Using SLIM-Based IMS-IMS Together with Cryogenic Infrared Spectroscopy for Glycan Analysis.

The isomeric heterogeneity of glycans poses a great challenge for their analysis. While combining ion mobility spectrometry (IMS) with tandem mass spectrometry is a powerful means for identifying and characterizing glycans, it has difficulty distinguishing the subtlest differences between isomers. Cryogenic infrared spectroscopy provides an additional dimension for glycan identification that is extremely sensitive to their structure. Our approach to glycan analysis combines ultrahigh-resolution IMS-IMS using structures for lossless ion manipulation (SLIM) with cryogenic infrared spectroscopy. We present here the design of a SLIM board containing a series of on-board traps in which we perform collision-induced dissociation (CID) at pressures in the millibar range. We characterize the on-board CID process by comparing the fragments generated from a pentapeptide to those obtained on a commercial tandem mass spectrometer. We then apply our new technique to study the mobility and vibrational spectra of CID fragments from two human milk oligosaccharides. Comparison of both the fragment drift times and IR spectra with those of suitable reference compounds allows us to identify their specific isomeric form, including the anomericity of the glycosidic linkage, demonstrating the power of this tool for glycan analysis.

Comparison in bioactivity and characteristics of Ginkgo biloba seed polysaccharides from four extract pathways.

The effects of hot-water extraction (HWE), ultrasound-treated extraction (UTE), enzyme-treated extraction (ETE) and ultrasound-enzyme treated extraction (UETE) on the α-glucosidase inhibitory activity, antioxidant activities and characteristics of Ginkgo biloba seed polysaccharides were investigated and compared in this study. Among the four extracted polysaccharides, the UETE-polysaccharide initially exhibited the highest α-glucosidase inhibitory activity and antioxidant activities. The HWE-polysaccharide showed a large number of small compact spherical structures, and the UTE-polysaccharide exhibited an irregular pleated porous shape; meanwhile, the ETE-polysaccharide and UETE-polysaccharide were spongy with smooth surface topography, as observed by scanning electron microscopy (SEM). The four polysaccharides varied in monosaccharide composition. The HWE-polysaccharide mainly consisted of homogeneous mannose; the UETE-polysaccharide was primarily composed of mannose, rhamnose, and glucose in a molar ratio of 8.25:1.00:1.53. The HWE-polysaccharide had the largest molecular weight (4.2 × 105 Da), reduced by the order of the UETE-polysaccharide (2.02 × 104 Da), ETE-polysaccharide (1.72 × 104 Da), and UTE-polysaccharide (1.34 × 104 Da). Thus, the four extract methods exerted significant effects on the bioactivity and characteristics of the polysaccharides. The UETE-polysaccharide from G. biloba seeds showed the highest bioactive activities and distinctive structural characteristics.

Multiple fingerprint and fingerprint-activity relationship for quality assessment of polysaccharides from Flammulina velutipes.

Polysaccharides are known as one of the most important bioactive compounds in Flammulina velutipes. However, there is no accurate and comprehensive assessment method to evaluate and authenticate F. velutipes polysaccharides (FVPs) from different sources. In this study, a multiple fingerprint analysis method including scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and high-performance liquid chromatography (HPLC) was established. The inhibitory activities of FVPs against HepG2 were measured and introduced into multiple linear regression (MLR) analysis to investigate fingerprint-activity relationship. The principal component analysis (PCA) scores showed that the polysaccharides extracted from 20 batches of different F. velutipes were highly similar, and substandard samples could be distinguished from the authentic polysaccharides clearly. The glucuronic acid could be considered as a marker for discrimination of white and yellow F. velutipes polysaccharides in HPLC fingerprints. Moreover, the HPLC fingerprint-growth inhibitory activity relationship illuminated that monosaccharides composition played an important role on the HepG2 growth inhibitory activity, and activity-associated markers (mannose, rhamnose, xylose, and galactose) were chosen to assess FVPs from different sources. The suggested HPLC fingerprint-activity relationship method provides an integrated strategy for the quality control of F. velutipes and its related products.

