Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody.

While characterizing a therapeutic IgG4 monoclonal antibody (mAb), we observed a variant with a mass 1177 Da larger than the predominant mAb form that could not be ascribed to previously described modifications. Through successive rounds of experimentation, we localized the mass addition to the C-terminus of the heavy chain (HC). During this process we observed that when the mAb was broken down into separate domains, the Fc and the 1177 Da-modified Fc could be chromatographically separated. Separation allowed collection of native and modified Fc fractions for LC/MS peptide mapping. A unique peptide present in the modified fraction was de novo sequenced and demonstrated to be a modified form of the HC C-terminus lacking two native residues (GK) and gaining twelve additional non-native residues (EAEAASASELFQ). Aware of other mAb variants with genetic origins, we sought to understand whether this modification too had a genetic basis. In silico translation of the expression vector encoding the mAb demonstrated that a normally non-coding section of nucleotides in the + 1 reading frame relative to the HC C-terminal coding region could have led to a transcript with the non-native C-terminal extension. Two potential mechanisms for how this nucleotide sequence might have fused to the native HC coding region and led to expression of the extension product are presented.

Stage Dependence, Cell-Origin Independence, and Prognostic Capacity of Serum Glycan Fucosylation, ß1-4 Branching, ß1-6 Branching, and α2-6 Sialylation in Cancer.

Glycans represent a promising but only marginally accessed source of cancer markers. We previously reported the development of a molecularly bottom-up approach to plasma and serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, ß1-6 branching, and core fucosylation as single analytical signals. Based on the behavior of P/S glycans established to date, we hypothesized that the alteration of P/S glycans observed in cancer would be independent of the tissue in which the tumor originated yet exhibit stage dependence that varied little between cancers classified on the basis of tumor origin. Herein, the diagnostic utility of this bottom-up approach as applied to lung cancer patients (n = 127 stage I; n = 20 stage II; n = 81 stage III; and n = 90 stage IV) as well as prostate (n = 40 stage II), serous ovarian (n = 59 stage III), and pancreatic cancer patients (n = 15 rapid autopsy) compared to certifiably healthy individuals (n = 30), nominally healthy individuals (n = 166), and risk-matched controls (n = 300) is reported. Diagnostic performance in lung cancer was stage-dependent, with markers for terminal (total) fucosylation, α2-6 sialylation, ß1-4 branching, ß1-6 branching, and outer-arm fucosylation most able to differentiate cases from controls. These markers behaved in a similar stage-dependent manner in other types of cancer as well. Notable differences between certifiably healthy individuals and case-matched controls were observed. These markers were not significantly elevated in liver fibrosis. Using a Cox proportional hazards regression model, the marker for α2-6 sialylation was found to predict both progression and survival in lung cancer patients after adjusting for age, gender, smoking status, and stage. The potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.

Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of ß-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli ß-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which ß-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

Characterization of additional vitamin D binding protein variants.

The gene (GC) for the vitamin D binding protein (DBP) shows significant genetic variation. Two missense variants, p.D432E and p.T436K, are common polymorphisms and both may influence vitamin D metabolism. However, less common variants, identified biochemically, have been reported previously. This study aimed to identify the underlying mutations by molecular screening and to characterize the mutant proteins by mass spectrometry. Denaturing high performance liquid chromatography (DHPLC) was used for screening genetic variants in GC exons and exon/intron boundaries of genomic DNA samples. Sanger sequencing identified the specific mutations. An immuno-capture coupled mass spectrometry method was used to characterize protein variants in serum samples. Initial molecular screening identified 10 samples (out of 761) containing an alanine deletion at codon 246 in exon 7 (p.A246del, c.737_739delCTG), and 1 sample (out of 97) containing a cysteine to phenylalanine substitution at codon 311 in exon 8 (p.C311F, c.932G>T). The mutant allele proteins and posttranslational modified products were distinguishable from the wild-type proteins by mass spectrum profiling. Loss of a disulfide bond due to loss of cysteine-311 was accompanied by the appearance of a novel mixed disulfide species, consistent with S-cysteinylation of the remaining unpaired cysteine-299 in the mutant protein. We confirm earlier biochemical studies indicating that there are additional deleterious GC mutations, some of which may be low-frequency variants. The major findings of this study indicate that additional mutant proteins are secreted and can be identified in the circulation. By combining molecular screening and mass spectrometric methods, mutant DBP species can be identified and characterized.

Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry.

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.

Mass Spectrometric Immunoassay for the qualitative and quantitative analysis of the cytokine Macrophage Migration Inhibitory Factor (MIF).

