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ATR/FT-IR Spectroscopic Technique for the Estimation of the Methoxy Polyethylene Glycol 5000 Content of the Methoxy Polyethylene Glycol 5000 Modified Protein Bovine Cupri-Zinc Superoxide Dismutase

Volume 50, Number 12 (Dec. 1996) Page 1603-1605

Severdia, Anthony G.; Bullock, John; Johnston, David


The modification of the protein bovine cupri-zinc superoxide dismutase (SOD) by methoxy polyethylene glycol 5000 (MeO-PEG 5000) has been shown to increase the effective pharmacological lifetime of SOD in vivo while maintaining physiological activity. Polyethylene glycol modification has been extensively employed, for similar purposes, in protein chemistry, peptide chemistry, and polymeric drug development. The modification of SOD is accomplished by the reaction of a number of its lysine residues with an active ester of MeO-PEG 5000. The extent of the modification of the SOD must be ascertained for a number of reasons: optimization of activity, control of in vivo half-life, and manufacturing uniformity, as well as regulatory concerns. The classical method used to determine the extent of protein modification by polyethylene glycols has been a titration with trinitrobenzene sulfonic acid (TNBS). Other techniques which have been employed to determine SOD modification include MALDI mass spectrometry, capillary zone electrophoresis (CZE), and ester hydrolysis with subsequent determination of protein and MeO-PEG 5000 by size-exclusion chromatography (SEC). This report describes an attenuated total reflectance (ATR) infrared technique which estimates the MeO-PEG 5000 content of the modified SOD by measuring absorbance intensity of the C-O-C stretch near 1090 cm-1 in its infrared spectrum. The spectroscopic technique requires no preparation of the modified protein sample, and the spectrum is determined in the saline/phosphate buffer drug formulation.