Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.     

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry

Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.     

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6–9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.     

Investigation of the correlation between charge and glycosylation of IgG1 variants by liquid chromatography–mass spectrometry

A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.     

Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry

Antibody-drug conjugation strategies are continuously evolving as researchers work to improve the safety and efficacy of the molecules. However, as a part of process and product development, confirmation of the resulting innovative structures requires new, specialized mass spectrometry (MS) approaches and methods, as compared to those already established for antibody-drug conjugates (ADCs) and the heightened characterization practices used for monoclonal antibodies (mAbs), in order to accurately elucidate the resulting conjugate forms, which can sometimes have labile chemical bonds and more extreme chemical properties like hydrophobic patches. Here, we discuss practical approaches for characterization of ADCs using new methodologies and ultrahigh-resolution MS, and provide specific examples of these approaches. Denaturing conditions of typical liquid chromatography (LC)/MS analyses impede the successful detection of intact, 4-chain ADCs generated via cysteine site-directed chemistry approaches where hinge region disulfide bonds are partially reduced. However, this class of ADCs is detected intact reliably under non-denaturing size-exclusion chromatography/MS conditions, also referred to as native MS. For ADCs with acid labile linkers such as one used for conjugation of calicheamicin, careful selection of mobile phase composition is critical to the retention of intact linker-payload during LC/MS analysis. Increasing the pH of the mobile phase prevented cleavage of a labile bond in the linker moiety, and resulted in retention of the intact linker-payload. In-source fragmentation also was observed with typical electrospray ionization (ESI) source parameters during intact ADC mass analysis for a particular surface-accessible linker-payload moiety conjugated to the heavy chain C-terminal tag, LLQGA (via transglutaminase chemistry). Optimization of additional ESI source parameters such as cone voltages, gas pressures and ion transfer parameters led to minimal fragmentation and optimal sensitivity. Ultrahigh-resolution (UHR) MS, combined with reversed phase-ultrahigh performance (RP-UHP)LC and use of the FabRICATOR® enzyme, provides a highly resolving, antibody subunit-domain mapping method that allows rapid confirmation of integrity and the extent of conjugation. For some ADCs, the hydrophobic nature of the linker-payload hinders chromatographic separation of the modified subunit/domains or causes very late elution/poor recovery. As an alternative to the traditionally used C₄ UHPLC column chemistry, a diphenyl column resulted in the complete recovery of modified subunit/domains. For ADCs based on maleimide chemistry, control of pH during proteolytic digestion is critical to minimize ring-opening. The optimum pH to balance digestion efficiency and one that does not cause ring opening needed to be established for successful peptide mapping.     

Epitope characterization of an anti‐PD‐L1 antibody using orthogonal approaches

The binding of programmed death ligand 1 protein (PD‐L1) to its receptor programmed death protein 1 (PD‐1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD‐L1/PD‐1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD‐L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD‐L1. Hydrogen–deuterium exchange mass spectrometry (HDX‐MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD‐1 binding interface, consistent with a desired PD‐1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD‐L1 variants confirmed that the region identified by HDX‐MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD‐L1‐mediated immune evasion. This is the first report of an epitope for any antibody targeting PD‐L1 and demonstrates the power of combining orthogonal epitope mapping techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine,a lysine-linked antibody drug conjugate

Antibody–drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla®). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin®), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1.     

Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain

A unique, late-eluting “basic peak” (relative to the “main peak”) was observed by weak cation exchange-HPLC (WCX) for a recombinant monoclonal antibody (mAb) sample. Peak fractions were collected, desalted, and analyzed by high-resolution MS using a top-down characterization approach that provided accurate masses of intact mAb charge isoforms and a comprehensive profile of the structural heterogeneity. The individual light (L) and heavy (H) chain subunits from the main and basic peaks were analyzed by reversed-phase (RP) HPLC/MS after disulfide bond reduction and cysteine alkylation. Three mAb isoforms were detected, and their modifications were localized to H chain. Bottom-up characterization using RP-HPLC/MS peptide mapping and accurate mass measurements identified three distinct H chain C-terminal peptides ending in glycine, lysine, or α-amidated proline. The combined analyses showed that the main WCX peak mAb isoform contained two unmodified L chains and two H chains terminating in glycine. Each mAb isoform that coeluted in the basic peak consisted of two unmodified L chain subunits and a single H chain ending in glycine, but the second H chain terminated in lysine for one isoform and α-amidated proline for another isoform. The WCX elution positions of the isoforms were consistent with their respective net charge. To the best of our knowledge, the occurrence of C-terminal α-amidation in mAbs has not been reported previously.     

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA1c sample

Glycated hemoglobin (HbA_1c) is a ‘gold standard’ biomarker for assessing the glycemic index of an individual. HbA_1c is formed due to nonenzymatic glycosylation at N-terminal valine residue of the β-globin chain. Cation exchange based high performance liquid chromatography (CE–HPLC) is mostly used to quantify HbA_1c in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE–HPLC-based quantification, resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA_1c (52.1%) in the CE–HPLC method. The observed HbA_1c did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA_1c and Hb Beckman might have resulted in the coelution of the variant with HbA_1c in CE–HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA_1c, it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA_1c must be estimated using an alternative method.     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.     

An automated,high‐throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA‐HR/AM)

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC‐MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi‐automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.     

Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

ABSTRACT

Determination of the impact of individual antibody glycoforms on Fc?RIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated Fc?RIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure–function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.     

Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

Heparin and heparin-derived components are widely applied anticoagulant drugs used for amongst other applications medical treatment of deep vein thrombosis and pulmonary embolism. Depolymerisation of native heparin results in complex mixtures of sulfated linear oligosaccharides that are usually not well characterised. In order to further characterise such mixtures, two on-line ion-pairing reverse-phased chromatography electrospray ionisation (ESI) mass spectrometry methods have been developed. One of the systems allows the determination of more than 200 components in a medium molecular weight heparin preparation, whereas the other system can be used to separate isomeric heparin oligosaccharides after previous separation according to size. This latter system allows semi-preparative isolation of isomeric heparin oligosaccharides. The experimental setup includes on-line cation exchange in order to prevent the ion-pairing reagent from entering the mass spectrometer.