The application of capillary electrophoresis for monitoring effects of excipients on protein conformation

Journal of Pharmaceutical and Biomedical Analysis
K A McIntoshS A Charman

Abstract

Studies were conducted to assess the utility of free solution capillary electrophoresis (CE) for monitoring the effects of selected excipients on the thermal denaturation of a model protein (Ribonuclease A, RNase A) at low pH. Thermal denaturation/unfolding experiments were conducted via temperature-controlled CE using a run buffer of 20 mM citric acid in the pH range of 2.3-3.1, with a marker peptide incorporated to correct for temperature-induced changes in endoosmotic flow. The effects of selected excipients on the thermal unfolding of RNase A were then evaluated by adding either sorbitol, sucrose, polyethylene glycol 400 (PEG 400) or 2-methyl-2,4-pentanediol (MPD) to the electrophoretic run buffer (pH 2.3). Confirmatory denaturation experiments were conducted under the same solution conditions using circular dichroism (CD) spectropolarimetry. Using temperature-controlled CE, an increase in solution pH from 2.3 to 2.7 and 3.1 resulted in an increase in transition temperatures of RNase A by approximately 8 and 13 degrees C, respectively. Similar shifts in transition temperatures were observed when thermal denaturation transitions were monitored by far-UV CD. Sorbitol (0.55-1.1 M) and sucrose (0.55 M) each shifted the denatura...Continue Reading

References

Feb 21, 1978·Biochemistry·E P Pittz, S N Timasheff
Jan 1, 1979·Advances in Protein Chemistry·P L Privalov
Jul 5, 1974·Journal of Molecular Biology·P L Privalov, N N Khechinashvili
May 1, 1974·Biophysical Chemistry·E I Tiktopulo, P L Privalov

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Citations

Mar 29, 2002·Electrophoresis·Ersilia De LorenziGabriele Caccialanza
May 18, 1999·Journal of Chromatography. a·E StellwagenP G Righetti
Aug 26, 1999·International Journal of Pharmaceutics·W Wang
Apr 10, 2007·Analytical Biochemistry·Earle StellwagenNancy C Stellwagen
Sep 5, 2002·Proteins·Earle StellwagenPier Giorgio Righetti
Nov 25, 2004·Journal of Chromatography. a·Hui XuHong-Yuan Chen

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