Andras Guttman

University of Pannonia, Veszprém · Research Institute of Chemical and Process Engineering

SDS capillary gel electrophoresis is a widely used in the biopharma and the biomedical fields for rapid size separation of proteins. However, very limited information is available on the use of dilute and ultra-dilute sieving matrices for SDS-protein analysis. Here, background electrolytes (BGEs) containing 1%-0% dextran were used in borate-based B...

A simple and widely applicable coaxial sheath flow reactor interface (CSFRI) is introduced for easy and robust connection of liquid-phase microseparation methods to mass spectrometric detection, especially for capillary gel electrophoresis analysis of proteins and peptides including SDS-protein complexes. The interface readily accommodated post-col...

This review summarizes the fundamental principles, basic methodologies, strength and weaknesses of capillary gel electrophoresis of proteins by providing both a short historical overview and highlighting new developments and applications in biopharmaceutical, biomedical as well as food and agriculture fields. The subsets of the method including nat...

In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Singl...

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure–function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly i...

The role of nitrite (NO2−) and nitrate (NO3−) is essential in the global nitrogen cycle. Monitoring their concentration in environmental and industrial aqueous samples, surface water, soil, food and agricultural products are of high importance. Especially, the effect of anthropogenic emission, i.e., intensified agriculture is essential due to the o...

Characterization of glycans, glycoproteins, and their alterations that have been associated with various diseases has proven to be a promising approach in biomarker discovery. Glycosylation is one of the most common posttranslational modifications of proteins and has high relevance in biological processes. The use of capillary electrophoresis (CE)...

The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340-640 mM) caused electromigration dispersion shifts for...

Full characterization of the attached carbohydrate moieties of glycoproteins is of high importance for both the rapidly growing biopharmaceutical industry and the biomedical field. In this paper we report the design and production of three important 6HIS tagged exoglycosidases (neuraminidase, β-galactosidase and hexosaminidase) to support rapid sol...

N-glycosylation of therapeutic antibodies starts as a co translational step followed by a set of post-translational modifications and is considered as one of the critical quality attributes because of its impact on biological functions as well as therapy outcome. In addition to detailed product characterization of these glycans by the manufacturers...

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...

Capillary sodium dodecyl sulfate gel electrophoresis has long been used for the analysis of proteins, mostly either with entangled polymer networks or translationally cross-linked gels. In this paper capillary agarose gel electrophoresis is introduced for the separation of low molecular weight immunoglobulin subunits. The light (LC~24 kDa) and heav...

This chapter discusses the instrumentation details including sample introduction methods, detection systems, and the effects of operation variables. In addition to the regularly used electrokinetic and hydrodynamic injection methods, sample stacking techniques and injection-related artifacts are also conferred. The most often used capillary electro...

This chapter is devoted to the basic principles of capillary gel electrophoresis with the corresponding theoretical treatments on the fundamentals of the electromigration process of biopolymers. Efficiency, selectivity, and resolution terms are all thoroughly conferred. Band broadening in capillary gel electrophoresis is an important issue influenc...

The application chapter gives numerous examples of the use of capillary gel electrophoresis (CGE) of nucleic acids, proteins, and complex carbohydrates also having a capillary affinity gel electrophoresis section. Separation examples of single- and double-stranded DNA molecules include DNA sequencing applications and restriction fragment analysis,...

This chapter provides a deep dive into separation matrix and capillary column technology. First, gels and polymer network systems are discussed, paying close attention to the structures and properties of cross-linked (chemical) gels as well as transitionally cross-linked sieving matrices. Another important approach in capillary gel electrophoresis...

Capillary electrophoresis-related microseparation techniques include ultrathin-layer gel electrophoresis and microchip gel electrophoresis. Since this latter can be a subject of another book, in this chapter we only discuss the former one. Analysis of DNA molecules was accomplished by ultrathin-layer gel electrophoresis in early sequencing applicat...

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 adminis...

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis...

We present in this study a novel strategy to drastically improve the detection sensitivity and peak capacity for capillary electrophoresis with laser induced fluorescent detection (CE-LIF) of glucose oligomers and released glycans. This is based on a new approach exploiting a polymer-free background electrolyte (BGE) for CE-LIF of glycans. The best...

Glycomics has a growing interest in the biopharmaceutical industry and biomedical research requiring new high-performance and high-sensitivity bioanalytical tools. Analysis of N-glycosylation is very important during the development of protein therapeutics and it also plays a key role in biomarker discovery. The most frequently used glycoanalytical...

