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The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Current proteomic technologies focus on the quantification of protein levels, while little effort is dedicated to the development of system approaches to simultaneously monitor proteome variability and abundance. Protein variants may display different immunogenic epitopes detectable by monoclonal antibodies. Epitope variability results from alternative splicing, posttranslational modifications, processing, degradation, and complex formation and possesses dynamically changing availability of interacting surface structures that frequently serve as reachable epitopes and often carry different functions. Thus, it is highly likely that the presence of some of the accessible epitopes correlates with function under physiological and pathological conditions. To enable the exploration of the impact of protein variation on the immunogenic epitome first, here, we present a robust and analytically validated PEP technology for characterizing immunogenic epitopes of the plasma. To this end, we prepared mAb libraries directed against the normalized human plasma proteome as a complex natural immunogen. Antibody producing hybridomas were selected and cloned. Monoclonal antibodies react with single epitopes, thus profiling with the libraries is expected to profile many epitopes which we define by the mimotopes, as we present here. Screening blood plasma samples from control subjects (n = 558) and cancer patients (n = 598) for merely 69 native epitopes displayed by 20 abundant plasma proteins resulted in distinct cancer-specific epitope panels that showed high accuracy (AUC 0.826-0.966) and specificity for lung, breast, and colon cancer. Deeper profiling (≈290 epitopes of approximately 100 proteins) showed unexpected granularity of the epitope-level expression data and detected neutral and lung cancer-associated epitopes of individual proteins. Biomarker epitope panels selected from a pool of 21 epitopes of 12 proteins were validated in independent clinical cohorts. The results demonstrate the value of PEP as a rich and thus far unexplored source of protein biomarkers with diagnostic potential., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5-10% hospitalization and 2-3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin - converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N- linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1 ) size heterogeneity analysis to determine the presence/absence of the non - glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2 ) capillary gel isoelectric focusing for possible N -glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3 ) capillary gel electrophoresis for N- glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N -glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N- linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Szabo, Sarkozy, Szigeti, Farsang, Kardos, Kozma, Csanky, Chung, Szekanecz and Guttman.)

Omalizumab, a glycoprotein based biotherapeutics, is one of the most frequently used targeted antibody biopharmaceutical to reduce asthma exacerbations, improve lung function and reduce oral corticosteroid use. The effector function and clearance time of such glycoprotein drugs is affected by their N-glycosylation, that defines the required administration frequency to improve the quality of life in appropriately selected patients. Therefore, the glycosylation of biologics is an important critical quality attribute (CQA). The profile of asparagine linked carbohydrates is greatly dependent on the manufacturing process. Even a small deviation may have a major effect on the structure and therefore the function of the biotherapeutic product. For this reason, comprehensive N-glycosylation analysis is of high importance during production and release. Capillary electrophoresis (CE) is one of the frequently used tools to characterize protein therapeutics and utilized by the biopharmaceutical industry for protein and glycan level analysis, which are key parts both for drug development and quality control. To reveal important structure - function relationships, characterization of omalizumab is presented using capillary SDS gel electrophoresis with UV detection at the protein level and capillary gel electrophoresis with laser induced fluorescent detection at the N-linked carbohydrate level. This latter technique was also used for oligosaccharide sequencing for glycan structure validation. The results suggested no ADCC function - structure relationship due to the mostly core fucosylated biantennary glycans found. However, the presence of the high mannose structures probably affects the clearance rate of the drug., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Multiple myeloma (MM) is an immedicable malignancy of the human plasma cells producing abnormal antibodies (also referred to as paraproteins) leading to kidney problems and hyperviscosity syndrome. In this paper, we report on the N-glycosylation analysis of paraproteins from total human serum as well as the fragment crystallizable region (F c ) and fragment antigen binding (F ab ) κ/λ light chain fractions of papain digested immunoglobulins from multiple myeloma patients. CE-LIF detection was used for the analysis of the N-glycans after endoglycosidase (PNGase F) mediated sugar release and fluorophore labeling (APTS). While characteristic N-glycosylation pattern differences were found between normal control and untreated, treated and remission stage multiple myeloma patient samples at the global serum level, less distinctive changes were observed at the immunoglobulin level. Principal component analysis adequately differentiated the four groups (control and three patient groups) on the basis of total serum N-glycosylation analysis. 12 N-glycan features showed statistically significant differences (p <0.05) among various stages of the disease in comparison to the control at the serum level, while only six features were identified with similar significance at the immunoglobulin level, including the analysis of the partitioned F c fragment as well as the F ab κ and F ab λ chains., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Comprehensive carbohydrate analysis of glycoproteins from human biological samples and biotherapeutics are important from diagnostic and therapeutic points of view. This review summarizes the current state-of-the-art liquid phase separation techniques used in N-glycosylation analysis. The different liquid chromatographic techniques and capillary electrophoresis methods are critically discussed in detail. Miniaturization of these methods is also important to increase throughput and decrease analysis time. The sample preparation and labeling methods for asparagine linked oligosaccharides are also addressed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by progressive airflow limitation and significant extrapulmonary (systemic) effects that lead to co-morbid conditions, though the pathomechanism of COPD is largely undetermined. Alveolar macrophages (AM) derived from peripheral monocytes (MO) appear to play a key role in initiating and/or sustaining disease progression., Objectives: To identify disease- and cell type-specific gene expression profiles and potential overlaps in those in order to diagnose COPD, characterize its progression and determine the effect of drug treatment., Method: Global gene expression analysis was used for primary screening in order to obtain expression signatures of AMs and circulating MOs of COPD patients and healthy controls. The results of microarray analyses of AMs (20 controls and 26 COPD patients) and MOs (16 controls and 22 COPD patients) were confirmed and validated by real-time quantitative polymerase chain reaction., Results: We have identified gene sets specifically associated with COPD in AMs and MOs. There were overlapping genes between the two cell types. Our data also show that COPD-specific gene expression signatures in AMs and MOs correlate with percent of predicted FEV(1)., Conclusion: Disease-specific and overlapping gene expression signatures can be defined in lung-derived macrophages and also in circulating monocytes. Some of the validated expression changes in both cell types correlate with lung function and therefore could serve as biomarkers of disease progression., (Copyright © 2011 S. Karger AG, Basel.)

