Abstract:

Cervical cancer is the fourth most common cancer among women. Human papilloma virus (HPV) is the most common cause of cervical cancer which accounts for 5% of all human cancers and results in about 528000 cases and 266000 deaths every year. HPV vaccines are considered the most effective strategy for the prevention of HPV infection and cervical carcinoma. Since 2006, three prophylactic vaccines against HPV have been available on the market, including bivalent vaccines, quadrivalent vaccines, and nine-valent vaccines. Among them, nine-valent vaccines have been reported to be the most effective. They can prevent 97% of the high-grade pre-cancer lesions. Virus-like particles (VLPs), which are arranged as 360 copies of capsid proteins L1, are the only antigens of the HPV vaccine. Nine-valent HPV vaccines are prepared by mixing nine types of VLPs with adjuvants. Thus, the quality of the VLPs, including their stability and content in the HPV bulk, is very important for developing HPV vaccines. In this study, a method was developed for the determination of the nine types of VLPs (HPV6/11/16/18/31/33/45/52/58) in HPV bulk by size exclusion chromatography (SEC). The parameters of this method were optimized in terms of column brand, pore size of stationary phase particles, buffer concentration, and pH value. SHIMSEN Ankylo SEC-300 column (300 mm×7.8 mm, 3 μm) combined with a buffer aqueous solution containing 300 mmol/L NaCl and 50 mmol/L phosphate (pH 7.0) was utilized to separate the VLPs from the matrix since a narrow peak shape and good repeatability for VLPs could be obtained with this column and mobile phase. The optimized method had a wide linear range, good repeatability (RSDs of peak area were not more than 5.0%), and a satisfactory sensitivity (LOQs in the range of 4.58-15.24 μg/mL). The optimized method was used to determine the VLPs in the HPV bulk. The LOQs of the current method were much lower than the content of the nine types of VLPs in the HPV bulk, indicating that this method was sensitive enough for the determination of the nine types of VLPs in the HPV bulk. The method was also used to determine the VLPs in an HPV bulk that had been stored at 4 ℃ for one week. A decrease in the nine types of VLPs in the range of 10.0%-62.6% was observed after they were stored at 4 ℃ for one week. An HPV vaccine was prepared by mixing the VLPs with an adjuvant. Thereafter, the VLPs were adsorbed on the surface of the adjuvant. The developed method was applied to determine the free VLPs in twelve batches of HPV vaccines from three different manufacturers. No obvious free protein was detected in the twelve batches of the HPV vaccines from the three manufacturers, indicating that VLPs from these manufactures react well with their aluminum adjuvant. Folin-phenol (Lowry assay) is commonly used for the determination of proteins in vaccines. It is based on the reduction of phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 650 nm. The Lowry method was sensitive to interfering substances. Most interfering substances caused a lower color yield, while some detergents caused a slight increase in color. To reduce the effect of the interfering substances, a procedure for precipitating the proteins was usually required before the sample was tested. Thus, the Lowry assay is complex, time-consuming, and of low selectivity. Compared to the Lowry method, the method we developed is simpler and more automatic. It is a high-throughput method of determining VLPs. It can be used to determine VLPs in HPV bulk and free VLPs in HPV vaccines.

Key words: size exclusion chromatography (SEC), quantitative analysis, human papilloma virus (HPV), virus-like particle (VLP), vaccine