Objective: To establish an enzyme-linked immunosorbent assay (ELISA) for the detection of the concentration of antibody-drug conjugate [ADC006M, recombinant anti-human epidermal growth factor receptor 2 (HER2) domain Ⅱ humanized monoclonal antibody-monomethylauristatin E (MMAE) conjugate] in rat plasma, and to study its pharmacokinetic characteristics of single intravenous injection. Methods: Based on the test principle of ELISA, 96-well plate was pre-coated with anti-MMAE antibody, after adding the samples, detected with HRP-labeled antibody B1G7 (B1G7-HRP). Then, TMB substrate solution was added for color display. After the reaction was quenched,the absorption(A)was read at double wavelength of 450 nm/630 nm by using a microplate reader. With reference to the relevant regulations, the method had been validated. Meanwhile, SD rats were given 5 mg·kg-1 ADC006M intravenously to determine the plasma concentration,and the curve fitting was performed tocalculate the pharmacokinetic parameters. Results: The linear range of the method was 31.25-2 000 ng·mL-1, with lower limit of quantitation (LLOQ) of 31.25 ng·mL-1. The accuracy RE of intra-assay and inter-assay were -14.6% to 12.6% and -13.1% to -0.7%. The intra- and inter- precision RSD were both ≤12.6%, respectively. The selectivity (matrix effect), specificity, dilution linearity, hook effect, and stability of the method were all good. The pharmacokinetic parameters of ADC006M were determined by plasma concentration of SD rats, the Cmax was (114 218±17 845) μg·L-1 for female and (119 900±12 259) μg·L-1 for male, respectively, AUC0-∞ was (90 546±6 481) μg·L-1·d-1and (89 648±13 735) μg·L-1·d-1, t1/2 was (1.46±0.33) d and (1.70±0.28) d, respectively. Conclusion: An ELISA method was established to detect the concentration of ADC006M in the plasma of SD rats,its specificity, accuracy, precision, stability and so on all conform to the requirements of the research guidelines of biological preclinical pharmacokinetics. There was no gender difference in the pharmacokinetic characteristics of ADC006M in SD rats. It is basically consistent with the metabolic characteristics of similar ADC drugs and has great pharmacokinetic characteristics.
PENG Jia-bao, PENG Ying
. Determination of antibody-drug conjugate in rat plasma by ELISA and study of its pharmacokinetics[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(6)
: 1037
-1044
.
DOI: 10.16155/j.0254-1793.2022.06.16
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