Providing Bionic Glycome as internal standards by glycan reducing and isotope labeling for reliable and simple quantitation of N-glycome based on MALDI- MS.

Accurate, simple and economical methods for quantifying N-glycans are continuously required for discovering disease biomarkers and quality control of biopharmaceuticals. Quantitative N-glycomics based on MS using exogenous isotopic labeling internal standards is promising as it is simple and accurate. However, it is largely hampered by the lack of available glycan internal standard libraries with good coverage of the natural glycan structural heterogeneity as well as broad dynamic mass and ion abundance range. To overcome this limitation, we developed a novel method, providing 'Bionic Glycome' as internal standards for glycan quantitation by MALDI-MS. Bionic Glycome was produced using N-glycome from pooled samples to be analyzed as substrate by one step of glycan reducing and isotope labeling (Glycan-RAIL). Each bionic glycan has 3 Da mass increment over its corresponding glycan analyte based on hemiacetals/alditols and H/D mass difference. In addition, Bionic Glycome has the same glycome composition and similar glycome profile in abundance with N-glycome to be analyzed from biological sample. Through the investigation of single glycan standard and complex glycans released from model glycoprotein and serum, the results demonstrate that the method has good quantitative accuracy and high reproducibility. Lastly, this method was successfully used for discovery of lung cancer specific glycan markers by comparing the serum glycans from each sample in lung cancer group (n = 16) and healthy controls (n = 16), indicating its potential in clinical applications.

Effects of Ultrasound Treatment on Extraction and Rheological Properties of Polysaccharides from Auricularia Cornea var. Li.

Auricularia cornea var. Li. is an edible fungi and polysaccharides in Auricularia cornea var. Li. may have bioactive activities. Polysaccharides from Auricularia cornea var. Li. (ACP) was extracted using ultrasound-assisted extraction (UAE) method and compared with hot water extraction (HWE) for extraction yield, extraction rate, purity of polysaccharides, microstructure of residues after extraction, preliminary structure and rheological properties of polysaccharides. Optimum conditions for UAE (particle size of 150⁻200 mesh, water to raw material ratio of 70:1, extraction temperature at 70 °C for 40 min, ultrasonic amplitude of 40%) and HWE (particle size of 150⁻200 mesh, water to raw material ratio of 60:1, extraction temperature at 90 °C for 3.0 h) were obtained via single-factor experiment. Under optimum conditions, extraction yield of polysaccharides by UAE was 30.99 ± 1.93% which showed no significant difference with that by HWE (30.35 ± 1.67%) (P > 0.05). Extraction rate (29.29 ± 1.41%) and purity (88.62 ± 2.80%) of polysaccharides by UAE were higher than those by HWE (extraction rate of 24.95 ± 2.78% and purity of 75.33 ± 6.15%) (P < 0.05). Scanning Electron Microscopy (SEM) images of residues by UAE showed more broken cells than those by HWE. Fourier Transform Infrared (FTIR) spectra showed that the dialyzed ACP extracted by HWE and UAE (DACP-HWE and DACP-UAE) had similar characteristic absorption peaks of polysaccharides. Both DACP-HWE and DACP-UAE solutions showed typical shear thinning and temperature-independent behaviors (25⁻90 °C) and UAE resulted in polysaccharides with remarkably lower viscosity in comparison with HWE. DACP-UAE solutions exhibited more liquid-like state while DACP-HWE solutions solid-like system. Data indicated that ultrasound treatment may be a useful means for extraction of polysaccharides from Auricularia cornea var. Li.

Collaborative study on saccharide quantification of the Haemophilus influenzae type b component in liquid vaccine presentations.

