High yield transient expression in mammalian cells using unique pairing of high density growth

Mammalian protein expression systems are the best choice for the production of eukaryotic proteins, especially when correct folding and post-translational modification is required. They produce eukaryotic recombinant proteins in the most natural state, with native tertiary structure, physiochemical characteristics and bioactivities. They have been successfully applied in the biopharmaceutical production of cytokines, monoclonal antibodies, growth factors and so on. Email:marketing@medicilon.com.cn web:www.medicilon.com

The present invention is directed generally to cell culture media (particularly serum free, non animal derived, and/or chemically defined media) which are useful for introducing macromolecules and compounds (e.g., nucleic acid molecules) into cells (e.g., eukaryotic cells). According to the invention, such introduction can take place in the presence of said medium. Cells containing such introduced materials can then be cultured in the medium and the effect of the introduced materials on the cells can be measured or determined. In particular, the invention allows introduction of nucleic acid molecules (e.g., vectors) into cells (particularly eukaryotic cells) and expression of proteins encoded by the nucleic acid molecules in the cells. The invention obviates the need to change the cell culture medium each time a different procedure is performed with the cells (e.g., culturing cells vs. transfecting cells). The invention also relates to compositions and kits useful for culturing and transforming/transfecting cells.

The present invention provides a cell culture and transfection system, whereby the system supports introduction by way of transfection and subsequent expression of one or more macromolecules (such as, e.g., expressible nucleic acids) into a plurality of eukaryotic cells in culture, and further supports the cultivation and growth of the cells subsequent to the introduction/transfection, wherein growth of the at least one cell continues in the medium in the absence of the medium being supplemented with fresh medium.

In some embodiments, it is not necessary to remove, replenish or replace the medium used during the introduction/transfection of the cells from the presence of the cells to support the further growth thereof. In another preferred embodiment, after the introduction/transfection, growth of the cells and production of an expressed protein from the expressible nucleic acid can be accomplished in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which the introduction/transfection occurred. Using the medium of the present invention, it is not necessary to replenish, replace or supplement the medium after one has introduced nucleic acid into cells, and before cells into which nucleic acid has been introduced are further cultured to express the nucleic acid.

Transient expression is fast becoming the system of choice for rapid mammalian protein production. The flexibility of transient transfection enables a rapid realization time from concept to protein-in-hand and many different proteins can be produced simultaneously, or serially. The next key advance in transient transfection technology is to approach or equal expression levels attained using stable expression systems without losing the speed and flexibility of the transient format. We report for the first time the development of a novel transient transfection system that utilizes high density 293F cell cultures to generate expression levels of >1 g/L (up to about 2 g/L) of human IgG and anon-IgG proteins within 7 days after cells are transfected.

To attain such high levels of protein expression, a novel cell culture system which includes a new high density growth culture medium in combination with a population of suspension cells that are adapted for high density growth in such a media was developed that allows certain populations of mammalian cells to reach viable cell densities of up to 20×106 cells/ml (more typically up to about 15×106 cells/ml). These ultra-high density cultures enable transfection at higher cell densities than traditional protocols, significantly increasing the volumetric yield of protein. Additive The addition of one or more expression enhancer formulations following or during transfection was also found to boost protein expression level to levels up to 10- to 12-fold higher than the expression levels seen with current commercially available transient transfection systems. Parental suspension culture mammalian cells were adapted for improved growth and viability characteristics under high density culture conditions, and were then further selected for increased protein production. The resulting high density adapted cells have an increased growth rate, increased cell size, and increased specific productivity compared to the parental cell line. Finally, the transfection method was optimized through the use of one or more transfections reagents that are used in combination with one or more expression enhancer formulations to further increase overall protein yield.

When all of these improvements were combined into a single expression system, protein levels were increased up to 10-fold for both IgG and non-IgG recombinant proteins compared to the commercially available FreeStyle™ 293 expression system and expression levels of >1 g/L were attained for multiple proteins. Additionally, protein functionality was demonstrated to be comparable for several proteins expressed in the high yield expression system of the present invention when compared to the popular commercially available FreeStyle™ 293 system. Together, these results indicate that significant increases in functional protein yields can be attained using a novel transient mammalian expression system that incorporates numerous advances in protein expression technology into a single, easy to use format.

The present invention also provides a method of cultivating eukaryotic cells comprising: (a) contacting the cells with the cell culture medium of the present invention; (b) maintaining the cells under conditions suitable to support cultivation of the cells in culture; and (c) optionally expressing a nucleic acid to form a protein product.

The present invention also provides a method for introducing one or more macromolecules into at least one eukaryotic cell in culture, the method comprising: (a) culturing at least one eukaryotic cell in the medium of claim 1 in culture; (b) introducing at least one macromolecule into the culture under conditions sufficient to cause one or more of the at least one macromolecule to be introduced in the at least one cell; and (c) cultivating the at least one cell in the medium to produce a product whose production is controlled by the at least one molecule, wherein growth of the at least one cell continues in the medium in the absence of the medium being with fresh medium, wherein it is not necessary to remove medium used during the introduction from the presence of the at least one cell to support growth of the at least one cell, and/or wherein after the introduction, growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which the introduction occurred.