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. 2024 Mar 26;16(9):385–400. doi: 10.4155/bio-2023-0230

Development and validation of bioanalytical methods to support clinical study of disitamab vedotin

Baiyang Wu 1,‡, Qiaoning Li 2,‡, Ling Wang 2, Fang Chen 3, Jing Jiang 1,2,*
PMCID: PMC11216245  PMID: 38530234

Abstract

Disitamab vedotin (RC48), a humanized anti-HER2 antibody conjugated with monomethyl auristatin E (MMAE), is the first antibody–drug conjugate in China with an approved biological license application. A bioanalytical method was established for three analytes (total antibody, conjugate antibody and free payload) to help characterize their pharmacokinetic behavior in clinical settings. The bioanalytical methods were validated according to M10 guidance. Electrochemiluminescence assay methods were used for the quantitative measurement of total antibody and conjugated antibody in human serum. A LC–MS/MS method was used to quantify the concentration of MMAE in human serum. The method had high specificity and sensitivity with a quantitative range of 19.531–1250.000 ng/ml (total antibody), 39.063–5000.000 ng/ml (conjugated antibody) and 0.04–10.0 ng/ml (MMAE), respectively.

Keywords: : bioanalytical method, conjugated antibody, disitamab vedotin (RC48), ECLA, LC–MS/MS, MMAE, total antibody

Plain language summary

Executive summary.

  • The complexity of the structures and mechanisms of action of antibody–drug conjugates (ADCs), combining the molecular characteristics of small-molecule agents and macromolecular biological therapeutic drugs, brings fundamental challenges to pharmacokinetic bioanalysis.

  • A specific and highly sensitive bioassay for the precise determination of pharmacokinetic parameters in human serum matrices, particularly for well-established ADCs, is needed.

  • The accurate measurement of total antibody, conjugated antibody and monomethyl auristatin E (MMAE) levels in ADCs plays a pivotal role in investigating the in vivo pharmacokinetic characteristics of disitamab vedotin and evaluating its drug exposure-response relationship.

  • Bioanalytical methods were developed and validated to quantify total antibody, conjugated antibody and MMAE for disitamab vedotin in human serum.

  • The method used electrochemiluminescence assay and liquid chromatography-mass spectrometry to detect three analytes of disitamab vedotin.

  • During the optimization of the electrochemiluminescence assay method, crucial parameters such as coating conditions, culture duration, detection reagent concentration and minimum sample dilution were meticulously investigated and enhanced through a comprehensive chessboard combination experiment.

  • A stable and sensitive assay was developed following the guidelines on bioanalytical method validation provided by the US FDA and EMA, exhibiting a broad dynamic range of 19.531 to 1250.000 ng/ml for total antibody, 39.063 to 5000.000 ng/ml for conjugated antibody and 0.04 to 10 ng/ml for MMAE.

  • Bioanalytical methods are currently being used in clinical trials for pharmacokinetic analysis of disitamab vedotin.

The antibody–drug conjugate (ADC) represents an approach to targeted cancer therapy involving the conjugation of a monoclonal antibody (mAb) specifically targeting tumors with a cytotoxic payload through a sophisticated chemical linker. ADCs facilitate precise targeting and enhance the overall efficacy of treatment [1–3]. As of June 2023, there have been 15 ADC drug approvals for hematological malignancies and solid tumors worldwide [4]. Once the mAbs of the ADCs specifically bind to the cancer cells' target antigens, the ADCs are internalized by cells and form early endosomes. These early endosomes then mature into late endosomes before ultimately fusing with lysosomes. Cytotoxic payloads are subsequently released within the lysosomes through either chemical- or enzyme-mediated mechanisms, leading to apoptosis or cell death by targeting DNA or microtubules [5–7].

