Stability Investigation Of Virus-like Particle By Asymmetrical Flow Field-Flow Fractionation

Virus-like particles are self-assembled macromolecules consisting of virus capsid proteins but devoiding of genetic materials. They mimic the morphology of the true virus and could act as vaccine. For example, hepatitis B surface antigen (HBsAg), an important virus-like particle, has been used for vaccination to block the transmission of the hepatitis B virus. In the production and transportation processes, HBsAg would face several harsh environmental factors such as change of the salt solution, temperature or repeated freezing-thawing, which could induce aggregation and deactivation of HBsAg and influence badly the safety and validity of the vaccine. Unfortunately, until now, there is no systemic report concerning the stability of HBsAg. In this work, we employed asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle laser light scattering (MALLS), as a first attempt, to characterize the HBsAg virus like particles and studied the stabilities of two important HBsAg VLP products (expressed respectively by Chinese Hamster Ovary (CHO) cell and Hansenula polymorpha (Hans) yeast cell) under different environmental conditions which may occur during their purification, production, transportation and storage processes. The main results are following:1) The AF4 fractionation method for HBsAg and its aggregates was optimized and established as:① injection and focus step:injection flow was 0.2 ml/min, focus flow was 1.8 ml/min, crossflow was 1.0 ml/min, time was 4 min, the transition time between focus step and elution step was 1 min, detector flow was constant at 1.0 ml/min; ② elution step:crossflow was constant at 1.0 ml/min for 40 min and then decreased to zero linearly in 5 min; ③ rinse step:kept channel flow of 1.0 ml/min for 5 min. The spacer height was 350μm and the membrane was regenerated cellulose with cutoff Mw of 10 kDa. The molecular weight and hydrodynamic diameter of CHO-HBsAg and Hans-HBsAg characterized by AF4-MALLS was 4727 kDa and 3039 kDa,29.4 nm and 22.8nm respectively.2) The aggregation behaviours of CHO-HBsAg and Hans-HBsAg in different solution environment were studied by AF4-MALLS and the antigenicy was analyzed by ELISA kit. Under condition of 2 M ammonium sulfate, CHO-HBsAg was stable with antigenicity keeping above 95%, while Hans-HBsAg aggregated heavily to large particles with molecular weight about 105-106 kDa and kept only 25% antigenicity. During freeze-thaw cycles, CHO-HBsAg had little change in structure and antigenicity. By contrast, Hans-HBsAg aggregated heavily and kept only 26.9% antigenicity. After the experiment of high temperature at 60℃, CHO-HBsAg was still stable but the antigenicity of Hans-HBsAg was below 5%.3) The stability of Hans-HBsAg in NaCl and (NH4)2SO4 was researched and the aggregation behavior was discussed, based on which a model concerning aggregation of Hans-HBsAg was established. Hans-HBsAg aggregated reversibly into oligomeric particle (OP) in NaCl solution, accompanied by a slight loss of antigenicity. After removing NaCl by dialysis, both particle form and antigenicity could recover to original state. The aggregation of HBsAg in (NH4)2SO4 solution was more profound and complicated. At low concentration (<0.5M) and short incubation time (<1 hour), most aggregates of HBsAg VLP were in OP form, and could be disaggregated when the salt was removed. However, as the concentration increased, the OP further aggregated into polymeric particles (PP) with greater size, accompanied by severe loss of antigenicity. Removal of (NH4)2SO4 by dialysis could not restore the PP into original HBsAg form, and the antigenicity was lost irreversibly.4) Effect of several additives on the stability and antigenicity of Hans-HBsAg was studied. Arginine could suppress the aggregation of Hans-HBsAg in salt solution and the suppressing effect was related with the concentration of Arginine. When the concentration of Arginine reached 1M, the suppressing effect was satisfactory. Urea and GdmHCl would not prevent the aggregation of Hans-HBsAg. Conversely, they can promote Hans-HBsAg to aggregate. Polyethylene (PEG) had almost no apparent effect on the antigenicity of Hans-HBsAg. Different surfactant exhibited different effect in antigenicity of Hans-HBsAg, Tween 80 could improve the antigenicity of Hans-HBsAg while Triton 100 could decrease the antigenicity of Hans-HBsAg.