Development of a sensitive LC-MS/MS method for quantitation of trastuzumab emtansine in human serum


Lakshmanan Deenadayalan1, Sashank Pillai1, Rahul Baghla2 and Eshani Galermo2
1SCIEX, India and 2SCIEX, USA

Abstract


This technical note demonstrates a sensitive method for the quantitation of trastuzumab emtansine (TEx) in human serum using the SCIEX 7500+ system. A lower limit of quantitation (LLOQ) of 0.025 µg/mL (38.3 pg on column) and limit of detection (LOD) of 0.0125 µg/mL (19.1 pg on column) was achieved in extracted human serum samples (Figure 1).

TEx was licensed by the FDA in 2013 for the treatment of breast cancer.1 TEx works by attaching itself to the HER2 of the cancerous cells and producing targeted DNA damage in the cancer cells. This allows a large dosage of medication to target the cancerous cells directly.2 TEx serves 2 purposes: the antibody aids in microtubule targeting and makes use of the monoclonal antibody’s (mAb’s) full capacity. To meet safety and efficacy standards, it is therefore essential to precisely assess levels of ADCs, such as TEx, in biological matrices for toxicokinetic and pharmacokinetic profiles.

Figure 1: Representative extractrd ion chromatograms [XICs] of matrix blank, 0.0125 µg/mL [LOD], 0.025 µg/mL [LLOQ] and 0.05 µg/mL for TEx in extracted human serum.

Key benefits for analysis of trastuzumab emtansine using the SCIEX 7500+ system
 

  • Sensitive quantitation of TEx: Achieve 0.025 µg/mL LLOQ and 0.0125 µg/mL LOD for quantitation of TEx in human serum

  • Low serum consumption: Low-level quantitation was achieved using 50 µL human serum

  • Effortlessly meet critical quantitative performance criteria: Achieve accurate quantitative performance with %CV <11 at all concentration levels across a linear dynamic range (LDR) of 3.9 orders of magnitude

  • Streamlined data management: SCIEX OS software, a 21 CFR Part 11-compliant platform, simplifies data acquisition and processing

Introduction


ADCs are the most promising targeted cancer treatment with excellent target specificity. The structure of ADC includes a mAb attached to a cytotoxic drug via a chemical linker. The mAb targets explicitly the tumor antigen, while the payload can be delivered to the site to initiate a cytotoxic effect. Because of their selectivity towards cancer cells, ADCs have a high level of efficacy with minimal adverse effects. TEx is the anti-HER2 ADC with a specific target for breast cancer.

Due to its high potency in treating cancers, highly sensitive assays are necessary to ensure precise and accurate detection and quantitation when assessing pharmacokinetic and pharmacodynamic effects .

Methods
 

Standard preparation: 1 mg of TEx stock was procured from Medchem Express and dissolved in water.  

Sample preparation: Dilutions were performed in human serum to prepare spiked sample concentrations ranging from 0.025 µg/mL to 100 µg/mL. Samples were prepared using 50 µL of spiked human serum, 200 µL of protein A beads and 200 µL of PBS. Protein A beads were washed 3 times with PBS before use. Samples were gently shaken for 45 minutes at room temperature, followed by 2 PBS washes. The beads were resuspended in 150 µL of digestion buffer containing 150mM ammonium bicarbonate and 1mM calcium chloride before denaturation at 95°C for 10 minutes. After allowing samples to cool to room temperature, 10 µg of trypsin was added to each sample, followed by on-bead digestion for 2 hours at 50°C. Digestion was stopped by adding 3 µL of formic acid. Samples were separated from the beads and transferred to vials for LC-MS/MS analysis.

Chromatography: Analytical separation was performed on the ExionLC AE system using a Phenomenex Kinetex C18 (2.1 × 100 mm, 1.7 μ.m) column at a 0.3 mL/min flow rate. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was 0.1% (v/v) formic acid in acetonitrile. The column temperature was set to 55°C. The gradient conditions used are summarized in Table 1. A 5 μL sample was used for LC-MS/MS analysis.

Table 1: LC gradient conditions for TEx.

Mass spectrometry: The optimized source and gas parameters are listed in Table 2 and the MRM parameters are included in Table 3.

Table 2: Source, gas and SCIEX 7500+ system conditions.

Table 3: MRM parameters used for quantitation.

Data processing: Analysis was performed using SCIEX OS software, version 3.4.0. Peaks were integrated using the MQ4 algorithm, and a weighting of 1/x was used for TEx quantitation.

Method development


Trastuzumab emtansine in human serum was prepared using an immunoaffinity capture protocol followed by an on-bead digestion as described in the Methods section.

