
1. Испытуемые образцы 1–3: иммуноглобулин против лихорадки Эбола из сыворотки крови лошадей, очищенный (серии No 2–4, иммуноглобулин Эбола), раствор, содержащий 20 ммоль натрия дигидрофосфата моногидрата и 0,2 % глицина. 3. Стандартный образец 2 — раствор человеческого нормального иммуноглобулина (фармакопейный стандарт), кат. Растворы маркеров молекулярных масс и стандартных образцов готовили согласно инструкциям по применению. 7. Определение молекулярных параметров испытуемых образцов проводили в соответствии с требованиями Европейской фармакопеи 9.6 и Государственной фармакопеи Российской Федерации 6. Испытуемые образцы доводили до концентрации 5 мг/мл раствором, содержащим 20 ммоль натрия дигидрофосфата моногидрата и 0,2 % глицина. Последовательность ввода проб: П‐С1‐С2‐О1‐О2‐О3‐С1‐С2, где П — плацебо, С1 и С2 — растворы стандартных образцов лошадиного и человеческого иммуноглобулинов соответственно, О1, О2, О3 — испытуемые образцы иммуноглобулинов (серии No 2–4).
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