Purification method of virus-like particles
Inventors
Okada, Masahiro • MUKAI, Akiko • Nishino, Tomonori • ARINOBU, Daisuke • Ito, Hiroyuki • Satoh, Mamoru
Assignees
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Publication Number
US-10023844-B2
Publication Date
2018-07-17
Expiration Date
Abstract
The present invention aims at establishing a novel means for purifying norovirus VLPs. There is provided a purification method of norovirus virus-like particles, comprising: contacting solution containing norovirus virus-like particles with support for hydroxyapatite chromatography, binding the virus-like particles to the support, subsequently washing the support with buffer, and then eluting the virus-like particles from the support with buffer containing phosphate, wherein the phosphate concentration of the buffer used for elution is less than 10 mM.
Core Innovation
The invention relates to a purification method of norovirus virus-like particles using hydroxyapatite chromatography. A solution containing norovirus virus-like particles is contacted with support for hydroxyapatite chromatography to bind the virus-like particles, the support is washed with buffer, and the virus-like particles are then eluted with buffer containing phosphate.
The core innovation is that the phosphate concentration of the elution buffer is less than 10 mM. The disclosure also indicates low-phosphate washing buffer with phosphate concentration less than 10 mM, and a preferred phosphate concentration range for elution of 3 to 7 mM.
The background problem addressed is that prior methods using much higher phosphate in elution led to contaminating proteins co-eluting with the norovirus VLPs. By using elution phosphate below 10 mM, impurities are largely removed away from the VP1 elution fractions, supporting effective VLP purity for vaccine production.
Claims Coverage
The document includes one independent claim covering a hydroxyapatite chromatography purification method for norovirus virus-like particles with phosphate in the elution buffer constrained to below 10 mM, plus dependent claims that refine the phosphate concentrations and further define the starting solution and encoded norovirus structural protein.
Low phosphate elution for hydroxyapatite chromatography purification of norovirus VLPs
Contacting a solution containing norovirus virus-like particles with support for hydroxyapatite chromatography to bind the virus-like particles, washing the support with buffer, and eluting the virus-like particles from the support with buffer containing phosphate, wherein the phosphate concentration of the elution buffer is less than 10 mM.
Preferred elution phosphate range of 3 to 7 mM
The phosphate concentration of the buffer used for elution is 3 to 7 mM.
Low phosphate washing buffer of less than 10 mM
The buffer used for washing has a phosphate concentration of less than 10 mM.
Low phosphate washing phosphate range of 3 to 7 mM
The phosphate concentration of the washing buffer is 3 to 7 mM.
Culture supernatant starting solution expressing norovirus nucleic acid sequence
The solution containing norovirus virus-like particles is culture supernatant of microorganisms or cells expressing a nucleic acid sequence of norovirus.
Norovirus nucleic acid encoding VP1 structural protein
The norovirus nucleic acid sequence encodes norovirus structural protein VP1.
Overall, the claim set centers on hydroxyapatite chromatography purification of norovirus VLPs with elution phosphate concentration below 10 mM, preferably 3 to 7 mM, with optional low-phosphate washing constraints, and it narrows the starting material to culture supernatant from expressing cells or microorganisms, further specifying VP1 encoded by the norovirus nucleic acid sequence.
Stated Advantages
Reduces co-elution of contaminating proteins compared with prior higher-phosphate elution methods.
Improves removal of impurities away from VP1 elution fractions, supporting effective VLP purity for vaccine production.
Documented Applications
VLP purity for vaccine production (industrial applicability described in the partial content).
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