LC-MS Peptide Mapping Research Articles

Improved Mass Accuracy and Precision for Multi-Attribute Methods Using a New Internally Calibrated High Resolution Orbitrap Mass Detector.

In the development of biotherapeutics, a thorough understanding of a molecule's product quality attributes (PQAs) and their effect on structure-function relationships and long-term stability is essential for ensuring the safety and efficacy of the product. First published in 2015, the multi-attribute method (MAM), based on LC-MS peptide mapping and automation principles, can be used to support biotherapeutic process and product development. The MAM provides simultaneous site-specific detection, identification, quantitation, and quality control monitoring of selected PQAs. In this article, a low-maintenance MAM-ready mass detector with a small footprint was evaluated for its ability to monitor PQAs on proteolytically digested proteins with high mass accuracy and precision. Optimized source parameters enable robust relative quantitation of attributes with high sensitivity and minimal in-source fragmentation. A combination of a built-in one-point mass calibration procedure prior to data acquisition and Scan-to-Scan on-the-fly mass correction allows monitoring of most peptides for at least 54 days with sub-1 ppm mass accuracies at high-resolution (180,000 at m/z 200). This enables the use of <3 ppm mass tolerances for peptide monitoring, supporting high method specificity and robustness. LC-MS based MAM data from this instrument compares well to data collected by earlier MAM systems and conventional HPLC profile-based drug substance release assays.

Fully Unattended Online Protein Digestion and LC-MS Peptide Mapping.

LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC-MS peptide mapping. The method generates LC-MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC-MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC-MS analysis.

Cutting-edge mass spectrometry strategy based on imaged capillary isoelectric focusing (icIEF) technology for characterizing charge heterogeneity of monoclonal antibody

Imaging capillary isoelectric focusing (icIEF) technology has been becoming the gold criteria of monitoring monoclonal antibody (mAb) charge heterogeneity that is one of the major product-related variants in recombinant biopharmaceuticals, since the first commercial instrument developed twenty years ago. However, the protein identification in icIEF separation is just based on isoelectric point (pI) measurement of protein. Although high resolution mass spectrometry (HRMS) is currently the most powerful means of qualitative protein analysis, traditional icIEF cannot compatibly be used in conjunction with MS due to the use of less volatile reagents. In addition, protein heterogeneity characterization in depth such as peptide mapping by high performance liquid chromatography (HPLC) requires the focused protein bands to be collected as fractions after the icIEF separation, which is a great challenge in biopharmaceutical discovery. In this work, pembrolizumab was employed as targeting mAb (a highly selective anti-PD-1 humanized mAb), an integrated icIEF platform was developed including analytical profiling, MS coupling and fraction collections for charged variant preparation. Multiple operation modes can be rapidly and flexibly switched just by changing customized capillary separation cartridges without more configurations. Main component, four acidic variants (A1-A4) and three basic variants (B1–B3) were baseline separated then directly detected by icIEF-HRMS online coupling for rapid screening of intact protein heterogeneity where reliable and accurate molecular weight of protein charged variants were obtained. Next, by installing preparative capillary separation cartridge, fractions of major charge variants (A2-3 and B1-2) and main component were collected for following LC-MS peptide mapping characterization. The whole workflow of icIEF-based MS strategy for protein heterogeneity is straight forward, reliable and accurate, which provides a comprehensive and revolutionary technology for protein drug quality control (QC) monitoring, MS coupling for fingerprinting intact protein and HPLC-MS peptide mapping in depth.

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants

ABSTRACT Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68–81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68–61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Discrimination of Isoleucine and Leucine by Dimethylation-Assisted MS3

Protein sequencing by mass spectrometry has transformed the field of biopharmaceutical analysis, but a missing part in the analytical toolkit is the ability to distinguish between the isomeric residues isoleucine and leucine because it is a requisite for efficient analysis of the primary structure of proteins. To address this need, we have developed a novel mass spectrometric method that combines reductive dimethylation and MS3 fragmentation with LCMS peptide mapping. The dimethylation of peptide N-termini leads to intense a1-ions upon collision-induced fragmentation, and further fragmentation of the isoleucine/leucine a1-ion leads to informative spectra with fragments that can discriminate between the two isomers. The methodology of a1-directed MS3 was applied to two antibodies in combination with the proteases trypsin, thermolysin, chymotrypsin, and pepsin to generate peptides exposing N-terminal I/L residues.

