Purification of recombinant EV71 virus-like particle and method for preparing vaccine thereof
Inventors
LI, Guoshun • GU, Meirong • Liu, Jiankai • Guo, Lin • Chen, Lei • ZHANG, Gaimei • XU, Yingzhi • Li, Jin • XIAO, Haifeng
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10987416-B2
Publication Date
2021-04-27
Expiration Date
Abstract
The present invention provides a method for purifying an EV71 virus-like particle and a method for preparing a vaccine thereof. The virus-like particle is obtained by performing high density fermentation cultivation on recombinantly engineered bacteria; inducing expression of the EV71 virus-like particle protein expression using methanol; collecting the bacteria by centrifugation and performing high-pressure homogenization for disruption; performing precipitation on the supernatant with ammonium sulfate; and purifying by redissolution, ultrafiltration, ion exchange chromatography, molecular sieve chromatography, hydroxyapatite chromatography, etc.
Core Innovation
The disclosed approach provides a method for purifying a recombinant EV71 virus-like particle. The method includes fermentation of engineered recombinant Hansenula yeast that contains an EV71 capsid protein P1 gene and a 3CD protease gene, followed by disrupting the engineered yeast and carrying out precipitation, redissolution and ultrafiltration of the recombinant EV71 VLP.
After precipitation-based recovery and ultrafiltration, the purification workflow further includes ion exchange chromatography and then molecular sieve chromatography or hydroxyapatite chromatography. The purification sequence is directed to obtaining a purified recombinant EV71 VLP suitable for subsequent quality evaluation and formulation.
The document also describes downstream vaccine-related processing and characterization of the EV71 VLP. It states assessment of VLP size and structure by DLS and TEM, evaluation of vaccine quality tests, and measurement of immunogenicity using neutralizing antibody ED50, together with animal safety observations including absence of abnormal toxicity in KM mice and BALB/c mice.
Claims Coverage
The independent claim set contains one independent claim covering a complete purification method for a recombinant EV71 virus-like particle, including the engineered host, the core recovery operations, and two chromatographic steps with an explicit selection between molecular sieve chromatography and hydroxyapatite chromatography. The independent claim is supplemented by dependent claims that refine specific purification operations and provide a defined vaccine end-use.
Purification workflow using engineered Hansenula and EV71 capsid P1 plus 3CD protease
A method for purifying a recombinant EV71 virus-like particle comprising performing fermentation on engineered recombinant Hansenula yeast containing an EV71 capsid protein P1 gene and a 3CD protease gene.
Precipitation, redissolution and ultrafiltration after yeast disruption
A method for purifying a recombinant EV71 virus-like particle comprising disrupting the engineered yeast, and performing precipitation, redissolution and ultrafiltration of the recombinant EV71 virus-like particle.
Sequential ion exchange chromatography and molecular sieve or hydroxyapatite chromatography
A method for purifying a recombinant EV71 virus-like particle comprising performing ion exchange chromatography, and performing molecular sieve chromatography or hydroxyapatite chromatography.
Ammonium sulfate precipitation step with centrifugation and defined concentration
The step of precipitating the recombinant EV71 virus-like particle comprises pouring cell solution after disruption into a centrifuge bowl, performing centrifugation at 6,000 to 8,000 rpm for 40 to 60 min, collecting a supernatant, and adding ammonium sulfate into the collected supernatant to a final concentration of 20 to 28%.
Vaccine against hand-foot-mouth disease using purified EV71 VLP
A vaccine against hand-foot-mouth disease prepared by purifying a recombinant EV71 virus-like particle according to the method of claim 1.
Overall, the claim coverage centers on a purification method for recombinant EV71 VLP produced in engineered Hansenula yeast, with recovery through precipitation, redissolution and ultrafiltration and final purification through ion exchange followed by molecular sieve chromatography or hydroxyapatite chromatography, and includes a dependent end-use directed to a hand-foot-mouth disease vaccine.
Stated Advantages
Provides purified EV71 virus-like particles with stated characterization of VLP size and structure by DLS and TEM and vaccine quality test results.
States immunogenicity by measuring neutralizing antibody ED50.
States absence of abnormal toxicity in animal studies (KM mice and BALB/c mice).
States purification simplicity and recovery for large-scale production.
Documented Applications
Use as a vaccine against hand-foot-mouth disease prepared by purifying recombinant EV71 virus-like particles according to the claimed method.
Interested in licensing this patent?