Cell lines for high level production of protein-based pharmaceuticals
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Publication Number
US-10329594-B1
Publication Date
2019-06-25
Expiration Date
Abstract
This invention provides improved cell lines for manufacture of protein-based pharmaceutical agents, considerably reducing the cost of commercial production. The cell lines are obtained by fusing cells from one or more parental cell populations. The hybrid cells are then selected 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 hybrids or parental cells in the starting mixture. A gene encoding a therapeutic protein is 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 as much as eight grams or more of protein per liter of culture fluid.
Core Innovation
The invention provides methods of obtaining a cell line adapted for high-level production of protein-based pharmaceuticals by culturing a mixture of cells under conditions that form one or more cell hybrids. Each hybrid comprises two or more cells from the mixture, so the resulting population includes hybrids and parental cells for comparative selection. The invention links productive capacity to intracellular secretory pathway organelles by using endoplasmic reticulum and/or Golgi apparatus as the selection basis.
In one approach, the invention sorts the mixture of cells according to the density of endoplasmic reticulum and/or Golgi apparatus per cell, and selects and recovers hybrids having a relatively high density of endoplasmic reticulum and/or Golgi apparatus per cell. This thereby obtains a producer cell line that supports increased production and/or secretion of protein compared with other hybrids or parental cells in the starting mixture.
In another approach, the invention expresses a fusion protein in cells in the mixture in which a fluorescent or bioluminescent peptide generates an optical signal fused with a peptide processed by endoplasmic reticulum and/or Golgi apparatus. Cells that express the optical signal at a higher level relative to other cells in the mixture are selected and recovered, thereby obtaining a producer cell line supporting increased production and/or secretion of protein compared with other hybrids or parental cells.
Claims Coverage
The document includes three independent claims (clm-00001, clm-00017, clm-00018) directed to organelle-density sorting and hybrid recovery, optical-signal reporter fusion selection during hybrid mixtures, and a corresponding hybrid cell line characterized by genomic contribution, increased secretory organelle concentration, and high protein output. Across these independent claims, the inventive features focus on using endoplasmic reticulum and/or Golgi apparatus as a productivity-enrichment criterion for obtaining producer cell lines adapted for high-level production of protein-based pharmaceuticals.
Organelles based sorting of formed cell hybrids
Culturing a mixture of cells to form one or more cell hybrids, sorting the mixture according to the density of endoplasmic reticulum and/or Golgi apparatus per cell, and selecting and recovering cell hybrids having a relatively high density of endoplasmic reticulum and/or Golgi apparatus per cell to obtain a producer cell line supporting increased production and/or secretion of protein compared with other hybrids or parental cells in the starting mixture.
Optical reporter fusion selecting hybrids by ER/Golgi processing
Culturing a mixture of cells to form one or more cell hybrids, expressing a fusion protein that contains a fluorescent or bioluminescent peptide generating an optical signal fused with a peptide processed by endoplasmic reticulum and/or Golgi apparatus, and selecting and recovering cells expressing the optical signal at a higher level relative to other cells in the mixture to obtain a producer cell line supporting increased production and/or secretion of protein compared with other hybrids or parental cells in the starting mixture.
Hybrid cell line with increased ER/Golgi concentration and high protein capacity
Providing a hybrid cell line comprising part or all of the genome of two or more parental cell lines, having a higher concentration of endoplasmic reticulum and/or Golgi apparatus relative to any of the parental cell lines, and having a capacity to produce at least eight grams of protein per liter of culture fluid from one or a combination of recombinantly inserted genes.
Overall, the claims cover obtaining producer cell lines by forming cell hybrids and enriching for higher protein production by selecting hybrids with relatively high endoplasmic reticulum and/or Golgi apparatus by density sorting or by an ER/Golgi-processed fluorescent/bioluminescent optical reporter, and they also cover a hybrid cell line defined by increased ER/Golgi concentration plus at least eight grams of protein per liter from recombinantly inserted genes.
Stated Advantages
Increased production and/or secretion of protein compared with other hybrids or parental cells in the starting mixture.
Producer cell lines adapted for high-level production of protein-based pharmaceuticals.
A hybrid cell line capable of producing at least eight grams of protein per liter of culture fluid.
Documented Applications
Obtaining cell lines adapted for high-level production of protein-based pharmaceuticals.
Producing a hybrid-cell-line protein intended for compounding as a biopharmaceutical [procedural detail omitted for safety].
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