PROTEIN GLYCOSILATION ANALYSIS BY AN INTEGRATED PNGASE-F-IMER-LC-ESI-Q-TOF APPROACH

Protein glycosylation is acknowledged to be one of the major post-translational modifications, which significantly affects protein folding, conformation, stability and activity. Since more than one-third of approved biotherapeutics are glycoproteins [1], glycosylation state is a key issue in the production of biotherapeutics and biosimilars. Indeed, the presence and type of glycans dramatically affect efficacy and safety of this class of drugs. Thus, the increasing use of biotherapeutics translates into an urgent need to characterize glycosylation state and detect its alteration, in view of quality control reliable analytical methods. In this context, mass spectrometry (MS) coupled with liquid chromatography (LC) has already been shown to be an effective tool for analyzing glycoproteins and glycosylation sites. However, glycosylation analysis still remains highly challenging. The basic pipeline for glycosylation analysis is time consuming and includes protein deglycosylation and/or glycoprotein digestion (or combination of) followed by the analysis of intact proteins, glycopeptides and free glycans (either not labeled or labeled). To overcome low automation rate and long analysis time, glycan release by an immobilized enzyme reactors, i.e., enzymes immobilized on a suitable solid support (IMERs), can be used as a valuable alternative to the in solution assay. This presentation focuses on the development of a novel Peptide-N-glycosidase F (PNGase F) immobilized enzyme reactor, its characterization and its integration into a LC-ESI-Q-ToF platform, which enables the sequential analysis of both deglycosilated proteins and released glycans. PNGase F is one of the most frequently used endoglycosidases employed to release N-linked glycans, the most common form of glycans. The PNGase F-IMER was obtained by oriented covalent immobilization of the target enzyme onto the surface of a short bed, high performance monolithic column (epoxy CIMac™ Analytical column), which has been purposely fabricated for the analysis of large biomolecules. The monolith material, which has excellent permeability and fast mass transfer, has previously been shown to be a suitable support for IMER preparation when integration into a separative system is required. The LC-MS integrated platform consisted of a series of two analytical columns able to selectively trap and separate in parallel both the proteins and the free glycans released by the IMER. In the proposed set up, glycoproteins were first on-line deglycosylated by the PNGaseF-IMER, the deglycolysalted proteins were subsequently collected by a small C4 CIMac™ analytical column (0.1 mL bed volume), and analysed by ESI-Q-ToF. In parallel, released glycans were trapped onto a Hypercarb porous graphitic carbon (PGC) column and sent to ESI-Q-ToF for their analysis and identification. Each step was optimized to be perfectly interfaced with the subsequent MS analysis as well as with the enzyme requirements for stability and activity. [1] G. Walsh, R. Jefferis, Post-translational modifications in the context of therapeutic proteins, Nat. Biotechnol. 24 (2006) 1241-1252.