Quantiﬁcation for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Afﬁnity Capture Liquid Chromatography-Mass Spectrometric Method

: Trastuzumab emtansine (T-DM1, brand name: Kadcyla ® ) is the ﬁrst FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-afﬁnity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantiﬁed as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax 2 + bx + c, was used to ﬁt the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantiﬁcation run met the in-house acceptance criteria of ± 25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-afﬁnity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.

From the perspectives of the full pharmacokinetic (PK) properties of ADC, three main components including (1) total antibody (tAb), (2) antibody-conjugated drug (acDrug)/(or conjugated antibody), and (3) free payload should be quantitated from in vivo samples [15]. Among these three main analytes, acDrug demonstrates the concept that ADC exists in an active form as a conjugate [16]. The acDrug may also play an important role in the doseresponse analysis of ADCs. However, it was very difficult to evaluate the acDrug of the first generation ADCs without cleavable linkers such as trastuzumab emtansine (T-DM1, brand name: Kadcyla ® ) by LC-MS, and therefore it was reported that the conjugated antibody instead of acDrug were quantitated by ELISA assay (https://www.accessdata.fda.gov/ drugsatfda_docs/nda/2013/125427Orig1s000PharmR.pdf (accessed on 1 September 2021)) for Kadcyla. Recently, more attention is paid to the importance of the in vivo acDrug levels of ADCs regarding the efficacy, in vivo drug-antibody ratio (DAR) evaluation, and toxicity. Currently, only ADCs with cleavable linkers are easily applicable to the quantitation of acDrug by the immuno-affinity capture method followed by on-bead digestion using specific enzymes such as cathepsin B or beta-glucuronidase for the LC-MS analysis of acDrug [16][17][18]. Due to lack of specific sites in the linker, this method was not applicable to Kadcyla, which contains non-cleavable linkers.
In this paper, we developed an immuno-affinity capture LC-MS/MS assay using previously reported chemical desulfuration pretreatment [19] to quantify acDrug in rat preclinical PK samples even for trastuzumab emtansine (T-DM1, brand name: Kadcyla ® ), a representative ADC with non-cleavable linkers. T-DM1 is the first FDA-approved ADC to treat metastatic human epidermal growth factor receptor 2 (HER2)-positive breast cancer and consists of the monoclonal antibody trastuzumab, maleimidomethyl cyclohexane-1carboxylate (MCC) as a non-cleavable linker, and maytasinoid as a payload [20]. This developed assay was applied to evaluate in vitro plasma linker stability and preclinical PK studies in rats. To our best knowledge, this is the first approach to quantitatively evaluate the acDrug of T-DM1 in vivo rat PK samples.

Materials
T-DM1 was purchased from Dongwon Pharmaceutical (Daejeon, Korea). Verapamil for internal standard (ISTD) was purchased from Sigma-Aldrich Korea (Seoul, Korea). Sodium borohydride (NaBH 4 ) and nickel chloride hexahydrate (NiCl 2 ·6H 2 O) for the chemical desulfuration pretreatment were also purchased from Sigma-Aldrich Korea (Seoul, Korea). The protein A magnetic bead was purchased from Millipore Korea (Seoul, Korea). Rat blank plasma (Sprague Dawley (SD) rat, fasted male, Na-heparin anticoagulant) was purchased from Biomedex (Seoul, Korea). Other reagents were commercially purchased for analytical purposes or reagent grading and were used without further purification.

Preparation of Stocks, Standard (STD) and Quality Control (QC) Samples
A stock solution (1 mg/mL) of T-DM1 was prepared in phosphate buffered solution (PBS) and stored at 4 • C. A stock solution was diluted with PBS and spiked into rat blank plasma to make STD and QC samples. The calibration curve was made up of eight calibrated STD samples with a final plasma concentration range of 17.09~1709.44 ng/mL. QC samples in the final plasma concentration were 42.74 (low QC), 427.36 (medium QC) and 854.72 (high QC) ng/mL.

