Ákos Szekrényes

High-Throughput Profiling of the Serum N-Glycome on Capillary Electrophoresis Microfluidics Systems: Toward Clinical Implementation of GlycoHepatoTest

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE-202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use.

Chip‐based CE for rapid separation of 8‐aminopyrene‐1,3,6‐trisulfonic acid (APTS) derivatized glycans

Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid – lectin affinity enrichment. After fluorescent labeling with 8‐aminopyrene‐1,3,6‐trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470 nm) LED. The separation was completed in 40 s in a microfluidic channel of 14 mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20‐mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip‐based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35 cm separation length and microfluidic chips with a separation distance of only 14 mm.

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

The IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)-UPLC, reversed phase (RP)-UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC-UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.

Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.

Analysis of haptoglobin N‐glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis

A CE‐based method was introduced to compare the N‐glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease, and lung cancer patients by means of two haptoglobin‐specific monoclonal antibodies. Haptoglobin N‐glycans were then enzymatically released, fluorescently labeled, and profiled by CE. Disease‐associated changes of core and antennary fucosylation were identified by targeted exoglycosidase digestions and their levels were compared in the different patient groups. Terms such as core‐ and arm‐fucosylation degree, as well as branching degree, were introduced for easier characterization of the changes and statistical analysis was used to examine which structures were responsible for the observed differences. Increased level of α1–6 fucosylated tri‐antennary glycans was found in all disease groups compared to the control. Elevated amounts of core‐ and arm‐fucosylation on tetra‐antennary glycans were detected in the lung cancer group compared to the chronic obstructive pulmonary disease group. A larger scale study is necessary to confirm and validate these preliminary findings in the glycosylation changes of haptoglobin, so could then be used as biomarkers in the diagnosis of malignant and inflammatory lung diseases.

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.

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.

High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling

Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.

Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery

mAb proteomics, a reversed biomarker discovery approach, is a novel methodology to recognize the proteins of biomarker potential, but requires subsequent antigen identification steps. While in case of high‐abundant proteins, it generally does not represent a problem, for medium or lower abundant proteins, the identification step requires a large amount of sample to assure the proper amount of antigen for the ID process. In this article, we report on the use of combined chromatographic and precipitation techniques to generate a large set of fractions representing the human plasma proteome, referred to as the Analyte Library, with the goal to use the relevant library fractions for antigen identification in conjunction with mAb proteomics. Starting from 500 mL normal pooled human plasma, this process resulted in 783 fractions with the average protein concentration of 1 mg/mL. First, the serum albumin and immunoglobulins were depleted followed by prefractionation by ammonium sulfate precipitation steps. Each precipitate was then separated by size exclusion chromatography, followed by cation and anion exchange chromatography. The 20 most concentrated ion exchange chromatography fractions were further separated by hydrophobic interaction chromatography. All chromatography and precipitation steps were carefully designed aiming to maintain the native forms of the intact proteins throughout the fractionation process. The separation route of vitamin D‐binding protein (an antibody proteomics lead) was followed in all major fractionation levels by dot blot assay in order to identify the library fraction it accumulated in and the identity of the antigen was verified by Western blot.

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.