Disitamab vedotin received approval from China's National Medical Products Administration in 2021 for the treatment of locally advanced or metastatic gastric or gastroesophageal junction cancer [8,9]. Disitamab vedotin is a newly developed ADC targeting HER2 comprised of hertuzumab coupling monomethyl auristatin E (MMAE) via a cleavable linker [10]. Current clinical indications for disitamab vedotin include gastric carcinoma, gastroesophageal junction adenocarcinomas, breast carcinoma and urothelial cancer [11] Understanding the pharmacokinetics (PKs) of disitamab vedotin is critical to explaining its clinical efficacy. The primary intricate mechanism of action involves the alternative delivery of the anticancer agent MMAE to HER2-expressing cancer cells, thereby anchoring the HER2 protein on the tumor surface. Additionally, disitamab vedotin exhibits precise recognition and binding to tumor cells, subsequently inducing their demise by penetrating their cell membranes [11–13]. According to relevant literature reports, a comprehensive analysis of total antibody (TAb), ADC and small-molecule partial loading in vivo is required to comprehensively evaluate the absorption, distribution, metabolism and excretion characteristics of ADCs [14,15]. Accurate quantification of TAb, conjugated antibody (CAb) and payload loading is crucial for investigating the PK properties of ADCs in vivo and evaluating their exposure-response relationship [16].

Ligand-binding assays (LBAs) and LC–MS are the preferred platforms for ADC bioanalysis [17,18]. In previous studies, LBAs have consistently been regarded as the gold standard for protein quantification and are frequently employed in antibody determination. LC–MS/MS, on the other hand, is commonly utilized for quantifying small molecules [19–27]. In the LBA platform, ELISA and electrochemiluminescence assays (ECLAs) are commonly used to quantify total and conjugated antibodies [28,29]. Compared with ELISA, the ECLA platform offers several advantages, including a wider dynamic range, higher sensitivity and fewer sample volume requirements [30–32]. Using the ECLA method, the capture reagent was placed directly on the solid-phase carbon electrode, and the analyte was specifically bound to the detection antibody conjugated with the electrochemical luminescent agent, ruthenium terpyridine. The TAb and CAb of disitamab vedotin in human serum were quantitatively analyzed according to the luminescence intensity on the ECLA. LC–MS/MS method analysis with a multiple reaction monitoring (MRM) mode was used to quantitatively analyze the concentration of MMAE by liquid–liquid extraction (LLE) of analyte from human serum [21]. These bioassay methods were developed, validated and used in PK studies of disitamab vedotin for clinical application. This paper presents the development of a bioassay aimed at quantifying the concentrations of TAb, CAb (ADC) and MMAE in human serum to elucidate the PK behavior of ADCs [33]. Additionally, it includes methodological validation, which has rarely been reported in previous studies.

Methods

Chemicals & reagents

Disitamab vedotin (RC48) was generated by RemeGen Co., Ltd (Yantai, China). Rabbit anti-RC48 polyclonal antibody (biotin), mouse anti-MMAE mAb (biotin) were generated by RemeGen Co., Ltd and marked by United-Power Pharma Tech Co., Ltd (Beijing, China). Recombinant human HER2 extracellular domain (HER2-ECD) was purchased from G&P Biosciences (MO, USA). SULFO-TAG™ Streptavidin, Read Buffer T (4x) with surfactant, Blocker A kit and QuickPlex 96-well high-bind plate were purchased from Meso Scale Discovery (MD, USA). The internal standard (IS) deuterated (D8)-MMAE was obtained from Med Chem Express (NJ, USA). Blank human serum was purchased from BioIVT (AZ, USA). MMAE was obtained from Research Chemicals (Toronto, Canada). Methanol, acetonitrile, formic acid, methyl tertbutyl ether, isopropanol and N-hexane (LC–MS grade) were purchased from Thermo Fisher Scientific (MA, USA).

