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
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.