Sara Tengattini

Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography.

In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.

Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity

In this paper a series of 2-iminomethoxyethyl mannose-based mono- and disaccharides have been synthesized by a chemoenzymatic approach and used in coupling reactions with e-amino groups of lysine residues in a model protein (ribonuclease A, RNase A) to give semisynthetic neoglycoconjugates. In order to study the influence of structure of the glycans on the conjugation outcomes, an accurate characterization of the prepared neoglycoproteins was performed by a combination of ESI-MS and LC-MS analytical methods. The analyses of the chymotryptic digests of the all neoglycoconjugates revealed six Lys-glycosylation sites with a the following order of lysine reactivity: Lys 1 ≫ Lys 91 ≅ Lys 31 > Lys 61 ≅ Lys 66. A computational analysis of the reactivity of each lysine residue has been also carried out considering several parameters (amino acids surface exposure and pKa, protein flexibility). The in silico evaluation seems to confirm the order in lysine reactivity resulting from proteomic analysis.

Liquid chromatography-mass spectrometry structural characterization of neo glycoproteins aiding the rational design and synthesis of a novel glycovaccine for protection against tuberculosis.

Hereby we describe a pilot study for the rational design and synthesis of a glycoconjugate vaccine against Tuberculosis (TB) by site-specific coupling of well-defined glycans to non-antigenic amino acids in a selected protein carrier. A combination of ESI-MS and LC-MS analytical methods was applied for the systematic characterization of the reactivity of the surface amino acids in the glycosylation reaction with monosaccharides towards 2-iminomethoxyethyl or homobifunctional (4-nitrophenyl ester) linkers, both on the model protein, ribonuclease A (RNase A) and on TB10.4, the simplest antigenic protein isolated from Mycobacterium tuberculosis (MTB). Intact protein analysis was carried out to quantify the glycosylation degree and profile the glycoform composition of all the prepared neo glycoconjugates, while pronase and chymotriptic digests were analyzed to map and rank the reactivity of protein residues. Neo glycopeptides were purified by on-line porous graphitized carbon solid-phase extraction, separated by hydrophilic interaction liquid chromatography and analyzed by electrospray mass spectrometry (ESI-MS(n)). Significantly, different site specificity and glycosylation efficiency were demonstrated for the two linkers, resulting in structurally diverse glycoconjugates. A computational analysis of the amino acids involved in the epitope formation in TB10.4 addressed the choice to 2-iminomethoxyethyl-saccharide activation, that resulted in a more targeted and selective conjugation preserving the protein antigenicity. Additionally, a rational design of experiments lead to the identification of suitable experimental conditions for the preparation of highly pure and homogeneous neo glycoconjugates.

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo-glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins

Improved methods for detailed characterization of complex glycoproteins are required in the growing sector of biopharmaceuticals. Hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution (HR) time-of-flight mass spectrometric (TOF-MS) detection was examined for the characterization of intact neo-glycoproteins prepared by chemical conjugation of synthetic saccharides to the lysine residues of selected recombinant proteins. The separation performances of three different amide HILIC columns (TSKgel Amide-80, XBridge BEH and AdvanceBio Glycan Mapping) were tested. Water-acetonitrile gradients and volatile eluent additives have been explored. Addition of 0.05% (v/v) trifluoroacetic acid to the mobile phase appeared to be essential for achieving optimum resolution of intact glycoforms and minimal ion suppression effects. Gradient elution conditions were optimized for each protein on every column. HILIC stationary phases were evaluated for the analysis of highly heterogeneous semi-synthetic derivatives of the same protein (ribonuclease A), and in the enhanced characterization of TB10.4 and Ag85B glycoconjugates, selected antigens from Mycobacterium tuberculosis (MTB). HILIC-MS results indicated that the HILIC selectivity is predominantly governed by size of the conjugated glycans and number of glycans attached, providing efficient glycoform separation. Moreover, HILIC separation prior to HRMS detection allowed assignment of several product impurities. Additional top-down MS/MS experiments confirmed conjugation at the N-terminus of TB10.4 next to its lysine residue. Overall, the obtained results demonstrate that amide-stationary-phase based HILIC coupled to MS is highly useful for the characterization of intact neo-glycoproteins allowing assessment of the number, identity and relative abundance of glycoforms present in the semi-synthetic products.

