Monoclonal antibodies: Development of universal (platform) methods for the assessment of high and low-molecular weight impurities

INTRODUCTION. The development of unified techniques for quality control monoclonal antibodies (mAb) is one of the challenges for mAbs quality standardization.

AIM. This study aimed to develop and analyse the possibility of using platform (universal) techniques for estimating the impurity content of high molecular weight compounds and fragments, non-glycosylated molecules in various mAbs by methods of size-exclusion high performance liquid chromatography (SE-HPLC) and capillary electrophoresis methods.

MATERIALS AND METHODS. MAbs-containing medicinal products from 28 different international nonproprietary names of Russian and foreign origin. Studies by SE-HPLC were carried out on Agilent Technologies 1200 series instruments equipped with UV detectors, with data processed via OpenLab software. Capillary gel electrophoresis (CGE) was conducted under both reducing and non-reducing conditions using a PA 800 plus system (Beckman Coulter) with UV diode array detection, and Beckman 32Karat software.

RESULTS. Universal sample preparation protocols and separation conditions were established and validated across 28 mAbs. Retention times and relative migration times of target compounds peaks were determined for each mAb. Comparative analysis demonstrated concordance between platform methods and manufacturers' proprietary methods. Validation of methods for determination of the content of high-molecular weight compounds in mAbs samples by the SE-HPLC method, fragments of mAbs and non-glycosylated variants of heavy chains of mAbs under reducing and non-reducing conditions by the CGE method was performed. The accuracy, precision, and sensitivity of the methods met the established requirements. The validation characteristics obtained for the SE-HPLC method were as follows: precision (RSD) of peak areas was no more than 0.4% for the monomer, up to 8% for aggregate groups, no more than 0.4% for total peak areas, and 0.02% for the relative peak area corresponding to the monomer. The method's linearity was confirmed within the concentration range of 0.5–120%, accuracy within 99.1–102.1%, and the limit of quantification (LOQ) was 0.1%. For the CGE method, the validation characteristics showed precision of no more than 1% for intact immunoglobulin content or heavy and light chain peaks, and no more than 1% for absolute migration times of main peaks. Linearity was confirmed from LOQ to 300% concentration range. The accuracy for both methods ranged from 97.6–103.7%, with LOQ values of 0.5% and 0.75%, respectively.

CONCLUSIONS. The developed methods for assessing the purity are universal for unconjugated mAbs products. Their validation characteristics — including specificity, precision, limit of quantification, analytical range, linearity, and accuracy — meet all established acceptance criteria. These methods can be reliably implemented at any stage of the product lifecycle for this category of medicinal products.

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