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European Biopharmaceutical Review

The Sugar is the Key

Protein biologics now represent a significant share of pharmaceutical sales and future growth potential, particularly in an era of increasing patent expirations. A range of analytical methods is required to determine the purity, identity and integrity of protein biologics at multiple points along the manufacturing process, from cell culture to downstream purification, product characterisation and lot release. Most protein drugs developed to date are glycoproteins, including monoclonal antibodies (mAbs).

These glycoprotein pharmaceuticals contain complex oligosaccharide moieties whose presence, absence and profile can have a significant impact on therapeutic efficacy, pharmacokinetics, immunogenicity, folding and stability of the biologic drug. For example, certain glycan structures are known to cause aggregation and decrease drug efficacy.

The proportion and types of glycans present on a recombinant protein are determined by the expression system (cell types), the cell culture conditions used, and can be influenced throughout protein engineering and process development. A strong understanding of which glycans can contribute to both positive and negative drug performance has been built. Therefore, glycan profiles are determined early in the discovery and development of recombinant proteins so they can be optimised. Once the best profile is determined, it is monitored throughout development and manufacturing to ensure that the drug product is consistent and stable.

Different Methods

A variety of approaches can be used to characterise glycoproteins and their glycan moieties, including Highperformance liquid chromatography (HPLC) followed by quadrupole timeof- flight (QTOF) mass spectrometry (MS). In addition, glycosylation sites can be elucidated by digesting the protein with trypsin and using liquid chromatography (LC)/QTOF MS to separate and identify the resulting glycopeptides.

Comparison of the masses of these peptides to those generated by a theoretical digestion of the desired glycan form of the protein can determine if the protein is properly glycosylated. Capillary electrophoresis (CE) coupled to QTOF MS can also be used for this purpose, while delivering excellent separation efficiency, short run times, and minimal sample/ solvent consumption.

Analysis of the glycan moieties attached to a protein is most commonly done by enzymatic deglycosylation. Protein N-Glycosidase F (PNGase F), an amidase, is used to cleave asparagine-linked (N-linked) oligosaccharides from glycoproteins. However, current analysis techniques often lack the sensitivity required to detect low-abundance glycoforms, are labour-intensive and subject to human error, and can take as long as two days to complete due to long enzymatic reactions and labeling steps. This article focuses on three new technologies that are improving the sensitivity, accuracy, speed and ease of glycan analysis.

Traditional Glycan Analysis

While there are several techniques for analysing unlabelled glycans, the removed glycans are most commonly derivatised, labelled, and analysed by fluorescence detection. HPLC can be used to separate and identify glycans fluorescently labelled with 2-aminobenzamide (2-AB) using a hydrophilic interaction chromatography (HILIC) column or other separation media. This technique has been used to characterise the glycan moieties on mAbs and other glycoproteins. However, it can be time-consuming and laborious, and does not provide mass information or have the ability to separate all glycan isomers.

Sample Prep Automation

A rapid, highly reproducible, automated cartridge-based sample preparation workflow has been developed for producing labelled N-linked glycans from glycoproteins for analysis by CE, HPLC or LC/MS. It begins with an optional affinity purification step using a chromatography cartridge (for example, protein A or G). The purified glycoprotein is then denatured and immobilised on a hydrophobic cartridge and reacted with PNGase F to selectively release the N-glycans, which are eluted into a microplate well. The eluted glycans can be labelled with a number of different available labels (including 2-AB) using rapid chemistries optimised for automation. The labelled glycans are then cleaned up on an HILIC cartridge to remove excess free label and other reactants for downstream analysis. Multiple parameters such as sample volume, deglycosylation time and temperature, and labelling time and temperature can be quickly adjusted to optimise the glycan preparation process. This versatility is useful for the analysis of glycans that can be degraded by the deglycosylation process, such as sialic acid. This technology reduces a very complex workflow to three to five hours total assay time with less than one hour of hands-on time and two pipetting steps, automating sample preparation to increase throughput and decrease variability and potential human error (1).

Total Workflow Automation

The ability to characterise glycans rapidly has been limited by the sample preparation steps and structural complexity of the glycoproteins. A microfluidic chip technology has been developed to address this problem (2). It performs the rapid online cleavage of glycans from monoclonal antibodies, captures the released glycans, and then separates them prior to nanospray ionisation in a mass spectrometer; the entire run time is 12 minutes.