ECLA methods for TAb & CAb

A summary of the method of quantification for the TAb and CAb levels of disitamab vedotin is shown in Figure 1. Recombinant human HER2-ECD was coated on the QuickPlex 96-well high-bind plate at 0.5 µg/ml and incubated at 4°C overnight. Plates were blocked with a solution (3% Blocker A buffer in phosphate-buffered saline [PBS]) of 300 ul at room temperature (RT) for 120 min. The experimental samples, quality control (QC) samples and standards were diluted tenfold with 1% Blocker A buffer in PBS, added to the plate and incubated for 120 min. Rabbit anti-RC48 polyclonal antibody (biotin) at 5.2 µg/ml and mouse anti-MMAE mAb (biotin) at 17.25 µg/ml were used as the TAb and CAb detection antibodies, respectively, and incubated at RT for 90 min. Finally, the streptavidin-coupling chromogenic agent (SULFO-TAG), which binds to the detection antibody, was added and incubated at RT for 1 h. Read Buffer T (2×) was added and the SULFO-TAG was energized to emit light and detected on the MSD-QuickPlex SQ120 (Meso Scale Discovery) reader. Between each step, the plates were washed six times with 1% PBS-Tween wash buffer (0.05% Tween 20 in 1% PBS, no pH adjustment). The instrument signal value detected by the QuickPlex SQ120 was directly proportional to the concentration of disitamab vedotin TAb and CAb in the sample.

Figure 1.

Figure 1.

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Summary of electrochemiluminescence assay methods quantification.

(A) Total antibody and (B) conjugated antibody.

CAb: Conjugated antibody; ECD: Extracellular domain; MSD: Meso Scale Discovery; TAb: Total antibody.

LC–MS/MS for free MMAE

LC–MS/MS method

To conduct quantitative analysis using LC–MS/MS, the system consisted of LC-20AD (Shimadzu Corporation, Kyoto, Japan) and AB SCIEX 4000 (AB SCIEX, Toronto, Canada) mass spectrometers (equipped with an electron spray ionization ion source). For the detection of MMAE, a XSelect® CSHTM C18 (2.1 × 50 mm column, 3.5 μm; Waters, MA, USA) was used with a (A) mobile phase of 0.1% formic acid in water and (B) acetonitrile with 0.1% formic acid at a flow rate of 0.5 ml/min at 4–15°C. The following gradient elution program: 0–0.5 min: 5% B, 0.5–1.2 min: 5–95%, 1.2–2.0 min: 95%, 2.0–2.1 min: 95–5% and 2.1–3 min: 5%; the total analysis time was 3 min where two MRM scans (718.5/686.6 and 718.5/154.1 amu) were monitored, and samples were analyzed by positive ion electrospray under MRM modes. Figure 2 displays the representative ion fragment peaks observed for MMAE and D8-MMAE. The general parameters for MMAE and D8-MMAE were: curtain gas set at 10, collision gas set at 12, ion spray voltage set at 5500, temperature set at 600, ion gas source 1 set at 50 and ion gas source 2 set at 55.

Figure 2.

Figure 2.

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Identification and fragmentation.

(A) MMAE and (B) D8-MMAE.

Sample preparation

Standards and QC samples were prepared by LLE through serial dilution of MMAE in human serum. The standards encompassed eight concentration points, ranging from 0.04 to 10.0 ng/ml, ensuring a comprehensive calibration range for accurate quantification. Concentrations of QC samples were divided into four levels: high-QC (HQC) at 8.0 ng/ml, medium-QC (MQC) at 4.0 ng/ml, low-QC (LQC) at 0.1 ng/ml and lower limit of quantification (LLOQ) at 0.04 ng/ml. Approximately 50 μl of standards and QC samples were meticulously prepared from human serum and carefully transferred into centrifuge tubes. Each sample was supplemented with 600 μl of MTBE containing 20 ng/ml D8-MMAE. The mixture was vigorously vortexed for 1 min and subjected to centrifugation at 4861 × g for 15 min at 4°C. Following centrifugation, the resulting supernatant (440 μl) was carefully transferred to a well plate consisting of 96 wells. The collected supernatant was subsequently dried using nitrogen gas and combined with a 150-μl solution comprising acetonitrile in water 1:1 (v/v) solution containing 0.1% formic acid. LC–MS/MS analysis was performed using an aliquot size of 10 μl from the reconstituted supernatant.

