principal scientist Waters Corporation Milford, Massachusetts, United States
Purpose: Antibody-oligo conjugates (AOCs) are a similar class of biotherapeutics which build upon the fundamentals of antibody-drug conjugates (ADCs). The purpose of both AOCs and ADCs is the targeted delivery of a payload (oligonucleotide moiety or cytotoxic drug, respectively) using a full-length monoclonal antibody (mAb) or fragment thereof. AOCs will likely provide opportunities for targets thought to be inaccessible by other therapies. mAbs themselves are large and complex, requiring extensive characterization and monitoring. Conjugated mAbs require the same characterization as mAbs, plus analysis of oligo-to-antibody ratio (OAR), characterization of linker-payload building blocks, as well as in-process monitoring. This study builds upon previous analysis with light scattering techniques, to incorporate high resolution mass spectrometry into the toolbox of approaches for AOC characterization.
Methods: Samples of unconjugated “free” mAb, two AOCs, and siRNA building blocks were used for this case study. The accurate mass of the unconjugated mAb was obtained through reversed phase (RP) LC-MS (denaturing technique). The AOC samples require a non-denaturing, non-reducing approach, as the AOC will dissociate under typical denaturing RPLC-MS conditions. For OAR value and intact mAb & AOC characterization, we employ MS-compatible size-exclusion chromatography (SEC)-MS and strong cation exchange (SCX)-MS methods. To analyze the siRNA building blocks, we use denaturing (high temperature) and non-denaturing (low temperature) hydrophilic interaction chromatography (HILIC)-MS and denaturing ion-pairing reversed phase (IPRP) LC-MS methods. All data was acquired using the same UHPLC with a benchtop TOF MS.
Results: RPLC-MS (denaturing) and SEC-MS (non-denaturing) both provided accurate mass values for the free mAb sample, down to the granular detail of the various N-glycoforms present, which was not possible with SEC-MALS analysis. The SEC-MS analysis of the AOC samples confirms the mass shifts for one- and two- conjugation sites occupied (~14.8 kDa & ~29.7 kDa, respectively), as expected for OAR1 & OAR2 samples. Small amounts of unconjugated mAb were also detected in the OAR1 sample, which agrees with SEC-MALS results. An alternative approach for OAR analysis is SCX-MS (also non-denaturing), in the form of strong cation exchange chromatography. The use of a combined pH and salt gradient of MS-compatible mobile phases allowed for more flexible method development than SEC-MS (via gradient optimization), which led to an improvement in chromatographic separation of OAR species. Additionally, since the mobile phases are MS-compatible, we can confirm species with MS detection. We also chose to test SCX-MS separation with protease-generated mAb subunit samples to partially localize the conjugation sites. This analysis confirms the conjugation site is indeed above the hinge region of the mAb, as expected. HILIC LC-MS (denaturing & non-denaturing) and IPRP LC-MS (denaturing) were evaluated for the characterization of the free siRNA and linker + siRNA that would be used to synthesize the AOC. The siRNA is a non-covalently associated double stranded moiety (estimated molecular weight of 14.4 kDa). Both IPRP LC-MS and denaturing HILIC-MS confirmed the masses of each of the two oligo strands, as well as the strand with the MCC linker for that sample. Non-denaturing HILIC-MS provides a clearer picture of linker + siRNA sample composition, including a significant amount of unconjugated siRNA and a dimer species present.
Conclusion: A variety of optimized and flexible LC-MS techniques can be successfully employed for detailed characterization of AOCs and their oligo payloads.
