Cell-based expression systems: parallel reiterative design and scalable platforms

Adnexus Therapeutics (acquired by Bristol-Myers Squibb in 2007) has developed an E. coli-based platform for producing adnectins. Adnectins are derived from human fibronectin and many trillions of adnectin variants can be generated to represent a screenable library for desirable therapeutic properties. Scale-up from a selected lead is rapid, albeit with the requirement for PEGylation for clinical candidate manufacture (7).

Fabrus has recently addressed the rapid make-test cycle approach for biologics using arrays of predefined Fab antibody sequences produced in a high throughput expression system based on production of proteins in E coli, cell-lysis, and subsequent protein purification (8). The large volumes of cell culture required for high-yield parallel production of Fabs requiring a significant investment in specialized robotics equipment. The method allows production of hundreds of variant Fab proteins over a one-week production cycle to begin variant testing in high-throughput biochemical-binding assays.

Although cell-free protein synthesis has been practiced for decades as a research tool, only recently have advances suggested its feasibility for commercial biologics drug development and production as an alternative to conventional cell-based expression systems (9). An ideal cell-free protein production platform would produce fully soluble and correctly folded proteins at high volumetric productivities at any scale. The platform would be rapidly and predictably optimized by systems-level process design and control without the demanding requirements for maintaining cell viability and be readily adapted to high-throughput methods, including in vitro evolution of proteins to allow incorporation of nnAA into polypeptides. The platform would be based on simple batch systems using standard bioreactors that are known to scale to thousands of liters for both cell fermentation and subsequent cell-free protein production (10, 11).

Early efforts at developing such a system focused on projected costs that were much too high, as well as on proteins with disulfide bonds that could not be folded effectively. By focusing on basic biochemical reactions and controlling cell-free metabolism, these limitations have been methodically addressed (12). Amino-acid supply has been stabilized, and metabolism activated to dramatically reduce substrate costs by requiring only the addition of nucleotide monophosphates to drive energy production. Commercially available in vitro transcription translation kits based on E. coli, wheat germ, rabbit reticulocytes, and insect-cell extracts do not offer this advantage and are suitable only for research exploration at small scale. Control of the sulfhydryl redox potential has been gained and a robust disulfide isomerase added to facilitate oxidative protein-folding (13). These advances not only suggest production feasibility for pharmaceutical proteins containing the 20 natural amino acids, but they also provide enabling technology for incorporation of nnAAs at commercial scale.

A recent publication demonstrates that this open cell-free system (OCFS) developed by Swartz and collaborators can be optimized for high-level production of proteins to allow for scale-up to commercial levels once the target protein is identified (14). The authors expressed a multidisulfide-bonded protein, biologically active granulocyte-macrophage colony-stimulating factor (rhGM-CSF), at titers of 700 mg/L in 10 h. Importantly, they could show that the product was linearly scalable from starting materials in 96-well plates up to 100-L culture volume (14). The open nature of the system allows mass spectrometry-based profiling of the cell-free metabolome and proteome. Rapid testing of the effects of addition and subtraction of various components for system optimization can be modeled without the requirements for tuning more complex cellular networks required for maintaining cell viability commonly encountered in mammalian cell-line development.

Difficult-to-express proteins

The lack of a membrane-barrier in the OCFS provides the opportunity to express and study proteins that are difficult to express in cell-based systems. Many proteins that can't be readily expressed by in vivo expression systems due to poor folding, inclusion body accumulation, or due to toxicity can be expressed in an E. coli cell-free lysate.