Development and validation of a semi-automated hybrid immunocapture liquid chromatography-mass spectrometry method to...

Biotherapeutic antibodies are increasingly being developed and, various strategies have recently been used to maximize their potential therapeutic efficacy. The crystallizable fragment (Fc) region of therapeutic monoclonal antibodies (mAbs) is often engineered to tailor their effector functions and pharmacokinetic (PK) properties by introducing point mutations. Notably, most of these mutations are in the hinge and constant domains of the heavy chain, which may silence antibody effector functions. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been published to quantify biotherapeutics with a canonical human Fc portion. This work presents a rapid and sensitive hybrid immunocapture liquid chromatography-tandem mass spectrometry (IC-LC-MS/MS) method for quantifying total antibody concentration, specifically targeting the LALA-mutated peptide (L234A/L235A). The sample preparation process, which includes immunocapture, as well as trypsin and Glu-C digestion, is efficiently completed within two days through automation. The developed method was validated according to the ICH M10 guideline and white papers recommendation, focusing on the following parameters - accuracy, precision, dilution linearity, selectivity, stability, recovery- and using a humanized IgG1 LALA-mutated antibody, teplizumab, as analytical standard. The method demonstrated linearity for total antibody detection in mouse plasma samples, with a dynamic range from 150 ng/mL (lower limit of quantification, LLOQ) to 15,000 ng/mL (upper limit of quantitation, ULOQ). All validation parameters tested in mouse plasma met the predefined acceptance criteria, demonstrating the method's reliability and robustness. Additionally, the qualified method was successfully used to characterize the pharmacokinetic profile in mice of an antibody-drug conjugate (ADC-1) containing the LALA mutation in its Fc region. This work provides a valuable foundation for the quantification of new biological entities (NBEs) and antibody-drug conjugates (ADCs) in pharmaceutical development, as it enables the measurement of engineered Fc biotherapeutics using a unique and highly selective peptide, irrespective of the type of biological matrix and even in the presence of other biomolecules of similar IgG isotype.