Application of a rapid HILIC-UV method for synthesis optimization and stability studies of immunogenic neo-glycoconjugates

HIGHLIGHTSHILIC‐UV assists the development of new potential glycoconjugate vaccine.The method validation allows quantitative determination of recombinant protein.HILIC‐UV provides information on (glyco)‐protein antigen stability.Protein glycosylation is monitored by a simple HILIC‐UV method. ABSTRACT Proteins and glycoproteins with therapeutic activity are susceptible to environmental factors, which can cause their degradation and the loss of their activity. Thus, the maintenance of their stability during the production process is a critical factor. In this work, a simple and rapid hydrophilic interaction liquid chromatography HILIC‐UV method was validated in terms of accuracy, precision, linearity, LOD, LOQ and specificity and applied to the investigation of the stability of intact proteins and their neo‐glycoconjugates with antigenic activity against tuberculosis. The method proved to be suitable for the estimation of the degradation of the proteins under critical conditions (i.e. freeze‐thaw cycles) and for the monitoring of their coupling reaction with saccharidic moieties, without the need of sample preparation. In addition, the chromatographic analysis allowed to calculate the yields of the protein glycosylation reaction.

High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry

Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-β - 1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2 μL. HILIC-MS of rhIFN-β - 1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72.

A Proteomic-Based Approach to Study the Mechanism of Cytotoxicity Induced by Interleukin-1α and Cycloheximide

The exposure of HeLa cells to interleukin-1 alpha (IL-1α) in the presence of cycloheximide (CHX) leads to the release of active tumor necrosis factor alpha (TNF-α), eliciting cytocidal effect on these cells. A mass spectrometry (MS)-based analysis of the qualitative proteomic profiles of the HeLa cells treated only with IL-1α, CHX or simultaneously with IL-1α and CHX, in comparison to an untreated control, enabled to distinguish protein candidates possibly involved in this process. Among them protein disulphide isomerase (PDI) seemed to be particularly interesting for further research. Therefore, we focused on quantitative changes of PDI levels in HeLa cells subjected to IL-1α and CHX. Enzyme-linked immunosorbent assay (ELISA) was employed for determination of PDI concentrations in the investigated, differently treated HeLa cells. The obtained results confirmed up-regulation of PDI only in the cells stimulated with IL-1α alone. In contrary, the PDI levels in HeLa cells exposed to both IL-1α and CHX, where apoptotic process was intensive, did not increase significantly. Finally, we discuss how different expression levels of PDI together with other proteins, which were detected in this study, may influence the induction of cytotoxic effect and modulate sensitivity to cytotoxic action of IL1.Graphical Abstract

A new MS compatible HPLC-UV method for Teicoplanin drug substance and related impurities, part 1: Development and validation studies

&NA; Teicoplanin is a glycopeptide antibiotic prepared by fermentation from cultures of Actinoplanes teichomyceticus, used as drug of last resort for the treatment of bacterial infections in humans. This study, which is the first in a series of two parts, describes the development of a LC method for the separation of Teicoplanin drug substance and its related impurities compatible with MS detection. The separation conditions for Teicoplanin were set on a LiChrospher 100 RP‐18 column under gradient elution with a mobile phase composed of ammonium formate 25 mM at pH 6.00 and ACN. The new method was shown equivalent in terms of selectivity to the one reported in the European Pharmacopoeia Teicoplanin monograph, and was validated according to ICH Q2 R1 guidelines for the drug substance assay. The new method offers similar performance to the compendial one but has the advantage of being fully compatible with MS and it can be proposed as a useful tool also for controlling the quality of Teicoplanin fermentation batches and the occurrence of potential impurities. HighlightsA new RP‐LC method for teicoplanin drug substance and related impurities is developed.The method offers similar performance to the compendial and compatible with MS.Method was validated according to ICH Q2 R1 guidelines for the drug substance assay.The LC–MS method is a useful tool for teicoplanin fermentation batches quality control.

Rational design, preparation and characterization of recombinant Ag85B variants and their glycoconjugates with T-cell antigenic activity against Mycobacterium tuberculosis

Tuberculosis is the deadliest infectious disease in the world. The variable efficacy of the current treatments highlights the need for more effective agents against this disease. In the past few years, we focused on the investigation of antigenic glycoconjugates starting from recombinant Ag85B (rAg85B), a potent protein antigen from Mycobacterium tuberculosis. In this paper, structural modifications were rationally designed in order to obtain a rAg85B variant protein able to maintain its immunogenicity after glycosylation. Lysine residues involved in the main T-epitope sequences (namely, K30 and K282) have been substituted with arginine to prevent their glycosylation by a lysine-specific reactive linker. The effectiveness of the mutation strategy and the detailed structure of resulting neo-glycoconjugates have been studied by intact mass spectrometry, followed by peptide and glycopeptide mapping. The effect of K30R and K282R mutations on the T-cell activity of rAg85B has also been investigated with a preliminary immunological evaluation performed by enzyme-linked immunospotting on the different variant proteins and their glycosylation products. After glycosylation, the two variant proteins with an arginine in position 30 completely retain the original T-cell activity, thus representing adequate antigenic carriers for the development of efficient glycoconjugate vaccines against tuberculosis.