title: The application of capillary electrophoresis for monitoring effects of excipients on protein conformation.
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 denaturation transition temperatures of RNase A to higher values, whereas PEG 400 and MPD had minimal effect on the unfolding transition midpoint at the concentrations evaluated (0.55 M for each). The observed changes in the transition temperatures for RNase A as a function of pH and selected excipients were similar when measured by either CE or far-UV CD. These results support the utility of CE for monitoring the effects of neutral excipients on the thermal denaturation of a model protein under selected conditions. The widespread utility of the technique may be limited by the narrow temperature range of most commercial CE instruments and the need to use extreme pH conditions to monitor the complete denaturation transition.
authors.affiliation: Department of Pharmaceutics, Victorian College of Pharmacy, Monash University, Parkville, Victoria, Australia.
authors.forename: K A
authors.initials: KA
authors.lastname: McIntosh
authors.forename: W N
authors.initials: WN
authors.lastname: Charman
authors.forename: S A
authors.initials: SA
authors.lastname: Charman
mesh_terms: D002021:Buffers; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary; D005079:Excipients / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D002021:Buffers; D005079:Excipients; D012259:Ribonuclease, Pancreatic