BACKGROUND: The cytokine MIF (Macrophage Migration Inhibitory Factor) has diverse physiological roles and is present at elevated concentrations in numerous disease states. However, its molecular heterogeneity has not been previously investigated in biological samples. Mass Spectrometric Immunoassay (MSIA) may help elucidate MIF post-translational modifications existing in vivo and provide additional clarity regarding its relationship to diverse pathologies. RESULTS: In this work, we have developed and validated a fully quantitative MSIA assay for MIF, and used it in the discovery and quantification of different proteoforms of MIF in serum samples, including cysteinylated and glycated MIF. The MSIA assay had a linear range of 1.56-50 ng/mL, and exhibited good precision, linearity, and recovery characteristics. The new assay was applied to a small cohort of human serum samples, and benchmarked against an MIF ELISA assay. CONCLUSIONS: The quantitative MIF MSIA assay provides a sensitive, precise and high throughput method to delineate and quantify MIF proteoforms in biological samples.

Elevated plasma albumin and apolipoprotein A-I oxidation under suboptimal specimen storage conditions.

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC-electrospray ionization-MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at -80 °C, -20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at -20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze-thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress-including acute myocardial infarction and prostate cancer-demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above -30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.

Parallel workflow for high-throughput (>1,000 samples/day) quantitative analysis of human insulin-like growth factor 1 using mass spectrometric immunoassay.

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 µg/L and 5 µg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications.

Multiplexed surrogate analysis of glycotransferase activity in whole biospecimens.

Dysregulated glycotransferase enzymes in cancer cells produce aberrant glycans--some of which can help facilitate metastases. Within a cell, individual glycotransferases promiscuously help to construct dozens of unique glycan structures, making it difficult to comprehensively track their activity in biospecimens--especially where they are absent or inactive. Here, we describe an approach to deconstruct glycans in whole biospecimens then analytically pool together resulting monosaccharide-and-linkage-specific degradation products ("glycan nodes") that directly represent the activities of specific glycotransferases. To implement this concept, a reproducible, relative quantitation-based glycan methylation analysis methodology was developed that simultaneously captures information from N-, O-, and lipid linked glycans and is compatible with whole biofluids and homogenized tissues; in total, over 30 different glycan nodes are detectable per gas chromatography-mass spectrometry (GC-MS) run. Numerous nonliver organ cancers are known to induce the production of abnormally glycosylated serum proteins. Thus, following analytical validation, in blood plasma, the technique was applied to a group of 59 lung cancer patient plasma samples and age/gender/smoking-status-matched non-neoplastic controls from the Lung Cancer in Central and Eastern Europe (CEE) study to gauge the clinical utility of the approach toward the detection of lung cancer. Ten smoking-independent glycan node ratios were found that detect lung cancer with individual receiver operating characteristic (ROC) c-statistics ranging from 0.76 to 0.88. Two glycan nodes provided novel evidence for altered ST6Gal-I and GnT-IV glycotransferase activities in lung cancer patients. In summary, a conceptually novel approach to the analysis of glycans in unfractionated human biospecimens has been developed that, upon clinical validation for specific applications, may provide diagnostic and/or predictive information in glycan-altering diseases.

Building multidimensional biomarker views of type 2 diabetes on the basis of protein microheterogeneity.

BACKGROUND: In 2008, the US Food and Drug Administration (FDA) issued a Guidance for Industry statement formally recognizing (during drug development) the conjoined nature of type 2 diabetes (T2D) and cardiovascular disease (CVD), which has precipitated an urgent need for panels of markers (and means of analysis) that are able to differentiate subtypes of CVD in the context of T2D. Here, we explore the possibility of creating such panels using the working hypothesis that proteins, in addition to carrying time-cumulative marks of hyperglycemia (e.g., protein glycation in the form of Hb A(1c)), may carry analogous information with regard to systemic oxidative stress and aberrant enzymatic signaling related to underlying pathobiologies involved in T2D and/or CVD. METHODS: We used mass spectrometric immunoassay to quantify, in targeted fashion, relative differences in the glycation, oxidation, and truncation of 11 specific proteins. RESULTS: Protein oxidation and truncation (owing to modified enzymatic activity) are able to distinguish between subsets of diabetic patients with or without a history of myocardial infarction and/or congestive heart failure where markers of glycation alone cannot. CONCLUSION: Markers based on protein modifications aligned with the known pathobiologies of T2D represent a reservoir of potential cardiovascular markers that are needed to develop the next generation of antidiabetes medications.

Cysteine sulfenic acid as an intermediate in disulfide bond formation and nonenzymatic protein folding.