Before release onto the market, it must be demonstrated that the total and free polysaccharide (poly ribosyl-ribitol-phosphate, PRP) content of Haemophilus influenzae type b (Hib) vaccine complies with requirements. However, manufacturers use different methods to assay PRP content: a national control laboratory must establish and validate the relevant manufacturer methodology before using it to determine PRP content. An international study was organised by the World Health Organization (WHO), in collaboration with the Biological Standardisation Programme (BSP) of the Council of Europe/European Directorate for the Quality of Medicines & HealthCare (EDQM) and of the European Union Commission, to verify the suitability of a single method for determining PRP content in liquid pentavalent vaccines (DTwP-HepB-Hib) containing a whole-cell pertussis component. It consists of HCl hydrolysis followed by chromatographic separation and quantification of ribitol on a CarboPac MA1 column using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The unconjugated, free, PRP is separated from the total PRP using C4 solid-phase extraction cartridges (SPE C4). Ten quality control laboratories performed two independent analyses applying the proposed analytical test protocol to five vaccine samples, including a vaccine lot with sub-potent PRP content and very high free PRP content. Both WHO PRP standard and ribitol reference standard were included as calibrating standards. A significant bias between WHO PRP standard and ribitol reference standard was observed. Study results showed that the proposed analytical method is, in principle, suitable for the intended use provided that a validation is performed as usually expected from quality control laboratories.

Prospective randomized study evaluating the effects of PerClot® (Polysaccharide Hemostatic System) application in patients with high bleeding risk undergoing cardiac rhythm device implantation.

BACKGROUND: Thus far, the topic hemostatic agent PerClot® is used for surgical procedures. Data about the use of PerClot® for cardiac-rhythm-devices (CRD) implantation are missing. The aim of this study was to evaluate the safety and efficacy of PerClot® in patients with high bleeding risk. METHODS AND RESULTS: In this prospective randomized study we planned to include 150 patients admitted for CRD-Implantation receiving anticoagulation and/or dual-antiplatelet-therapy. Participants were randomized to receive PerClot® versus standard-of-care. The primary endpoint was the incidence of pocket hematoma. Safety endpoint was pocket infection. After a planned safety-interim-analysis the study was terminated early because of safety concerns. 51 patients were included. The two groups were comparable with regard to age (73±11years vs. 74±10years; p=0.71), CHA2DS2VASc (3.6±1.5 vs. 4.0±1.5; p=0.27) and HASBLED-Score (2.4±1.1 vs. 2.5±1.0; p=0.98), CRD or procedure type, anticoagulant or anti-platelet therapy. The use of PerClot® resulted in a higher incidence of postoperative fever (7 (28%) vs. 0 (0%); p=0.004), higher C-Reactive Protein (66.1±50.5mg/l vs. 25.9±22.5mg/l; p=0.002); and higher postoperative white blood cell count (13.5±4.3/nl vs. 8.8±2.6/nl; p<0.001). Hematoma formation did not differ significantly (p=0.14). Reoperation was not necessary in any patient. CONCLUSION: This first randomized controlled study for the topical use of the hemostatic agent PerClot® in CRD implantation was terminated early by the safety monitoring board because of an augmented rate of fever and inflammatory markers in the PerClot® group. The addition of PerClot® does not suggest a benefit with regard to the frequency of pocket hematoma.

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

International collaborative study for establishment of the 2nd WHO International Standard for Haemophilus influenzae type b polysaccharide.

In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â†’ 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.

Quality control of sweet medicines based on gas chromatography-mass spectrometry.

Sweet medicines are a relatively untapped source of new drugs. Their biological activities are closely correlated to their chemical characteristics. However, accurately defining the chemical characteristics of glycans is a challenge due to their chemical heterogeneity and diversity. Gas chromatography-mass spectrometry (GC-MS) is an excellent technique for the analysis of glycans even though the preparation of adequate derivatives is necessary. We reviewed and discussed the most important methodologies currently used for glycan analysis in sweet medicines based on GC-MS, including the derivatization for monosaccharide analysis, hydrolysis methods for polysaccharide analysis, glycosidic linkage analysis based on methylation, and pyrolysis gas chromatography in carbohydrate analysis. Finally a strategy for quality control of sweet medicines based on quantification analysis is proposed.

