Capillary Electrophoresis–Mass Spectrometry for the Analysis of Heparin Oligosaccharides and Low Molecular Weight Heparin

Heparins, highly sulfated, linear polysaccharides also known as glycosaminoglycans, are among the most challenging biopolymers to analyze. Hyphenated techniques in conjunction with mass spectrometry (MS) offer rapid analysis of complex glycosaminoglycan mixtures, providing detailed structural and quantitative data. Previous analytical approaches have often relied on liquid chromatography (LC)–MS, and some have limitations including long separation times, low resolution of oligosaccharide mixtures, incompatibility of eluents, and often require oligosaccharide derivatization. This study examines the analysis of glycosaminoglycan oligosaccharides using a novel electrokinetic pump-based capillary electrophoresis (CE)–MS interface. CE separation and electrospray were optimized using a volatile ammonium bicarbonate electrolyte and a methanol–formic acid sheath fluid. The online analyses of highly sulfated heparin oligosaccharides, ranging from disaccharides to low molecular weight heparins, were performed within a 10 min time frame, offering an opportunity for higher-throughput analysis. Disaccharide compositional analysis as well as top-down analysis of low molecular weight heparin was demonstrated. Using normal polarity CE separation and positive-ion electrospray ionization MS, excellent run-to-run reproducibility (relative standard deviation of 3.6–5.1% for peak area and 0.2–0.4% for peak migration time) and sensitivity (limit of quantification of 2.0–5.9 ng/mL and limit of detection of 0.6–1.8 ng/mL) could be achieved

GlycCompSoft: Software for Automated Comparison of Low Molecular Weight Heparins Using Top-Down LC/MS Data

<div><p>Low molecular weight heparins are complex polycomponent drugs that have recently become amenable to top-down analysis using liquid chromatography-mass spectrometry. Even using open source deconvolution software, DeconTools, and automatic structural assignment software, GlycReSoft, the comparison of two or more low molecular weight heparins is extremely time-consuming, taking about a week for an expert analyst and provides no guarantee of accuracy. Efficient data processing tools are required to improve analysis. This study uses the programming language of Microsoft Excel^™ <i>Visual Basic for Applications</i> to extend its standard functionality for macro functions and specific mathematical modules for mass spectrometric data processing. The program developed enables the comparison of top-down analytical glycomics data on two or more low molecular weight heparins. The current study describes a new program, GlycCompSoft, which has a low error rate with good time efficiency in the automatic processing of large data sets. The experimental results based on three lots of Lovenox^®, Clexane^® and three generic enoxaparin samples show that the run time of GlycCompSoft decreases from 11 to 2 seconds when the data processed decreases from 18000 to 1500 rows.</p></div

Relatively quantitative comparison of identified enoxaparin oligosaccharides without 1,6-anhydro reducing end from five commercialized LMW heparin products.

<p>Oligosaccharide compositions are given as [ΔHexA = 0 or 1, HexA, GlcN, Ac, SO₃].</p

Flowchart of the Deletion Module for screening of the GlycCompSoft.

<p>Flowchart of the Deletion Module for screening of the GlycCompSoft.</p

The Overall Flowchart of the GlycCompSoft from text file(.txt) input to true glycans data screened and merged.

<p>The Overall Flowchart of the GlycCompSoft from text file(.txt) input to true glycans data screened and merged.</p

The overall workflow and illustration of GlycCompSoft.

<p>The overall workflow and illustration of GlycCompSoft.</p

Total workflow of enoxaparin intact chain LC-MS analysis coupling with software processing.

<p>Total workflow of enoxaparin intact chain LC-MS analysis coupling with software processing.</p

Time efficiency of GlycCompSoft based on Lovenox^®, Clexane^®, and generic enoxaparin 1,2 and 3 with different amounts of data rows.

<p>Time efficiency of GlycCompSoft based on Lovenox^®, Clexane^®, and generic enoxaparin 1,2 and 3 with different amounts of data rows.</p

Introduction of the Mass Spread Function for Characterization of Protein Conjugates

Traditionally, characterization of protein molecules conjugated with molecular probes is performed by UV–vis spectroscopy. This method determines the average incorporation ratio but does not yield information about the label distribution. Electrospray ionization mass spectroscopy (ESI-MS) allows direct measurement of the fraction of protein containing a given number of labels. However, for a glycosylated protein, this analysis can be severely limited due to spectral overlap of the labels and carbohydrates. To address this problem, we introduce the mass spread function (MSF) for conjugation analysis. By treating the ESI-MS spectrum of conjugated protein as the spectrum before conjugation convolved with the MSF, we are able to quantify the labeled protein population using a binomial distribution function. We first applied this procedure for characterization of labeled antibody F­(ab′)₂ fragments which do not contain carbohydrates. We then apply the MSF to fit spectra of entire conjugated monoclonal antibodies and quantify the distribution of labels in the presence of glycans

Merging procedure Module Algorithm of GlycCompSoft.

<p>Merging procedure Module Algorithm of GlycCompSoft.</p