Background: In addition to smoking, genetic predisposition is believed to play a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Genetic association studies of new candidate genes in COPD may lead to improved understanding of the pathogenesis of the disease., Methods: Two proposed casual single nucleotide polymorphisms (SNP) (rs1051740, rs2234922) in microsomal epoxide hydrolase (EPHX1) and three SNPs (rs1801282, rs1800571, rs3856806) in peroxisome proliferator-activated receptor gamma (PPARG), a new candidate gene, were genotyped in a case-control study (272 COPD patients and 301 controls subjects) in Hungary. Allele frequencies and genotype distributions were compared between the two cohorts and trend test was also used to evaluate association between SNPs and COPD. To estimate the strength of association, odds ratios (OR) (with 95% CI) were calculated and potential confounding variables were tested in logistic regression analysis. Association between haplotypes and COPD outcome was also assessed., Results: The distribution of imputed EPHX1 phenotypes was significantly different between the COPD and the control group (P = 0.041), OR for the slow activity phenotype was 1.639 (95% CI = 1.08- 2.49; P = 0.021) in our study. In logistic regression analysis adjusted for both variants, also age and pack-year, the rare allele of His447His of PPARG showed significant association with COPD outcome (OR = 1.853, 95% CI = 1.09-3.14, P = 0.0218). In haplotype analysis the GC haplotype of PPARG (OR = 0.512, 95% CI = 0.27-0.96, P = 0.035) conferred reduced risk for COPD., Conclusions: The "slow" activity-associated genotypes of EPHX1 were associated with increased risk of COPD. The minor His447His allele of PPARG significantly increased; and the haplotype containing the minor Pro12Ala and the major His447His polymorphisms of PPARG decreased the risk of COPD.

One-step global profiling of analyte (mRNA, protein, metabolite) biomarkers may soon replace conventional blood and histological/biopsy diagnostics technologies. It is important to establish whether the numerous blood and other body fluid-derived potential novel diagnostics will be sufficiently efficacious and precise to replace, for example, imaging and functional diagnostic tests. Currently, imaging technologies and spirometry are indispensable for the diagnosis and management of chronic obstructive pulmonary disease (COPD). To validate the concept of using body fluid biomarkers in COPD and to address the question of whether biomarker levels correlate with lung function, we measured the level of a number of biologically relevant lipids and metabolites in the bronchoalveolar lavage (BAL) fluid of COPD and control subjects and examined whether these correlate with numeric parameters of lung function. Both the diagnosis and management of COPD rely on costly and labor intensive lung function tests. Thus, there is an imminent need to replace the current diagnostic approaches with simpler clinical assays. As a first step, we demonstrate proof of principle; the correlation of lipid biomarkers as measured by LC-MS with lung function. In the apparently BAL-accessible fluid compartment, the total recovered lipid metabolite amount, particularly prostaglandin D(2) and eicosapentaenoic acid show a remarkable linear correlation with lung function (R(2)>0.7). The study outcome is encouraging for the continuation of the work toward the measurement of lipid metabolite levels in more easily obtainable biological fluids such as sputum, exhaled air condensate, urine and plasma.

