Cell lines for high level production of protein selected for rapid proliferation
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Publication Number
US-12723244-B2
Publication Date
2026-09-01
Expiration Date
Abstract
This disclosure provides improved cell lines for manufacture of protein, considerably reducing the cost of commercial production. The cell lines are obtained by selecting cells from a mixed population for one or more characteristics that support cell growth, compared with other cells in the starting mixture. Particularly effective producer cell lines can be obtained by preparing the cells for functional selection by making cell hybrids. A gene encoding a therapeutic protein of interest may be transfected into the cells before or after one or more cycles of fusion and selection. Depending on the protein product being expressed, cell lines may be obtained that produce eight grams or more of protein per liter of culture fluid.
Core Innovation
The disclosure describes obtaining a derivative cell line for industrial production of a therapeutic protein by providing a starting culture of eukaryotic cells adapted to produce a protein encoded in a transgene. Hybrid cells are formed in which each hybrid cell comprises two or more cells from the starting culture, and selected and recovered hybrid cells are used to establish the derivative cell line.
The disclosure further describes selecting hybrid cells that proliferate more rapidly than other hybrid cells and the starting culture, including enrichment using successive dilution culture to dilute out slower-growing cells and enrich for faster-growing cells. Enriched hybrid cells may also be obtained by incubating with a vital dye that stains endoplasmic reticulum and/or Golgi and sorting hybrid cells based on the amount of vital dye associated with each hybrid cell.
The disclosure also describes engineered cells in which a therapeutic-protein gene is provided by transfection at different times relative to fusion and selection, using marker genes such as secreted alkaline phosphatase or luciferase followed by marker replacement. The document reports that selected lines can achieve high protein production, and that examples using CHO-K1 demonstrate improved SEAP specific productivity in fused cells with higher endoplasmic reticulum levels than parental CHO.
Claims Coverage
The partial disclosure contains three independent claims. Across these independent claims, the main inventive features are focused on forming hybrid cells from eukaryotic starting cells, selecting for faster proliferation including successive dilution enrichment, optionally enriching for endoplasmic reticulum and/or Golgi using fusion-associated strategies, and establishing the resulting derivative or hybrid cell line for industrial production of transgene-encoded protein or therapeutic protein.
Hybrid cell-derived derivative line for industrial therapeutic protein production
A method of obtaining a derivative cell line for industrial production of a therapeutic protein comprising providing a starting culture of eukaryotic cells adapted to produce a protein encoded in a transgene, forming hybrid cells each comprising two or more cells from the starting culture, selecting and recovering hybrid cells that proliferate more rapidly than other hybrid cells and the starting culture, and establishing the derivative cell line from the hybrid cells selected and recovered.
Single cell line starting mixture for hybrid cell line production
A method of obtaining a hybrid cell line adapted for industrial production of protein encoded in a transgene comprising providing a starting mixture of eukaryotic cells, forming hybrid cells each comprising two or more cells from the starting mixture, selecting and recovering hybrid cells that proliferate more rapidly than other hybrid cells formed in step (b), and establishing the hybrid cell line from the hybrid cells selected and recovered, wherein the starting mixture provided in step (a) consists essentially of cells from a single cell line.
ER/Golgi enrichment combined with successive-dilution proliferation selection for hybrid cell lines
A method of obtaining a hybrid cell line for production of a therapeutic protein comprising providing a starting culture of cells or progeny thereof made by fusing cells together to form a population of hybrid cells and enriching the hybrid cells for a higher concentration of endoplasmic reticulum and/or Golgi apparatus compared with other hybrid cells in the population, then selecting and recovering hybrid cells that proliferate more rapidly from other cells in the culture by successive dilution, wherein the successive dilution comprises culturing the hybrid cells at a starting concentration, diluting and reculturing the hybrid cells periodically, and increasing the extent of the diluting and/or decreasing the time of the reculturing so as to dilute out slower growing cells and enrich for faster growing cells.
Together, the independent claims cover hybrid-cell-based generation of derivative or hybrid cell lines for industrial production, with selection centered on faster proliferation. The claims also require, in one independent claim, a starting mixture consisting essentially of cells from a single cell line, and in another independent claim, an enrichment step for higher endoplasmic reticulum and/or Golgi content combined with successive dilution to enrich faster-growing hybrid cells.
Stated Advantages
Enriching producer lines for higher densities of endoplasmic reticulum and/or Golgi apparatus.
Selecting and recovering hybrid cells that proliferate more rapidly than other hybrid cells and the starting culture.
Enriching for faster growing cells by diluting out slower growing cells through successive dilution.
Improving therapeutic protein production, including reported high protein output and improved SEAP specific productivity in fused cells with higher ER levels than parental CHO.
Documented Applications
Industrial production of a therapeutic protein encoded in a transgene using derivative cell lines obtained from selected hybrid cells.
Production of a therapeutic protein using hybrid cell lines obtained by fusing cells, enriching hybrid cells for higher endoplasmic reticulum and/or Golgi, and selecting faster-proliferating hybrid cells by successive dilution.
Example cell-line engineering using CHO-K1 demonstrating PEG-assisted fusion, ER-Tracker staining/sorting, and improved SEAP specific productivity in fused cells compared with parental CHO.
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