Capillary electrophoresis-mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals

Abstract

In this work, the usefulness of capillary electrophoresis–electrospray ionization time-of-flight–mass spectrometry for the analysis of biopharmaceuticals was studied. Noncovalently bound capillary coatings consisting of Polybrene-poly(vinyl sulfonic acid) or Polybrene-dextran sulfate-Polybrene were used to minimize protein and peptide adsorption, and achieve good separation efficiencies. The potential of the capillary electrophoresis-mass spectrometry (CE-MS) system to characterize degradation products was investigated by analyzing samples of the drugs, recombinant human growth hormone (rhGH) and oxytocin, which had been subjected to prolonged storage, heat exposure, and/or different pH values. Modifications could be assigned based on accurate masses as obtained with time-of-flight–mass spectrometry (TOF-MS) and migration times with respect to the parent compound. For heat-exposed rhGH, oxidations, sulfonate formation, and deamidations were observed. Oxytocin showed strong deamidation (up to 40%) upon heat exposure at low pH, whereas at medium and high pH, mainly dimer (>10%) and trisulfide formation (6–7%) occurred. Recombinant human interferon-β-1a (rhIFN-β) was used to evaluate the capability of the CE-MS method to assess glycan heterogeneity of pharmaceutical proteins. Analysis of this N-glycosylated protein revealed a cluster of resolved peaks which appeared to be caused by at least ten glycoforms differing merely in sialic acid and hexose N-acetylhexosamine composition. Based on the relative peak area (assuming an equimolar response per glycoform), a quantitative profile could be derived with the disialytated biantennary glycoform as most abundant (52%). Such a profile may be useful for in-process and quality control of rhIFN-β batches. It is concluded that the separation power provided by combined capillary electrophoresis and TOF-MS allows discrimination of highly related protein species.

Analytical characterization of NOTA-modified somatropins

“…The CE-MS analysis was performed using fused-silica capillaries (800 mm × 50 µm I.D., Polymicro Technologies, Phoenix, AZ, USA) coated with a bilayer of Polybrene and poly (vinyl sulfonic acid) as described previously [37]. Sample injections were performed at 1 psi for 12 s. Molecular weight determinations of proteins were performed using the "Charge Deconvolution" utility of the Data Analysis software.…”

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].…”

“…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].…”

“…An alternative high resolution technique is capillary zone electrophoresis (CZE), which separates analytes based on differences in charge and hydrodynamic volume. CZE has been demonstrated to be a high efficiency separation method for the analysis of proteins [25,26,274,275]. In contrast to LC (except low resolution SEC), CZE does not involve interaction of the analyte with the stationary phase, thus representing a complementary separation approach.…”

“…An alternative approach is the use of a sheathless junction, in which the background electrolyte (BGE) is electrosprayed directly from the separation capillary without dilution from the sheath liquid [65,67]. A commercial sheathless system (CESI) has been introduced and shown to be highly sensitive for the analysis of peptides and proteins, including glycoproteins [1, 25,26]. A recent study from our laboratory using the CESI interface has demonstrated the separation of recombinant human interferon beta-1 (rhINF-β1, ~23,000 Da), where 138 proteoforms were detected and 55 quantitated [1].…”