Featuring the intact protein analysis workflow using the ZenoTOF 7600 system and Biologics Explorer software from SCIEX
Rashmi Madda, Haichuan Liu and Zoe Zhang
SCIEX, USA
This technical note highlights an efficient intact protein analysis workflow for determining drug-to-antibody ratios (DARs) of antibody-drug conjugates (ADCs)1 . This workflow leverages high-resolution mass spectrometry (MS) for accurate intact mass measurement in addition to automated protein deconvolution and DAR calculation provided by Biologics Explorer software. Average DAR values of 3.6 and 3.5 were obtained for the glycosylated (native) and deglycosylated forms of trastuzumab emtansine (T-DM1), respectively.
ADCs―a new generation of biotherapeutics―combine potent cytotoxic drugs with monoclonal antibodies (mAbs) that target specific cells.1 The drug and mAb are connected through a cysteine-based, lysine-based or site-specific linker. Depending on the type of linkage used in an ADC, DAR values as high as 8 might be observed. 2 This variability underscores the need for accurate and reliable DAR measurements to optimize ADC performance and efficacy. 3 The mean quantity of drugs conjugated to antibodies is a critical parameter for ADCs. Therefore, DAR measurement is pivotal in determining essential properties, such as drug clearance, pharmacokinetics and biodistribution, which are vital considerations for developing targeted therapies.
In this work, the glycosylated and deglycosylated forms of T-DM1 were characterized using the ZenoTOF 7600 system, followed by automated data analysis using Biologics Explorer software (Figure 1), leading to accurate DAR measurements in a streamlined workflow.
Sample preparation: Lyophilized T-DM1 was reconstituted in deionized (DI) water to a final concentration of 5 mg/mL. For the intact ADC analysis, the reconstituted T-DM1 was diluted with 0.1% formic acid (FA) in water to 1 mg/mL prior to LC/MS analysis. Deglycosylation of T-DM1 was performed by adding 1 µL of 50 units/µL PNGase F (Sigma-Aldrich) in 20mM Tris-HCl buffer (pH = 8.2) to 100 µL of 1 µg/µL T-DM1 (100 µg). The mixture was incubated overnight at 37°C. Finally, 5 µL samples of T-DM1 were injected for LC-MS analysis.
Chromatography: Intact glycosylated and deglycosylated ADC samples were separated using the LC gradient displayed in Table 1, using a Waters ACQUITY BEH C4 column (2.1 mm x 50 mm, 1.7 µm, 300 Å, Waters). The flow rate was set to 0.3 mL/min for all LC runs. The column was kept at 60°C in the column oven of an ExionLC AD system (SCIEX). Mobile phases A and B consisted of 0.1 % FA in water and 0.1% FA in acetonitrile, respectively.
Mass spectrometry: Intact LC-MS data of glycosylated and deglycosylated ADCs were acquired using the ZenoTOF 7600 system (SCIEX). The source parameter and TOF MS settings are listed in Tables 2 and 3.
Data processing: Intact T-DM1 LC-MS data were analyzed using the intact protein analysis workflow template in Biologics Explorer software. The DM1 and linker were added to the conjugate table.
To determine the DAR of T-DM1, intact mass measurements of the glycosylated and deglycosylated forms of T-DM1 were performed using the ZenoTOF 7600 system. Complex charge state distributions were observed for the 2 forms of T-DM1 in the high-resolution TOF MS spectra (Figure 2A and 2D). The results of intact protein deconvolution from Biologics Explorer software revealed that the complex MS profile of the glycosylated T-DM1 consists of different glycoforms―mainly G0F, G1F and G2F―conjugated with as many as 8 molecules of the payload DM1 (Figure 2B and 2C). By comparison, the removal of N-linked glycosylation led to a simpler MS profile (Figure 2D–F), where the deglycosylated T-DM1 carrying up to 8 DM1 was detected. The 2 forms of T-DM1 were identified with a <10 ppm mass accuracy and were automatically integrated through Biologics Explorer software. Figure 3 shows the DAR distributions of the glycosylated and deglycosylated forms of T-DM1. In both cases, the major T-DM1 species had a DAR value of 2–4 (Figure 3).
The average DARs of the glycosylated and deglycosylated forms of T-DM1 can be calculated from the DAR distributions within Biologics Explorer software. Average DAR values of 3.6 and 3.5 were measured for the glycosylated and deglycosylated forms of T-DM1, respectively. The measured DAR values agree with those reported for T-DM1 in the literature.5, 6
The analysis of intact ADC data was streamlined in 1 workflow using Biologics Explorer software, as shown in Figure 4. This compelling software displays the deconvoluted spectrum (Figure 4A) and detailed information about the annotated peaks (Figure 4B–D) on a single page for easy results review. Moreover, the payload and linker composition can be conveniently defined in the conjugate table within the protein mapping activity (Figure 4E), which provides an automated average DAR measurement of the glycosylated T-DM1 (Figure 4F). This intuitive template highlights the ability of Biologics Explorer software to seamlessly guide users through accurate measurements and informative data analysis.Â