Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

Abstract

Carbohydrate‐based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life‐threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T‐cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide–protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid‐state NMR spectroscopy. As a test case, a large protein assembly, l‐asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined.

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

“…The analysis of the conjugation pattern and the evaluation of the conjugation degree of each lysine residue has been achieved by integrating solution and solid‐state NMR spectroscopy . The spectra were collected on a PEGylated sample of ANSII where only Lys residues were isotopically enriched in 13 C and 15 N [U‐ 2 H, Lys‐ 1 H, 13 C, 15 N].…”

“…The analysiso ft he conjugation pattern and the evaluation of the conjugation degree of each lysine residue has been achieved by integratings olution ands olid-state NMR spectroscopy. [19] The spectra were collected on aP EGylated sample of ANSII where only Lysr esiduesw ere isotopically enriched in 13 C and 15 N[ U-2 H, Lys-1 H, 13 C, 15 N].T he H z -N z new cross-peaks corresponding to eight lysine residues functionalized with PEG chains could be assigned in the 2D 1 H- 15 NT ROSY-HSQC of PEG-ANSII in solution,f rom the analysis of 3D NOESY spectra. The intensities of the signals have been used as ap arameter to evaluatet he functionalization degree and combined with the changesi nt he relative intensity of the cross-peaks correlating the C a and the C e of the lysine sidechains in the 2D 13 C-13 C DARR SSNMR spectrum of the native and PEGylated protein.…”

“…[26,26] SSNMR experiments were performed both on the crystalline preparation of native ANSII and on the PEGylated preparation of the protein, obtained with rehydration of freeze-dried material as previously reported. [19,20] All the spectra were recorded at % 280 Ko na Bruker AvanceIII 850 MHz wide-bore spectrometer (20 T, 213.6 MHz 13 CL armor frequency), equipped with 3.2 and 1.3 mm DVT MAS probe heads in triple-resonance mode. The inter-scan delay was set to 2.2 si na ll the experiments.…”

Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside

“…As expected, the protein residues experiencing the largest effects are the lysines and residues in their close proximity (Figures S6 and S7). For a semi‐quantitative evaluation of the conjugation degree of each lysine residue, the relative intensity of the cross‐peaks corresponding to the reacted and unreacted species within the same spectrum has been analyzed in detail and graphically reported in Figure …”

“…Moreover, several studies demonstrated that glycosylation induces various effects on the stability of protein pharmaceuticals . Therefore, substantial efforts are being made for the efficient glycosylation of proteins, and for developing biophysical methodologies to characterize these protein derivatives . In fact, glycosylation of proteins is not a trivial task as it requires a chemoselective chemical ligation that introduces the carbohydrate moieties under conditions that are compatible with the protein environments such as room temperature and aqueous medium, as well as the absence of a metal catalyst.…”