Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins
Graphical abstract
Introduction
Biopharmaceutical products and drugs of biological origin represent an important class of therapeutics. In particular, glycoproteins are among the most commercially successful biological drugs due to their numerous applications and indications ranging from cancer to inflammation to infectious diseases (glycovaccines). It is well known that glycans attached to a protein can influence the therapeutic efficacy of protein drug and the protein physicochemical and pharmacological properties. Thus, there is a need for the production of well-defined glycoproteins and elegant synthetic strategies have been explored for the preparation of homogeneous glycoproteins with particular emphasis on glycovaccines.
An interesting approach, often used for the production of glycoconjugate vaccines, entails the extraction or the synthesis of saccharides (antigens) and their covalent linkage to a carrier protein produced by recombinant DNA technology. The conjugation is commonly achieved by chemical activation of the glycan portion allowing targeting of nucleophilic functional groups of specific amino acid residues (lysines, aspartic/glutamic acids or cysteines) in the protein [1], [2].
The characterization of neo-glycoprotein products, obtained as described above, is quite challenging. In fact, in addition to the inevitable heterogeneity of recombinant proteins due to the biotechnological manufacturing, the heterogeneity related to the carbohydrate component (structure, number, and position) has to be considered. Similarly to native glycoproteins, also for semisynthetic glycosylated proteins, the inherent structural complexity (i.e. sequence mutation, oxidation, deamidation, N- and C-terminal alteration, glycoform variety due to multiple conjugation sites) necessitates the development of analytical strategies for their characterization [3].
Liquid chromatography (LC) coupled to electrospray ionization mass spectrometry (ESI-MS) is widely used for glycoprotein characterization by bottom-up approaches, in which the glycoproteins are first digested into peptides, providing structural information and allowing localization of glycosylation sites [4], [5]. Nevertheless, quality control of the intact glycoproteins is of great added value in biopharmaceutical analysis, as it provides specific information on the presence of proteoforms as well as on the exact combinations of multiple modifications, which cannot be revealed by merely bottom-up approaches. Moreover, intact protein analysis is relatively fast, requires minimum sample treatment and prevents undesired modifications induced by enzymatic treatments [6], [7].
Hydrophilic interaction liquid chromatography (HILIC) has demonstrated to be a highly useful technique for analysis of amino acids, released glycans and glycopeptides [8]. In contrast to reversed-phase (RP) LC, HILIC has the ability to retain and resolve (highly) polar compounds, based on a complex retention mechanism, involving hydrophilic partitioning and polar interactions. As a consequence, glycans and glycopeptides show stronger HILIC retention with an increasing number and size of sugar units [9], [10].
Only few studies have been published on the use of HILIC methods for the characterization of intact proteins [8], [11], [12] and for the determination of their glycoform composition [11], [12], [13], [14], indicating HILIC to be complementary and orthogonal to RPLC. So far, the application of HILIC for the separation of native glycoforms of intact proteins has been limited to the model glycoprotein ribonuclease B (RNase B), containing only one glycosylation site, and monoclonal antibodies [11], [12]. Recently, we have reported a preliminary work where HILIC-UV was used for the monitoring of the glycosylation reaction of a model protein ribonuclease A (RNase A), with different glycans [15]. The developed analytical method was not compatible with MS detection, however, it was suitable for the analysis of intact neo-glycoproteins, allowing discrimination of non-glycosylated protein from glycoproteins and separation of different glycoforms, including isomers, as demonstrated by off-line MS analysis [15].
On-line MS detection after HILIC separation of glycoforms, would offer many advantages, such as the assignment of glycoform structures and other potential protein modifications and degradations. Moreover, following a top-down approach, HILIC-MS/MS could provide information on N-terminal sequence, including glycosylation occurrence at the α-amino group. Nevertheless, on-line HILIC-MS of intact glycoproteins has been reported only for RNase B and for IdeS-digested and reduced monoclonal antibodies [11], [12], [14].
In order to expand the applicability of HILIC with UV and HRMS detection to the characterization of intact neo-glycoproteins, we studied the chromatographic performances of three different amide HILIC columns (TSKgel Amide-80, XBridge BEH and AdvanceBio Glycan Mapping). Neo-glycoproteins, obtained by chemical conjugation of synthetic mono-, di- and tri-mannose to lysine residues of RNase A and of the two recombinant tuberculosis antigens TB10.4 and Ag85B [16], were considered as representative real-life proteins in this work. TB10.4 and Ag85B have been evaluated by our research group for the development of new effective vaccines against tuberculosis [3], [17]. Their semi-synthetic glycoconjugates comprise a large number of glycoforms with multiple conjugation sites, each carrying short carbohydrate chains. This intrinsic heterogeneity combined with the relatively small hydrophilic contribution of every individual carbohydrate chain, makes chromatographic resolution and characterization of these neo-glycoproteins an interesting case for the application of the three amide HILIC columns. MS-compatible mobile phase composition, gradients and column temperature were studied in order to achieve good separation of the semi-synthethic glycoprotein products. Moreover, the HILIC-MS results obtained aid in understanding structure-retention relationships of glycoproteins. The potential of the developed HILIC-MS(/MS) methods for the detection of glycoconjugate product impurities and for the confirmation of the glycan position in conjugated TB10.4 was also examined.
Section snippets
Reagents
RNase A and RNase B from bovine pancreas were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. Formic acid (FA), ammonium hydroxide (30%), sodium tetraborate, trifluoroacetic acid (TFA), phosphate-buffered saline (PBS), benzamidine chloride were from Sigma-Aldrich (St. Louis, MO, USA). Water was obtained from a Direct-QTM Millipore system Millipore (Millipore, Billerica, MA, USA). Acetonitrile (ACN) MS grade was purchased from Sigma-Aldrich (St.
Results and discussion
In this work, HILIC-UV-MS was evaluated for the characterization of neo-glycoproteins. Three different commercial HILIC columns were investigated for their capacity to reduce heterogeneity complexity by resolving intact glycoforms as well as specific proteoforms to facilitate their assignment. Initial chromatographic optimization was carried out by HILIC-UV using MS-compatible conditions and RNase B as model glycoprotein. Then, HILIC-MS methods were applied to the analysis of semi-synthetic
Conclusion
HILIC-UV-MS(/MS) methods for the characterization of intact neo-glycoproteins were developed. Under optimized separation conditions, resolution and assignment of proteoforms and individual glycoforms of semi-synthetic intact glycoproteins were achieved. HILIC retention and selectivity appeared to be dominated by the size and number of the saccharides conjugated to the carrier protein. Three tested amide HILIC stationary phases showed similar selectivity for the glycosylated proteins. However,
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Contributed equally.