The novel prenyl transferase-mediated, site-specific, antibody–drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2), with the equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 19.17~958.67 ng/mL for acDrug. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions (short-term: 1 day, long-term: 10 months, Freeze/Thaw (F/T): 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats.

A simple liquid chromatography–quadrupole-time-of-flight–mass spectrometric assay (LC-TOF-MS/MS) has been developed for the evaluation of metabolism and pharmacokinetic (PK) characteristics of monomethyl auristatin F (MMAF) in rat, which is being used as a payload for antibody-drug conjugates. LC-TOF-MS/MS method was qualified for the quantification of MMAF in rat plasma. The calibration curves were acceptable over the concentration range from 3.02 to 2200 ng/mL using quadratic regression. MMAF was stable in various conditions. There were no significant matrix effects between rat and other preclinical species. The PK studies showed that the bioavailability of MMAF was 0% with high clearance. Additionally, the metabolite profiling studies, in vitro/in vivo, were performed. Seven metabolites for MMAF were tentatively identified in liver microsome. The major metabolic pathway was demethylation, which was one of the metabolic pathways predicted by MedChem Designer. Therefore, these results will be helpful to understand the PK, catabolism, and metabolism behavior of MMAF comprehensively when developing antibody-drug conjugates (ADCs) in the future.

MMAE is a potent antimitotic drug used as payload of an antibody‐drug conjugate which shows potent activity in preclinical and clinical studies against a range of lymphomas, leukemia and solid tumors. Liquid chromatography‐high resolution mass spectrometric method was developed for the quantification of MMAE and its preclinical pharmacokinetics. The method consisted of protein precipitation using acetonitrile (ACN) for sample preparation and liquid chromatography – quadrupole – time‐of‐flight – tandem mass spectrometry (LC‐qTOF‐MS/MS) analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 1.01–2,200 ng/mL for MMAE. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. Recovery was 42.84%. The dilution integrity was determined for 5‐fold dilution and the accuracy and precision ranged within ±25%. The stability results indicated that MMAE was stable for the following conditions: short‐term (4 h), long‐term (4 weeks), freeze/thaw (3 cycles) and post‐preparative stability (12 h). This qualified method was successfully applied to a pharmacokinetic study of MMAE in rat as a preclinical animal model. The PK results suggest that MMAE has moderate CL and low BA.Also, these results would be helpful in having a comprehensive understanding of the PK characteristics of MMAE and developing ADC in future.

A liquid chromatography–quadrupole time-of-flight (Q-TOF) mass spectrometric method was developed for early-stage research on adalimumab in rats. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC-QTOF-MS/MS analysis of specific signature peptides of adalimumab in the positive ion mode using electrospray ionization. This specific signature peptide is derived from the complementarity-determining region (CDR) of adalimumab. A quadratic regression (weighted 1/concentration), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 1–100 μg/mL for adalimumab. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. This qualified LC-QTOF-MS/MS method was successfully applied to a pharmacokinetic study of adalimumab in rats as a case study. This LC-QTOF-MS/MS approach would be useful as a complementary method for adalimumab or its biosimilars at an early stage of research.

A single hybrid affinity-captured-LC-TOF-MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine-citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65-613.00 ng/mL with an equation y = ax + bx + c for the antibody-conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on-bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00-100.00 μg/mL with an equation y = ax + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC-TOF-MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01-2200 ng/mL with an equation y = ax + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity-captured-LC-TOF-MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine-citrulline linker at the early drug discovery stage.