Method validation

The method underwent validation following the guidelines set forth by the US FDA [34] and the International Conference on Harmonization [35]. Bioanalytical methods for quantifying TAb and CAb using LBA and free-MMAE using LC–MS/MS were developed and validated. Following the regulations of the US FDA and EMA [36], validation for the detection methods for TAb, CAb and free MMAE was carried out from various perspectives. Briefly, ECLA methods were validated for linearity and range, accuracy and precision, dilutional linearity and hook effect, selectivity, experiment and storage stability and specificity. Similarly, the LC–MS/MS method was validated for linearity and range, selectivity, carryover, accuracy and precision, matrix effect and stability. Summaries of the validating experiments are outlined in subsequent sections. Data analysis was performed using Prism 8.0 (GraphPad Software, CA, USA).

ECLA method validation for detection of TAb & CAb

Linearity & range

Freshly prepared analytes of varying concentrations and blanks were analyzed in duplicate. The labeled sample was tested in six independent runs, and the nonzero calibrator was expected to calculate the concentration in the range of ±20% of the nominal (theoretical) concentration, except the LLOQ and upper limit of quantification (ULOQ), where the calibrator should be ±25% of the nominal concentrations in each validation run. The anchor calibrators (<LLOQ and >ULOQ) did not require acceptance criteria because they are beyond the quantifiable range of the curve.

Accuracy & precision

In each of the six assay runs, QC samples were duplicated at five concentrations: LLOQ (19.531 ng/ml), LQC (50 ng/ml), MQC (600 ng/ml), HQC (1000 ng/ml) and ULOQ (1250 ng/ml) in ECLAs to detect TAb; and LLOQ (39.063 ng/ml), LQC (100 ng/ml), MQC (600 ng/ml), HQC (4000 ng/ml) and ULOQ (5000 ng/ml) in ECLAs to detect CAb. ECLA methods were used to assess the intra/interbatch precision and accuracy in control serum. The overall within-run and between-run accuracy was required to be ±20% for nominal values and ±25% for LLOQ and ULOQ.

Dilutional linearity & hook effect

Dilution linearity samples were generated from disitamab vedotin (10 mg/ml) at five different concentrations from 100.0 to 0.2 µg/ml (1000.0–2.0 µg/ml) for method validation for the detection of TAb (CAb). Samples were processed and analyzed in three independent runs. Dilution effect verification samples were diluted by 1000-,100-, 10-, 5- and 2-fold, respectively. Dilution linearity was evaluated by calculating the sample concentrations of TAb (100 ng/ml) and CAb (1000 ng/ml). The accuracy of the back concentration of the validation samples after dilution correction for each concentration level was within the range of ±20%, and the precision of the back concentration of all validation samples was not more than 20%. Validation samples above the ULOQ were used for investigation of the hook effect with response values higher than ULOQ or the back-calculated concentration as acceptable quality limit.

Selectivity

Selectivity refers to the ability to accurately determine the analyte in the presence of unrelated material in the matrix and was determined by analyzing (healthy volunteer) blank serum samples from 10 individuals (5 male, 5 female). Selectivity was tested by spiking serum (LLOQ, LQC and blank) from 10 individual serum samples. At least 80% of the individually formulated validation samples had accuracy in the range of ±25% (LLOQ) and ±20% (LQC) for the back-calculated concentrations, and the measured values for substrates without added analytes should be below the LLOQ (BQL).

Experiment & storage stability

The stability of QCs (LQC and HQC, n = 3) was evaluated under different conditions. The stability of the QCs was tested after storage at RT, after nine freeze–thaw cycles and long-term storage at -20°C and -80°C. The mean concentrations obtained from the calculations were mandated to fall within a 20% range of the nominal values.

