A new alternative tool to analyse glycosylation in pharmaceutical proteins based on infrared spectroscopy combined with nonlinear support vector regression.

Almost 60% of commercialized pharmaceutical proteins are glycosylated. Glycosylation is considered a critical quality attribute, as it affects the stability, bioactivity and safety of proteins. Hence, the development of analytical methods to characterise the composition and structure of glycoproteins is crucial. Currently, existing methods are time-consuming, expensive, and require significant sample preparation steps, which can alter the robustness of the analyses. In this work, we suggest the use of a fast, direct, and simple Fourier transform infrared spectroscopy (FT-IR) combined with a chemometric strategy to address this challenge. In this context, a database of FT-IR spectra of glycoproteins was built, and the glycoproteins were characterised by reference methods (MALDI-TOF, LC-ESI-QTOF and LC-FLR-MS) to estimate the mass ratio between carbohydrates and proteins and determine the composition in monosaccharides. The FT-IR spectra were processed first by Partial Least Squares Regression (PLSR), one of the most used regression algorithms in spectroscopy and secondly by Support Vector Regression (SVR). SVR has emerged in recent years and is now considered a powerful alternative to PLSR, thanks to its ability to flexibly model nonlinear relationships. The results provide clear evidence of the efficiency of the combination of FT-IR spectroscopy, and SVR modelling to characterise glycosylation in therapeutic proteins. The SVR models showed better predictive performances than the PLSR models in terms of RMSECV, RMSEP, R2CV, R2Pred and RPD. This tool offers several potential applications, such as comparing the glycosylation of a biosimilar and the original molecule, monitoring batch-to-batch homogeneity, and in-process control.

Analysis of Glycoproteins by ATR-FTIR Spectroscopy: Comparative Assessment.

FTIR spectroscopy has been widely used to characterize biopharmaceuticals for many years, in particular to analyze protein structure. More recently, it was demonstrated to be a useful tool to study and compare protein samples in terms of glycosylation. Based on a spectral region specific to carbohydrate absorption, we present here a detailed protocol to compare the FTIR spectra of glycoproteins in terms of global glycosylation level and in terms of glycan composition. This FTIR information is compared to MS information. Both approaches yield consistent results but it appears FTIR analysis is easier and more rapid to perform comparisons.

FTIR spectroscopy as an analytical tool to compare glycosylation in therapeutic monoclonal antibodies.

Glycosylation is the most common protein post-translational modification (PTM), especially in biopharmaceuticals. It is a critical quality attribute as it impacts product solubility, stability, half-life, pharmacokinetics and pharmacodynamics (PK/PD), bioactivity and safety (e.g. immunogenicity). Yet, current glycan analysis methods involve multiple and lengthy sample preparation steps which can affect the robustness of the analyses. The development of orthogonal, direct and simple method is therefore desirable. In this study, we suggest use of FTIR spectroscopy to address this challenge. Use of this technique, combined with statistical tools, to compare samples or batches in terms of glycosylation or monosaccharide profile, has three potential applications: to compare glycosylation of a biosimilar and the original (innovator) molecule, for monitoring of batch-to-batch consistency, and for in-process control. Fourteen therapeutic monoclonal antibodies (mAbs), one Fc-fusion protein and several other common glycoproteins have been used to demonstrate that FTIR spectra of glycoproteins display spectral variations according to their glycan and monosaccharide compositions. We show that FTIR spectra of glycoproteins provide a global but accurate fingerprint of the glycosylation profile. This fingerprint is not only sensitive to large differences such as the presence or absence of several monosaccharides but also to smaller modifications of the glycan and monosaccharide content.

Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.

Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34.

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.