Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles.

Recombinant hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells were stored at various conditions for 12-18 months in their naked form or adsorbed to alum (HBV vaccine). The physical, chemical and immunological parameters during storage at -20 degrees C, 4 degrees C, room temperature and 37 degrees C were investigated. HBsAg particles fully retained the original peptide composition when stored for 6 months, as a dispersion, at -20 degrees C and 4 degrees C; and as lyophilized powder, at all four temperatures. Ten percent sucrose preserved the size, shape and protein content of the naked particles stored at 4 degrees C and -20 degrees C for 18 months as a dispersion or lyophilized. Lyophilization in the presence of glucose, sucrose and trehalose, but not mannitol, further improved the 4 degrees C stability of size, shape and the protein content during 2 years of storage. Stability of the particle's lipid components was inferior to that of the protein components. Dispersions of naked HBsAg and of particles lyophilized in the presence of sucrose and stored at -20 degrees C were the only forms in which the lipid content and composition, including the lipid polyunsaturated acyl chains, were preserved for at least 18 months storage. The level of phospholipid and free cholesterol were most stable in the HBV vaccine which was stored at 4 degrees C; they did not change after 1 year of storage. Preservation of immunogenicity was evaluated according to dose-dependent changes in S-specific antibody titers in sera obtained from immunized BALB/c mice. The ED50 for achieving seroconversion was 0.07 microg/ml/mouse, indicating that the vaccine is very immunogenic. Freezing or freeze-drying of the HBV vaccine results in the total loss of vaccine immunogenicity (in spite of the good chemical stability), while full immunological potency was retained for at least 2.5 years at 4 degrees C. Storing formulated vaccine at 25 and 37 degrees C for 4 and 2 weeks, respectively, did not alter the vaccine potency. This study suggests that the vaccine's physical, chemical and immunological characteristics are sufficiently stable at high temperatures to reduce the need for 'cold chain' transportation.

Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): composition, structure and immunogenicity.

The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared. The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols). Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions. CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide. The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality. Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg. The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure. Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes. The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.