Marton Szigeti

Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns

N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.

Fully Automated Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Therapeutics

There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead–based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum–centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead–based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead–based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences.

Triple-Internal Standard Based Glycan Structural Assignment Method for Capillary Electrophoresis Analysis of Carbohydrates

Despite the ever growing use of capillary electrophoresis in biomedical research and the biopharmaceutical industry, the development of data interpretation methods is lagging behind. In this paper we report the design and implementation of a coinjected triple-internal standard method to alleviate the need of an accompanying run of the maltooligosaccharide ladder for glucose unit (GU) calculation. Based on the migration times of the coinjected standards of maltose, maltotriose, and maltopentadecaose (bracketing the peaks of interest), a data processing approach was designed and developed to set up a virtual ladder that was used for GU calculation. The data processing was tested in terms of the calculated GU values of human IgG glycans, and the resulting relative standard deviation was ≤1.07%. This approach readily supports high-throughput capillary electrophoresis systems by significantly speeding up the processing time for glycan structural assignment.

High-Resolution Glycan Analysis by Temperature Gradient Capillary Electrophoresis

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 to 45 °C at 5 °C temperature intervals. It was found that within this temperature range, the separation selectivity was carbohydrate structure dependent. The resolution between some glycan structures was greater at elevated temperatures, while others separated better at lower temperatures. More interestingly, the temperature of resolution maximum was different for most structures. On the basis of this observation, a temperature gradient was designed and optimized to fully resolve all the glycans in the mixture. Our results demonstrate how temperature is a critical separation parameter that can be utilized for selectivity manipulation in the analytical glycomics field.

Enzymatic removal of N-glycans by PNGase F coated magnetic microparticles.

Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N‐glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high‐throughput, and reproducible methods to analyze protein N‐glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N‐glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme‐coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in‐solution enzyme reactions using standard glycoproteins possessing the major N‐glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in‐solution based method in 10‐min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture: Part 1 Design and feasibility

Design, fabrication, integration, and feasibility test results of a novel microfluidic cell capture device is presented, exploiting the advantages of proton beam writing to make lithographic irradiations under multiple target tilting angles and UV lithography to easily reproduce large area structures. A cell capture device is demonstrated with a unique doubly tilted micropillar array design for cell manipulation in microfluidic applications. Tilting the pillars increased their functional surface, therefore, enhanced fluidic interaction when special bioaffinity coating was used, and improved fluid dynamic behavior regarding cell culture injection. The proposed microstructures were capable to support adequate distribution of body fluids, such as blood, spinal fluid, etc., between the inlet and outlet of the microfluidic sample reservoirs, offering advanced cell capture capability on the functionalized surfaces. The hydrodynamic characteristics of the microfluidic systems were tested with yeast cells (similar size as red blood cells) for efficient capture.

N‐Glycosylation analysis of formalin fixed paraffin embedded samples by capillary electrophoresis

In this study, N‐linked glycans from intact, formalin treated and formalin fixed paraffin embedded (FFPE) standard glycoproteins, human serum and mouse tumor tissue samples were investigated in respect to their susceptibility for formaldehyde treatment mediated changes. FFPE samples were first deparaffinized, followed by solubilization in radioimmunoprecipitation assay buffer and treated with PNGase F for N‐glycan release. The released glycans were labeled with a charged fluorophore and analyzed by capillary electrophoresis with laser induced fluorescent detection. No significant alterations were found in the N‐glycome profile at any of the investigated complexation levels (i.e., glycoprotein, serum and tissue samples) of the study. These results suggest that FFPE samples can be readily used for global N‐glycome analysis holding the promise to find novel carbohydrate biomarkers in prospective and retrospective studies. Exoglycosidase based carbohydrate sequencing was also applied to reveal some basic structural information about the N‐linked carbohydrates of the mouse tumor tissue samples.

GUcal: An integrated application for capillary electrophoresis based glycan analysis

Recent emergence in the use of monoclonal antibody therapeutics and other glycoprotein biopharmaceuticals requires high‐throughput, robust, and automated techniques for their glycosylation analysis. Capillary electrophoresis is one of the high‐performance methods of choice; however, while the necessary instrumentation is well developed, the related bioinformatics tools are lacked behind. In this paper, we introduce an integrated toolset dubbed as GUcal, to automatically calculate the glucose unit (GU) values for all sample components of interest in an electropherogram with a concomitant database search for structural assignment. The database comprises CE GUs and suggested structures of N‐glycans released from human IgG. The app is freely available online (www.lendulet.uni‐pannon.hu/gucal) and readily facilitates CE‐based glycan analysis.

Automated N-Glycosylation Sequencing Of Biopharmaceuticals By Capillary Electrophoresis

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 one, albeit introduced decades ago, still mostly practiced by following tedious and time consuming manual processes. In this paper we introduce an automated carbohydrate sequencing approach using the appropriate exoglycosidase enzymes in conjunction with the utilization of some of the features of a capillary electrophoresis (CE) instrument to speed up the process. The enzymatic reactions were accomplished within the temperature controlled sample storage compartment of a capillary electrophoresis unit and the separation capillary was also utilized for accurate delivery of the exoglycosidase enzymes. CE analysis was conducted after each digestion step obtaining in this way the sequence information of N-glycans in 60 and 128 minutes using the semi- and the fully-automated methods, respectively.

Computational Fluid Dynamics-Based Design of a Microfabricated Cell Capture Device

A microfluidic cell capture device was designed, fabricated, evaluated by numerical simulations and validated experimentally. The cell capture device was designed with a minimal footprint compartment comprising internal micropillars with the goal to obtain a compact, integrated bioanalytical system. The design of the device was accomplished by computational fluid dynamics (CFD) simulations. Various microdevice designs were rapidly prototyped in poly-dimethylsiloxane using conventional soft lithograpy technique applying micropatterned SU-8 epoxy based negative photoresist as moulding replica. The numerically modeled flow characteristics of the cell capture device were experimentally validated by tracing and microscopic recording the flow trajectories using yeast cells. Finally, we give some perspectives on how CFD modeling can be used in the early stage of microfluidics-based cell capture device development.