High Resolution CZE-MS Quantitative Characterization of Intact Biopharmaceutical Proteins: Proteoforms of Interferon-β1

Abstract

New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-β1 (Avonex, rhIFN-β1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms.

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

“…[12][13][14] Mass spectrometry techniques are often coupled with chromatographic separation techniques producing methods that have very high analyte resolution which are often the best means of characterizing complex biological samples in detail. Again, these combined MS/chromatography technologies are widely adopted and are the subject of much scientific literature (e.g., HPLC-MS, 15,16 UHPLC-MS, 17,18 CE-MS 19,20 ). Chromatographic and MS-based techniques are well understood, very effective, and widely used; they do, however, have several significant drawbacks.…”

The glycosylation aspects of biosimilarity

“…N-glycosylation analysis of therapeutic proteins can be performed at three different levels (intact protein, glycopeptide, and released glycans), as depicted in Figure 3 [64]. Intact glycoproteins can be analyzed by mass spectrometry (MS) via direct infusion or coupling with separation techniques, such as liquid chromatography (LC)-MS or capillary electrophoresis (CE)-MS [65,66]. Such top-level analysis also offers information about glycan pairing on the two heavy chains of an IgG molecule.…”

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

“…As a result, comprehensive characterization of a single isolated protein -let alone a complex mixture -is highly challenging, requiring separation prior to high-resolution high mass accuracy MS analysis. We and others have recently demonstrated the power of CZE-MS in high-resolution separations of pharmaceutical glycoproteins [297][298][299]. In our study, a sheathless cross-linked polyethyleneimine-coated capillary coupled to the Orbitrap Elite MS was used to separate proteoforms of recombinant human interferon-β1 (Avonex), resulting in the detection of 138 proteoforms and the separation of deamidated, sialylated, truncated species, and positional glycoform isomers [299].…”

“…We and others have recently demonstrated the power of CZE-MS in high-resolution separations of pharmaceutical glycoproteins [297][298][299]. In our study, a sheathless cross-linked polyethyleneimine-coated capillary coupled to the Orbitrap Elite MS was used to separate proteoforms of recombinant human interferon-β1 (Avonex), resulting in the detection of 138 proteoforms and the separation of deamidated, sialylated, truncated species, and positional glycoform isomers [299]. Nanoflow (<1 µL/min) and, especially, ultra-low flow (≤25 nL/min) separation techniques coupled to nanoESI are highly desirable for native and top-down analyses of protein structures where an MS signal is divided into multiple channels corresponding to ion species of different charge states [300,301].…”