Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

Abstract

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…However, these techniques offer insufficient sensitivity for the detection of PTMs and differentiation of many proteoforms. Studies have shown that even highly pure isolates of a single protein pharmaceutical -recombinant human interferon-β1 (rhINF-β1; Avonex, Biogen) -exist as hundreds of individual proteoforms distributed across a broad dynamic range [1, 25,26]. Such proteoforms may even be combinations of several different PTMs, differentially affecting biological activity [1].…”

“…Because there is no sample dilution, as occurs in sheath-flow systems, the sheathless interface is more sensitive [235][236][237]. We [238] and others [229,239] have demonstrated the potential of high resolution CZE coupled to high resolution MS to analyze intact complex glycoproteins. Recently, groups have shown that CZE-MS of intact proteins (MS 1 ) can be used to analyze complex protein mixtures [233,[240][241][242], but much development remains in this area as well as top-down analysis of glycoproteins.…”

“…We use a capillary positive coating, generating between 350,000 and 450,000 total plates, with high migration reproducibility, based on apparent mobility of solutes. Although intact protein analysis of 80 proteoforms of rhIFN-β1 by CZE-MS has been reported [229], in the present study, we have significantly improved the separation and analysis of the proteoforms, leading to the determination of 138 individual proteoforms, some of which were identified via the selective digestion by several exoglycosidases. Besides elucidating the main glycoforms of Avonex, we have observed intact separation of glycan positional isomers of the biantennary mono-sialic acid glycan as well as triantennary glycans with the third antenna attached to either the α(1-3) or α(1-6) arm.…”

“…Efforts are being made to develop LC columns suitable for high resolution intact protein separation [228]. At the same time, capillary zone electrophoresis (CZE) represents an alternative and complementary separation approach based on charge and hydrodynamic volume, with the potential for ultra-high efficiency [229][230][231].…”

“…RhIFN-β1 has known modifications, such as complex glycosylation, deamidation and succinimide, as well as oxidation [208,229,244,245]. Here, CZE-MS is demonstrated to successfully characterize and quantitate individual molecular proteoforms with high-resolution accurate mass analysis.…”