Sample Preparation
Each 24 µL of STD and QC plasma sample was mixed with 350 µL of 0.1% tween 20 in PBS and 30 µL of protein A magnetic bead. In addition, 20 µL of all study samples were also mixed with 354 µL of 0.1% tween 20 in PBS and 30 µL protein A magnetic bead (https://www.merckmillipore.com/KR/ko/product/PureProteome-Protein-A-Magnetic-Bead-System,MM_NF-LSKMAGA10#anchor_BRO (accessed on 1 September 2021)) [8]. After vortexing, the mixture samples were incubated gently for about 2 h at room temperature and then the samples were fixed on a magnetic rack and washed with 200 µL of 0.1% tween 20 treated PBS and 200 µL of PBS.
For the chemical desulfuration pretreatment, the washed samples were treated with 90 µL of 0.2 M glycine hydrochloric acid (Gly-HCl) and incubated for 20 min at 60 • C. After incubation, samples were chilled for 5 min at 4 • C. Then, 100 µL of methanol, 40 µL of NiCl 2 ·6H 2 O (100 mg/mL) and 30 µL of NaBH 4 (40 mg/mL) were added to the chilled samples [19]. Figure 1 shows the chemical desulfuration pretreatment mechanism of the thioether bond between the MCC linker and the DM1 of T-DM1.
The above mixture samples were gently shaken at room temperature overnight and centrifuged at 12,000 rpm for 10 min. The supernatant samples were transferred into another Eppendorf tube and evaporated to dryness under vacuum in a centrifugal evaporator (CVE-3110, Eyela, Tokyo, Japan) connected to a cold trap (UT-1000, Eyela, Tokyo, Japan). The dried residue samples were reconstituted using 30% acetonitrile (ACN) solution dissolved with 100 ng/mL of verapamil as an ISTD solution, vortexed, and then centrifuged at 12,000 rpm for 10 min. The supernatant samples were transferred into LC vials for LC-MS/MS analysis.

Method Qualification
Method qualification was carried out with a fit-for-purpose approach [8]. The qualification run contained duplicate STDs at eight concentrations and QCs at three different level concentrations. The in-house acceptance criteria for STDs and QCs in the qualification run were within ±25% of the precision and accuracy values. A quadratic regression (weighted 1/concentration), with an equation y = ax 2 + bx + c, was used to fit the calibration curves. In addition, two blank plasma samples were run. The intra-and inter-day run for the accuracy and precision assays were calculated at each QC concentration.
Preliminary stability tests were conducted in rat plasma samples under different conditions, such as short-term, long-term, and freeze-thaw stability at three different levels of QC. The short-term stability was determined at room temperature for 6 h. The long-term stability was determined by analyzing QC samples kept frozen at −80 • C for 3 months. For the freeze-thaw stability, the samples were subjected to three freeze-and-thaw cycles at −80 • C. The in-house acceptance criteria for all preliminary stability tests were also within ±25% of the precision and accuracy values.

Application for In Vitro Linker Stability Test and Preclinical Pharmacokinetic (PK) Study in Rats
For the in vitro linker stability test, 10 µL of 500 µg/mL T-DM1 in PBS was spiked into 50 µL of rat blank plasma to make an incubation sample. After incubation at 37 • C for 0, 1, 3, 5, and 7 days, the incubated samples were stored at −80 • C until analysis.
The preclinical PK studies were conducted in SD rats. T-DM1 was administered to rats via single intravenous bolus injection (3 mg/kg). Blood samples were collected at 3 min; 1, 4 and 7 h; 1, 2, 4, 7, 14, and 21 days post-dose in heparinized tubes. The blood samples were centrifuged, and the supernatant plasma were stored at −80 • C until analysis. Animal experiments followed the animal care protocol (No. 202003A-CNU-024) approved by the Chungnam National University. All procedures related to animal experiments were also performed in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). PK parameters were calculated by non-compartmental analysis (NCA) using WinNonlin ® version 8.0.0 (Pharsight Corporation, Mountain View, CA, USA).
For the chemical desulfuration pretreatment, the washed samples were treated with 90 µL of 0.2 M glycine hydrochloric acid (Gly-HCl) and incubated for 20 min at 60 °C. After incubation, samples were chilled for 5 min at 4 °C. Then, 100 µL of methanol, 40 µL of NiCl2•6H2O (100 mg/mL) and 30 µL of NaBH4 (40 mg/mL) were added to the chilled samples [19]. Figure 1 shows the chemical desulfuration pretreatment mechanism of the thioether bond between the MCC linker and the DM1 of T-DM1. The above mixture samples were gently shaken at room temperature overnight and centrifuged at 12,000 rpm for 10 min. The supernatant samples were transferred into another Eppendorf tube and evaporated to dryness under vacuum in a centrifugal evaporator (CVE-3110, Eyela, Tokyo, Japan) connected to a cold trap (UT-1000, Eyela, Tokyo, Japan). The dried residue samples were reconstituted using 30% acetonitrile (ACN) solution dissolved with 100 ng/mL of verapamil as an ISTD solution, vortexed, and then centrifuged at 12,000 rpm for 10 min. The supernatant samples were transferred into LC vials for LC-MS/MS analysis.

