Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products

Abstract

Mass spectrometry performed in non-denaturing conditions (native MS), and its hyphenation to ion mobility spectrometry (IM-MS), have gained interest for the qualitative and quantitative characterization of intact monoclonal antibody-related (mAb) products. However, one main drawback is the manual sample preparation, which hampers its routine use in high throughput automated environments. Size exclusion chromatography (SEC) appears as an interesting technique to perform online buffer exchange in an automated way. We present here an exhaustive and systematic evaluation of the possibilities and versatility of SEC direct hyphenation to native MS or IM-MS (SEC-nativeMS/IM-MS) for the characterization of a variety of mAb-formats (IgGs, ADCs, bispecific mAbs and Fc-fusion proteins). First, online SEC-native MS allows automated sample preparation, resulting in high resolution mass spectra and improved mass accuracies (<80 ppm) compared to manual buffer exchange procedures. When hyphenated to ion mobility, SEC-native IM-MS can deliver conformational characterization through collision cross section (CCS) measurements within few minutes without affecting mAb structures. Finally, benefits of online SEC-nativeIM-MS compared to standalone SEC-UV or native MS techniques are demonstrated for higher order structure characterization of mAb forced degraded samples. While SEC provides separation of high/low molecular weight species from the main mAb peak along with precise quantification of the species, native MS affords complementary unambiguous identification of SEC peaks, even when poor SEC separation is achieved. The synergic online coupling of SEC to native MS/IM-MS is envisioned to definitely push native MS approaches at the forefront of mAb characterization in quality-controlled environments and as multiple monitoring method.

Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing

“…The theoretical and experimental mass comparison and the mass deviations over the different charge states, ranging from 0 to 1.3 Da depending on the glycoform, showed consistent performance of the high-resolution MS instrument and confident mass assignments (ESM Table S2). The satisfying mass accuracies can also attribute to the implementation of SEC which provides inline removal of sample salts that might lower MS resolution [36]. The mass spectrum allows estimation of the relative abundance of each glycoform which was in agreement with values commonly observed for trastuzumab formulations [37].…”

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

“…Since the initial publication of our work on the intact mass determination of interchain cysteine-linked ADCs, 25 additional publications have emerged describing similar nSEC-MS methods that have been deployed for unique and interesting purposes. 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.…”

Glycosylation of biosimilars: Recent advances in analytical characterization and clinical implications

“…Moreover, the use of a complementary dimension provides 2D peak patterns that facilitate the identification of unknown protein variants. Additionally, a second dimension can be used to increase the compatibility of separation techniques with non-volatile mobile phase components in the first dimension to MS by providing a rapid on-line desalting procedure [117].…”

“…The use of IM-MS for the analysis of originator mAb and biosimilar products has recently emerged as an interesting analytical technique for global conformational characterization. IM-MS allows to create a 3D fingerprint of the higher order structure of the protein and is easy to integrate in routine analysis, due to the limited (manual buffer exchange) or no sample preparation (automated removal of salts) [117,131,132]. Structural information of mAbs regarding the disulfide bridge heterogeneity as well as the presence of monomer and dimer conformations can be rapidly obtained via IM-MS [133].…”