Background: It is well known that more than 90% of cancer deaths are due to metastases. However, the entire tumorigenesis process is not fully understood, and it is evident that cells spreading from the primary tumor play a key role in initiating the metastatic process. Tumor proliferation and invasion also elevate the concentration of regular and...

One of the most extensively utilized rapid characterization, release and stability testing methods of therapeutic proteins in the biopharmaceutical field today is capillary SDS gel electrophoresis using borate cross-linked high molecular weight dextran. In spite of its widespread use, however, the gel composition dependent separation characteristic...

Capillary electrophoresis (CE) has emerged as a powerful technique for comprehensive physicochemical characterization of monoclonal antibodies (mAbs) as well as other therapeutic modalities. The method provides high resolution separation and high sensitivity characterization for analysis of therapeutic biomolecules. CE based techniques such as sodi...

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we r...

Capillary gel electrophoresis-based methods were applied to comprehensively characterize two development phase new modality monoclonal antibodies including a glycoengineered and a bispecific test compound. The samples were subject to multilevel characterization at the intact (both by SDS-SGE and cIEF) as well as the reduced protein and the released...

There is recently growing interest towards synthesized human milk oligosaccharides (HMOs) as baby formula additives, and interestingly also as dietary supplements for adults. Currently quite a few manufacturers synthesize HMOs, however, their analysis is challenging, both in resolution and speed. In this paper an ultrafast high-resolution method is...

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Ca...

With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently u...

Capillary electrophoresis-based glycan separation and structure identification is one of the most frequently used tools in almost all fields of biological-medical research and biopharmaceutical manufacturing. In this chapter, recent advances in database search assisted glycan structure identification techniques are discussed. The underlying theory...

Capillary Gel Electrophoresis and Related Microseparation Techniques covers all theoretical and practical aspects of capillary gel electrophoresis. It also provides an excellent overview of the key application areas of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microse...

The utilization of N-glycan profiling recently gained high importance both in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In th...

he human serum N-glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the N-glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the N-glycosylation pattern of their serum samp...

The market segment of new biological drugs (monoclonal antibodies, fusion proteins, antibody-drug conjugates and new modality protein therapeutics) is rapidly growing, especially after the patent expiration of the original biologics, initiating the emergence of biosimilars. N-glycosylation of therapeutic proteins has high importance on their stabil...

Human milk is a complex, dynamically changing biological fluid, which contains a large amount of non-conjugated carbohydrates, referred to as human milk oligosaccharides (HMOs). These HMOs are very important for the infants as they play important roles in the formation of the gut microbiome, the immune system and support brain development. HMOs sho...

Prostate cancer has the highest malignancy rate diagnosed in men worldwide. Albeit, the gold standard serum prostate-specific antigen (PSA) assays reduced the mortality rate of the disease, the number of false positive diagnoses steeply increased. Therefore, there is an urgent need for complementary biomarkers to enhance the specificity and selecti...

Determination of complex type free, non-conjugated oligosaccharide glucose unit values in tomato xylem sap for early detection of nutrient deficiency Although knowledge on glycan biosynthesis and processing is continuously maturing, there are still a limited number of studies that examine biological functions of N-glycan structures in plants, which...

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and les...

A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr⁴⁺ modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ult...

Adeno-associated virus (AAV) is one of the most promising viral gene delivery vectors with long-term gene expression and disease correction, featuring high efficiency and excellent safety in human clinical trials. During the production of AAV vectors, there are several quality control (QC) parameters that should be rigorously monitored to comply wi...

Background: Oral and enteral mucositis due to high-dose cytostatic treatment administered during autologous and allogeneic stem-cell transplantation increases mortality. Salivary secretory immunoglobulin A (sIgA) is a basic pillar of local immunity in the first line of defense. Altered salivary sialoglycoprotein carbohydrates are important in the...

Background Type 2 diabetes (T2DM) and colorectal cancer (CRC) are both known to modulate gene expression patterns in peripheral blood leukocytes (PBLs). Objective As T2DM has been shown to increase the incidence of CRC, we were prompted to check whether diabetes affects mRNA signatures in PBLs isolated from CRC patients. Methods 22 patients were...

Background Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for bulk release of plasmid products; thus it should be analyzed accordingly. M...

Background Immunoglobulin G and A, transferrin, haptoglobin and alpha-1-antitrypsin are representing approximately 85% of the human serum glycoproteome and their N-glycosylation analysis may lead to discover important molecular disease markers. However, due to the labile nature of the sialic acid residues, the desialylated subset of the serum N-gly...

Aims Demonstrating the capabilities of our new capillary electrophoresis – mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. Background Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic in...

