LC-MS based methods for ADCs: Maximizing the capabilities of hgh resolution MS using the SWATH Acquisition Mode

In the first two blogs of this three-part blog series, we explored the application of high resolution mass spectrometry (HRMS) and the SelexION™ differential mobility device to provide intact molecular weight information, in addition to examining peptide mapping techniques to determine the exact location of the drug and linker within the ADC. In this third and final installment, we discuss MS/MS with SWATH™ Acquisition using the TripleTOF® 5600+ system, a unique scan function which brings together data-independent acquisition for comprehensive spectra collection and targeted quantification.

Bioanalytical studies of ADCs typically pose three questions: what should be measured, when, and by which method? As most individual assays have limitations, [1] a combination of bioanalytical tools is used to characterize the ADC in vitro/in vivo, understand payload delivery to the intended site of action, and to establish a dose-response relationship.

Most often, either ligand binding assays (LBA) or liquid chromatography-mass spectrometry (LC-MS) based methods are used for performing bioanalytical studies of biotherapeutics. Biotherapeutics or protein based molecules owing to their well-defined tertiary structures respond very well to LBAs. For biotherapeutic bioanalysis, LBA and LC-MS are complementary to one another, where the LBA platforms offer ease-of-use, cost effectiveness, and faster turnaround time. However, LC-MS is an indispensable analytical solution for monitoring and profiling various ADC molecular entities. Regardless of the analyte measured, precautionary measures should be taken when preparing ADC samples. Conventional protocols need to be re-evaluated to ensure minimal effect on conjugation integrity during preparation. This assessment needs to be performed on each ADC, as the outcome may be affected by conjugation chemistry, linkers, and drug. Any pH alteration of the matrix, such as that used for sample preparation or LC separation, may lead to unintended loss of drug and changes in the drug-antibody ratio. For tissue analysis, protease inhibitors should be added to the homogenization buffer to prevent any degradation of antibody and/or linker-drug. [2] Typically, bioanalysis of ADCs can be approached in two ways: quantification of unconjugated or free drug, its metabolites and any other related released entities, using a triple-stage quadrupole LC-MS/MS system or characterization of the remaining conjugated form in a semi-quantitative manner using high resolution mass spectrometry (HRMS).[3] Selectivity, sensitivity, and the ability to develop generic MS methods in an expeditious manner are some of the important criteria that should be considered when selecting the LC-MS platform for ADC bioanalysis.

Bioanalytical multi-taskers

The main goal for ADC bioanalysis is to quantify all possible circulating species with cytotoxic drug, which include drug Phase I, II, and glutathione (GSH) adducts, drug + linker + Phase I, II, and GSH adducts, peptide + drug + linker Phase I, II, GSH adducts, and several others.3 Amidst such complicated species requiring highly sensitive quantitation, it is important to utilize one LC-MS platform with a single acquisition method capable of addressing all circulating species containing cytotoxic drug. SCIEX’s solution using the TripleTOF® System and SWATH™Acquisition mode offers the unique ability to acquire data on total antibody, conjugated antibody/drug and free drug + linker within a single raw data file (Figure 1).

Figure 1
Figure 1. ADC PK profile showing multi-component quantification in a single injection using SWATH™ Acquisition on TripleTOF® 5600+ system

In MS/MS with SWATH™ Acquisition mode, the Q1 quadrupole of the TripleTOF® 5600+ system is stepped at 25 amu increments across the mass range of interest, passing a 25 amu window into the collision cell. The transmitted ions are fragmented and the resulting product ions detected in the TOF MS analyzer at high resolution with accurate mass assignment. This sequential windowed acquisition is termed SWATH™ Acquisition since a large mass range can be interrogated in an LC-MS time frame due to the larger Q1 mass steps. Although this yields a more complicated MS/MS spectrum at each step, the high resolution capability of the TripleTOF 5600+ enables tighter extraction windows to maintain high specificity. Therefore, the key benefits of the SWATH™Acquisition mode for ADC analysis include:

  • Ability to use a generic MS method, particularly in discovery and development phases
  • Comprehensive qualitative and quantitative analysis
  • Ultimate safety net for capturing both predicted and unpredicted metabolites/catabolites
  • Retrospective data analysis for ADC catabolite and metabolite identification

Some of the above-mentioned benefits are highlighted in Figure 2, where in the same spectrum individual peptide fragments, the cytotoxic drug, and the product ion can be observed. While the TripleTOF®5600 in SWATH™Acquisition mode delivers exceptionally comprehensive data in one acquisition, it also allows for sensitive quantitative analysis over a broad dynamic range (Figure 3).

Figure 2
Figure 2. SWATH™ spectrum of drug + linker + peptide in one acquisition

Figure 3
Figure 3. Quantitative analysis of an ADC using a TripleTOF®5600 system in SWATH™Acquisition mode

The SelexION™ differential ion mobility technology described in the first blog post of this series can offer several additional advantages to ADC analysis, including enhanced specificity/selectivity through the elimination of isobaric background interferences, high quality MS/MS data for accurate identification of ADC components, and simultaneous quantitative/qualitative analytical capabilities (Figure 4).

<pFigure 4
Figure 4. Reduced complexities in the spectrum using SelecxION™ differential ion mobility technology

ADC bioanalysis can be extremely complicated owing to the nature and type of fragments that are formed in biological matrices. Considering the challenges faced by scientists who are either transitioning from small to large molecule bioanalysis, or those initiating their studies with ADCs, it is extremely important to have a single easy, robust, reproducible, and sensitive assay that offers comprehensive information of all ADC fragments. SCIEX’s solution with its TripleTOF®systems, SWATH™ Acquisition, and easy-to-use BiopharmaView™ Software enable bioanalytical scientists to achieve their goals with a single workflow solution.

The complexity of ADCs creates several challenges in the entire biotherapeutics workflow. Each of the stages of development, bioanalytical assay design, and process monitoring are essential for the understanding and selection of ADCs, and has a significant contribution not only towards the achievements of an ADC program, but also towards organizational success.


  1. Stephan JP, Kozak KR, Wong WL et al., Challenges in developing bioanalytical assays for characterization of antibody-drug conjugates, Bioanalysis., 2011; 3(6):677-700 
  2. Clark T, Han X, King L, Barletta F et al., Insights into antibody-drug conjugates: bioanalysis and biomeasures in discovery, Bioanalysis., 2013; 5(9):985-987 
  3. Xu K, Liu L, Saad OM et al., Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary LC-MS, Anal. Biochem., 2011;412:56-66