Physicochemical studies on the stability of influenza haemagglutinin in vaccine bulk material.
The relative unknown conformational stability of monovalent bulks of influenza virus haemagglutinin (HA) from three different strains (B/Guangdong, A/New Caledonia and A/Panama) was investigated with fluorescence and circular dichroism (CD) spectroscopy. Various stress conditions (concentration of denaturant, freeze-thawing, pH and temperature) affected the spectroscopic properties of the haemagglutinin proteins differently. Unfolding experiments revealed a poor stability of Guangdong haemagglutinin (GD-HA) in comparison with New Caledonia (NC-HA) and Panama haemagglutinin (P-HA). Freeze-thawing altered the secondary and tertiary structure of Guangdong haemagglutinin and only the tertiary structure of Panama haemagglutinin. From pH 4.6-9.2 the tertiary structures of Guangdong, New Caledonia and Panama haemagglutinin were all affected to a different extent. The secondary structure was only altered at low pH. Incubation of haemagglutinin at 60 degrees C resulted in denaturation of the protein and a dramatic change of the fluorescence spectrum, indicative of oxidised tryptophan (Trp). In conclusion, fluorescence and circular dichroism spectroscopy are highly suitable techniques to monitor the stability of haemagglutinin in a straightforward and fast way.
Center for Biological Medicines and Medical Technology, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands. dion.kuykx@rivm.nl
Dion M A M
DM
Luykx
Marco G
MG
Casteleijn
Wim
W
Jiskoot
Janny
J
Westdijk
Peter M J M
PM
Jongen
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D004355:Drug Stability; D005615:Freezing; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007202:Indicators and Reagents; D007252:Influenza Vaccines / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007202:Indicators and Reagents; D007252:Influenza Vaccines