Antibody-Drug conjugates (ADCs) are an emerging class of biotherapeutics that hold enormous promise for effective targeted therapy in oncology and beyond. They combine the strengths of antibodies and small molecule drugs - high specificity and high potency - in a single format. ADCs consist of three elements: antibody, payload drug, and linker. The conjugation of payload and antibody via a linker is a critical aspect that defines ADC quality, safety and efficacy. The linker is designed to be stable in circulation but facilitate effective release of the payload at the site of action. Still, its attachment to the antibody may affect the specificity and stability of the ADC compared to the antibody. Mass spectrometry (MS) is a powerful tool for the characterization of molecular details of large biomolecules. It is particularly useful for monitoring the attachment of linker/payload to an antibody and for assessing the stability of an ADC.
Advanced MS Approaches for the Characterization of ADC Heterogeneity and Stability
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We apply high resolution mass spectrometric methods for the characterization of antibodies and ADCs, including:
- Determination of drug-to-antibody ratio (DAR) and ADC heterogeneity by size-exclusion chromatography and mass spectrometry (SEC-MS)
- Mapping of the sites of drug attachement and quantification of their occupancy by mass spectrometric peptide sequencing
- Assesment of ADC stability by forced degradation studies
- Comparision of ADC vs antibody stability
Dr. Jürgen Kast, Team Leader Biotherapeutical Analytics
Determination of DAR and ADC Heterogeneity
The drug-to-antibody ratio (DAR) is a key metric to define ADC potency (drug load per ADC molecule). DAR determination can be used to monitor the conjugation reaction and to evaluate different experimental conditions during ADC development.
During ADC production, it is a useful readout of the quality of a batch and batch-to-batch variation. SEC-MS is the tool of choice for MS-based DAR determination. Additional steps such as removal of N-glycans by PNGase F to eliminate the heterogeneity of N-glycosylated antibodies may help with peak assignment and DAR determination. SEC-MS can also be used to assess ADCheterogeneity by displaying the range of the number of drug molecules attached to the antibody. It alsoreveals the modality of drug incorporation, e.g.the pairwise conjugation of drug molecules in Cys-conjugated ADCs due to prior reduction of inter-chain disulfide bonds.
Assessment of conjugation site distribution and occupancy
MS analysis of intact ADCs is limited to the wholemolecule level. For true in-depth assessment of ADCs at the level of individual conjugation sites, an MS based peptide sequencing approach is necessary. Peptides carrying only a single reactive conjugation site can be generated by enzymatic digestion of the ADC. They can be used to identify and quantify sites of drug attachment via peptide sequencing by tandem mass spectrometry (MS/MS).
This approach is widely used for mapping of a variety of posttranslational modifications (PTMs). Indeed, the attachment of a drug/linker moiety in itself represents a post-translational modification. For each peptide and site, the MS/MS spectra are interrogated for evidence of conjugation, apparent as conjugate specific mass shift. Determining the ratio of the conjugated vs non-conjugated forms then provides a simple means to determine the relative site occupancy. Applying this approach to an entire ADC then provides a map of the conjugation site distribution. As the workflow is compatible with most types of PTMs, it can also be used to screen for other PTMs that may be induced during conjugation, most notably oxidation and deamidation.
Assessment of ADC stability by forced degradation
Antibodies, while generally stable in vitro and in circulation, are still susceptible to slow degradation. For example, post-translational modifications such as oxidation and deamidation can accumulate over time. Forced degradation studies are widely employed to identify such vulnerable sites on an accelerated time scale. Typical conditions used in such studies are high pH, low pH, and elevated temperature. Exposing an antibody to high pH can induce deamidation of asparagine and glutamine residues, which also occurs at low pH in addition to fragmentation at amide bonds. Elevated temperature elicits a wide range of changes by accelerating the underlying chemical reactions, including deamidation and oxidation of methionine residues.
SEC-MS reveals the impact of stress on an ADC via DAR determination. In a model study, low and high pH stress resulted in only minor changes in the DAR. In contrast, elevated temperature stress led to a noticeably lower DAR and a shift to fewer drug molecules incorporated, and broader signals due to higher heterogeneity due to increase in PTMs. Subsequent mapping of drug conjugation sites confirmed the more pronounced drop in drug load upon elevated temperature stress. It also demonstrated that the loss of conjugated drug occurred across all conjugation sites, while pH stress resulted in only minor changes. This indicated an accelerated release of linker and drug from lysine residues. Deamidation and oxidation of selected sites was also assessed to monitor the stress treatment. Increased deamidation was observed for all stress conditions, while the pattern was characteristic for the type of stress. Methionine oxidation was only observed for elevated temperature stress. Comparing the effect of each stress condition on lysine conjugation, deamidation, and oxidation indicated that the stability of the linker and drug is susceptible to temperature, but not pH-induced elimination. Mapping the results of the site-specific analysis onto the ADC revealed possible quality attributes.
Complementary Approaches for the characterization of ADC heterogeneity and stability also available at PPS:
- Hydrophobic interaction chromatography (HIC) to determine DAR and heterogeneity
- Higher order structure methods such as infrared (FT-IR) and CD spectroscopy, fluorescence analysis, and differential scanning calorimetry (DSC) to assess ADC vs antibody stability
- Cysteine linkage analysis to monitor disulfide bond reduction and re-closing during conjugation
- Glycosylation analysis to track N-glycan editing during glycan conjugation