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies.

Abstract 3770: CAR-T cell harboring a camelid single domain antibody as a targeting agent to kill tumors expressing VEGFR2

Abstract Modulation of the immune system is showing tremendous promise in the treatment of malignancies. In addition to checkpoint inhibitors that re-activate T cells present in the tumor microenvironment, exogenously transduced chimeric antigen receptor (CAR) T cells are providing excellent responses in clinical trials for the treatment of leukemias. In this study, we describe CAR-T cells that target VEGFR2-expressing tumors. Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor (VEGF), which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFR). As VEGFR2 is also expressed by a variety of tumors, we investigated the utility of anti-VEGFR2 CAR-T cells as a method to treat VEGFR2-expressing tumors. Camelid antibodies are small (14 kD) single chain antibodies. To generate a camelid antibody targeting the extracellular domain of VEGFR2, a llama was immunized with recombinant VEGFR2/Fc. A phage display library was generated and screened to identify an antibody with high binding affinity to VEGFR2. The selected antibody was expressed in the E. coli. BL21 (DE3) pT7 system. The purified antibody was characterized by SEC, LC-MS peptide mapping and ELISA. CAR-T cells were engineered to express the camelid anti-VEGFR2 antibody in combination with the CD28 and 4-1BB costimulatory molecules and the CD3 zeta chain. Tumor cells were screened for expression of VEGFR2, and the HL-60 acute promyelocytic leukemia, ZR-75-30 breast ductal carcinoma and NCI-H23 non-small cell lung adenocarcinoma were identified. Co-incubation of anti-VEGFR2 CAR-T cells with the VEGFR-2-expressing cell lines resulted in dose-dependent target cell toxicity as measured by LDH release. In addition, T cell activity was confirmed, as high levels of IL-2 and IFN-γ were detected in the cell culture media. These results suggest that anti-VEGFR2 CAR-T may be useful in directly targeting VEGFR2-expressing tumors. We previously showed the utility of camelid antibodies in CAR-T constructs as anti-CEACAM6 CAR-T cells show both in vitro and in vitro efficacy against the pancreatic tumor Bx-PC3. The use of the a camelid V21 antibody to target VEGFR2-expressing tumors provides hope that camelid single domain antibodies can be developed for CAR-T therapies. Citation Format: Heman Chao, Baomin Tian, Marni Uger, Wah Wong. CAR-T cell harboring a camelid single domain antibody as a targeting agent to kill tumors expressing VEGFR2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3770. doi:10.1158/1538-7445.AM2017-3770

Characterization of succinimide stability during trypsin digestion for LC-MS analysis

LC-MS peptide mapping is the most commonly used method to analyze protein modifications. The proteins are generally digested using trypsin at a slightly basic pH at 37 °C from several hours to overnight. Assay-induced artifacts can be generated during this procedure, potentially causing false-positive or false-negative results for a given modification. Unfortunately, for the analysis of succinimide, both false-negative and false-positive results can be generated within the same procedure. This study evaluates the stability of succinimide during the peptide mapping procedure and has demonstrated that up to 13% of pre-existing succinimide was lost during a 4 h trypsin digestion at pH 5.0 which was previously determined to be optimal for the detection of succinimide. The same procedure was able to simultaneously generate approximately 3% succinimide. Using the optimized procedure, it was also found that two aspartate residues that are followed by glycine residues in the conserved Fc region of a recombinant monoclonal antibody were not prone to isomerization. On the other hand, an aspartate residue followed by a glycine in the heavy chain variable domain was highly susceptible to isomerization. Interestingly, the antibody containing the succinimide eluted from an SEC column after the monomer peak.

Effect of acrylodan conjugation and forced oxidation on the structural integrity, conformational stability, and binding activity of a glucose binding protein SM4 used in a prototype continuous glucose monitor.

