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

Abstract 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

Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines

Effect of glycosylation on the affinity of the MTB protein Ag85B for specific antibodies: towards the design of a dual-acting vaccine against tuberculosis

Background: To create a dual-acting vaccine that can fight against tuberculosis, we combined antigenic arabino-mannan analogues with the Ag85B protein. To start the process, we studied the impact of modifying different parts of the Ag85B protein on its ability to be recognized by antibodies. Results: Through our research, we discovered that three modified versions of the protein, rAg85B-K30R, rAg85B-K282R, and rAg85B-K30R/K282R, retained their antibody reactivity in healthy individuals and those with tuberculosis. To further test the specificity of the sugar AraMan for AraMan antibodies, we used Human Serum Albumin glycosylated with AraMan-IME and Ara3Man-IME. Our findings showed that this specific sugar was fully and specifically modified. Bio-panning experiments revealed that patients with active tuberculosis exhibited a higher antibody response to Ara3Man, a sugar found in lipoarabinomannan (LAM), which is a major component of the mycobacterial cell wall. Bio-panning with anti-LAM plates could eliminate this increased response, suggesting that the enhanced Ara3Man response was primarily driven by antibodies targeting LAM. These findings highlight the importance of Ara3Man as an immunodominant epitope in LAM and support its role in eliciting protective immunity against tuberculosis. Further studies evaluated the effects of glycosylation on the antibody affinity of recombinant Ag85B and its variants. The results indicated that rAg85B-K30R/K282R, when conjugated with Ara3Man-IME, demonstrated enhanced antibody recognition compared to unconjugated or non-glycosylated versions. Conclusions: Coupling Ara3Man to rAg85B-K30R/K282R could lead to the development of effective dual-acting vaccines against tuberculosis, stimulating protective antibodies against both AraMan and Ag85B, two key tuberculosis antigens

Chromatographic tools for plant-derived recombinant antibodies purification and characterization

In the last two decades, plants became an interesting alternative for the production of recombinant proteins for human therapy and several antibodies expressed in plants have reached the clinical development stage. Plants are capable of post-translational modifications (PTMs) necessary for protein activity and pharmacokinetics, such as glycosylation. However, there are important kingdom-specific modifications that have to be considered when expressing recombinant proteins. Therefore, there is a need for efficient analytical methods for deep protein characterization starting from the expression platform design until the product approval to guarantee product authenticity, quality and efficacy. Literature lacks of reviews dealing with plant-derived proteins purification and characterization by chromatographic methods, thus the focus of the present review is on this topic for the most representative biotechnological drugs i.e. monoclonal antibodies (mAbs). In the first part, a comprehensive discussion of the methods applied in dowstream processes (extraction and clarification) and a detailed overview of the chromatographic techniques useful for the purification of plant-made mAbs are reported. Among purification techniques, Protein A affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, hydrophobic charge induction chromatography or mixed mode chromatography are described. In the second part, we will discuss analytical platforms based on chromatographic techniques (reverse phase, size exclusion chromatography, ion-exchange chromatography, hydrophilic interaction liquid chromatography) coupled with different detection systems (UV, Fluorescence, MS) used at protein, peptide and glycan level to characterize plant-made mAbs with their unique features

β-Mannosidase from <i>Cellulomonas fimi</i>: Immobilization Study and Application in the β-Mannoside Synthesis

The β-d-mannopyranoside linkage is found in a number of biological structures, in particular, in the core trisaccharide of N-linked glycoproteins, as well as within the antigenic polysaccharides of Salmonella, yeasts, and glycolipids. The construction of this glycosydic bond by chemical approach is very challenging and requires cumbersome protection and activation steps prior to glycosylation. In this context, β-mannosidase from Cellulomonas fimi (Cf-β-Man) was immobilized for the first time, and it was employed in the synthesis of β-mannosides. Cf-β-Man immobilized on IDA-Co2+-agarose allows the synthesis of the disaccharide, cyanomethyl β-d-mannopyranosyl-(1→6)-2-acetamido-2-deoxy-1-thio-β-d-glucopyranoside, with a higher conversion compared to the soluble enzyme (20% vs. 5%) after 6 h under best conditions. This explorative work opens new scenarios concerning the design of engineered Cf-β-Man mutants and their immobilization in order to obtain a robust and recyclable biocatalyst for applications in chemoenzymatic glycan synthesis

Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically pro- duced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient sepa- ration of the antigen proteoforms, a polyionic multilayer coat- ing of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time- of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth

: Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A-coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production

Chromatographic profiling of silk sericin for biomedical and cosmetic use by complementary hydrophylic, reversed phase and size exclusion chromatographic methods

Silk sericin (SS) is, together with silk fibroin (SF), one of the two proteins forming the silkworm cocoon. SS is ideal ingredient for cosmetic applications in the formulation of specific products for skin care and hair due to its peculiar physical-chemical composition. SS also showed a great potential in different pharmacological and biotechnological applications, as anticancer drug, anticoagulant, cell culture additive, wound healing agent and drug delivery carrier. Reasons for SS use in biomedical applications derive from its physical-chemical composition. As a consequence, a detailed characterization of SS in terms of average molecular weight, molecular weight distribution and hydro/lipophilic character is crucial to properly address and assess its quality, cosmetic or pharmacological use. In this study, the application of different and complementary chromatographic modes allows a detailed investigation of SS protein isolated from wastewater using two diverse extraction methods. Hydrophilic interaction liquid chromatography (HILIC using an AdvanceBio Glycan Map column) and reverse phase (RP using Symmetry300 C18 column) were applied to intact protein characterization to derive data on protein hydrophilicity and on hydrophobic components of the two SS preparations (SS#1 and SS#2). A higher hydrophilic character of SS#1 was observed by HILIC trace, coherently with the preparation method used, while no significant differences in hydrophobicity were detectable in the RPLC separations. Size distribution was also defined by using a SEC-UV-MS method (using TSKgel SuperSW2000 column) properly optimized to maximize both the size selectivity and the method sensitivity. Taken together, the chromatographic data allowed to better characterize the SS samples obtained by different extraction methods, and the structural properties were correlated to their biological activities

Silk Fibroin Bioink for 3D Printing in Tissue Regeneration: Controlled Release of MSC extracellular Vesicles

Sodium alginate (SA)-based hydrogels are often employed as bioink for three-dimensional (3D) scaffold bioprinting. They offer a suitable environment for cell proliferation and differentiation during tissue regeneration and also control the release of growth factors and mesenchymal stem cell secretome, which is useful for scaffold biointegration. However, such hydrogels show poor mechanical properties, fast-release kinetics, and low biological performance, hampering their successful clinical application. In this work, silk fibroin (SF), a protein with excellent biomechanical properties frequently used for controlled drug release, was blended with SA to obtain improved bioink and scaffold properties. Firstly, we produced a printable SA solution containing SF capable of the conformational change from Silk I (random coil) to Silk II (β-sheet): this transition is a fundamental condition to improve the scaffold’s mechanical properties. Then, the SA-SF blends’ printability and shape fidelity were demonstrated, and mechanical characterization of the printed hydrogels was performed: SF significantly increased compressive elastic modulus, while no influence on tensile response was detected. Finally, the release profile of Lyosecretome—a freeze-dried formulation of MSC-secretome containing extracellular vesicles (EV)—from scaffolds was determined: SF not only dramatically slowed the EV release rate, but also modified the kinetics and mechanism release with respect to the baseline of SA hydrogel. Overall, these results lay the foundation for the development of SA-SF bioinks with modulable mechanical and EV-release properties, and their application in 3D scaffold printing