Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins and a bi-specific antibody (bsAb) were investigated using size exclusion chromatography (SEC). Their levels of high molecular weight species (HMWS) were experimentally estimated between 0.1% and 13.1%. Except for blinatumomab, etanercept and pembrolizumab, the HMWS amount for the other antibodies was well below the limit of 5% usually set a specification for therapeutic mAbs in the biopharmaceutical industry. The main chromatographic peak shape of 24 therapeutic antibodies and the NIST mAb [1] was found suitable (0.8<As<1.5) with a generic SEC method involving potassium-based salts mobile phase. Conversely, only acidic therapeutic proteins (pI<7) could be successfully analyzed with a mass spectrometry (MS) compatible mobile phase containing 100mM ammonium acetate. This study aimed to provide HMWS data for 30 therapeutic proteins covering a wide range of physico-chemical properties with molecular weights between 54 and 153kDa, pI values comprised between 6.1 and 9.4 and hydrophobic interaction chromatography (HIC) retention factors ranging from 1.2 to 6.0 for the mAbs.
Keywords: Aggregates; Antibody-drug conjugates; Bi-specific antibodies; Fc-fusion proteins; Monoclonal antibodies; SEC-MS; Size exclusion chromatography.
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