Validation of Pretreatment Methods for the In-Process Quantification of Foot-and-Mouth Disease Vaccine Antigens

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...

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Published inVaccines (Basel) Vol. 9; no. 11; p. 1361
Main Authors Kim, Ah-Young, Park, Sun Young, Park, Sang Hyun, Jin, Jong Sook, Kim, Eun-Sol, Kim, Jae Young, Park, Jong-Hyeon, Ko, Young-Joon
Format Journal Article
LanguageEnglish
Published Basel MDPI AG 19.11.2021
MDPI
Subjects
Antibodies
Antigens
Chloroform
Chromatography
Foot & mouth disease
foot-and-mouth disease (FMD)
Fractionation
High performance liquid chromatography
Infections
Liquid chromatography
Polyethylene glycol
Pretreatment
Proteins
Quality control
quantification
SE-HPLC
Sucrose
vaccine antigen
Vaccines
Viruses
United States > US
Japan
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Summary: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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ISSN:2076-393X
2076-393X
DOI:10.3390/vaccines9111361