Control strategy for the limit of aldehydes in polysorbates based on genotoxicity and influence on the aggregation of monoclonal antibody

An HPLC pre-column derivatization detection method was established to detect and analyze the formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 from different manufacturers.The effects of aldehyde and acetaldehyde on the aggregation of adalimumab under different conditions were monitored.Based on the control of genotoxic impurities and the influence on the stability of monoclonal antibody preparations, the control limits of the two chemicals were preliminarily obtained.2, 4-dinitrophenylhydrazine (2, 4-DNPH) was applied as the derivatization reagent in HPLC pre-column derivatization; acetonitrile and water were used as mobile phase to perform a gradient elution on a C₈ (4.6 mm × 150 mm, 5 μm) chromatographic column.The detection wavelength was 360 nm, and the external standard method was used for quantification.Verification results showed that the method was suitable for the quantitative analysis of trace formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 . The detection and analysis of formaldehyde or acetaldehyde in different batches of polysorbate 80 and polysorbate 20 from different manufacturers showed that the content of formaldehyde and acetaldehyde were quite different. The content of formaldehyde and acetaldehyde in polysorbate 80 were significantly higher than those of polysorbate 20. After monitoring the changes of adalimumab aggregates treated by formaldehyde and acetaldehyde by size exclusion chromatography (SEC), it was found that the effect of formaldehyde on adalimumab aggregation was significantly higher than that of acetaldehyde.According to the requirements of ICH M7 (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk), the impurity limits of formaldehyde and acetaldehyde in polysorbate 80 and polysorbate 20 for monoclonal antibody preparations were calculated from the perspective of risk assessment.Combined with the influence on the aggregation stability of monoclonal antibodies, the preliminary limis for acetaldehyde and acetaldehyde were recommended to be ≤ 7 μg/g and ≤ 765 μg/g, respectively.