Sodium dodecyl sulfate‐capillary gel electrophoresis of polyethylene glycolylated interferon alpha

Abstract

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution.

Application of microchip CGE for the analysis of PEG‐modified recombinant human granulocyte‐colony stimulating factors

“…The MWs determined by MALDI-TOF MS were relatively consistent with the calculated MWs based on molecular information. On the other hand, the overestimation of MW of PEGylated proteins in MCGE is caused by the large hydrodynamic radius of PEG molecule, which leads to delayed migration of PEGylated proteins compared with the globular protein, as reported previously in SDS-PAGE and SDS-CGE of PEGylated proteins [12]. The reproducibility of the molecular sizing by MCGE was good with relative standard deviations of less than 0.7 and 3.4% (n 5 3) in Protein 80 and 230 Labchips, respectively.…”

“…MALDI-TOF MS was carried out using a Voyager-DE STR Biospectrometry Workstation (Applied Biosystems, Foster City, CA, USA), as described previously [12,13]. Linear, positive-ion TOF detection was performed and a saturated solution of sinapinic acid in 70% acetonitrile containing 0.1% trifluoroacetic acid as the final concentration was used as the matrix solution.…”

PEGylation of Octreotide: I. Separation of Positional Isomers and Stability Against Acylation by Poly(D,L-lactide-co-glycolide)

“…In general, PEGylation results in heterogeneity of the molecule with respect to distribution in both the number and position of the attached PEG molecules as well as the inherent polydispersity of PEG itself (15,16). To prepare a homogeneous PEGylated peptide, Felix et al proposed the sitespecific conjugation of PEG to the growth hormone-releasing factor 1-29 by solid-phase peptide synthesis (17).…”

Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

“…Both techniques are widely used to separate PEGylated proteins on the laboratory scale, but are mainly used for analytical purposes [26]. PAGE has been used to analyze and characterize PEGylation reactions using mixtures of stains to visualize reaction products and unreacted PEG [27][28][29]. The technique has been extremely useful in the separation of conjugates for subsequent mass and structure analysis.…”