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. 2021 Nov 19;9(11):1361.
doi: 10.3390/vaccines9111361.

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

Ah-Young Kim  1 , Sun Young Park  1 , Sang Hyun Park  1 , Jong Sook Jin  1 , Eun-Sol Kim  1 , Jae Young Kim  1 , Jong-Hyeon Park  1 , Young-Joon Ko  1
Affiliations

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

Ah-Young Kim et al. Vaccines (Basel). .

Abstract

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.

Keywords: SE-HPLC; foot-and-mouth disease (FMD); pretreatment; quantification; vaccine antigen.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Purity of 146S antigen peak fractions collected by SE-HPLC from the 10× concentrates for the crude virus infection supernatant (CVIS) of FMDV O SKR/Boeun/2017: (a) original chromatograms from the SE-HPLC of CVIS (10×) without pretreatment (NPC); (b) original chromatogram from SE-HPLC of CVIS (10×) with benzonase digestion (B+); (c) original chromatogram from SE-HPLC of CVIS (10×) with combinational pretreatment of chloroform and benzonase (C+B+); (d) original chromatogram from SE-HPLC of CVIS (10×) with chloroform extraction (C+). Yellow backgrounds indicate the collected target peak fractions; (e) a silver-stained gel after SDS-PAGE of SE-HPLC target peak fraction dependent on each pretreatment method; (f) Western blot result against FMDV type O VP1 on SE-HPLC target peak fraction dependent on each pretreatment method; (g) dsDNA removal rate (%) of each target peak fraction of CVIS (10×) pretreated with various methods. Groups that do not share a letter are significantly different (p < 0.05). Abbreviations: SE-HPLC, size-exclusion high-performance liquid chromatography; M, marker.
Figure 1
Figure 1
Purity of 146S antigen peak fractions collected by SE-HPLC from the 10× concentrates for the crude virus infection supernatant (CVIS) of FMDV O SKR/Boeun/2017: (a) original chromatograms from the SE-HPLC of CVIS (10×) without pretreatment (NPC); (b) original chromatogram from SE-HPLC of CVIS (10×) with benzonase digestion (B+); (c) original chromatogram from SE-HPLC of CVIS (10×) with combinational pretreatment of chloroform and benzonase (C+B+); (d) original chromatogram from SE-HPLC of CVIS (10×) with chloroform extraction (C+). Yellow backgrounds indicate the collected target peak fractions; (e) a silver-stained gel after SDS-PAGE of SE-HPLC target peak fraction dependent on each pretreatment method; (f) Western blot result against FMDV type O VP1 on SE-HPLC target peak fraction dependent on each pretreatment method; (g) dsDNA removal rate (%) of each target peak fraction of CVIS (10×) pretreated with various methods. Groups that do not share a letter are significantly different (p < 0.05). Abbreviations: SE-HPLC, size-exclusion high-performance liquid chromatography; M, marker.
Figure 2
Figure 2
Purity of 146S antigen peak fractions collected by SE-HPLC from the 10× concentrates for PEG precipitate (PEG-P) of FMDV O SKR/Boeun/2017: (a) original chromatograms from SE-HPLC of PEG-P (10×) without pretreatment (NPC); (b) Original chromatogram from SE-HPLC of PEG-P (10×) with benzonase digestion (B+); (c) original chromatogram from SE-HPLC of PEG-P (10×) with combinational pretreatment of chloroform and benzonase (C+B+); (d) original chromatogram from SE-HPLC of PEG-P (10×) with chloroform extraction (C+). Yellow backgrounds indicate the collected target peak fractions; (e) a silver-stained gel after SDS-PAGE of SE-HPLC target peak fraction dependent on each pretreatment method; (f) Western blot result against FMDV type O VP1 on SE-HPLC target peak fraction dependent on each pretreatment method; (g) dsDNA removal rate (%) of each target peak fraction of PEG-P (10×) pretreated with various methods. Groups that do not share a letter are significantly different (p < 0.05). Abbreviations: SE-HPLC, size-exclusion high-performance liquid chromatography; M, marker.
Figure 2
Figure 2
Purity of 146S antigen peak fractions collected by SE-HPLC from the 10× concentrates for PEG precipitate (PEG-P) of FMDV O SKR/Boeun/2017: (a) original chromatograms from SE-HPLC of PEG-P (10×) without pretreatment (NPC); (b) Original chromatogram from SE-HPLC of PEG-P (10×) with benzonase digestion (B+); (c) original chromatogram from SE-HPLC of PEG-P (10×) with combinational pretreatment of chloroform and benzonase (C+B+); (d) original chromatogram from SE-HPLC of PEG-P (10×) with chloroform extraction (C+). Yellow backgrounds indicate the collected target peak fractions; (e) a silver-stained gel after SDS-PAGE of SE-HPLC target peak fraction dependent on each pretreatment method; (f) Western blot result against FMDV type O VP1 on SE-HPLC target peak fraction dependent on each pretreatment method; (g) dsDNA removal rate (%) of each target peak fraction of PEG-P (10×) pretreated with various methods. Groups that do not share a letter are significantly different (p < 0.05). Abbreviations: SE-HPLC, size-exclusion high-performance liquid chromatography; M, marker.
Figure 2
Figure 2
Purity of 146S antigen peak fractions collected by SE-HPLC from the 10× concentrates for PEG precipitate (PEG-P) of FMDV O SKR/Boeun/2017: (a) original chromatograms from SE-HPLC of PEG-P (10×) without pretreatment (NPC); (b) Original chromatogram from SE-HPLC of PEG-P (10×) with benzonase digestion (B+); (c) original chromatogram from SE-HPLC of PEG-P (10×) with combinational pretreatment of chloroform and benzonase (C+B+); (d) original chromatogram from SE-HPLC of PEG-P (10×) with chloroform extraction (C+). Yellow backgrounds indicate the collected target peak fractions; (e) a silver-stained gel after SDS-PAGE of SE-HPLC target peak fraction dependent on each pretreatment method; (f) Western blot result against FMDV type O VP1 on SE-HPLC target peak fraction dependent on each pretreatment method; (g) dsDNA removal rate (%) of each target peak fraction of PEG-P (10×) pretreated with various methods. Groups that do not share a letter are significantly different (p < 0.05). Abbreviations: SE-HPLC, size-exclusion high-performance liquid chromatography; M, marker.
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