Simple and rapid quality control of sulfated glycans by a fluorescence sensor assay--exemplarily developed for the sulfated polysaccharides from red algae Delesseria sanguinea.

Sulfated polysaccharides (SP) from algae are of great interest due to their manifold biological activities. Obstacles to commercial (especially medical) application include considerable variability and complex chemical composition making the analysis and the quality control challenging. The aim of this study was to evaluate a simple microplate assay for screening the quality of SP. It is based on the fluorescence intensity (FI) increase of the sensor molecule Polymer-H by SP and was originally developed for direct quantification of SP. Exemplarily, 65 SP batches isolated from the red alga Delesseria sanguinea (D.s.-SP) and several other algae polysaccharides were investigated. Their FI increase in the Polymer-H assay was compared with other analytical parameters. By testing just one concentration of a D.s.-SP sample, quality deviations from the reference D.s.-SP and thus both batch-to-batch variability and stability can be detected. Further, structurally distinct SP showed to differ in their concentration-dependent FI profiles. By using corresponding reference compounds, the Polymer-H assay is therefore applicable as identification assay with high negative predictability. In conclusion, the Polymer-H assay showed to represent not only a simple method for quantification, but also for characterization identification and differentiation of SP of marine origin.

Analytical platform for glycomic characterization of recombinant erythropoietin biotherapeutics and biosimilars by MS.

BACKGROUND: Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell production. The quality, safety and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell culture conditions can significantly affect the glycosylation, and therefore the efficacy, of recombinant erythropoietins. Thus, detailed glycomic analyses are necessary to assess biotherapeutic quality. We have developed a platform for qualitative and quantitative glycomic analysis of recombinant erythropoietins. RESULTS: The platform was used to profile native N-glycans from three production batches of darbepoetin alfa (also known as NESP), a common form of recombinant erythropoietin. Darbepoetin alfa was found to contain an abundance of large, multi-antennary N-glycans with high levels of sialylation, O-acetylation and dehydration. Results were verified by independent orthogonal analysis with both MALDI-TOF and nano-LC/Q-TOF MS. CONCLUSION: This platform may be applied to QC and batch analysis of not only recombinant erythropoietin, but also other complex, glycosylated biotherapeutics and biosimilars.

Industry and regulatory experience of the glycosylation of monoclonal antibodies.

We surveyed 23 antibody-related marketing applications for glycoform analytical and functional information. Our database analysis shows a clear trend of increasing sophistication of analytical methods used to identify and quantify glycans. These have revealed a high degree of complexity and heterogeneity of glycans attached to antibody products. The nature of the complexity is influenced by product type and expression system, and may be associated with functional consequences in some but not all cases.

The effects of abundant plasma protein depletion on global glycan profiling using nanoLC FT-ICR mass spectrometry.

We report the results of abundant plasma protein depletion on the analysis of underivatized N-linked glycans derived from plasma proteins by nanoLC Fourier-transform ion cyclotron resonance mass spectrometry. N-linked glycan profiles were compared between plasma samples where the six most abundant plasma proteins were depleted (n = 3) through a solid-phase immunoaffinity column and undepleted plasma samples (n = 3). Three exogenous glycan standards were spiked into all samples which allowed for normalization of the N-glycan abundances. The abundances of 20 glycans varying in type, structure, composition, and molecular weight (1,200-3,700 Da) were compared between the two sets of samples. Small fucosylated non-sialylated complex glycans were found to decrease in abundance in the depleted samples (greater than or equal to tenfold) relative to the undepleted samples. Protein depletion was found to marginally effect (less than threefold) the abundance of high mannose, hybrid, and large highly sialylated complex species. The significance of these findings in terms of future biomarker discovery experiments via global glycan profiling is discussed.

[Study on quality evaluation of Radix Isatis based on analysis of biothermic activity].

