The impact of antibody glycoforms on Fc gamma RIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring... Show moreThe impact of antibody glycoforms on Fc gamma RIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed and applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess the binding of different antibody glycoforms to the Fc gamma IIa receptor without the need of glycoengineering. The approach required only low microgram amounts of antibody and receptor and enables assessing the binding of high and low-abundance glycoforms. The approach indicated clear differences in binging between doubly-, hemi-glycosylated and non-glycosylated antibodies as well as for mutated (Leu234Ala, Leu235Ala - Pro329-Gly (LALA-PG)) IgG1 antibodies silenced for Fc gamma binding. The LALA-PG mutated antibody showed no binding to the Fc gamma IIa receptor (excluding potential non-specific binding effects) while the non-glycosylated IgG1 showed a strongly reduced, but still minor binding. The highest binding affinity was for the antibody carrying two complex-type glycans. Man5 glycans resulted in decreased binding compared to complex-type glycans, with the lowest binding for the IgG containing two Man5. For complex-type glycans, galactosylation showed a subtle increase in binding to the Fc gamma IIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the Fc gamma IIa receptor. Finally, the assay was evaluated for the two variants of the Fc gamma RIIa receptor (allotypes H131 and R131) showing highly comparable glycoform selectivity. Overall, the proposed approach allows the direct comparison of binding affinities of different antibody species in mixtures promising a fast establishment of their structure-function relationships. Show less
Schaick, G. van; Dominguez Vega, E.; Gstottner, C.; Berg-Verleg, J.H. van den; Schouten, O.; Akeroyd, M.; ... ; Franc, V. 2022
Monoclonal antibody (mAb) pharmaceuticals consist of a plethora of different proteoforms with different functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their... Show moreMonoclonal antibody (mAb) pharmaceuticals consist of a plethora of different proteoforms with different functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their individual assessment. Current binding techniques do not distinguish between coexisting proteoforms requiring tedious production of enriched proteoforms. Here, we have developed an approach based on mobility shift-affinity capillary electrophoresis-mass spectrometry (ACE-MS), which permitted us to determine the binding of coexisting mAb proteoforms to Fc receptors (FcRs). For high-sensitivity MS analysis, we used a sheathless interface providing adequate mAb sensitivity allowing functional characterization of mAbs with a high sensitivity and dynamic range. As a model system, we focused on the interaction with the neonatal FcR (FcRn), which determines the half-life of mAbs. Depending on the oxidation status, proteoforms exhibited different electrophoretic mobility shifts in the presence of FcRn, which could be used to determine their affinity. We confirmed the decrease of the FcRn affinity with antibody oxidation and observed a minor glycosylation effect, with higher affinities for galactosylated glycoforms. Next to relative binding, the approach permits the determination of individual K-D values in solution resulting in values of 422 and 139 nM for double-oxidized and non-oxidized variants. Hyphenation with native MS provides unique capabilities for simultaneous heterogeneity assessment for mAbs, FcRn, and complexes formed. The latter provides information on binding stoichiometry revealing 1:1 and 1:2 for antibody/FcRn complexes. The use of differently engineered Fc-only constructs allowed distinguishing between symmetric and asymmetric binding. The approach opens up unique possibilities for proteoform-resolved antibody binding studies to FcRn and can be extended to other FcRs and protein interactions. Show less
Pot, S.; Gstottner, C.; Heinrich, K.; Hoelterhoff, S.; Grunert, I.; Leiss, M.; ... ; Dominguez Vega, E. 2021
Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid... Show moreCharacterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 mu g). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy. (C) 2021 The Author(s). Published by Elsevier B.V. Show less
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine... Show moreAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type O-glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison. Show less
Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4... Show moreHuman immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed "Fab-arm exchange" in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severemyasthenicmuscleweakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects onMuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders. Show less
Chemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large hold tanks and batchwise production of media, we developed continuous on-demand... Show moreChemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large hold tanks and batchwise production of media, we developed continuous on-demand reconstitutions directly from solids consisting of a hopper and a screw conveyor capable of feeding dry powdered media with the required precision +/- 5% at low dosing rates of 0.171 g min(-1). A commercially available dry powdered cell culture medium was continuously fed over a duration of 12 h into a mixer which was connected to a UV-cell for monitoring and the media were compared to a batchwise production. A comparable amino acid, carbohydrate, and osmolality profile to a batchwise reconstitution could be obtained. Cell cultivation showed comparable performance of batch and continuous reconstitution for two CHO cell lines producing the antibodies adalimumab and trastuzumab on a small and benchtop scale. In-depth analysis of the produced antibodies showed the same glycosylation pattern, other posttranslational profiles such as methionine oxidation and deamidation compared to batchwise reconstitution. Therefore, we conclude a continuous reconstitution of the medium results in the same quality of the product. A continuous on-demand media reconstitution will impact the supply chain and significantly reduce the floor space necessary for preparation and storage. Show less
Schaick, G. van; Gstottner, C.; Buttner, A.; Reusch, D.; Wuhrer, M.; Dominguez Vega, E. 2021
Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion... Show moreAssessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N-ace-tyllactosamine units, was possible while providing additional information on other modifications such as O-acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs. (C) 2020 The Authors. Published by Elsevier B.V. Show less
Gstottner, C.; Nicolardi, S.; Haberger, M.; Reusch, D.; Wuhrer, M.; Dominguez Vega, E. 2020
Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of... Show moreBispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd'1, Fd'2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches. (C) 2020 Elsevier B.V. All rights reserved. Show less
Gstottner, C.; Vergoossen, D.L.E.; Wuhrer, M.; Huijbers, M.G.M.; Dominguez Vega, E. 2020
Bispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and their involvement in IgG4 mediated autoimmune diseases. While the... Show moreBispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and their involvement in IgG4 mediated autoimmune diseases. While the production of BsAbs is getting more accessible, their analytical characterization remains challenging. We explored the potential of sheathless CE-MS for monitoring exchange efficiency and stability of in-house produced bispecific antibodies. Two IgG4 bispecific antibodies with different molecular characteristics were prepared using controlled Fragment antigen binding (Fab)-arm exchange. Separation of BsAbs from their parent monospecific antibodies was achieved using a polyethyleniimine (PEI)-coated capillary and acidic background electrolytes permitting reliable assessment of the exchange efficiency. This was especially valuable for a Fab-glycosylated BsAb where the high glycan heterogeneity resulted in an overlap of masses with the monospecific parent antibody, hindering their discrimination by MS only. The method showed also good capabilities to monitor the stability of the generated BsAbs under different storage conditions. The levels of degradation products were different for the studied antibodies indicating pronounced differences in stability. Overall, the proposed method represents a useful analytical tool for exchange efficiency and stability studies of bispecific antibodies. Show less
Gstottner, C.; Reusch, D.; Haberger, M.; Dragan, I.; Veelen, P. van; Kilgour, D.P.A.; ... ; Nicolardi, S. 2020
Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for... Show moreBispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS. Show less
