Annika A. M. van der Zon

Antibody function depends on the molecular pairing of antigen-binding Fab regions with Fc domains, linking antigen specificity to effector potential. However, antibody repertoire methods analyze these subunits separately, creating an inference problem in which Fc-Fab connectivity is lost. Here, we introduce a multi-level HPLC-MS workflow integrating native nanoflow cation-exchange chromatography-MS (nCEC-MS) of intact serum IgG with middle-up Fab and Fc/2 profiling. Native nCEC-MS reduces spectral congestion compared with denaturing reversed-phase HPLC-MS, enabling detection of 56-74 intact IgG mass features from 2 μg of purified IgG per donor. Middle-up analysis provided complementary information on Fab diversity, subclass, and allotype, revealing donor-specific profiles. Reconstructing intact IgG1 masses from independently measured Fab and Fc/2 subunits partially recovers Fc-Fab molecular connectivity and supports interpretation of native intact mass features. Together, this framework provides an integrated view of the human IgG repertoire and establishes a foundation for studying its molecular topology in health and disease.

Minor deviations in biopharmaceutical manufacturing processes, such as those used for monoclonal antibodies (mAbs), can introduce structural modifications that alter protein efficacy and safety. Monitoring these changes is critical to ensure product consistency and efficacy. Ion-exchange chromatography (IEC) is widely employed to assess product quality by separating charge variants. However, the use of non-volatile salts in IEC makes direct identification and characterization of these variants difficult. In this study, we developed a multi-heart-cut IEC–-reversed-phase liquid chromatography (RPLC) platform that enables the separation, online concentration and desalting, and fractionation of mAb charge variants. The system integrates a 10-port valve and a six-column selector valve, allowing automated collection of up to five charge variants within a single analytical workflow. Moreover, the platform can be directly coupled to mass spectrometry (MS) for the characterization of charge heterogeneity and glycosylation. Systematic optimization of the capture and buffer-exchange conditions, including evaluation of stationary phase types and mobile phase compositions, was performed to maximize overall recovery. This platform can also be used for other chromatographic modes, such as hydrophobic interaction chromatography or size-exclusion chromatography. This integrated multiple heart-cut IEC–RPLC platform provides a high-resolution and efficient platform for detailed characterization of mAb charge variants, facilitating the characterization of critical product quality attributes.

Positron emission tomography (PET) using radiolabeled antibodies (radioimmunoconjugates) is a powerful imaging technique to track the distribution of therapeutic antibodies. Accurate determination of the average number of chelators per antibody (CAR) is essential for (radio)immunoconjugate characterization. Radiometric titration, although widely used, is labor-intensive and requires approximately half a day per sample. To overcome this analytical bottleneck, we developed a microscale size exclusion chromatography – mass spectrometry (SEC-MS) analysis. Thanks to the microscale flow applied, high concentrations of volatile salts, up to 1000 mM ammonium acetate, could be used to minimize secondary interactions. The method was optimized using model radioimmunoconjugates based on intact trastuzumab, with a particular focus on the impact of buffer concentration on both chromatographic effects and CAR determination. A final buffer concentration of 600 mM ammonium acetate was selected. The method was compared to radionuclide titration for the analysis of the same immunoconjugate model, reporting similar results. Finally, we demonstrated the broad applicability of the method by testing diverse immunoconjugates (ipilimumab), including cysteine and lysine-linked chelator samples, as well as other smaller protein models, such as nanobodies. With a 20-min run time, the averageCAR, dispersity index, glycosylation profile, and impurities could all be determined. This approach offers a broadly applicable alternative for radiometric titration for the characterization of immunoconjugates.

Glycosylation significantly impacts the pharmacokinetics and efficacy of monoclonal antibody (mAb) biotherapeutics. Characterizing mAbs' glycoform profiles is crucial for optimizing therapeutic outcomes, and intact antibody analysis provides key information about the glycoform combinations present. While state-of-the-art RPLC-MS methods are commonly used for intact mAb analysis, they lack the selectivity to resolve glycoforms and, therefore, may not detect lower-abundance glycoforms. In contrast, HILIC methods have demonstrated good resolving power for middle-up mAb glycoform analysis. However, to date, no application of HILIC has been described to characterize mAb glycoforms at the intact level. This study describes the development of acrylamide monoliths for HILIC-MS intact mAb glycoprofiling. We studied how the porogen composition (octanol and DMSO ratio) in the polymerization mixture affects the column permeability and separation performance. Our findings indicated that increasing the DMSO content increased retention and decreased the peak widths. The optimized HILIC-MS method was applied to analyze five reference intact mAbs (IgG₁ and IgG₄). The method demonstrated glycoform selectivity at the intact protein level, achieving baseline separations between single and double Fc glycosylation (e.g., for trastuzumab, resolution (Rs) of 3.62 for G0F vs G0F/G0F) and partial separations between glycoforms differing by one glycan unit (e.g., for trastuzumab, Rs of 1.06 between G0F/G0F and G0F/G1F). Compared to state-of-the-art RPLC-MS, acrylamide-monolith HILIC-MS enabled the measurement of low-abundance glycoforms (e.g., single G0F and M5/M5). The selectivity and sensitivity (ng of sample injection) of this method open opportunities for studies of IgG heterogeneity in bioanalytical applications.