Method Qualification
Method qualification was carried out with a fit-for-purpose approach [8]. The qualification run contained duplicate STDs at eight concentrations and QCs at three different level concentrations. The in-house acceptance criteria for STDs and QCs in the qualification run were within ±25% of the precision and accuracy values. A quadratic regression (weighted 1/concentration), with an equation y = ax 2 + bx + c, was used to fit the calibration curves. In addition, two blank plasma samples were run. The intra-and inter-day run for the accuracy and precision assays were calculated at each QC concentration.
Preliminary stability tests were conducted in rat plasma samples under different conditions, such as short-term, long-term, and freeze-thaw stability at three different levels of QC. The short-term stability was determined at room temperature for 6 h. The longterm stability was determined by analyzing QC samples kept frozen at −80 °C for 3 months. For the freeze-thaw stability, the samples were subjected to three freeze-and- The pumping mode of two Shimadzu LC-20AD pumps was binary flow and the composition of mobile phases were as follows: mobile phase A was distilled and deionized water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The mobile phase B percent of gradient elution increased from 10% mobile phase B to 95% mobile phase B during run time. The LC gradient for this analysis is summarized in Table 1. The injection volume was 10 µL and LC flow rate was 0.4 mL/min. The desulfurated DM1 was separated through a Phenomenex Kinetex XB-C18 column (2.1 × 50 mm, 2.6 µm). The TOF-MS scan mass spectra and the product ion scan mass spectra were recorded in the positive ion mode. The scan range was at m/z 100~900 in the TOF-MS scan and at m/z 100~750 in the product ion scan. For the quantification, [M+H] + ions of desulfurated DM1 and verapamil were selected at m/z 706.3 and m/z 455.3, respectively and their product ions at m/z 547.2 and m/z 165.1 were used for quantitative analysis, respectively. The temperature of the ion source was 500 • C and the ion spray voltage was at 5500 V. For the desulfurated DM1 and verapamil of ISTD, the declustering potential was 10 V and 125 V, respectively, and the collision energy was 29 V and 30 V, respectively. The analytical run was performed after sufficient system equilibrium was performed. In addition, before the corresponding analytical run, calibration was performed on the TOF-MS and a positive mode scan using a calibration solution standard for TripleTOF™ 5600, and the error ppm was less than 1 ppm.

Method Development and Qualification
An immuno-affinity capture LC-MS/MS assay was used for the quantification of desulfurated DM1 as acDrug of T-DM1. Desulfurated DM1 is the cleaved form of the sulfur element from DM1, and the MS/MS spectrum is shown in Figure 2. The parent [M+H] + ion was detected at m/z 706.3, and the most abundant product ion was at m/z 547.2. desulfurated DM1 and verapamil of ISTD, the declustering potential was 10 V and 125 V respectively, and the collision energy was 29 V and 30 V, respectively. The analytical run was performed after sufficient system equilibrium was performed. In addition, before the corresponding analytical run, calibration was performed on the TOF-MS and a positive mode scan using a calibration solution standard for TripleTOF™ 5600, and the error ppm was less than 1 ppm.

Method Development and Qualification
An immuno-affinity capture LC-MS/MS assay was used for the quantification o desulfurated DM1 as acDrug of T-DM1. Desulfurated DM1 is the cleaved form of the sul fur element from DM1, and the MS/MS spectrum is shown in Figure 2. The parent [M+H] ion was detected at m/z 706.3, and the most abundant product ion was at m/z 547.2. The calibration curves with eight points in the range of 17.09~1709.44 ng/mL in du plicate were freshly prepared for all data sets. The quadratic regression using the ratios o peaks versus concentrations was weighted by 1/concentration. The acceptance of the curve was ≥0.99 for the desulfurated DM1. Figure 3 shows the calibration curve of desul furated DM1. Representative chromatograms of the limit of quantification (LLOQ) and high QC are also shown in Figure 4. The peak retention time was the same in all samples. The calibration curves with eight points in the range of 17.09~1709.44 ng/mL in duplicate were freshly prepared for all data sets. The quadratic regression using the ratios of peaks versus concentrations was weighted by 1/concentration. The acceptance of the curve was ≥0.99 for the desulfurated DM1. Figure 3 shows the calibration curve of desulfurated DM1. respectively, and the collision energy was 29 V and 30 V, respectively. The analytical run was performed after sufficient system equilibrium was performed. In addition, before the corresponding analytical run, calibration was performed on the TOF-MS and a positive mode scan using a calibration solution standard for TripleTOF™ 5600, and the error ppm was less than 1 ppm.

