Quantification of active and total adalimumab using affinity purification coupled with tryptic digestion-based liquid
Xiaoran Hou1, Yifei Liu1, Zengxuan Cai2
1Tianjin Medical University General Hospital, No. 154 Anshan Road, Heping District, Tianjin 300070, China; Zhejiang Key Laboratory of Artificial Organs and Computational Medicine, Zhejiang Shuren University, 8, Shuren Street, Hangzhou 310015, China.
Abstract:
Therapeutic drug monitoring is commonly employed to investigate the loss of response to adalimumab. However, existing methodologies focus exclusively on measuring active adalimumab, neglecting the presence of inactive forms, which may result in incomplete or misleading clinical evaluations. To address this limitation, a liquid chromatography-tandem mass spectrometry method incorporating affinity purification was developed for the simultaneous quantification of both active and total adalimumab concentrations in serum samples. A signature peptide, APYTFGQGTK, was selected from in silico-generated peptides derived from the Fab region based on its selectivity and sensitivity. An isotope-labeled analog, [13C9, 15 N]-APYTF*GQGTK, was used as the internal standard. Sample pretreatment steps, including acidic dissociation, competitive binding using biotinylated tumor necrosis factor alpha (TNFα), and affinity capture, were optimized to achieve maximal recovery of both active and total adalimumab. The method was validated in accordance with the U.S. Food and Drug Administration guidelines, demonstrating excellent recovery (89.4-111.4 %), precision (coefficient of variation < 9.7 %), and linearity (r² > 0.998) across a concentration range of 1-32 μg/mL. Clinical sample analysis revealed a strong correlation (r = 0.929) with the latex-enhanced turbidimetric immunoassay; however, a systematic bias of 14.1 % was observed. The ratio of active to total adalimumab concentrations varied between 53.5 % and 115.8 %. These results of clinical samples have generated significant interest, warranting further investigation into the quantitative methods and the concentration ratios, both of which may have clinical significance in guiding drug response. The results also emphasize the importance of conducting quantifications within the same analytical system to ensure data comparability. Moreover, the proposed methodology provides a robust and adaptable framework that can be extended to the quantification of other therapeutic monoclonal antibody drugs.
