The dominant system to produce therapeutic recombinant proteins over the last few decades has been mammalian cell-based expression systems.

Their capacity to handle complex post-translational modifications, folding and assembly of recombinant proteins and protein complexes is superior to other systems.

Cell Lines & Media

Traditional media formulations for mammalian cell culture relied on the addition of animal-serum from 1% to 20% of the total culture volume. However serum-free media has become the more popular choice driven principally by concerns about the introduction of adventitious agents in animal derived serum and the expense. Serum-free media formulations have been shown to perform equally as well as serum supplemented media.  Host cell lines have also been improved to enhance their ability to survive, grow and produce recombinant proteins, in culture. Introduction of antiapoptotic, growth factor, proto-onco and cell-cycle control genes into host cell lines have been shown to greatly enhance their capabilities. Commercially available cell lines carry many of these genetically engineered enhancements. Commonly used mammalian cell lines for protein production, in particular biotherapuetics are the Chinese hamster ovary (CHO), Human embryonic kidney (HEK-293) and Mouse myeloma (NS0) cell lines.

Expression Vectors

Mammalian expression vectors have been engineered to contain elements that improve expression and are generally more complex than vectors from other systems. Some common variable elements found in expression vectors are listed below:

  • Strong viral promoters and enhancers for recombinant protein expression

  • Alternate (sometimes weaker) promoter/enhancer for the selective gene

  • An intron sequence to enable splicing (Splicing has been shown to promote mRNA nuclear export)

  • Unique linearisation site (known to decrease disruption of the expression cassette during integration)

  • Directed integration into the host cell genome via a recombination system e.g. Cre/loxP

Transient vs Stable Cell Culture

There are two main methods for recombinant protein production in mammalian cells i.e. stably transfected and transiently transfected cell lines. Stably transfected cell lines enable continuous expression of the recombinant protein and have been shown to produce high yields.  The creation of a stable cell line can take up to several months to complete.

A transient transfection can be used for small- to large-scale expression cultures. Recent improvements to the method have been able to increase yields from milligram to gram quantities. The advantages of a transient transfection method lie in the shorter timeframe from DNA delivery to protein harvest.