Specificity

Specificity refers to the ability of an analytical method to determine an analyte accurately and specifically in the presence of relevant interfering substances in the sample. Specificity in this study was examined by the concentration of LQC, and a series of concentration gradients of Human-IgG (5.0, 50.0 µg/ml), Mouse-IgG (5.0, 50.0 µg/ml) and MMAE (5.0, 50.0 µg/ml) were added to the specificity verification samples of HQC and blank. Blank, LQC and HQC were used as baseline values in triplicate. The accuracy of the recovery of the tested substance at the concentration levels of LQC and HQC was within ±20% in the presence of interferences, and the back-calculated concentration of the blank sample should be lower than that of BQL. Baseline values should meet the acceptance criteria.

LC–MS/MS method validation for detection of MMAE

Linearity & range

The standard curve contained at least one double-blank sample, one zero concentration sample and eight concentration samples of 0.0400 to 10.0 ng/ml with a weighting factor of 1/x2 and equation: y = ax + b for detection-free MMAE, in duplicate for each concentration sample. In MRM mode, the ratio of the peak area of MMAE to the IS was used to establish a linear relationship with the concentration of MMAE. At least 75.0% of the nonzero points must meet the deviation within ±15.0% of the theoretical value, the LLOQ samples must be within ±20.0% of the theoretical value and at least 50.0% of the samples in each concentration must meet these requirements.

Selectivity

Double-blank samples (without MMAE and D8-MMAE) were prepared using serum from six individuals, while LLOQ samples were prepared using serum from a relevant source. The interference of MMAE and D8-MMAE in blank samples derived from different individual matrices and the interference of IS D8-MMAE in blank samples was investigated. In the six normal double-blank samples, the peak area of the interference peak at the retention time of the tested substance should be ±20% of the average peak area of the tested substance in the LLOQ sample that met the acceptance criteria in the standard curve. At the retention time of the IS, the peak area of the interference peak did not exceed 5.0% of the average peak area of the standard curve and the IS of the QC samples in the analytical batch. Blank and zero calibrators should be free of interference at the retention times of the analytes (MMAE) and the IS (D8-MMAE).

Carryover

To investigate potential effects on low-concentration samples after injection analysis of the high-concentration samples, double-blank samples were taken after ULOQ to investigate the residue. In three accuracy and precision analysis batches, the peak area of the residue in the double-blank sample should not exceed 20.0% of the average peak area of the tested substance in the LLOQ sample (5.0% of the IS response).

Accuracy & precision

QC samples for accuracy and precision experiments were prepared fresh on the same day. Each batch contained four control samples (0.0400, 0.100, 1.00 and 8.00 ng/ml), and each concentration level was run in six replicates. The quantity recovered from human serum was estimated using respective regression equations. The acceptable standards for accuracy and precision were as follows: the deviation of accuracy within and between batches of each concentration level should be less than 15.0% of the theoretical value, and the LLOQ should be less than 20.0%.

Matrix effect

To evaluate the effect of normal blank serum matrix, hemolysis matrix and high-fat matrix on MMAE extraction. Six different individual blank serum matrices, one hemolysis matrix and one high-fat matrix were extracted. MMAE was added at both low and high concentrations, along with ISs at corresponding concentrations, to the extracted blank samples to prepare the final sample with an equivalent concentration to that obtained under normal extraction conditions. Samples were determined using an established LC–MS/MS bioassay. The area ratio of MMAE to IS peaks was determined in samples with and without matrix. Matrix factor (MF) for each analyte and IS should be calculated for each batch of matrix by the ratio of peak area in the presence of matrix (blank matrix was extracted and added with analyte and IS) to the absence of matrix (pure solution of analyte and IS). The normalized MF of the IS should be calculated by dividing the MF of the analyte by the MF of the IS. The coefficient of variation (%CV) of the IS normalized MF calculated from six batches of matrix for the evaluation of MF should not be greater than 15.0% and should be investigated at LLOQ and HQC concentrations.

