Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry-mass spectrometry.

Trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1-6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins.

A monodispersed metal-complexing peptide-based polymer for mass cytometry enabling spectral applications.

We report the synthesis of a novel class of metal-complexing peptide-based polymers, which we name HyperMAPs (Hyper-loaded MetAl-complexed Polymers). The controlled solid-phase synthesis of HyperMAPs' scaffold peptide provides our polymer with a well-defined molecular structure that allows for an accurate on-design assembly of a wide variety of metals. The peptide-scaffold features a handle for direct conjugation to antibodies or any other biomolecules by means of a thiol-maleimide-click or aldehyde-oxime reaction, a fluorogenic moiety for biomolecule conjugation tracking, and a well-defined number of functional groups for direct incorporation of metal-chelator complexes. Since metal-chelator complexes are prepared in a separate reaction prior to incorporation to the peptide scaffold, polymers can be designed to contain specific ratios of metal isotopes, providing each polymer with a unique CyTOF spectral fingerprint. We demonstrate the complexing of 21 different metals using two different chelators and provide evidence of the application of HyperMAPs on a 13 parameter CyTOF panel and compare its performance to monoisotopic metal-conjugated antibodies.

Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from a single sample injection - Intact protein workflow.

BACKGROUND: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes. RESULTS: The on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-µL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h. SIGNIFICANCE: The developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure.

Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes.

Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-µL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or "trap-and-elute" set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins.

Assessment of Macro- and Microheterogeneity of Monoclonal Antibodies Using Capillary Zone Electrophoresis Hyphenated with Mass Spectrometry.

This chapter focuses on the application of capillary zone electrophoresis hyphenated with mass spectrometry (CZE-MS) for the characterization of monoclonal antibodies (mAbs). mAbs are complex molecules comprising different glycoforms and many other posttranslational modifications. In addition to this inherent microheterogeneity, misassembling of antibodies can take place during production contributing to their macroheterogeneity. CZE-MS is a versatile and powerful technique which has demonstrated high potential for the assessment of both micro- and macroheterogeneity of mAbs. In this chapter, technical and practical considerations for the characterization of mAbs by CZE-MS are described. CE-MS interfacing, capillary coatings for the prevention of mAb adsorption, and sample preparation considerations are covered in detail. The assessment of the macro- and microheterogeneity is discussed and exemplified through three different approaches involving analysis of intact, enzymatically digested, and reduced antibodies. The examples also illustrate the use of two commercially available interfacing techniques (i.e., sheath liquid and sheathless) as well as different types of capillary coatings (positively charged and neutral coatings).

Studying protein structure and function by native separation-mass spectrometry.

Alterations in protein structure may have profound effects on biological function. Analytical techniques that permit characterization of proteins while maintaining their conformational and functional state are crucial for studying changes in the higher order structure of proteins and for establishing structure-function relationships. Coupling of native protein separations with mass spectrometry is emerging rapidly as a powerful approach to study these aspects in a reliable, fast and straightforward way. This Review presents the available native separation modes for proteins, covers practical considerations on the hyphenation of these separations with mass spectrometry and highlights the involvement of affinity-based separations to simultaneously obtain structural and functional information of proteins. The impact of these approaches is emphasized by selected applications addressing biomedical and biopharmaceutical research questions.

CE-MS for Proteomics and Intact Protein Analysis.

This chapter aims to explore various parameters involved in achieving high-end capillary electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides, and their posttranslational modifications. The structure of the topics discussed in this book chapter is conveniently mapped on the scheme of the CE-MS system itself, starting from sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical considerations, a variety of relevant applications for this analytical approach are presented, including posttranslational modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry.

Perceptions about Research Participation among Individuals at Risk and Individuals with Premanifest Huntington's Disease: A Survey Conducted by the European Huntington Association.

There has been great progress in Huntington's disease (HD) research. Yet, effective treatments to halt disease before the onset of disabling symptoms are still unavailable. Scientific breakthroughs require an active and lasting commitment from families. However, they are traditionally less involved and heard in studies. Accordingly, the European Huntington Association (EHA) surveyed individuals at risk (HDRisk) and with premanifest HD (PreHD) to determine which factors affect their willingness to participate in research. Questions assessed research experience and knowledge, information sources, reasons for involvement and noninvolvement, and factors preventing and facilitating participation. The survey included 525 individuals, of which 68.8% never participated in studies and 38.6% reported limited research knowledge. Furthermore, 52% trusted patient organizations to get research information. Reasons for involvement were altruistic and more important than reasons for noninvolvement, which were related to negative emotions. Obstacles included time/financial constraints and invasive procedures, while professional support was seen as a facilitator. PreHD individuals reported less obstacles to research participation than HDRisk individuals. Overall, a high motivation to participate in research was noted, despite limited experience and literacy. This motivation is influenced by subjective and objective factors and, importantly, by HD status. Patient organizations have a key role in fostering motivation through education and support.

Limited Lactosylation of Beta-Lactoglobulin from Cow's Milk Exerts Strong Influence on Antigenicity and Degranulation of Mast Cells.

