Capillary electrophoretic separation of poly(ethylene glycol)-modified granulocyte-colony stimulating factor

Abstract

We evaluated the utility of capillary electrophoretic methods for analyzing poly(ethylene glycol) (PEG)-modified granulocyte-colony stimulating factor (G-CSF), a long-acting form of GCSF for the treatment of cancer therapy-induced neutropenia. Low- and high-molecularweight PEG-G-CSF conjugates prepared with aldehyde-activated PEG-5K and PEG-20K were separated by high-performance size-exclusion chromatography (HP-SEC), capillary zone electrophoresis (CZE), and sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE). HPSEC showed low resolution for separating mono- and di-PEG-G-CSFs. SDS-CGE had higher resolution, but required a long analysis and had low peak efficiency. CZE could successfully separate both PEG-5K- and PEG-20K-conjugated G-CSFs with a running time of 20 min and high peak efficiency. In conclusion, CZE was better than SDS-CGE for separating PEG-G-CSF conjugates and will be useful for PEGylation studies, such as reaction monitoring for optimization of the PEGylation reaction, and purity and stability tests of PEG-G-CSF.

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

“…So far, various analytical methods, such as SDS-PAGE, reversed-phase HPLC, SEC, and MS, have been applied to characterize PEG-G-CSF [5,6]. In our pursuit of improved analytical methods with regard to separation capacity, speed, and sample consumption, we recently reported CE methods of PEG-G-CSF [7]. Although the CE methods showed satisfactory separation capacity, they were still time-consuming, requiring 10-40 min for separation and additional time between injections for rinsing and reloading the run buffer.…”

Development of a chamber system for rapid, high yield and cost-effective purification of deoxyribonucleic acid fragments from agarose gel

“…[678] Some documents developed electro elution into troughs excised ahead of band, filled with polyethylene glycol. [9] Disadvantages of this procedure are the need for constant vigilance and removing buffer from the trough frequently. Stabile and Wurtzel designed electro elution into wells cut out on the left side of the band.…”

Applications of capillary electrophoresis in characterizing recombinant protein therapeutics

“…Lee et al. carried out a study on the PEG‐modified granulocyte colony‐stimulating factor (G‐CSF). Size exclusion chromatography showed low resolution for separating mono‐ and di‐PEG‐G‐CSFs.…”