Virus purification
Inventors
Barbero Calzado, Jana • Nebenführ, Mario • Schlegl, Robert • Weber, Michael • HEINDL-WRUSS, Jürgen
Assignees
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Publication Number
US-10537630-B2
Publication Date
2020-01-21
Expiration Date
Abstract
Described herein are improved purification methods for virus vaccines and compositions. Also described are Zika, Chikungunya, dengue and yellow fever vaccines and methods of producing and administering said vaccines to subjects in need thereof.
Core Innovation
The invention relates to purification of infectious virus particles from a crude harvest containing virus particles and impurities generated from growing the virus particles on a cell substrate. It introduces reducing impurities by precipitation using an agent comprising protamine to obtain a virus preparation, and further purifying the virus preparation by sucrose density gradient centrifugation so that protamine can be completely or almost completely separated from the virus fraction.
In the sucrose density gradient, the gradient includes a virus-containing fraction in a 10%±1% (w/w) sucrose solution and three further layers of sucrose solutions with defined densities of 15%±1% (w/w), 35%±1% (w/w), and 50%±1% (w/w). The document states that this step reduces the protamine concentration such that the protamine concentration in the final drug substance is below 1 µg/ml.
The disclosure further states that protamine sulphate treatment reduces immature/non-infectious virus particles while preserving infectious virus particles, alongside removal of impurities such as host DNA and host cell proteins. It also links the purification strategy to virus vaccine manufacture and to inactivated or attenuated virus formulations.
Claims Coverage
The independent claims cover two related inventive methods centered on protamine removal and infectious virus particle purification using a specifically defined sucrose density gradient composition and its separation behavior, with one claim also requiring low residual protamine in the final drug substance and the preceding protamine precipitation impurity-reduction step.
Protamine removal using a multi-layer sucrose gradient centrifugation
A method for removal of protamine from purified infectious virus particles, comprising sucrose gradient centrifugation, wherein the sucrose gradient comprises a virus comprising fraction in a 10%±1% (w/w) sucrose solution and three further layers of sucrose solutions with different densities of 15%±1% (w/w), 35%±1% (w/w), and 50%±1% (w/w).
Infectious virus purification with protamine precipitation followed by protamine-separating sucrose gradient
A method for purification of infectious virus particles, comprising providing a crude harvest comprising virus particles and impurities generated from growing the virus particles on a cell substrate; reducing impurities by precipitation with an agent comprising protamine to obtain a virus preparation; and further purifying by sucrose density gradient centrifugation to obtain a virus preparation, wherein the sucrose gradient is provided such that the protamine can be completely or almost completely separated from the virus fraction and the protamine concentration in the final drug substance is below 1 µg/ml, using a virus-containing fraction in 10%±1% (w/w) sucrose and three layers of 15%±1%, 35%±1%, and 50%±1% (w/w) sucrose.
Across the independent claims, the inventive focus is the use of protamine precipitation for impurity reduction followed by sucrose density gradient centrifugation with a specified four-fraction sucrose configuration to separate protamine from the virus fraction and achieve very low residual protamine in the final drug substance.
Stated Advantages
Reduces impurities by using protamine precipitation and separates protamine from the virus fraction during sucrose density gradient centrifugation.
Reduces the protamine concentration in the final drug substance to below 1 µg/ml.
Reduces immature/non-infectious virus particles while preserving infectious virus particles.
Provides safety motivation by targeting low residual protamine related to protamine-related adverse reactions.
Enables complete or almost complete separation of protamine from the virus fraction.
Documented Applications
Purification approach for virus vaccine manufacture, including preparing vaccine formulations with inactivated or attenuated viruses.
Use with multiple viruses, including Zika virus, Chikungunya virus, yellow fever virus, Japanese encephalitis virus SA14-14-2, and related virus sequence examples.
Purification of infectious virus particles from a crude harvest containing impurities generated from growing virus particles on a cell substrate.
Removal of protamine from purified infectious virus particles.
Development of DSP for live attenuated Chikungunya E nsP3.
Production of an inactivated Zika vaccine H/PF/2013.
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