Stability

The stability of the LQC (0.102 ng/ml) and HQC (8 ng/ml) (n = 3) was evaluated under different conditions. The stability of the QCs was tested after storage at RT, 4°C in the sample injection device, after four freeze–thaw cycles and long-term storage at -20°C and -80°C.

Results

ECLA method validation

Linearity & range

Varying concentrations of vedotin were freshly prepared in human serum and analyzed twice in six assay runs. The standard curve was fitted using a four-parameter equation. The dynamic ranges for TAb and CAb are shown in Figure 3A and B, respectively (19.531–1250 ng/ml and 39.063–5000 ng/ml).

Figure 3.

Figure 3.

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Calibration standards showing the dynamic range of the detection.

(A) Total antibody and (B) conjugated antibody. Summary of quality controls for accuracy and precision assessment of (C) total antibody and (D) conjugated antibody.

HQC: High-quality control; LLOQ: Lower limit of quantification; LQC: Low-quality control; MQC: Mid-quality control; ULOQ: Upper limit of quantification.

Accuracy & precision

Six analytical runs containing at least five replicates of disitamab vedotin at LLOQ, ULOQ and QCs (LQC, HQC and MQC) were prepared in blank human serum. The maximum accepted values for both accuracy and precision were ±25% at LLOQ and ULOQ and ±20% for LQC, HQC and MQC. Accuracy and precision were acceptable under all conditions, as shown in Figure 3C and D.

Dilutional linearity & hook effect

The verification sample for dilution effect was diluted by 1000-fold, 100-fold, 10-fold, 5-fold and 2-fold, respectively. The results revealed no indication of the hook effect, and the accuracy and precision of serum dilution samples remained within an acceptable range of ≤20.0%, even when subjected to a high dilution factor as extreme as 1000-fold, as shown in Figure 4A and B.

Figure 4.

Dilution effects of TAb(A) and CAb(B) of Disitamab Vedotin in matrices. Selectivity of TAb (C) and CAb (D).

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Dilution effects of disitamab vedotin in matrices.

(A) Total antibody and and (B) conjugated antibody. Selectivity of (C) total antibody and (D) conjugated antibody.

CAb: Conjugated antibody; LQC: Low-quality control; LLOQ: Lower limit of quantification; TAb: Total antibody.

Selectivity

Selectivity was tested by spiking serum (LLOQ, LQC and blank) from ten individual serum samples. The concentration of 100% blank matrix was BQL for both TAb and CAb. The accuracies of 100% (TAb) and 80% (CAb) LLOQ were in the range of ±25%. The accuracy of 90% (TAb) and 100% (CAb) LQC back concentration was in the range of ±20%, as shown in Figure 4C and D.

Experiment & storage stability

The QCs remained stable at RT for 24 h. Additionally, long-term stability was maintained within predefined limits at -20°C and -80°C, as shown in Table 1. The samples showed stability throughout nine freeze–thaw cycles, with coefficients of variation for back-calculated HQC and LQC concentrations in TAb and CAb analysis in the range of ±20%, as shown in Figure 5.

Table 1.

Stability of qualy control solutions under different conditions for detected total and conjugated antibodies.

Condition   Detection Level Concentrations ng/ml Analysis calculated (mean)
%CV %RE
Room temperature (h) 24 TAb HQC 1000 0.3 -1.9
    LQC 50 2.2 6.7
  CAb HQC 4000 5.3 1.2
    LQC 100 1.0 -6.2
Long-term (-20°C) storage (days) 180 TAb HQC 1000 1.2 -7.7
    LQC 50 1.8 -3.8
30 CAb HQC 4000 5.3 -8.7
    LQC 100 2.2 -6.7
Long-term (-80°C) storage (days) 900 TAb HQC 1000 5.0 -7.0
    LQC 50 3.9 -1.3
780 CAb HQC 4000 -6.7 -10.3
    LQC 100 5.3 -5.8
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CAb: Conjugated antibody; CV%: Coefficient of variation; HQC: High-quality control; LQC: Low-quality control; RE%: Relative error; TAb: Total antibody.

