Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding.

The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb - FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.

A robust nanoscale RP HPLC-MS approach for sensitive Fc proteoform profiling of IgG allotypes.

The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes. To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness. This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs.

Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment.

After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.

Affinity capillary electrophoresis - mass spectrometry permits direct binding assessment of IgG and FcγRIIa in a glycoform-resolved manner.

The impact of antibody glycoforms on FcγRIIa 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 FcγIIa 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 Fcγ binding. The LALA-PG mutated antibody showed no binding to the FcγIIa 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 FcγIIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the FcγIIa receptor. Finally, the assay was evaluated for the two variants of the FcγRIIa 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.

Chemical Synthesis and Immunological Evaluation of Fragments of the Multiantennary Group-Specific Polysaccharide of Group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their different capsular polysaccharides. The Group B carbohydrate (GBC) is shared by all serotypes and therefore attractive be used in a glycoconjugate vaccine. The GBC is a highly complex multiantennary structure, composed of rhamnose rich oligosaccharides interspaced with glucitol phosphates. We here report the development of a convergent approach to assemble a pentamer, octamer, and tridecamer fragment of the termini of the antennae. Phosphoramidite chemistry was used to fuse the pentamer and octamer fragments to deliver the 13-mer GBC oligosaccharide. Nuclear magnetic resonance spectroscopy of the generated fragments confirmed the structures of the naturally occurring polysaccharide. The fragments were used to generate model glycoconjugate vaccine by coupling with CRM197. Immunization of mice delivered sera that was shown to be capable of recognizing different GBS strains. The antibodies raised using the 13-mer conjugate were shown to recognize the bacteria best and the serum raised against this GBC fragment-mediated opsonophagocytic killing best, but in a capsule dependent manner. Overall, the GBC 13-mer was identified to be a highly promising antigen for incorporation into future (multicomponent) anti-GBS vaccines.

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).

Affinity Capillary Electrophoresis-Mass Spectrometry as a Tool to Unravel Proteoform-Specific Antibody-Receptor Interactions.

Monoclonal 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 KD 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.

Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography - Mass spectrometry.

Characterization 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 µg). 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.

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger.

The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in Aspergillus niger. EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.

Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells.

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 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.

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis.

Human 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 severe myasthenic muscle weakness, 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 on MuSK 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.

Media on-demand: Continuous reconstitution of a chemically defined media directly from solids.

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 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.

Anion exchange chromatography - Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals.

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 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-acetyllactosamine 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.

Sheathless CE-MS as a tool for monitoring exchange efficiency and stability of bispecific antibodies.

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 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.

Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS.

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 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.

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

Bispecific 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.

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics.

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS.

Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography-mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb1 is presented in this paper.