BACKGROUND: beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity. PRIMARY OBJECTIVE: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS). SECONDARY OBJECTIVE: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells. METHODS: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used. RESULTS: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)-K47, K60, K75, K77, K91, K138 and K141-have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding (p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells (p < 0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow's milk allergens provided that the process is well-controlled.

Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins.

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation.

Probing Polyester Branching by Hybrid Trapped Ion-Mobility Spectrometry-Tandem Mass Spectrometry.

Trapped ion-mobility spectrometry combined with quadrupole time-of-flight mass spectrometry (TIMS-QTOFMS) was evaluated as a tool for resolving linear and branched isomeric polyester oligomers. Solutions of polyester samples were infused directly into the ion source employing electrospray ionization (ESI). TIMS-MS provides both mobility and m/z data on the formed ions, allowing construction of extracted-ion mobilograms (EIMs). EIMs of polyester molecules showed multimodal patterns, indicating conformational differences among isomers. Subsequent TIMS-MS/MS experiments indicated mobility differences to be caused by (degree of) branching. These assignments were supported by liquid chromatography-TIMS-MS/MS analysis, confirming that direct TIMS-MS provided fast (500 ms/scan) distinction between linear and branched small oligomers. Observing larger oligomers (up to 3000 Da) using TIMS required additional molecular charging to ensure ion entrapment within the mobility window. Molecular supercharging was achieved using m-nitrobenzyl alcohol (NBA). The additional charges on the oligomer structures enhanced mobility separation of isomeric species but also added to the complexity of the obtained fragmentation mass spectra. This complexity could be partly reduced by post-TIMS analyte-decharging applying collision-induced dissociation (CID) prior to Q1 with subsequent isolation of the singly charged ions for further fragmentation. The as-obtained EIM profiles were still quite complex as larger molecules possess more possible structural isomers. Nevertheless, distinguishing between linear and symmetrically branched oligomers was possible based on measured differences in collisional cross sections (CCSs). The established TIMS-QTOFMS approach reliably allows branching information on isomeric polyester molecules up to 3000 Da to be obtained in less than 1 min analysis time.

Asymmetrical flow field-flow fractionation to probe the dynamic association equilibria of ß-D-galactosidase.

Protein dynamics play a significant role in many aspects of enzyme activity. Monitoring of structural changes and aggregation of biotechnological enzymes under native conditions is important to safeguard their properties and function. In this work, the potential of asymmetrical flow field-flow fractionation (AF4) to study the dynamic association equilibria of the enzyme ß-D-galactosidase (ß-D-Gal) was evaluated. Three commercial products of ß-D-Gal were investigated using carrier liquids containing sodium chloride or ammonium acetate, and the effect of adding magnesium (II) chloride to the carrier liquid was assessed. Preservation of protein structural integrity during AF4 analysis was essential and the influence of several parameters, such as the focusing step (including use of frit-inlet), cross flow, and injected amount, was studied. Size-exclusion chromatography (SEC) and dynamic light scattering (DLS) were used to corroborate the in-solution enzyme oligomerization observed with AF4. In contrast to SEC, AF4 provided sufficiently mild separation conditions to monitor protein conformations without disturbing the dynamic association equilibria. AF4 analysis showed that ammonium acetate concentrations above 40 mM led to further association of the dimers ("tetramerization") of ß-D-Gal. Magnesium ions, which are needed to activate ß-D-Gal, appeared to induce dimer association, raising justifiable questions about the role of divalent metal ions in protein oligomerization and on whether tetramers or dimers are the most active form of ß-D-Gal.

Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry.

Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.

Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms.

Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization.

Experimental design and measurement uncertainty in ligand binding studies by affinity capillary electrophoresis.

In all life sciences ligand binding assays (LBAs) play a crucial role. Unfortunately these are very error prone. One part of this uncertainty results from the unavoidable random measurement uncertainty, another part can be attributed to the experimental design. To investigate the latter, uncertainty propagation was evaluated as a function of the given experimental design. A design space including the normalized maximum response range (nMRR), the data point position (DPP), the data point range (DPR) and the number of data points (NoDP) was defined. Based on ten measured ms ACE source data sets 20 specific parameter sets were selected by Design of Experiments. Monte Carlo simulations using 100 000 repeats for every parameter set were employed. The resulting measurement uncertainty propagation factors (measurement uncertainty multiplier: MUM) were used to describe the whole design space by polynomial regression. The resulting 5-dimensional response surface was investigated to evaluate the design parameter's influence and to find the minimal uncertainty propagation. It could be shown, that the nMRR is of highest importance, followed by DPP and DPR. Interestingly, the NoDP is less relevant. However, the interactions of the four parameters need to be carefully considered during design optimization. Using at least five data points which cover over 40% of the upper part of the binding hyperbola (DPP > 0.57) the MUM will be minimized (MUM approximately 1.5) when the nMRR is appropriate. It is possible to reduce the measurement uncertainty propagation more than one order of magnitude.

Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry.

Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab)2 and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab)2 part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18-33 proteoforms) of the three analyzed mAbs.

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

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2. Graphical abstract ᅟ.