Figure 5.

Figure 5.

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Stability of quality control solutions under freeze–thaw different cycle times of detected (A) total antibody and (B) conjugated antibody.

CAb: Conjugated antibody; HQC: High-quality control; LQC: Low-quality control; TAb: Total antibody.

Specificity

The results showed that the calculated concentrations of TAb and CAb blank samples were all BQL and the relative deviations of the calculated concentrations of LQC and HQC concentration level verification samples were less than 20%, indicating no significant endogenous interference, proving that selectivity was acceptable.

LC–MS/MS method validation

Linearity & range

The results shown in Figure 6 show the linear relationship between concentration and response was good, in the range of 0.04 to 10.0 ng/ml (R2 ≧0.99). The accuracy of each sample in the standard curve was -1.2 to 1.5%. The quantification range of this method was 0.04 to 10.0 ng/ml.

Figure 6.

Figure 6.

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Calibration standards showing the dynamic range of the detection MMAE.

Selectivity

A representative chromatogram from a double-blank human serum sample (without MMAE and D8-MMAE) is shown in Figure 7A. A representative chromatogram for MMAE at 0.04 ng/ml (LLOQ) in human serum is shown in Figure 7B. The results showed that interference of the test substance and IS in the blank sample was 0.0%, which met the acceptance standard. The interference of the tested substance in the zero sample was determined to be 0.0%, thereby satisfying the acceptance criterion.

Figure 7.

Figure 7.

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Selectivity of representative chromatogram.

Blank of human serum without MMAE and D8-MMAE (A) and (B) D8-MMAE (internal standard) with LLOQ at 0.04 ng/ml MMAE in human serum.

Carryover

Results showed that, in the first blank sample after ULOQ, the peak area of MMAE was within 20.0% of the LLOQ in the standard curve, and the peak area of IS D8-MMAE was within 5.0% of the corresponding IS.

Accuracy & precision

The range of deviation of intra-assay accuracy was -11.0 to 2.1% and the range of precision was 2.0 to 11.6%, as shown in Table 2.

Table 2.

Accuracy and precision of liquid chromatography-mass spectrometry method for quality control samples of free monomethyl auristatin E.

Level Concentration ng/ml Intra-assay Inter-assay
    Accuracy (%) Precision (%) Accuracy (%) Precision (%)
LLOQ 0.04 -11.0 to -6.0 6.3 to 9.1 -9.2 8.0
LQC 0.10 -8.9 to 2.1 4.6 to 11.6 0.3 8.8
MQC 1.00 -1.6 to 1.9 3.3 to 3.5 0.1 3.3
HQC 8.00 -2.4 to 0.1 2.0 to 3.5 -0.3 3.1
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HQC: High Quality Control; LLOQ: Lower Limit of Quantification; LQC: Low Quality Control; MQC: Middle Quality Control.

Matrix effect

The IS normalized MF %CVs for HQC, MQC and LQC in normal blank serum matrix were 1.8, 1.4 and 8.3%, respectively. At HQC and LQC concentrations, deviations from the theoretical concentrations of hemolysis matrix and high-fat samples were -11.4 to -13.6% and -1.0 to -2.1%, respectively. Therefore, the obtained results were verified and no matrix interference of MMAE in serum samples was observed.

Stability

QCs were stable at RT for up to 24 h and 4°C in the sample injection device for up to 58 h. Samples were stable during four freeze–thaw cycles. Long-term stability at -20°C and -80°C was within predefined limits, as shown in Table 3.

Table 3.

Stability of quality control solutions under different conditions of different detection monomethyl auristatin E.