An accurate mass glycan database is used for quick assignment and identification of the glycans. Deglycosylation is performed using an online PNGase F reactor, and the resulting glycans are concentrated on a graphitised carbon enrichment column and separated on a graphitised carbon analytical column. Porous graphitised carbon has the ability to separate all potential glycan structures, including isomers, which are often present (see Figure 1). All of these steps are performed on a single HPLC-chip that interfaces with a QTOF mass spectrometer. The analyst simply dilutes and centrifuges the mAb sample and then places it in the autosampler.

The LC/QTOF data file is processed using an algorithm that extracts unique compounds, taking into account LC retention times, accurate masses, charge states, and adducts of the glycans. The compounds are identified using an accurate mass glycan database. Figure 2 illustrates the screen display for the results from the analysis of a mAb standard, showing the list of identified glycans in the left upper panel, as well as their overlaid extracted ion chromatograms. The structure for the highlighted glycan on the left is shown in the upper right panel. The m/z values, charge states and isotopes are shown in the lower panel. Data processing, including reporting, can be completed in less than five minutes per sample.

Glycan analysis using this automated chip technology typically results in the detection of a higher number of low abundance glycans, which can be due to several factors. Higher ionisation efficiency of glycosylamines is achieved with this technology. Also, traditional analysis approaches can convert one glycosylamine into a pair of reducing glycan anomers, resulting in loss of detection of one or both. Finally, traditional techniques are vulnerable to potential losses of low abundance glycans at each step of the multi-step process.

This new chip-based technology thus enables automated glycan cleavage, separation and identification in fewer than 20 minutes, for an analysis that could traditionally take days to complete, with highly sensitive and reproducible results.

Simultaneous Glycan Analysis and Peptide Mapping

CE has gained much attention in analysing glycans, delivering highefficiency separations in short run times. Enzymatically released glycans are labelled with the fluorescent chromophore APTS (8-aminopyrene- 1,3,6-trisulfonic acid) and subjected to CE separation with high-sensitivity laser-induced fluorescence or MS detection. The APTS labelling imparts negative charge to glycans, enabling enhanced electrophoretic separations and electrospray ionisation (negative mode) for identification by QTOF MS. Accurate mass determinations facilitate identification of unknowns and accurate detection of glycan modifications.

However, a recent technological advancement enables simultaneous peptide mapping and glycan analysis (3). Coupling of a CE system to a QTOF MS with a coaxial sheath liquid interface enables analysis of the glycopeptides generated from an mAb by tryptic digestion (see Figure 3). The glycopeptides are assigned using accurate mass measurement. At the same time, oxonium ions generated from the glycan moieties are used to identify the specific glycans attached to each glycopeptide, using CE/MS/MS and powerful data processing software. This new approach significantly shortens the process for characterising glycoproteins.

Conclusion

Monitoring the types and relative amounts of N-linked glycans which modify a protein biopharmaceutical is absolutely critical to successful development and manufacture, from fermentation to packaging. Fortunately, new technologies have been developed to assure the accuracy of the process while reducing the time and hands-on effort required to do so. Automation of sample preparation, as well as the entire workflow of glycan analysis using HPLC-chip technology, is now available for this purpose, as is simultaneous peptide mapping and glycan analysis. Continued technology advances in glycan analysis will ensure rapid development of vital and effective protein biopharmaceuticals in the future.

References

1. Murphy S, Bovee M, Van Den Heuvel Z, Krahenbuhl A, Reich J and Fulton S, Characterize N-glycans using a new quantitative, automated sample preparation platform, 2013. Visit: www.agilent.com

2. Staples G, Yin H and Killeen K, Glycan quantitation strategies based on high sensitivity Nano LC/MS, J Biomol Tech, 24 (Suppl): S36-237, 2013

3. Babu S, Tang N, Gudihal R, Preckel T and Greiner M, Capillary electrophoresis-mass spectrometry (CE-MS) analysis of glycopeptides in monoclonal antibodies, 2011. Visit: www.agilent.com


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Taegen Clary is the Marketing Director for Agilent Technologies Pharmaceutical Segment within the Life Sciences Group. Prior to joining Agilent, Taegen was a product manager at Bio-Rad Laboratories for lab-scale protein purification solutions and a DMPK LC/MS Analyst at Berlex Biosciences. He holds a BSc Biotechnology from UC Davis and an MBA, Marketing from St Mary’s College of California.
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