Aims To apply capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions. Background Altered N-glycosylation of body fluid glycop...

Background: The protective/inhibitory B subunits of coagulation factor XIII (FXIII-B) is a ~80 kDa glycoprotein containing two N-glycosylation sites. Neither the structure nor the functional role of the glycans on FXIII-B has been explored. Objective: To reveal the glycan structures linked to FXIII-B, to design a method for deglycosylating the n...

Chronic obstructive pulmonary disease (COPD) and lung cancer are two major diseases of the lung with high rate of mortality, mostly among tobacco smokers. The glycosylation patterns of various plasma proteins show significant changes in COPD and subsequent hypoxia, inflammation and lung cancer, providing promising opportunities for screening aberra...

The temperature dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate capillary gel electrophoresis in the interval of 15oC to 60oC using borate cross-linked dextran sieving matrix. Arrhenius plots were generated to calculate the respective activation energy values...

Capillary electrochromatography (CEC) is a powerful hybrid separation technique that combines capillary electrophoresis and capillary chromatography, capable to address the analytical challenges of proteomics and glycomics. The focus of this paper is to review the recent developments in capillary electrochromatography of proteins and carbohydrates....

Absztrakt A krónikus obstruktív tüdőbetegség (COPD) világszerte előkelő helyet foglal el a morbiditási és mortalitási statisztikákban. A COPD megelőzhető és kezelhető betegség, kialakulásáért döntően a dohányzás tehető felelőssé. A prevenció kulcsfontosságú, de korlátozottan kivitelezhető, így a rizikó meghatározása és a korai noninvazív diagnoszti...

Protein glycosylation is the most complex and prevalent post-translation modification in terms of the number of proteins modified and the diversity generated. To understand the functional roles of glycoproteins it is important to gain an insight into the repertoire of oligosaccharides present. The comparison and relative quantitation of glycoforms...

GUcal is a standalone application for automatically calculating the glucose unit (GU) values for separated N-glycan components of interest in an electropherogram and suggests their tentative structures by utilizing an internal database. We have expanded the original database of GUcal by integrating all publicly available capillary electrophoresis d...

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to t...

Glycosylation is considered as one of the crucial critical quality attributes of therapeutic glycoproteins and for their biosimilar counterparts. Carbohydrate moieties of such biopharmaceuticals should be closely monitored during all stages of development and manufacturing and studied accordingly during comparability or similarity exercises. In the...

Background A number of human inflammatory diseases and tumors have been shown to cause alterations in the glycosylation pattern of plasma proteins in a specific manner. This highly variable and versatile post-translational modifications fine-tunes protein functions by influencing sorting, folding, enzyme activity and subcellular localization. Howev...

We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS...

By coupling a sample pretreatment technique of sample clean up and enrichment power with capillary electrophoresis (CE) of high-performance separation, the task of analyzing trace analytes in a complex matrix such as a biological sample can be carried out successfully with ease. This review aims for providing an overview of strategies to couple sam...

Formalin‐fixed, paraffin‐embedded (FFPE) samples are generally used for histology studies, however, they also possess important molecular diagnostics information. While it has been reported that the N‐glycan moieties of glycoproteins are not affected by the formalin fixation, paraffin embedding process, no information is available about the effect...

As glycomics research is gaining momentum in the biopharmaceutical industry, there is an increasing need for reproducible high throughput glycoanalytical methods to monitor and characterize the N-glycosylation of therapeutic glycoproteins. Since the glycosylation pattern of glycobiotherapeutics influences their important biological functions, appro...

Mátyás et al. recently published their paper entitled as “Decision support algorithm for the selection of analytical methods in organic compounds detection for future extraterrestrial exploratory missions”, which we found interesting. However, there are some points of the developed method that need to be refined in order to get a practical tool for...

Laser induced fluorescence detection (LIF) is a powerful tool for the quantitative analysis of fluorescent molecules, widely used in glycan analysis with fluorophore labeled carbohydrates where each species has a common response factor. Electrospray ionization mass spectrometry (ESI-MS), on the other hand, while revealing important structural infor...

Lung adenocarcinoma is one of the leading causes of mortality among cancer patients worldwide and chronic obstructive pulmonary disease (COPD) is also high in death statistics. In addition, patients with chronic obstructive pulmonary disease (COPD) have high risk for developing primary lung cancer. Prevention, risk estimation and a non-invasive dia...

Multiple myeloma (MM) is characterized as the clonal proliferation of malignant plasma B-lymphocytes and even as of today, it is an incurable disease. MM accounts for approximately 10% of all hematologic cancers. Its molecular pathogenesis is poorly understood, but the bone marrow microenvironment of tumor cells and genetic factors have apparent ro...

