(W1030-06-36) Development of a Rapid, High Throughput Quantitative Bioanalysis of Conjugated Payloads of Protease-Cleavable Antibody-Drug Conjugate (ADC) in Rat Serum Using Automated Immunocapture Based UPLC-MS/MS

Purpose: Antibody Drug Conjugate (ADC) is a new therapy that combine the high targeting of monoclonal antibodies with the strong cytotoxicity of small molecule toxins. Due to its precise and powerful effects, it has become an important trend in the development of anti-tumor antibody drugs. ADC consists of three parts, including the antibody part, the small molecule payload and the linker that connects the two. The absorption, distribution, metabolism and excretion (ADME) of ADC drugs directly affect the efficacy and safety of ADC which is more complex and challenging than other drugs. Not only large molecule parts (total antibody and conjugated antibody) need to be analyzed, but also small molecule components such as free payload and conjugated payload. Quantitative bioanalysis of conjugated payloads, free payloads and related metabolites runs through all stages of ADC drug development, including early discovery, screening, preclinical and clinical stage. Among the 15 ADC drugs currently on the market, there are 8 ADC drugs with enzyme-cleavable linkers, and six of them have MC-VC-PABC linker. The linker of DS-8201 is MC-GGFG which is used in many drugs development. This study took these two enzyme-cleavable linkers with the highest proportion as examples to establish a rapid, high-throughput, sensitive and reliable conjugated payload quantification method, which was successfully applied to in vivo PK rat serum samples.

Methods: DS-8201 and RC48 (basic information were listed in Table 1) working solution were spiked into rat serum to prepare standards and quality controls. The samples were immunocaptured by using Hamilton with IMCS tips which were coated with streptavidin. Digestion with papain then conjugated payloads were analyzed by LC-MSMS. Instruments and mobile phase information were listed in Table 1.

Results: After automatic immunocapture and enzyme digestion, the linear range of DS-8201 was from 0.05 to 200 µg/mL with excellent linearity (r² > 0.999), converted to the corresponding conjugate payload (DXd) concentration was from 1.3 to 5045.3 ng/mL. The lower limit of quantitation (LLOQ) of DS-8201 was 50 ng/mL in rat serum. Equivalent to the LLOQ of DXd was 1.3 ng/mL in rat serum. (The chromatogram is shown in Figure 1). Results showed the within-run accuracy were from 5.00% to 17.0% and precision were from 9.98% to 13.9% (Table 2). The linear range of RC48 was from 0.01 to 200 µg/mL with a good linearity (r² > 0.994), converted to the corresponding conjugate payload (MMAE) concentration was from 0.2-3984 ng/mL. The LLOQ of RC48 was 10 ng/mL, equivalent to the LLOQ of MMAE was 200 pg/mL in rat serum. (The chromatogram is shown in Figure 1). Results showed the within-run accuracy were from 5.00% to 11.0% and precision were from -4.21% to 3.26% (Table 2). No matrix effect or carryover was observed for this method. Non-specific binding: we added 0.05% Triton to working solution and low-binding materials to avoid non-specific binding. The immunocapture time of traditional magnetic bead is about 8 hours, which is shortened to 2 hours by using automation. Compared with magnetic beads immunocapture, automated immunocapture method has the advantages of rapid, high throughput and reduced human error. The enzyme digestion efficiency is 92.1% for MC-GGFG linker and for MC-VC-PABC linker, the digestion efficiency is 78.0%. (Table 2).

Conclusion: In this work, we developed a rapid, high throughput automated immunocapture based UPLC-MS/MS bioanalysis method for the quantitation of conjugated payload in rat serum. The validated method shown efficient and reliable sensitivities, good linearity, intra-day precision and accuracy, as well as excellent stability. This method is applicable to enzyme-cleavable linkers such as MC-GGFG and MC-VC-PABC. Established methods are successfully applied to the in vivo rat serum PK study. The advantages of our approach: Automated Immuno-capture: Compared with the magnetic bead method, the pretreatment time is reduced from 8 h to 2 h. High digestion efficiency and immuno-capture efficiency. Sample volume:10 µL.

References: [1] Azar I , Alkassis S , Fukui J ,et al. Spotlight on Trastuzumab Deruxtecan (DS-8201,T-DXd) for HER2 Mutation Positive Non-Small Cell Lung Cancer[J].Lung Cancer: Targets and Therapy, 2021, Volume 12:103-114.
[2] Fan S , Yanli L, Xuexiao Z, et al. Disitamab vedotin: a novel antibody-drug conjugates for cancer therapy. Drug Delivery. (2022)29. 1335-1344.

Acknowledgements: We thank the Large Molecular and Biomarker Analysis team for sample analysis. Internal funding for this work was provided by WuXi AppTec. No funding was received from outside institutions or funding agencies.

Table 1. Basic information and LC-MS/MS condition for DS-8201 and RC48

Figure 1. Chromatogram of LLOQ for DXd and MMAE in rat serum
(A) LLOQ of DS-8201 conjugated payload DXd was 1.3 ng/mL (B) Calibration Curve of DS-8201 (C) LLOQ of RC48 conjugated payload MMAE was 200 pg/mL (D) Calibration Curve of RC48

Table 2 Accuracy and precision of DXd and MMAE in rat serum samples and digestion efficiency results