As a posttranslational protein modification, cysteine sulfenic acid (Cys-SOH) is well established as an oxidative stress-induced mediator of enzyme function and redox signaling. Data presented herein show that protein Cys-SOH forms spontaneously in air-exposed aqueous solutions of unfolded (disulfide-reduced) protein in the absence of added oxidizing reagents, mediating the oxidative disulfide bond formation process key to in vitro, nonenzymatic protein folding. Molecular oxygen (O(2)) and trace metals [e.g., copper(II)] are shown to be important reagents in the oxidative refolding process. Cys-SOH is also shown to play a role in spontaneous disulfide-based dimerization of peptide molecules containing free cysteine residues. In total, the data presented expose a chemically ubiquitous role for Cys-SOH in solutions of free cysteine-containing protein exposed to air.

Possibilities and pitfalls in quantifying the extent of cysteine sulfenic acid modification of specific proteins within complex biofluids.

BACKGROUND: Cysteine sulfenic acid (Cys-SOH) plays important roles in the redox regulation of numerous proteins. As a relatively unstable posttranslational protein modification it is difficult to quantify the degree to which any particular protein is modified by Cys-SOH within a complex biological environment. The goal of these studies was to move a step beyond detection and into the relative quantification of Cys-SOH within specific proteins found in a complex biological setting--namely, human plasma. RESULTS: This report describes the possibilities and limitations of performing such analyses based on the use of thionitrobenzoic acid and dimedone-based probes which are commonly employed to trap Cys-SOH. Results obtained by electrospray ionization-based mass spectrometric immunoassay reveal the optimal type of probe for such analyses as well as the reproducible relative quantification of Cys-SOH within albumin and transthyretin extracted from human plasma--the latter as a protein previously unknown to be modified by Cys-SOH. CONCLUSIONS: The relative quantification of Cys-SOH within specific proteins in a complex biological setting can be accomplished, but several analytical precautions related to trapping, detecting, and quantifying Cys-SOH must be taken into account prior to pursuing its study in such matrices.

Glycosylation status of vitamin D binding protein in cancer patients.

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

Elucidation of two major aggregation pathways in an IgG2 antibody.

Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 37 degrees C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH - 6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species (approximately 26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH - 5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH - 4.0 and 6.0. The dimer species formed at pH - 5.0 had a larger contribution from covalent species-both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH - 6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH - 4.0 have altered secondary and tertiary structure.

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

Automated tryptic digestion procedure for HPLC/MS/MS peptide mapping of immunoglobulin gamma antibodies in pharmaceutics.

The rapid growth of antibody drugs and drug candidates in the biopharmaceutical industry has created a demand for automated proteolytic digestion to assist in pharmaceutical stability studies, identity assays and quality control of these therapeutic proteins. Here, we describe the development of a fully automated proteolytic digestion procedure for monoclonal antibodies in solution, which requires a high concentration of denaturants for unfolding. The antibody samples were placed in a 96-well plate or in 0.5-mL Eppendorf tubes. The proteins were then reduced and alkylated in a denaturing solution of 6M guanidine HCl. The denaturing solution was replaced with a digestion buffer using a custom-designed 96-well size-exclusion plate for desalting. The sample was digested for 5 h with two additions of trypsin. The completeness and reproducibility of digestion were verified by reversed-phase high-performance liquid chromatography tandem mass spectrometry (HPLC/MS) analysis of the digestion products. The performance of the automatic digestion was comparable to the currently used manual digestion procedure, but saved time, reduced manual labor, and increased the reproducibility of the tryptic digests. Our method should be useful not only for high-throughput analysis of antibodies, but for other therapeutic protein samples as well. Other applications like gel-free proteomics, where the analysis of a large number of samples is often needed and the completeness of the liquid digestion is critical for the identification of a large number of different proteins, should also benefit from this fully automated liquid proteolytic digestion procedure.

Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity.

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain.

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody.

Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies.

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability.

An enhanced analytical RP-HPLC/MS method was developed for monitoring the stability and production of intact and fragmented monoclonal antibodies (MAbs). The use of high column temperatures (70-80 degrees C), organic solvents with high eluotropic strength coefficients (isopropyl and n-propyl alcohols), and Zorbax StableBond columns, were critical for good recovery and resolution of immunoglobulin G1 (IgG1) and IgG2 monoclonal antibodies. Using this method, cleavage products of a degraded IgG1 antibody were clearly separated and identified by in-line electrospray ionization time-of-flight (ESI-TOF) mass spectrometry generating exact masses and unique terminal ladder sequences. The glycosylation profile, including mapping of the terminal galactose and fucose heterogeneity of the N-linked sugars, was determined by mass spectrometry of intact MAbs. In addition, we discovered that several IgG2 MAbs exhibited greater structural heterogeneity compared to IgG1s. Mass spectral characterization data and reduction data suggested that the heterogeneity is disulfide related. This reversed-phase LC/MS method represents a key advancement in monitoring intact MAb production and stability.