By reading the commentary of Bevelacqua and Mortazavi regarding our recently published paper titled as "The effect of simulated space radiation on the N-glycosylation of human immunoglobulin G1"[1], we are afraid that some of the important messaging aspects of our paper might not have been articulated adequately to be fully understandable for a wid...

More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its p...

The attached carbohydrates at the highly conserved asparagine-linked glycosylation site in the CH 2 domain of the fragment crystallizable (Fc) region of monoclonal antibody therapeutics can play an essential role in their mechanism of action, including ADCC, CDC, anti-inflammatory functions, and serum half-life. Thus, this particular glycosylation...

As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol...

On a roundtrip to Mars, astronauts are expectedly exposed to an approximate amount of radiation that exceeds the lifetime limits on Earth. This elevated radiation dose is mainly due to Galactic Cosmic Rays (GCR) and Solar Particle Events (SPE). Specific patterns of the N‐glycosylation of human immunoglobulins have already been associated with vario...

Capillary electrophoresis connected to electrospray ionization mass spectrometry is a promising combination to analyze complex biological samples. The use of sheathless electrospray ionization interfaces, such as a porous nanoelectrospray capillary emitter, requires the application of forward flow (either by pressure or electroosmosis) to maintain...

The carbohydrate moieties on the polypeptide chains in most glycoprotein based biotherapeutics and their biosimilars play essential roles in such major mechanisms of actions as antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, anti-inflammatory functions and serum clearance. In addition, alteration in glycosylation m...

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence...

The recent expiration of several protein therapeutics opened the door for biosimilar development. Biosimilars are biologic medical products that are similar but not identical copies of already-authorized protein therapeutics. Critical quality attributes (CQA), such as post-translational modifications of recombinant biotherapeutics, are important fo...

Background: Drotaverine, a type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitor, blocks the degradation of 3',5'-cyclic adenosine monophosphate. However, published receptor binding data showed that drotaverin also binds to the L-type voltage-operated calcium channel (L-VOCC). Based on these molecular mechanisms of action, a direct and indir...

The effect of air plasma exposure time on the surface energies and acid-base characteristics of polydimethylsiloxane (PDMS) particles was studied. Polymerized PDMS powder was radio frequency induced air plasma irradiated for 2–10 s with the power of 500 W. The efficiency of the plasma treatments was investigated by a new generation inverse gas chro...

Continuous-flow processing in the manufacturing of modern biotherapeutics represents a great potential and could significantly improve productivity and product quality as well as reduce operating costs. Microfluidic perfusion systems are not only capable for producing therapeutic proteins but also suitable for organ-on-a-chip based drug testing and...

N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than...

We present the application of a smartphone anatomy based technology in the field of liquid phase bioseparations, particularly in capillary electrophoresis. A simple capillary electrophoresis system was built with LED induced fluorescence detection and a credit card sized minicomputer to prove the concept of real time fluorescent imaging (zone adjus...

Comprehensive analysis of the N-linked carbohydrates of glycoproteins is gaining high recent interest in both the biopharmaceutical and biomedical fields. In addition to high resolution glycosylation profiling, sugar residue and linkage specific enzymes are also routinely used for exoglycosidase digestion based carbohydrate sequencing. This latter...

Significance: Biopharmaceuticals have seen something of tremendous development in recent years, which governs the parallel blooming of analytical glycomics. One of the frequently used methods for the analysis of complex carbohydrates is capillary electrophoresis with laser induced fluorescent detection (CE-LIF). CE-LIF is a high resolution separat...

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer un...

One of the most frequently used high-resolution glycan analysis methods in the biopharmaceutical and biomedical fields is capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Glycans are usually labeled by reductive amination with a charged fluorophore containing a primary amine, which reacts with the aldehyde group at the...

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 solubilizatio...

Capillary coatings effectively improve the separation performance of proteins in capillary electrophoresis, mainly by reducing protein adsorption onto the inner capillary wall and by regulating the electroosmotic flow (EOF) to accommodate the separation problem in hand. In the first part of this review the newest trends in dynamic and permanent cap...

Temperature gradient capillary electrophoresis was introduced to enhance separation selectivities for branched glycans of biotherapeutic interest. A mixture of afucosylated, fucosylated and high mannose oligosaccharides was separated in the range of 15°C to 45°C at 5°C temperature intervals. It was found that within this temperature range the separ...

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 Fc and Fab κ/λ light chain fractions of pa...