We define mAb proteomics as the global generation of disease specific antibodies that permit mass screening of biomarkers. An integrated, high-throughput, disease-specific mAb-based biomarker discovery platform has been developed. The approach readily provided new biomarker leads with the focus on large-scale discovery and production of mAb-based, disease-specific clinical assay candidates. The outcome of the biomarker discovery process was a highly specific and sensitive assay, applicable for testing of clinical validation paradigms, like response to treatment or correlation with other clinical parameters. In contrast to MS-based or systems biology-based strategies, our process produced prevalidated clinical assays as the outcome of the discovery process. By re-engineering the biomarker discovery paradigm, the encouraging results presented in this paper clearly demonstrate the efficiency of the mAb proteomics approach, and set the grounds for the next steps of studies, namely, the hunt for candidate biomarkers that respond to drug treatment.

The TNF region within the MHC includes a number of immunologically important genes. Microsatellites TNFa and TNFb adjacent to TNF exhibit extensive polymorphism. Employing a PCR-based technique, we identified TNFab haplotypes and defined their distribution in 97 controls and 48 diabetics of Caucasoid origin in a search for other genes within the MHC potentially associated with IDDM. Twenty-five different TNFab haplotypes were identified. A significant difference (p < 0.0005) in frequency between patients and controls was found for TNFa1b5 (relative risk 53). However, no other TNFab microsatellites demonstrated significantly different frequencies. Among diabetics TNFa1b5 was found to be in linkage disequilibrium with HLA-DR3-B18, a haplotype known to be associated with IDDM. Thus the increased frequency of TNFa1b5 among diabetics could reflect a linkage disequilibrium with a gene within the TNF region or with other genes, including the HLAs, which characterize this haplotype. In both controls and diabetics TNFa2b3 and TNFa7b4 were in linkage disequilibrium with DR3-B8 and DR7, respectively. Among diabetics, TNFa2b1 and TNFa6b5 were in linkage disequilibrium with DR4-B62 and DR4-B44, respectively. It is intriguing that TNFab haplotypes, represented by a short piece of about 200 nucleotides in the untranslated region upstream of TNF beta gene, maintain strong linkage disequilibria with different HLA haplotypes extending over 1 million base pairs. The identification of TNFab microsatellites exhibiting a high polymorphic index in a region lacking known polymorphic markers may provide potentially important information regarding the association of HLA haplotypes with autoimmune diseases, as they are in close proximity to other genes of immunologic importance.

The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.

The effect of alkaline pH and alkaline hydrolysis on the physico-chemical and biological properties of endotoxin (ET) isolated from Serratia marcescens ATCC 13477 by the Biovin procedure was studied. Major emphasis was put on the ion exchange column chromatography and immune adjuvant activity (ADA) of the alkali treated samples. To measure changes in some endotoxicity parameters, Limulus lysate clotting (LAL), chick embryo lethality, Shwartzman skin reactivity and in vitro TNF release were measured. The toxic properties of ET, with the unique exception of the Shwartzman skin reactivity, rapidly diminished during alkaline treatment. As immunogen CBre3, a recombinant HIV glycoprotein which spans the C terminus of gp 120 and the N terminus of gp 41, was used in CD-1 mice, alkali treated and immediately neutralized ET samples (zero time) were inactive as adjuvants, in some cases immunosuppression could be clearly seen. But if the alkaline hydrolysis was continued for 6 h, the ADA became higher than it had been for the starting ET sample. Further alkaline hydrolysis eliminated the ADA of the samples. Both NaOH and propylamine acted similarly on the ET preparation. Reaction kinetic studies of the NaOH detoxification indicated the cleavage of ester bound acyl groups with low binding energy. Chemical analyses of the samples revealed that changes occurred in the fatty acid composition, characterized by a loss of approximately half of the 3-OH myristic acid content.