Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering.

Jianmin Hao; Youyan Liu; Zhiguo SU; Songping Zhang; Zhengjun LI

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Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering.

Author: Jianmin HAO ¹ ; Youyan LIU ¹ ; Zhiguo SU ² ; Songping ZHANG ² ; Zhengjun LI ²
Author Information

1. College of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, Guangxi, China.
2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
Publication Type:Journal Article
Keywords: inactivated avian influenza virus particles; ion-exchange chromatography; quantitative detection; size exclusion chromatography
MeSH: Animals; Reproducibility of Results; Influenza A Virus, H7N9 Subtype; Influenza in Birds; Chromatography, Gel; Virion; Lasers
From: Chinese Journal of Biotechnology 2023;39(10):4295-4307
CountryChina
Language:Chinese
Abstract: We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R²=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.