Methods and compositions for generating stable transfected cells
AssigneeMaxCyte, Inc.
InventorsWeili Wang, James P. Brady, Madhusudan V. Peshwa
Life of this patent
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Abstract
Methods and compositions are provided involving high producing cell lines. Embodiments concern efficient methods for screening for such cell lines and for creating such cell lines. These cell lines can be used to create large amounts of protein. To quickly generate large quantity of recombinant proteins or vaccines for both pre-clinical study and clinical trials, almost all drug development will face the same challenging obstacle of rapidly generating a high stable producer. Developing and identifying a stable cell line is a critical part of biopharmaceutical development.
Claims14
A method for producing a stable mammalian cell line that expresses an exogenous polypeptide comprising:
- transfecting an expression construct into cells using electroporation, wherein the expression construct comprises i) a selectable gene and ii) a sequence encoding an exogenous polypeptide;
- selecting for expression of the selection gene in transfected cells under culture conditions comprising a conditionally lethal concentration of at least 1.6 mg/ml of G418 for at least 6 days.
A method for producing a stable CHO cell expressing line comprising:
- a) transfecting into CHO cells an expression construct comprising a sequence i) encoding a polypeptide conferring G418 resistance and ii) encoding an exogenous polypeptide using a flow electroporation device;
- b) selecting transfected CHO cells in media comprising at least 1.6 mg/ml of G418 for at least 6 days;
- c) isolating selected transfected CHO cells by limiting dilution to obtain clonal cells;
- and,
- d) expanding isolated transfected CHO cells to produce a stable CHO cell expressing line.
A method for screening for high producing CHO cell lines comprising:
- a) transfecting into CHO cells an expression construct comprising a sequence i) encoding a polypeptide conferring G418 resistance and ii) encoding an exogenous polypeptide using a flow electroporation device;
- b) selecting transfected CHO cells in media comprising greater than about 1.6 mg/ml of G418 for at least 6 days;
- c) isolating selected transfected CHO cells by limiting dilution to obtain clonal cells;
- d) expanding isolated transfected CHO cells to produce a stable CHO cell expressing line;
- and,
- e) evaluating the cells for production of the exogenous polypeptide.
Dependencies are inferred from the claim text.