LC-MS based methods for ADCs: Peptide mapping techniques for site occupancy

In the first of this three-part blog series on LC-MS based methods for antibody drug conjugates (ADCs), we demonstrated the utility of high resolution mass spectrometry (HRMS) coupled with the SelexIon™ differential ion mobility device for providing intact molecular weight determination free of interferences. Today, we explore methods for determining the exact location of the drug and linker within the ADC, once again leveraging the high quality MS/MS data from the TripleTOF® 5600+ system.

As biotherapeutics are produced in cells, the sequences and post translational modifications (PTMs) can be altered owing to changing environmental conditions. It is also important to determine critical quality attributes (CQAs), which are necessary for drug safety and efficacy and should be monitored in every step of biologic development and production. Peptide mapping analysis provides product sequence, PTMs, glycosylation heterogeneity, and low-level sequence variants. In addition, simultaneous quantitation of each peptide from a protein product and its modified forms allow additional capability to detect changes over time.

The LC-MS analysis of ADCs at an intact level can provide data to calculate the average drug-antibody ratio; however, further analysis is typically continued by peptide mapping to determine the exact location of the drug and linker, and to also examine the variety of constructs. Peptide mapping produces a different level of complexity, as peptides with the drug and linker are often identified from a complex mixture of peptides that may be variants of each other. For scientists working with ADCs, being able to group these together as part of the same family provides:

  • More accurate representation of which variants are present
  • The ability to monitor variations in a coherent manner offering better product information
  • Accelerated product development with more comprehensive information for comparability
  • The ability to avoid unwanted variants earlier in the process

Here, we present a unique methodology, which uses the very nature of ADCs to determine the species that are present, allowing one to intelligently assign identity. Locating the sites of conjugation on the ADCs is greatly facilitated by using tools that produce high quality MS/MS spectra, thereby associating the fragmentation patterns with a variety of modified peptides. Standard ‘matching’ software to identify peptide modification is inadequate when the “Drug + Linker” mass is used as a discrete alteration. Typically, standard methodologies fail to provide information in cases of incomplete attachment, and would not identify adducts with the linker only and not the drug. In addition, such methodologies would ignore every case where the linker undergoes fragmentation, and therefore would deliver inadequate information on partial synthesis. In the example provided below, the fragmentation pattern of a linker molecule was used to identify 64 sites of a theoretically possible 90 sites in an ADC. The sites were shown to have specific linker attachments using the fragmentation patterns.

Most peptide mapping techniques for accessing the location of ADCs have relied primarily on separations and mass of the “peptide + linker + drug” construct to determine which peptide and “drug + linker” elements are bound. [1] Unless an exhaustive manual search through the raw data is undertaken, this may under-represent the species because the approach fails to identify fragmented species.

The technique adopted in this study considers the fragment species regardless of peptide attachment. The ability to use the exceptional high resolution and accurate mass MS/MS capability of the TripleTOF® 5600+ system operating in an unbiased manner offers an enormous efficiency gain, resulting in accurate profiling and confirmation of which species actually exist in a large theoretical set (Figures 1 and 2).

Figure 1 - Ladder sequences
Figure 1. Ladder sequences of the ‘CONJUGATION-DRUG’ drug/linker fragments are salient in the spectra.

Figure 1 - Spectra and peptides
Figure 2. Example of spectra demonstrating one of the peptides attached to the linker and fragments of the linker.

It is facile to assign the fragments of the known drug and linker molecules, and thereafter individually as modifications using peptide mapping software. At the outset of the experiment, the fragmentation pattern was not known, but could be easily determined from a visual examination of the spectra of known conjugated species and a rapid appraisal of the common fragments. In addition, Figure 2 shows evidence of the peptide sequence in each case and is superimposed under the X-axis for all of the ‘y’ ions in the sequence (blue, red, and orange, respectively). The ability to comprehensively provide such high quality MS/MS data without pre-knowledge allows an unbiased retrospective review of the data. A search of just the expected modified peptides would not have revealed that multiple combinations were present. The benefit of acquiring MS/MS spectra in an unbiased manner is that it provides a greater depth of coverage and proof of attachment.

The application of the presented peptide mapping methodology offers:

  • Greater security to organizations aiming to thoroughly characterize their ADC constructs
  • Accurate determination of linker and drug locations from the peptide map information in a straightforward manner, even when CID fragmentation is widespread
  • Better understanding of complex fragmentation patterns, thereby enhancing interpretation of MS/MS spectra
  • Acquisition of unbiased MS/MS data
  • Ability to data mine linker specific fragment ions
  • A reduction in the steps required to create an exhaustive list of all theoretical fragmentations, yielding a comprehensive set of peptides with or without attached construct

Tune in tomorrow for the final installment in our LC-MS/ADC series, where we investigate maximizing the capabilities of HRMS from identification to quantification and examine bioanalytical strategies for ADC PK profiling using SWATH™ Acquisitions.


  1. Wakankar A, Chen Y, Gokarn Y, Jacobson FS, Analytical methods for physicochemical characterization of antibody drug conjugates., MAbs., 2011;3:161–72. doi: 10.4161/mabs.3.2.14960