Hybrid yeast cell lines for high level production of recombinant protein

Inventors

Forman, Lawrence

Assignees

CHO Plus Inc

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 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 heterologous 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 obtaining a hybrid yeast cell line adapted for high-level production of protein. It addresses the generation of protein-producing hybrids by fusing and culturing individual yeast cells in a starting population to form a plurality of cell hybrids, and the resulting yeast cell line supports increased production and/or secretion of protein compared with parental cells in the starting population.

Cell hybrids are sorted according to the density of endoplasmic reticulum and/or Golgi apparatus per cell hybrid. Cell hybrids are then selected and recovered that have a relatively high density of endoplasmic reticulum and/or Golgi apparatus per cell hybrid compared with other cell hybrids sorted in the density-based sorting step, thereby enriching subcellular organelle features in the selected hybrid population.

A yeast cell line is also provided that comprises a transgene encoding a target protein, where the yeast cell line is prepared by fusing individual yeast cells to form cell hybrids, sorting hybrids according to the density of endoplasmic reticulum and/or Golgi apparatus per cell hybrid, selecting and recovering hybrids with relatively high organelle density, and establishing the yeast cell line from the selected and recovered cell hybrids. The enrichment of organelle density is associated with increased protein production and/or secretion and improved specific productivity, including improved quality such as glycosylation.

Claims Coverage

The document provides two independent claims. Across them, the coverage centers on preparing a hybrid yeast cell line via fusion and on enrichment by sorting and selecting cell hybrids based on endoplasmic reticulum and/or Golgi apparatus density, with the second independent claim adding an established transgene-carrying yeast cell line prepared using the same organelle-density-based process.

Overall, the inventive claim coverage is directed to producing an improved protein-secreting or protein-producing hybrid yeast cell line by enriching hybrids for relatively high endoplasmic reticulum and/or Golgi apparatus density through density-based sorting and selection, with transgene-carrying yeast cell lines established from those enriched hybrids.

Stated Advantages

Documented Applications