Condition   Analysis calculated
    %CV %RE
Room temperature (h) 24 8.1 7.3
10.4 17.0
Sample injection device (4°C; h) 58 11.1 6.0
5.1 -0.6
Freeze–thaw (-80°C; cycles) 4 4.2 -5.0
6.1 -3.1
Long-term storage (-20°C; days) 30 2.3 0.4
3.5 -1.6
Long-term storage (-80°C; days) 894 9.7 -0.9
3.4 -0.2
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%CV: Coefficient of variation; RE%: Relative error.

Discussion

Many ADCs have been launched and dozens have entered clinical trials, indicating they have good prospects in the field of tumor therapeutics [37]. ADCs face some challenges in tumor therapy; for example, the safety and resistance of payload, diversity of metabolism and differential distribution of targets in different individuals. The clinical PK study of ADCs is not just the simple sum of the PKs of antibody and payload molecules. According to the literature, some ADCs do not meet preclinical expectations due to fundamental changes in the PKs of small-molecule toxins conjugated to antibodies [38]. Therefore, the PK behavior of different analytes reflects different content and significance, constituting the overall picture of ADC metabolism in vivo, and its complex in vivo PK disposition is the focus of ADC drug research and development [39]. At the same time, there are new requirements for qualitative analytical methods for this kind of drug. It is necessary to select and establish effective biological analysis methods for different analytes.

This study was designed to establish and investigate an ECLA method for the quantitative detection of disitamab vedotin TAb and CAb and an LC–MS/MS method for the quantitative detection of payload in human serum. The methods have high specificity and sensitivity. In the process of establishing the ECLA method, some key conditions of the method were investigated and optimized. Coating conditions, incubation time, detection reagent concentration and minimum dilution of samples were optimized using the checkerboard combination test method, and the final, optimal analysis process was used. Standard curve, accuracy, precision, selectivity, specificity, dilution linearity, sample stability and other indicators were investigated in the method validation, which met the requirements of relevant regulations and guidelines for biological analysis [34–36]. An LC–MS/MS method for quantitative analysis of MMAE in human serum was also established. Chromatographic conditions, the method of sample pretreatment and other key method conditions were optimized. In the optimization of the chromatographic conditions, the components of the mobile phase and the elution procedure were optimized. In the process of sample pretreatment, matrix effect and recovery rate were used as the standard to compare the influence of different extraction reagents on the results, and the optimal analysis conditions were determined. The residual effect, selectivity, standard curve, precision and accuracy, stability, matrix effect, extraction recovery rate and others were investigated, and all met the requirements of relevant regulations and guidelines for biological analysis [36].

The established method can be efficiently applied to the detection of disitamab vedotin in clinical studies, providing a basis for clinical research on disitamab vedotin and a reference for the establishment of detection methods for similar drugs in ADCs. In future development, related analytical methods for ADCs will be more mature and QC methods will be gradually improved. Advanced analytical techniques will provide strong support for clinical PK research and clinical protocol design of ADCs.

Conclusion

Bioanalytical methods were developed and validated to analyze TAb, CAb and MMAE of disitamab vedotin in human serum. These methods, validated over three concentration ranges, 19.531 to 1250.000 ng/ml, 39.063 to 5000.000 ng/ml and 0.04 to 10.00 ng/ml, facilitate analysis of clinical PK samples. These methods demonstrated high accuracy, precision, sensitivity and specificity in determining the absolute concentrations of TAb, CAb and MMAE for disitamab vedotin in human serum.

Funding Statement

This work was financially supported by the Shandong Provincial Natural Foundation (no. ZR2021MH220) and Yantai Science and Technology Plan (no. 2021XDHZ083).

Author contributions

B Wu was primarily responsible for developing the experimental methodology, collating data and writing the paper. Q Li played a major role in data collation and experimental methodology development. L Wang was mainly responsible for data collation. F Chen was in charge of experimental methodology development. J Jiang was responsible for conceiving and guiding the study, reviewing and editing the article, supervising experiments and acquiring funding.

Financial disclosure

This work was financially supported by the Shandong Provincial Natural Foundation (no. ZR2021MH220) and Yantai Science and Technology Plan (no. 2021XDHZ083). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

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