Deanna C. Hurum

Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: the ABRF Glycoprotein Research Multi-Institutional Study 2012

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

High-performance anion-exchange chromatography with pulsed amperometric detection for carbohydrate analysis of glycoproteins

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an established technique for the carbohydrate analysis of glycoproteins. HPAE-PAD is routinely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This is true for both the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. This contribution reviews the fundamentals of HPAE-PAD, recent technological improvements, and advances in the last ten years in its application to carbohydrate analysis of glycoproteins. The application areas reviewed include monosaccharide determinations, sialic acid determinations, M-6-P determinations, sugar alcohol determinations, analysis of polysialic acids, neutral and charged oligosaccharide analysis, following glycosidase and glycosyltransferase reactions, and coupling HPAE-PAD to mass spectrometry (MS).

Five-minute glycoprotein sialic acid determination by high-performance anion exchange chromatography with pulsed amperometric detection.

Glycoprotein sialylation analysis is a common analytical step in characterizing biotherapeutic products and expression experiments to optimize production. In this article, a high-throughput (5-min) high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD)-based analytical method for glycoprotein sialic acid determination is described. Results from this method are compared with both published HPAE-PAD and 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization followed by ultra high-performance liquid chromatography fluorescence detection (UHPLC-FLD) assays. The quantified sialic acid amounts agree with prior HPAE-PAD analyses within replicate error and with UHPLC-FLD within an average of 24%, which are equivalent results based on assay reproducibility.

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α1-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.

Profiling N-linked oligosaccharides from IgG by high-performance anion-exchange chromatography with pulsed amperometric detection

Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics and cellular toxicity. Protein expression systems and reactor conditions can impact glycosylation, leading to potentially undesirable glycosylation. For example, high-mannose species may be present, which are atypical of human antibody glycosylation. Their presence in the Fc domain has been linked to increased serum clearance of immunoglobulin G (IgG) antibodies. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for determining glycans present in glycoprotein therapeutics. We report an improved HPAE-PAD method for IgG oligosaccharide separation. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations (RSDs) were between 0.63 and 4.0% while retention indices precision RSDs ranged from 0.27 to 0.56%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.