Several optimizations were performed for chromatographic separation to help separate the background from the peptide of interest. The tryptic digest of trastuzumab emtansine produced a complex peptide profile with numerous coeluting peaks, complicating quantitation.  Figure 2 shows the full XIC of the human serum matrix blank and highlights the retention time frame of the peptide peak of interest, IYPTNGYTR, along with several close eluting matrix components. To achieve baseline separation of these matrix components and the peptide of interest, IYPTNGYTRb, a longer gradient was applied.

Figure 2: Full XIC of human serum matrix blank highlighting the retention time frame of peptide peak of interest. Several peaks from close eluting matrix components are observed near the peptide peak of interest. Therefore, a longer chromatographic method was adopted to ensure proper separation of IYPTNGYTR from the matrix components.

Quantitative performance on the SCIEX 7500+ system


The method achieved excellent linearity, accuracy and precision from 0.0125 µg/mL to 100 µg/mL for TEx. The on-bead digestion immunoprecipitation workflow resulted in clean samples that enabled a sensitive quantitation of TEx.

Linearity was achieved across concentrations ranging from 0.0125 µg/mL to 100 µg/mL (Figure 3), achieving an LDR of 3.9 orders of magnitude. A coefficient of determination (r2) of >0.999 was reached, with a weighting factor of 1/x.

Figure 3: Calibration curve for quantitation of TEx with weighting factor 1/x. The linear range covers 0.0125 µg/mL to 100 µg/mL, demonstrating an LDR of 3.9 orders of magnitude.

Analytical performance was evaluated based on the requirement that the accuracy of the calculated mean should be between 80% and 120% at the LLOQ and between 85% and 115% at higher concentrations. The %CV of the calculated mean of the concentration should be below 20% at the LLOQ and below 15% at all higher concentrations.3

This assay's accuracy was within ±13% of the nominal concentration and the %CV was <11 for the quantitation of TEx in human serum (Figure 4). Calculated % accuracy and %CV values were within the acceptance criteria at each concentration level.

Figure 4: Quantitative performance for TEx (m/z 542.9 → m/z 405.1) analysis. Reproducibility and accuracy results were determined from the calibration curve standards across 3 replicates at each concentration. Statistical results were summarized using the Analytics module in SCIEX OS software.

Compliance-ready SCIEX OS software


Equivalent SCIEX OS software capabilities for regulated bioanalysis can be executed on the SCIEX 7500+ system, ensuring high fidelity when performing method transfers while retaining critical compliance features. SCIEX OS software is a closed system and requires records and signatures to be stored electronically, meeting the regulations outlined by 21 CFR Part 11. SCIEX OS software can open raw data files from any visible storage location within a closed network by using designated processing workstations.

Figure 5 illustrates the features of SCIEX OS software that are used to monitor the audit trail, acquire and process data, and configure user access. The audit trail feature enables users to audit critical user actions and locks in data integrity. The Central Administrator Console (CAC) feature allows users to centralize acquisition and processing using a single platform to maximize efficiency for multi-instrument laboratories, independent of compliance standards.

The configuration module allows users to assign roles and access as the administrator, method developer, analyst, and reviewer.

Figure 5: Features of SCIEX OS software for monitoring user access and evaluating the audit trail. The audit trail view allows users to filter for high-risk events easily and enables data integrity features to meet compliance requirements. The software features a Central Administrator Console (CAC) to manage users and groups, role definitions, workstations and projects across all systems. The CAC feature supports both regulated and non-regulated compliance standards. The configuration module enables users to quickly set up roles and levels of access for the administrator, method developer, analyst and reviewer levels.

Conclusions
 

  • An LLOQ of 0.025 µg/mL and LOD of 0.0125 µg/mL were achieved for the quantitation of TEx in human serum

  • Low-level quantitation was achieved using 50 µL human serum

  • Linearity was achieved at concentrations ranging from 0.0125 µg/mL to 100 µg/mL, achieving an LDR of 3.9 orders of magnitude

  • Comparable quantitative performance was demonstrated with accurate and highly reproducible (%CV <11) results on the SCIEX 7500+ system

  • A single platform for streamlined data acquisition, processing, and management with SCIEX OS software was presented

  • Retain data management and compliance-readiness (21 CFR Part 11) features using SCIEX OS software to support regulated bioanalysis on the SCIEX 7500+ system

References
 

  1. FDA approves ado-trastuzumab emtansine for early breast cancer. Jan 2019.
  2. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blattler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX: Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 2008, 68:9280–9290.10.1158/0008-5472.CAN-08-1776
  3. Bioanalytical Method Valication, May 2018
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