SIZE AND CONFORMATIONAL STABILITY OF THE HEPATITIS-A VIRUS USED TO PREPARE VAQTA, A HIGHLY PURIFIED INACTIVATED VACCINE

Citation
Db. Volkin et al., SIZE AND CONFORMATIONAL STABILITY OF THE HEPATITIS-A VIRUS USED TO PREPARE VAQTA, A HIGHLY PURIFIED INACTIVATED VACCINE, Journal of pharmaceutical sciences, 86(6), 1997, pp. 666-673
Citations number
41
Categorie Soggetti
Chemistry,"Pharmacology & Pharmacy
ISSN journal
00223549
Volume
86
Issue
6
Year of publication
1997
Pages
666 - 673
Database
ISI
SICI code
0022-3549(1997)86:6<666:SACSOT>2.0.ZU;2-3
Abstract
A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-i nactivated and adsorbed to aluminum salts for use as the vaccine VAQTA ). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformationa l stability of HAV was determined by a combination of intrinsic HAV pr otein fluorescence, fluorescent probes of both RNA and protein, and UV -visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by similar to 10 degrees C. Salt concentrati ons above 0.2 M, however, decrease the solubility of HAV. The effect o f pH on the physical properties of HAV particles was monitored by dyna mic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversibl e association/aggregation at pH values below 6 with the concomitant ex posure of previously buried hydrophobic surfaces below pH 4. These res ults are in good agreement with previous studies of HAV thermal stabil ity under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infec tivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thu s providing a molecular basis with which to monitor HAV stability duri ng purification and storage.