Continuous glucose monitoring (CGM) devices offer diabetes patients a convenient approach to assist in controlling blood glucose levels. A prototype CGM has been developed that uses the emission profile of a polarity-sensitive fluorophore (acrylodan) conjugated to a glucose/galactose-binding protein (SM4-AC) to measure the concentration of glucose in vivo. During development, a decrease in the devices signal intensity was observed in vivo over time, which was postulated to be result of oxidative degradation of SM4-AC. A comprehensive physicochemical analysis of SM4-AC was pursued to identify potential mechanisms of signal intensity loss in this CGM during in vitro forced oxidation studies. An assessment of the structural integrity and conformational stability of SM4-AC indicated a relatively decreased polarity and lower tertiary structure stability compared to unconjugated protein (SM4). The stability and polarity of SM4-AC was also altered in the presence of H2 O2 . Furthermore, a time-dependent loss in the fluorescence signal of SM4-AC was observed when incubated with H2 O2 . An LC-MS peptide mapping analysis of these protein samples indicated that primarily two Met residues in SM4-AC were susceptible to oxidation. When these two residues were genetically altered to an amino acid not prone to oxidation, the glucose binding ability of the protein was retained and no loss of acrylodan fluorescence was observed in the presence of H2 O2 . Genetic alteration of these two residues is proposed as an effective approach to increase the long-term stability of SM4-AC within this prototype CGM in vivo.

A conventional procedure to reduce Asn deamidation artifacts during trypsin peptide mapping.

Asn deamidation is a common post-translational modification of proteins with significant biological consequences. Asn deamidation can cause changes in structure, stability and function of proteins. LC-MS peptide mapping is the most widely used method to detect and quantify Asn deamidation. However, a significant amount of deamidation can occur during sample preparation for peptide mapping, making it challenging to accurately determine the original level of deamidation. Although several protocols to reduce procedure-induced deamidation have been reported, they either require special procedural steps or are not optimal for maintaining trypsin activity. In the current study, several commonly used buffers that are optimal for trypsin activity were evaluated. The results demonstrated that much lower levels of Asn deamidation artifacts were observed when Tris buffer was used, especially at lower concentrations. The addition of 10% acetonitrile further reduced the levels of Asn deamidation artifacts. The utility of the optimized procedure was demonstrated by the digestion of a recombinant monoclonal antibody. The proposed procedure can be readily applied to any laboratory settings as it does not require any special reagents or procedures.

Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC.

Bioconjugation by copper-catalyzed azide-alkyne cycloaddition (CuAAC) provides a powerful means to produce site-specifically modified proteins. However, the use of a copper catalyst brings about the possible generation of reactive oxygen species that could cause degradation of vulnerable amino acid residues. We investigated whether PEGylation by CuAAC caused any modifications to the therapeutic protein interferon beta-1b, which was produced via global amino acid substitution with azidohomo-alanine at the N-terminus and contains no methionine residues. Using previously reported reaction conditions, LC-MS peptide mapping detected +32 Da and +48 Da oxidation modifications of tryptic peptides 28-33 (LEYCLK) and 137-147 (EYSHCAWTIVR) in the protein post-PEGylation. The oxidative degradation increased with reaction time, whereas reducing the copper concentration slowed the PEGylation rate as well as the oxidation rate. Replacing dithiothreitol (DTT) with any of five different monothiol reducing agents in anaerobic conditions allowed efficient PEGylation in 2-4 h and abrogated oxidative degradation. Free cysteine provided reproducible reaction results as a reducing agent in this system and has been successfully applied to other protein conjugations. Monothiol reducing agents, such as cysteine, may be useful tools as protective reducing agents for CuAAC in some bioconjugation systems.

Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.

A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (<2%), and high purity (>95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis

Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography–mass spectrometry peptide mapping

Liquid chromatography mass spectrometry (LC–MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC–MS as intact glycopeptides in a single experiment. LC–MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (∼0.1 to 1.0 mol% level) was obtained by LC–MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC–MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.

Characterization of a novel modification on IgG2 light chain: Evidence for the presence of O-linked mannosylation

This paper describes the analysis of a novel modification identified on the light chain of a recombinant IgG2 antibody. This modification, a +162 Da adduct, suggestive of a single hexose addition, was observed by mass analysis of the reduced molecule. The modification was located on residue serine 66 of the light chain by investigation with LC–MS peptide mapping, mass spectrometry and N-terminal sequencing techniques. Location of the adduct on serine pointed the investigation toward O-linked glycosylation. Identification of the hexose residue was deduced from its elimination by action of α-mannosidase, providing evidence for the presence of an O-mannosylated light chain. This type of modification in the glycosylation profile of antibodies, to our knowledge, has not been reported for human IgG molecules.

Characterization of a Novel Modification to Monoclonal Antibodies: Thioether Cross-link of Heavy and Light Chains

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.