OBJECTIVE: To establish a novel pattern and method to evaluate the quality of Radix Isatis based on analysis of biothermic activity. METHODS: Chemical and biothermodynamic methods were used and compared to investigate the quality of different Radix Isatis samples. And a mathematic model was established by computer aided pattern recognition to evaluate the quality of Radix Isatis. RESULTS: The quality of Radix Isatis was partially related to the content of organic acids and polycose, but it could not be correctly recognized by both chemical determination and HPLC fingerprint. On the other hand, the mathematic model based on the main four parameters of biothermodynamic analysis was very correct (misjudgment ratio of 1.39%) to recognize the quality of Radix Isatis. CONCLUSION: The established model in this paper based on analysis of biothermic activity is more accurate and reliable than that of chemical methods to evaluate the quality of Radix Isatis.

Determination of total dietary fiber in selected foods containing resistant maltodextrin by a simplified enzymatic-gravimetric method and liquid chromatography: interlaboratory study in China.

An interlaboratory study was conducted in China to validate the modified AOAC Official Method 2001.03 for the determination of total dietary fiber (TDF) in foods containing resistant maltodextrin (RMD), which will be adopted as the National Standard Method of China. The kind of buffer solution, the volume of filtrate evaporation, the volume of eluent for desalting and residual solution after evaporation, etc. were modified, which had been proved to have acceptable accuracy and precision in the routine assay. TDF contents in 3 representative foods and 2 kinds of RMD ingredient (i.e., NUTRIOSE 06 and NUTRIOSE 10) were measured using the modified method in 6 eligible laboratories representing commercial, industrial, and governmental laboratories in China. The results of the interlaboratory study indicated that the intralaboratory repeatability, interlaboratory reproducibility, and precision of the modified method are adequate for reliable analysis of TDF in food containing RMD, as well as resistant dextrin. Compared to AOAC Official Method 2001.03, the modified method is time- and cost-saving.

[Comparative study on quality of Tongrentang red ginseng and Korean red ginseng--determination of ginsenosides and polysaccharides].

OBJECTIVE: To establish methods for quantitative determination of ginseng saponins, ginsenoside Rg1, Re, Rb1 and polysaccarides and compare the qualities of Tongrentang Red Ginseng and Korean Red Ginseng. METHOD: Macroreticular resin-colorimetric method was developed to determine ginseng saponins and a new HPLC method with gradient eluents was established for determination of ginsenoside Rg1, Re, Rb1. For ginseng polysaccharides, phenol-oil of vitriol colorimetric method was developed and some factors were also optimized. RESULT: The content of ginseng saponins in Tongrentang Red Ginseng was not lower than that of Korean Red Ginseng. Ginsenoside Rg1 and Rb1 in Tongrentang Red Ginseng were higher than those in Korean Red Ginseng, while Ginsenoside Re was slightly lower than that of Korean Red Ginseng. However, the amount of Ginseng Polysaccharides in Tongrentang Red Ginseng was greater than those in Korean Red Ginseng. CONCLUSION: The contents of ginseng saponins and ginsenoside Rg1, Re, Rb1 in Tongrentang Red Ginseng were not lower than that in Korean Red Ginseng. The methods for determination of ginsenosides and ginseng polysaccharides were quite accurate and reliable to the quality control of Ginseng.

GLYDE-an expressive XML standard for the representation of glycan structure.

The amount of glycomics data being generated is rapidly increasing as a result of improvements in analytical and computational methods. Correlation and analysis of this large, distributed data set requires an extensible and flexible representational standard that is also 'understood' by a wide range of software applications. An XML-based data representation standard that faithfully captures essential structural details of a glycan moiety along with additional information (such as data provenance) to aid the interpretation and usage of glycan data, will facilitate the exchange of glycomics data across the scientific community. To meet this need, we introduce GLYcan Data Exchange (GLYDE) standard as an XML-based representation format to enable interoperability and exchange of glycomics data. An online tool () for the conversion of other representations to GLYDE format has been developed.