Key result
Combined pretreatment with chloroform and benzonase was optimal for crude virus infection supernatant, while sole pretreatment with benzonase was beneficial for PEG-precipitated samples.
Why the study?
SE-HPLC quantification of 146S antigens in foot-and-mouth disease vaccines is limited to purified samples due to interfering signals, creating a need for optimal pretreatment methods in less purified samples.
Optimal pretreatment methods using chloroform and benzonase improve the SE-HPLC quantification of FMD vaccine antigens in less purified samples.
May facilitate FMD vaccine potency testing; leaves open full regulatory replacement of SDG.
Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is controlled by vaccine policy in many countries. For vaccine potency, the content of intact virus particles (146S antigens) is critical, and the sucrose density gradient (SDG) fractionation is the gold standard for the quantification of 146S antigens. However, this method has several drawbacks. Although size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace the classic method, its application is generally confined to purified samples owing to the interfering signals. Therefore, we aimed to develop optimal pretreatment methods for SE-HPLC quantification in less purified samples. Crude virus infection supernatant (CVIS) and semi-purified samples with PEG precipitation (PEG-P) were used. Chloroform pretreatment was essential to remove a high level of non-specific signals in CVIS, whereas it caused loss of 146S antigens without the distinctive removal of non-specific signals in PEG-P. Benzonase pretreatment was required to improve the resolution of the target peak in the chromatogram for both CVIS and PEG-P. Through spiking tests with pure 146S antigens, it was verified that the combined pretreatment with chloroform and benzonase was optimal for the CVIS, while the sole pretreatment of benzonase was beneficial for PEG-P.
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Kim et al. (2021) studied Foot-and-mouth disease (FMD) vaccine antigens. Pretreatment methods (chloroform and benzonase) was evaluated on Optimal pretreatment for SE-HPLC quantification of 146S antigens in CVIS and PEG-P samples. Combined pretreatment with chloroform and benzonase was optimal for crude virus infection supernatant, while sole pretreatment with benzonase was beneficial for PEG-precipitated samples.
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