Methods for making recombinant proteins using apoptosis inhibitors
AssigneeGenentech, Inc.
InventorsVishva Dixit, Robert W. Hamilton, Jana van de Goor
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Abstract
The invention provided improved methods of making and producing recombinant proteins in in vitro cultures of host cells using apoptosis inhibitors. The use of one or more apoptosis inhibitors in the methods can reduce apoptosis in the cell cultures and markedly improve yield of the desired recombinant proteins.
Claims21
A method of making recombinant proteins using one or more apoptosis inhibitors, comprising the steps of:
- (a) providing a vector comprising a gene encoding caspase-9 dominant negative protein,
- (b) providing a vector comprising a gene encoding a protein of interest,
- (c) providing a Chinese hamster ovary (CHO) host cell,
- (d) transforming or transfecting the host cell with the vector of steps (a) and (b),
- (e) providing cell culture media,
- (f) culturing the transformed or transfected host cell in the cell culture media under conditions sufficient for expression of the protein of interest and the caspase-9 dominant negative protein, and optionally
- (g) recovering or purifying the protein of interest from the host cell and/or the cell culture media.
A method of making recombinant proteins using one or more apoptosis inhibitors, comprising the steps of:
- (a) providing a vector comprising a gene encoding a protein of interest,
- (b) providing a Chinese hamster ovary (CHO) host cell comprising a gene encoding caspase-9 dominant negative protein,
- (c) transforming or transfecting the host cell with the vector of step (a),
- (d) providing cell culture media,
- (e) culturing the transformed or transfected host cell in the cell culture media under conditions sufficient for expression of the protein of interest and the caspase-9 dominant negative protein and optionally
- (f) recovering or purifying the protein of interest from the host cell and/or cell culture media.
A method of making recombinant proteins using one or more apoptosis inhibitors, comprising the steps of:
- (a) providing a vector comprising a gene encoding a protein of interest,
- (b) providing a Chinese hamster ovary (CHO) host cell,
- (c) transforming or transfecting the host cell with the vector of step (a),
- (d) providing cell culture media,
- (e) providing an amount of caspase inhibitor z-VAD-fmk,
- (f) admixing the caspase inhibitor into the cell culture media,
- (g) culturing the host cell in the cell culture media under conditions sufficient for expression of the protein of interest, and optionally
- (h) recovering or purifying the protein of interest from the host cell and/or the cell culture media.
A method of increasing yield of a protein of interest in a cell culture, comprising the steps of:
- (a) providing a vector comprising a gene encoding caspase-9 dominant negative protein,
- (b) providing a vector comprising a gene encoding a protein of interest,
- (c) providing a Chinese hamster ovary (CHO) host cell,
- (d) transforming or transfecting the host cell with the vector of steps (a) and (b),
- (e) providing cell culture media,
- (f) culturing the transformed or transfected host cell in the cell culture media under conditions sufficient for expression of the protein of interest and an amount of the caspase-9 dominant negative protein which is effective in increasing yield of the protein of interest, and optionally
- (g) recovering or purifying the protein of interest from the host cell and/or the cell culture media.
A method of prolonging host cell viability in a cell culture, comprising the steps of:
- (a) providing a vector comprising a gene encoding caspase-9 dominant negative protein,
- (b) providing a vector comprising a gene encoding a protein of interest,
- (c)providing a Chinese hamster ovary (CHO) host cell,
- (d) transforming or transfecting the host cell with the vector of steps (a) and (b),
- (e) providing cell culture media,
- (f) culturing the transformed or transfected host cell in the cell culture media under conditions sufficient for expression of the protein of interest and an amount of caspase-9 dominant negative protein which is effective for prolonging viability of the host cells in the cell culture, and optionally
- (g) recovering or purifying the protein of interest from the host cell and/or the cell culture media.
Dependencies are inferred from the claim text.