Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry

Abstract

Antibody-drug conjugation strategies are continuously evolving as researchers work to improve the safety and efficacy of the molecules. However, as a part of process and product development, confirmation of the resulting innovative structures requires new, specialized mass spectrometry (MS) approaches and methods, as compared to those already established for antibody-drug conjugates (ADCs) and the heightened characterization practices used for monoclonal antibodies (mAbs), in order to accurately elucidate the resulting conjugate forms, which can sometimes have labile chemical bonds and more extreme chemical properties like hydrophobic patches. Here, we discuss practical approaches for characterization of ADCs using new methodologies and ultrahigh-resolution MS, and provide specific examples of these approaches. Denaturing conditions of typical liquid chromatography (LC)/MS analyses impede the successful detection of intact, 4-chain ADCs generated via cysteine site-directed chemistry approaches where hinge region disulfide bonds are partially reduced. However, this class of ADCs is detected intact reliably under non-denaturing size-exclusion chromatography/MS conditions, also referred to as native MS. For ADCs with acid labile linkers such as one used for conjugation of calicheamicin, careful selection of mobile phase composition is critical to the retention of intact linker-payload during LC/MS analysis. Increasing the pH of the mobile phase prevented cleavage of a labile bond in the linker moiety, and resulted in retention of the intact linker-payload. In-source fragmentation also was observed with typical electrospray ionization (ESI) source parameters during intact ADC mass analysis for a particular surface-accessible linker-payload moiety conjugated to the heavy chain C-terminal tag, LLQGA (via transglutaminase chemistry). Optimization of additional ESI source parameters such as cone voltages, gas pressures and ion transfer parameters led to minimal fragmentation and optimal sensitivity. Ultrahigh-resolution (UHR) MS, combined with reversed phase-ultrahigh performance (RP-UHP)LC and use of the FabRICATOR® enzyme, provides a highly resolving, antibody subunit-domain mapping method that allows rapid confirmation of integrity and the extent of conjugation. For some ADCs, the hydrophobic nature of the linker-payload hinders chromatographic separation of the modified subunit/domains or causes very late elution/poor recovery. As an alternative to the traditionally used C UHPLC column chemistry, a diphenyl column resulted in the complete recovery of modified subunit/domains. For ADCs based on maleimide chemistry, control of pH during proteolytic digestion is critical to minimize ring-opening. The optimum pH to balance digestion efficiency and one that does not cause ring opening needed to be established for successful peptide mapping.

Qualitative analysis of antibody–drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs

“…There are a few publications utilizing MALDI for ADC analysis; however, it has yet to be evaluated as an option for DAR determination. [44][45][46][47] Herein we have evaluated its suitability for DAR determination of reduced ADCs.…”

Native size-exclusion chromatography-mass spectrometry: suitability for antibody–drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels

“…In these studies, researchers have used nSEC-MS for assessing the composition of ADCs recovered from in vivo studies in rats, 30 to gain a better understanding of the gas-phase conformation of ADCs in ion-mobility separations, 31,32 and as a component of a suite of MS-based assays that can be used for comprehensive ADC characterization. 33 While the particulars of the nSEC-MS methods described above vary, they all share fundamental key attributes: a microbore SEC column is used to exchange the ADC into ammonium acetate, thus facilitating online native electrospray ionization (ESI) analysis. While this approach has been adopted with the understanding that it is appropriate for enabling direct determination of ADC mass and provides a qualitative assessment of DAR and drug-distribution, it was not clear whether the DAR and drug-distribution information derived from nSEC-MS was quantitatively accurate and valid.…”

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

“…3,5,6 This proves especially useful for ADC characterization, as it is imperative to monitor and report CQAs related to the cytotoxic payloads. 4,7 For instance, the drug-to-antibody ratio (DAR), or the average number of drugs conjugated to the antibody, must be known since it can affect the potency and toxicity of the ADC. 4,7,8 Other CQAs, such as drug load distribution and residual drug concentration, are also important.…”

“…4,7 For instance, the drug-to-antibody ratio (DAR), or the average number of drugs conjugated to the antibody, must be known since it can affect the potency and toxicity of the ADC. 4,7,8 Other CQAs, such as drug load distribution and residual drug concentration, are also important. 4,7 The versatility of protein RPLC, especially when coupled to MS for accurate mass analysis, allows the characterization of these CQAs.…”

“…4,7,8 Other CQAs, such as drug load distribution and residual drug concentration, are also important. 4,7 The versatility of protein RPLC, especially when coupled to MS for accurate mass analysis, allows the characterization of these CQAs. However, as the biotherapeutic industry matures even further, protein RPLC must also improve to support the need for higher resolution, enhanced sensitivity and faster throughput characterization.…”