Process for purifying VLPs
Inventors
Arhancet, Juan Pedro Humberto • Arhancet, Juan P. • Delaney, Kimberly • Hall, Kathleen B. • Summers, Neena
Assignees
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Publication Number
US-9181531-B2
Publication Date
2015-11-10
Expiration Date
Abstract
Processes and compositions to produce, package, and purify virus like particles containing heterologous cargo molecules utilizing self assembling proteins and protease treatment coupled with simple precipitation and filtration methods are described.
Core Innovation
The invention relates to virus-like particles (VLPs) consisting of Enterobacteriophage MS2 capsid proteins (SEQ ID NO.: 3) enclosing at least one heterologous cargo molecule. The VLPs are used in a purification process where a plurality of the VLPs are present in a cell lysate. The central idea is to maintain intact VLPs while processing the lysate to hydrolyze non-VLP components.
The problem addressed is purification of VLPs from whole-cell lysate while removing cell lysis products. In the process, the cell lysate is contacted with a protease for a time and under conditions sufficient to hydrolyze cell lysis products other than the VLPs to form a hydrolysate. After hydrolysis, the VLPs are isolated from the hydrolysate.
The document further describes how protease treatment can preserve capsid integrity while producing hydrolysis of non-capsid lysis products, including evaluation metrics such as capsid integrity and the fraction of non-enclosed polypeptides cleaved. Additional aspects include using the MS2 capsid to enclose heterologous cargo such as oligoribonucleotide cargo, including siRNA, shRNA, sshRNA, lshRNA, or miRNA, and peptide, polypeptide, or protein cargo via RNA linkers.
Claims Coverage
The independent claim covers a protease-based purification process for Enterobacteriophage MS2 VLPs enclosing heterologous cargo from a cell lysate by selectively hydrolyzing non-VLP lysis products and then isolating the VLPs. The inventive features are further narrowed in dependent claims by specifying proteases, cargo types, hydrolysis robustness constraints, and particular isolation schemes and purity or amount constraints.
Protease hydrolysis of non-VLP lysis products in a cell lysate
A process comprising obtaining a cell lysate comprising a plurality of VLPs and contacting the cell lysate with a protease for a time and under conditions sufficient to hydrolyze cell lysis products other than the VLPs to form a hydrolysate.
Isolation of intact MS2 VLPs from the hydrolysate
Isolating the VLPs from the hydrolysate, wherein the VLPs consist of Enterobacteriophage MS2 capsid proteins (SEQ ID NO.: 3) enclosing at least one heterologous cargo molecule.
Protease selection among named proteases
The process wherein the protease is selected from Proteinase K, Protease from Streptomyces griseus, Protease from Bacillus licheniformis, pepsin, or papain.
Hydrolysis conditions preserving capsid integrity while cleaving non-capsid polypeptides
The process wherein the hydrolysis is for a time and under conditions sufficient to hydrolyze a fraction of polypeptides in whole cell lysate that are not enclosed by capsids, while maintaining a specified fraction of capsids intact after hydrolysis.
Oligoribonucleotide cargo restricted to selected RNA classes
The process wherein the heterologous cargo molecule is an oligoribonucleotide selected from siRNA, shRNA, sshRNA, lshRNA, or miRNA.
Minimum quantity threshold for cargo-enclosing VLPs
The process including producing VLPs that enclose at least one heterologous cargo molecule in an amount of at least ten milligrams.
Across the claims, the core coverage is the selective protease hydrolysis of cell lysis products other than MS2 VLPs, followed by isolating the VLPs from the resulting hydrolysate. Dependent claim refinements specify particular proteases, cargo categories, hydrolysis robustness constraints linking non-enclosed polypeptide cleavage with capsid integrity, and additional quantitative and isolation scheme constraints.
Stated Advantages
Selectively hydrolyzing cell lysis products other than the VLPs while maintaining intact VLPs or capsids for subsequent isolation.
Enabling isolation of VLPs from the hydrolysate after protease treatment of a cell lysate.
Documented Applications
Purification of virus-like particles (VLPs) containing heterologous cargo, including oligoribonucleotide cargo such as siRNA, shRNA, sshRNA, lshRNA, and miRNA, and peptide, polypeptide, or protein cargo, from a cell lysate using protease treatment followed by isolation.
Use of cargo-enclosing MS2 VLPs with documented resistance of the MS2 VLPs to various proteases and preservation of RNA and capsid genes after purification [procedural detail omitted for safety].
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