Hybrid cell lines for high level production of a target protein
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Publication Number
US-11649449-B2
Publication Date
2023-05-16
Expiration Date
Abstract
This disclosure provides improved cell lines for manufacture of protein-based pharmaceutical agents, 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 protein production on a non-specific basis, such as the level of endoplasmic reticulum, Golgi apparatus, and/or other desired phenotypic features, 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 invention relates to improved producer cell lines for production of a pharmaceutical product and/or a target protein. It addresses a starting population of cultured cells in which the cells are heterogeneous in their capability to express protein, and it aims to obtain a producer cell line that supports increased production and/or secretion of protein compared with other cells in the starting population.
The method forms a plurality of cell hybrids from the heterogeneous population and sorts the cell hybrids according to the density of endoplasmic reticulum and/or Golgi apparatus per cell. Cell hybrids having a relatively high density of endoplasmic reticulum and/or Golgi apparatus per cell are selected and recovered to obtain the producer cell line.
In related embodiments reflected by the claim set, genetically altered cells are provided by introducing a transgene encoding a target protein, and a subset of cells expressing the target protein is selected. In further embodiments, a transgene is introduced into the genome of the producer cells after hybrid selection/recovery.
Claims Coverage
The document includes two independent methods directed to preparing a producer cell line. Both independent claims share a core inventive structure consisting of forming cell hybrids from a heterogeneous cultured cell population and sorting/selecting hybrids based on endoplasmic reticulum and/or Golgi apparatus density, yielding increased protein production and/or secretion.
Hybrid formation from heterogeneous cultured cells
Providing a population of cultured cells wherein cells in the population are heterogeneous in their capability to express protein; forming a plurality of cell hybrids from the population of cultured cells, each comprising two or more of the cells from the population.
Sorting by endoplasmic reticulum and/or Golgi density per cell
Sorting the cell hybrids according to the density of endoplasmic reticulum and/or Golgi apparatus per cell.
Selecting high endoplasmic reticulum and/or Golgi density hybrids to obtain increased production
Selecting and recovering cell hybrids from the population that have a relatively high density of endoplasmic reticulum and/or Golgi apparatus per cell, thereby obtaining a producer cell line that supports increased production and/or secretion of protein compared with other cells in the starting population.
Adapting a producer cell line for increased secretion of a target protein
Preparing a cell line adapted for production of a target protein by providing a population of cultured cells heterogeneous in their capability to express protein, forming a plurality of cell hybrids comprising two or more of the cells, sorting according to endoplasmic reticulum and/or Golgi apparatus density per cell, and selecting/recovering hybrids having a relatively high density to obtain a producer cell line that supports increased production and/or secretion of the target protein.
Across the independent claims, the inventive coverage centers on creating cell hybrids from heterogeneous protein-expressing cells and using endoplasmic reticulum and/or Golgi apparatus density per cell as the sorting and selection basis to obtain a producer cell line that supports increased production and/or secretion (for a pharmaceutical product and/or a target protein).
Stated Advantages
Supports increased production and/or secretion of protein compared with other cells in the starting population.
Supports increased production and/or secretion of a target protein.
Documented Applications
Production of a pharmaceutical product using a producer cell line obtained by the disclosed method.
Production of a target protein using a producer cell line obtained by the disclosed method.
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