Bioreactor production of virus from adherent cells
Inventors
Cappello, Joseph • Aguilar, Richard J.
Assignees
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Publication Number
US-10851350-B1
Publication Date
2020-12-01
Expiration Date
Abstract
Methods for producing viruses from adherent cells are provided. The methods include releasing virus from adherent host cells grown in a bioreactor, and purifying released virus by ultrafiltration and/or diafiltration. The methods can be used to manufacture viruses, including for clinical use, at reduced cost relative to conventional virus manufacturing methods.
Core Innovation
The invention provides a method for producing a virus in a bioreactor using host cells that are cultured with a biocompatible matrix for growing adherent cells. The matrix has a density that entraps cells or to which cells adhere, so that the cells are entrapped in and/or adhere to the matrix while process culture medium flows through the matrix to support cell growth.
The method includes treating the cells entrapped in and/or adhered to the matrix to lyse them and release the virus into the process medium in the bioreactor. During this lysis treatment, the lysed cells remain adhered to and/or entrapped in the matrix, and the process medium is not further treated to remove cells or cell debris, while the released virus is retained in the process medium for downstream processing.
The process then purifies the released virus from the process medium without further treatment of the process medium to remove cells or cell debris, using only one or two purification steps. Purification is limited to ultrafiltration or diafiltration in one step, or ultrafiltration and diafiltration in two steps, enabling scalable virus manufacturing with purification confined to ultrafiltration and/or diafiltration.
Claims Coverage
The independent claim is clm-00001, which defines the core method and restricts purification to one or two steps using ultrafiltration and/or diafiltration without further removal of cells or cell debris. The claim set includes three inventive features, with dependent claims refining host cell types, virus types, lysis conditions, purification details, membrane characteristics, and recovery thresholds.
Entrapment/adherence matrix in a bioreactor for adherent cell growth
A bioreactor comprising a matrix for growing adherent cells, the matrix being biocompatible and of a density that entraps cells or is one to which cells adhere, such that cells are entrapped in and/or adhere to the matrix; and the density of the matrix is such that the cells remain entrapped and/or the matrix is one to which cells remain attached under conditions in which cells are lysed and treated to release the virus, and the flow of process culture medium through the matrix is sufficient for cell growth.
In situ lysis while lysed cells remain adhered/entrapped
Treating the cells that are entrapped in and/or adhered to the matrix to lyse them and release the virus into the process medium in the bioreactor, wherein the lysed cells remain adhered to and/or entrapped in the matrix.
One- or two-step virus purification using ultrafiltration and/or diafiltration without further cell/debris removal
Purifying the released virus from the process medium without further treatment of the process medium to remove cells or cell debris, wherein one step is ultrafiltration or diafiltration; and two steps are ultrafiltration and diafiltration.
Across the independent claim and its refinements, the claimed inventive concept centers on culturing adherent host cells within a biocompatible cell-retaining matrix in a bioreactor, performing in situ lysis that keeps lysed cells adhered/entrapped, and then purifying the released virus using only ultrafiltration and/or diafiltration in one or two steps without additional process-medium treatment to remove cells or cell debris.
Stated Advantages
Rapid purification with a duration reported as less than or equal to 1 day.
High recovery and yield, often greater than 50% and up to approximately 95% or more.
Scale-up capability, including considerations for large-scale production.
Reduction in protein and DNA impurities versus conventional multi-step methods, as reported in the partial content.
Documented Applications
Producing viruses using a bioreactor with adherent host cells and a cell-retaining matrix, including therapeutic or oncolytic viruses (as stated in the partial content).
Producing recombinant vaccinia virus (GLV-1h68/GL-ONC1) using CV-1 cells in the described workflow (as stated in the partial content).
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