Glycosylation significantly impacts the pharmacokinetics and efficacy of monoclonal antibody (mAb) biotherapeutics. Characterizing mAbs' glycoform profiles is crucial for optimizing therapeutic outcomes, and intact antibody analysis provides key information about the glycoform combinations present. While state-of-the-art RPLC-MS methods are commonly used for intact mAbs analysis, they lack the selectivity to resolve glycoforms and, therefore, may not detect lower abundance glycoforms. In contrast, HILIC methods have demonstrated good resolving power for middle-up mAbs glycoform analysis. However, to date, no application of HILIC has been described to characterize mAb glycoforms at the intact level. This study describes the development of acrylamide monoliths for HILIC-MS intact mAb glycoprofiling. We studied how the porogen composition (octanol and DMSO ratio) in the polymerization mixture affects the column permeability and separation performance. Our findings indicated that increasing the DMSO content increased retention and decreased the peak widths. The optimized HILIC-MS method was applied to analyze five reference intact mAbs (IgG1 and IgG4). The method demonstrated glycoform selectivity at the intact protein level, achieving baseline separations between single and double Fc glycosylation (e.g., for trastuzumab, resolution (Rs) of 3.62 for G0F vs G0F/G0F), and partial separations between glycoforms differing of one glycan units (e.g., for trastuzumab, Rs of 1.06 between G0F/G0F and G0F/G1F). Compared to state-of-the-art RPLC-MS, acrylamide-monolith HILIC-MS enabled the measurement of low-abundance glycoforms (e.g., single G0F and M5/M5). The selectivity and sensitivity (ng sample injection) of this method open opportunities for studies of IgG heterogeneity in bioanalytical applications.

Native size-exclusion chromatography (SEC) coupled with native mass spectrometry (nMS) enables the characterization of proteins and protein complexes by combining liquid-phase separation (SEC) and mass measurement of the complexes (nMS). This approach allows for an increase in the throughput of native MS experiments, reduces the bias that may be present due to the co-ionization of oligomers, and facilitates online sample buffer exchange. SEC-nMS uses volatile buffers and relatively wide-diameter columns (e.g., ≥ 1 mm), with flow rates in the tens of µL/minute. To ionize sample components under this flow regime, relatively harsh electrospray ionization (ESI) desolvation conditions are needed, potentially resulting in protein dissociation/denaturation. Also, relatively large amounts of samples are required (several micrograms). Herein, we describe the development of a nanoflow SEC-nMS method using 200 µm I.D. columns, operated at 500 nL min-1. This approach allows buffer exchange, oligomer separation, and mild ionization conditions (e.g., without the assistance of heated gas flow or temperature). Compared to microflow (1 mm I.D. column), the nanoflow method achieved a 4-fold increase in MS peak intensity, despite using a sample 20 times less concentrated (0.05 mg mL-1 for nanoflow vs. 1 mg mL-1 for microflow). Furthermore, we evaluated the impact of three injection approaches on sensitivity and separation efficiency: large-volume (1 μL), nano-volume (50 nL), and online mix-bed ion-exchange capillary trap injection. To demonstrate its performance and applicability for sample-limited analysis, the final method using nano-volume injection was applied to several model proteins, protein complexes, and a urine sample from a pregnant donor.

Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs), e.g., charge variants, oxidation, etc., during production and storage, necessitating evaluation of the resulting PTMs as critical quality attributes (CQA) for protein quality and safety. Charge variants PTMs can be separated using capillary zone electrophoresis (CZE). The CZE EACA method developed by He et al. (2011) with UV detection is applied routinely in the pharmaceutical industry for analyzing charge variants. However, the method cannot be directly hyphenated with mass spectrometry (MS), preventing direct identification of separated charge variants due to the non-volatile background electrolyte (BGE), which hinders reliable charge variant identification. This study presents a CZE-UV/MS method using a neutral static capillary coating of hydroxypropyl methylcellulose combined with a volatile BGE at pH 5.0 to allow MS-compatible mAb charge variant separations. The effect of several parameters, including pH and concentration of BGE, voltage, and injected mAb concentrations in terms of separation performance on a panel of mAbs was investigated. The final method was tested with mAbs of IgG subclasses (IgG1 and IgG4) with different pI (7.4-9.2), and degrees of heterogeneity. Basic and acidic variants were separated from the parent mAb using a BGE of 50 mM acetic acid adjusted at pH 5.0. A linear correlation in relative charge variant abundance was obtained between our method and the EACA method. CZE-MS coupling was accomplished using the nanoCEasy, a low-flow sheath liquid interface, which enabled the identification and quantitation of basic, acidic, and incomplete pyroglutamate variants, and glycoforms of the tested mAbs.This method can be a useful tool for in-depth charge variants characterization of mAbs.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans including microheterogeneity at the same amino acid residue (glycosite). Top-down mass spectrometry (MS) allows precise identification and localization of glycans on intact proteins, and coupling top-down MS with chromatography allows time-resolved characterization of glycoforms. Here, we couple ultraviolet photo-dissociation (UVPD) to hydrophilic interaction chromatography (HILIC) to advance the characterization of glycoproteins ranging from 15-34 kDa, offering site localization of glycans, providing sequence coverages up to 93% and relative quantitation of individual glycoforms.

Sample transformation can be used to provide insights into the properties of complex samples.