Next-Gen Expression Systems - Pharmaceutical Technology

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Next-Gen Expression Systems
More sophisticated biological expression systems expand the functionality of the traditional systems for protein synthesis.

Pharmaceutical Technology
Volume 35, Issue 6, pp. 36-39

California-based Sutro Biopharma has been attempting the same type of manipulation, but using a cell-free expression system. As a bench-scale tool, cell-free expression systems have been in existence for many years, but recent advances, in particular, identification of a cost-effective supply of energy to power the system, have made it possible to scale up these systems to achieve yields purported to be suitable for manufacturing. Sutro Biosciences has developed a cell-free expression system that can be scaled from 200 μl to 200 L. The system uses an extract of E. coli (KGK10) that contains the cell's protein synthetic machinery, but is free of bacterial DNA. The extract can be produced in bulk and frozen for up to a year before use. The reaction mix contains the bacterial extract, a source of energy, amino acids, and the DNA coding the protein to be produced (6). The time- consuming processes of cell transfection and clonal selection are eliminated.

The system can be used to produce large molecular weight, correctly folded proteins with multiple disulfide bonds as well as multi-subunit proteins, according to Dr. Trevor Hallam, the company's chief scientific officer. Using this system, it has been possible to produce a number of proteins that have been difficult to express in E. coli. For example, Sutro can now produce up to 1300 mg/L of a version of biologically active human recombinant granulocyte-macrophage colony stimulating factor. The in vitro rate of protein synthesis is around one-twentieth the in vivo rate, which seems to facilitate the correct folding of the proteins.

An open system allows for certain manipulations that would not be possible in vivo, notes Hallam. For instance, the stoichiometry of a multi-subunit protein can be controlled by adjusting the ratio of DNA templates that are added to the reaction. Temporal control of expression of a multi-subunit protein is also possible. When expressing antibodies, Sutro researchers have found that adding the DNA template for the light chain an hour in advance of that of the heavy chain prevents aggregation of the heavy chain protein.

The cell-free system offers the potential to do development and manufacturing in the same system. Working initially at 96-well scale, researchers would be able to rapidly screen many iterations of the protein of interest. Nonnatural amino acids can be easily introduced, since there is no barrier to uptake, with the amino-acylated tRNA required for insertion added to the reaction mix as a reagent. Developers can then vary the site of insertion of the amino acid, and explore structure–activity relationships just as they would when developing small-molecule therapeutics. Once the optimal structure is identified, scale-up to commercial levels would be straight-forward, says Hallam. By using the non-natural amino acid as a point of attachment for an antibody–drug conjugate, drug developers could control the number of drug conjugates per antibody to produce a single species of high quality.

The current dominance of just two expression systems, E. coli and CHO cells, for biopharmaceutical manufacturing is driven by the fact that both manufacturers and regulatory agencies are familiar with those systems. The increasing market in biosimilars should move manufacturers to explore and adopt alternative expression systems, if there are cost-savings to be realized by doing so. Additionally, biologics developers are becoming more sophisticated in the types of molecules they wish to design. The market has moved beyond simple protein therapeutics to encompass antibodies, antibody fragments, and drug–antibody conjugates, which will drive adoption of systems that allow more control over the finished product.


1. G. Walsh, Nat. Biotechnol. 29(9), 917–924 (2010).

2. FDA, "Celebrating a Milestone: FDA's Approval of First Genetically-Engineered Product." SelectionsFromFDLIUpdateSeriesonFDAHistory/ucm081964.htm, accessed May 2011

3. S.R. Hamilton et al., Science 313(5792), 1441–1443 (2006).

4. P. Hossler, S.F. Khattak, and Z.J. Li, Glycobiol. 19(9), 936–949 (2009).

5. Y. Kanda et al., Glycobiol. 17(1), 104–118, (2006).

6. Zawada et al., Biotech. Bioeng. 108(7), 1570–1578, (2011).


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