Characterization of polyethylene glycol modified proteins using charge-reversed capillary electrophoresis

Robert L. Cunico, Victoria Gruhn, Lilia Kresin, Danute E. Nitecki, John E. Wiktorowicz

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

A capillary electrophoretic method employing a charge reversal technique [J. E. Wiktorowicz and J. C. Colburn, Electrophoresis, 11 (1990) 769] has been developed to characterize polyethylene glycol (PEG)-proteins. A removable coating is applied to a standard fused-silica capillary in order to change the negative charge of the capillary surface to a positive charge. This prevents the adsorption of basic and PEG-proteins. The positive electrode is positioned at the detector end of the capillary, orienting electroosmotic flow towards the detector. Automated reconditioning procedures prior to each analysis give relative standard deviations in migration time and area of less than 2%, with most analysis times under 20 min. As the number of PEGs conjugated to a protein increases, the net positive charge and migration time of the copolymer decrease. Resulting peak widths are broad, reflecting the broad molecular mass distribution of PEGs and the heterogeneous nature of the PEG conjugates. This method can be used to monitor process steps, optimize reaction conditions, determine extent of modification, or assess product quality and consistency. Examples of PEG derivatized molecules characterized in our laboratory by charge-reversed capillary electrophoresis are tryptophan, Lys-Trp-Lys, lysozyme, myoglobin, RNase and immunoglobulin G; the size of the attached monomethoxy-PEG molecules varied from 0.15 kDa to 10 kDa (103 dalton).

Original languageEnglish (US)
Pages (from-to)467-477
Number of pages11
JournalJournal of Chromatography A
Volume559
Issue number1-2
DOIs
StatePublished - Oct 18 1991
Externally publishedYes

Fingerprint

Capillary electrophoresis
Capillary Electrophoresis
Proteins
lysyl-tryptophyl-alpha-lysine
Polyethylene glycols
Electroosmosis
Detectors
Molecules
Myoglobin
Molecular mass
Fused silica
Ribonucleases
Muramidase
Electrophoresis
Tryptophan
Silicon Dioxide
Adsorption
Electrodes
Copolymers
Immunoglobulin G

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry
  • Molecular Medicine

Cite this

Characterization of polyethylene glycol modified proteins using charge-reversed capillary electrophoresis. / Cunico, Robert L.; Gruhn, Victoria; Kresin, Lilia; Nitecki, Danute E.; Wiktorowicz, John E.

In: Journal of Chromatography A, Vol. 559, No. 1-2, 18.10.1991, p. 467-477.

Research output: Contribution to journalArticle

Cunico, Robert L. ; Gruhn, Victoria ; Kresin, Lilia ; Nitecki, Danute E. ; Wiktorowicz, John E. / Characterization of polyethylene glycol modified proteins using charge-reversed capillary electrophoresis. In: Journal of Chromatography A. 1991 ; Vol. 559, No. 1-2. pp. 467-477.
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