Method Development and Qualification
An immuno-affinity capture LC-MS/MS assay was used for the quantification of desulfurated DM1 as acDrug of T-DM1. Desulfurated DM1 is the cleaved form of the sulfur element from DM1, and the MS/MS spectrum is shown in Figure 2. The parent [M+H] + ion was detected at m/z 706.3, and the most abundant product ion was at m/z 547.2. The calibration curves with eight points in the range of 17.09~1709.44 ng/mL in duplicate were freshly prepared for all data sets. The quadratic regression using the ratios of peaks versus concentrations was weighted by 1/concentration. The acceptance of the curve was ≥0.99 for the desulfurated DM1. Figure 3 shows the calibration curve of desulfurated DM1. Representative chromatograms of the limit of quantification (LLOQ) and high QC are also shown in Figure 4. The peak retention time was the same in all samples. Representative chromatograms of the limit of quantification (LLOQ) and high QC are also shown in Figure 4. The peak retention time was the same in all samples.
The intra-and inter-day runs for the accuracy and precision assays were performed for method qualification and the results are shown in Table 2. The qualification run met the acceptance criteria of ±25% accuracy and precision for QC samples.  The intra-and inter-day runs for the accuracy and precision assays were performed for method qualification and the results are shown in Table 2. The qualification run met the acceptance criteria of ±25% accuracy and precision for QC samples.   Preliminary stability tests were conducted in rat plasma under various conditions, such as short-term, long-term, and freeze-thaw, and the results are shown in Table 3. The accuracy (%) and precision (%CV) of stability samples met the acceptance criteria of ±25%. As a result, the desulfurated DM1 in rat plasma was stable under the different experimental conditions.

Application to In Vitro Plasma Stability Test and Preclinical PK Studies in Rats
This qualified LC-MS/MS assay was applied to the in vitro plasma stability test and a preclinical PK study in rats. The results of the in vitro linker stability test are shown in Figure 5. The remaining (%) of acDrug in T-DM1 was calculated by relative ratio to the concentration of desulfurated DM1 at day 0, which was the initial incubation time. The results show that desulfurated DM1 as acDrug decreased dramatically to 36.97% at day 3, and then slowly decreased until day 7 in rat plasma.

Application to In Vitro Plasma Stability Test and Preclinical PK Studies in R
This qualified LC-MS/MS assay was applied to the in vitro plasma s a preclinical PK study in rats. The results of the in vitro linker stability te Figure 5. The remaining (%) of acDrug in T-DM1 was calculated by rela concentration of desulfurated DM1 at day 0, which was the initial incub results show that desulfurated DM1 as acDrug decreased dramatically to 3 and then slowly decreased until day 7 in rat plasma. Rat plasma samples obtained from preclinical PK study after single intravenous administration of 3 mg/kg for T-DM1 were also analyzed using this qualified LC-MS/MS assay. The PK profiles and parameters for desulfurated DM1 are shown in Figure 6 and Table 4, respectively.
As a result, the half-life (T 1/2 ) was 4.56 ± 1.11 days, the maximum plasma concentration (C max ) was 1396.50 ± 267.92 ng/mL, and the clearance (CL) was 22.55 mL/day/kg. From the PK perspective, the acDrug of T-DM1 as a desulfurated form appeared to show relatively higher CL than the total antibodies of conventional therapeutic monoclonal antibodies. As shown from the in vitro plasma stability results, it is assumed that the linker instability of T-DM1 in rat plasma might play a role to some extent regarding the higher CL in vivo. tively higher CL than the total antibodies of conventional therapeutic monoclona bodies. As shown from the in vitro plasma stability results, it is assumed that the instability of T-DM1 in rat plasma might play a role to some extent regarding the CL in vivo.  T1/2, half-life; Cmax, maximum plasma concentration; AUClast, area under the curve up to last measurable concentratio AUCINF, area under the curve from time zero to infinity; AUC%Extrap, the relative ratio of AUClast to AUCINF; CL, clearanc Vss, volume of distribution.

Conclusions
In this paper, an immuno-affinity capture LC-MS/MS method for the acDrug DM1 in rat plasma was successfully developed and applied to in vitro linker stabili preclinical PK assessment in rats. The chemical desulfuration pretreatment used study was able to specifically cleave thioether bonds between the MCC linker and of T-DM1. The method was qualified in a dynamic range of 17.09~1709.44 ng/mL quadratic regression with 1/concentration weighting, which was sufficient to co time courses for this rat PK study with a dose level of 3 mg/kg for the acDrug of T This immuno-affinity capture LC-MS/MS method was accurate and reproducible determination of acDrug concentration. This method would be also helpful for the a analysis of innovative ADCs with similar non-cleavable linker platforms.