Assay for recombinant hepatitis B surface antigen using reversed-phase high-performance liquid chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Accurate quantification of recombinant hepatitis B surface antigen (rHBsAg) is crucial for vaccine production and therapeutic monitoring.
• Existing methods for rHBsAg analysis in complex matrices like yeast cell lysates can be time-consuming or lack sensitivity.

Purpose of the Study:

• To develop and compare high-performance liquid chromatography (HPLC) assays for monitoring rHBsAg mass in yeast cell lysates.
• To establish sensitive and efficient analytical methods for rHBsAg quantification.

Main Methods:

• Development of two distinct HPLC assays utilizing either a phenyl-ligated polymeric resin column or a silica-based octadecyl micropellicular column.
• One assay involved pre-derivatization with monobromobimane for fluorescence detection and separation using a dual gradient of acetic acid and acetonitrile.
• The second assay employed a rapid separation on the micropellicular column with an isopropanol/acetonitrile gradient at elevated temperature and high flow rate, without pre-derivatization.

Main Results:

• The derivatized rHBsAg was resolved in 17 minutes on the polymeric column.
• The micropellicular column assay achieved rHBsAg separation within 5 minutes, enabling sample re-injection every 10 minutes.
• Both assays demonstrated linear ranges for rHBsAg analysis and achieved detection limits between 10 and 50 ng per injection.

Conclusions:

• Both developed HPLC assays are effective for monitoring rHBsAg mass in yeast cell lysates.
• The micropellicular column assay offers a significantly faster analytical turnaround time, suitable for high-throughput applications.
• These methods provide sensitive and reliable quantification of rHBsAg, supporting bioprocess development and quality control.