Effects of pH, Temperature, and Sucrose on Benzyl Alcohol-Induced Aggregation of Recombinant Human Granulocyte Colony Stimulating Factor

• Published in: Journal of pharmaceutical sciences Vol. 95; no. 7; pp. 1480 - 1497
• Main Authors: Thirumangalathu, Renuka, Krishnan, Sampathkumar, Brems, David N., Randolph, Theodore W., Carpenter, John F.
• Format: Journal Article
• Language: English
• Published: Hoboken Elsevier Inc 01.07.2006; Wiley Subscription Services, Inc., A Wiley Company; Wiley; American Pharmaceutical Association
• Subjects: aggregation; benzyl alcohol; Benzyl Alcohol - chemistry; Biological and medical sciences; Chromatography, Gel; Circular Dichroism; Deuterium Exchange Measurement; General pharmacology; Granulocyte Colony-Stimulating Factor - chemistry; Humans; Hydrogen-Ion Concentration; Medical sciences; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; rhGCSF; Spectrophotometry, Infrared; Sucrose - chemistry; Temperature; pH; temperature; aggregation; benzyl alcohol; rhGCSF; Aggregation; Human; Granulocyte colony stimulating factor; Temperature; Pharmaceutical technology; Sucrose; Temperature effect; Benzyl alcohol
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.20619

Antimicrobial preservatives (e.g., benzyl alcohol), which are required in multidose formulations, can induce protein aggregation. In this study, the mechanism of benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor (rhGCSF) was investigated by determining the effects of temperature, pH, and sucrose on this process. rhGCSF was incubated at 25 and 37°C and at pH 7.0 (phosphate-buffered saline, PBS) and pH 3.5 (HCl). Benzyl alcohol (0.9% w/v) accelerated aggregation of rhGCSF at pH 7.0, an effect that was much greater at 37°C than at 25°C and partially counteracted by 1.0M sucrose. At pH 3.5, benzyl alcohol did not induce aggregation of rhGCSF. Spectroscopic studies showed that 0.9% benzyl alcohol altered the tertiary structure of rhGCSF at both pH, without detectably altering secondary structure. Structural perturbation was greater at 37°C than at 25°C. At both pH 7.0 and 3.5, the hydrogen-deuterium (H–D) exchange rate for rhGCSF was increased by 0.9% benzyl alcohol. Sucrose (1.0 M) partially counteracted the benzyl alcohol-induced perturbation of tertiary structure and the increase in H–D exchange rate. Thus, benzyl alcohol accelerates aggregation of rhGCSF at pH 7.0, because it favors partially unfolded aggregation-prone conformations of the protein. Sucrose partially counteracts benzyl alcohol-induced rhGCSF aggregation by shifting the molecular population away from these species and towards more compact conformations. We postulate that the absence of aggregation at pH 3.5, even with benzyl alcohol-induced structural perturbation, is due to the unfavorable energetics of intermolecular interactions (i.e., colloidal stability) between rhGCSF molecules at this pH. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association.