12:00 AM
Feb 17, 2011
 |  BC Innovations  |  Tools & Techniques

Engineering protein stability

Two American teams have proposed distinct strategies to improve the stability of recombinant proteins. The techniques could be used to increase the manufacturing yield, shelf life and potentially the duration of efficacy of biologics, including antibodies and therapeutic cytokines.

Efforts to improve a protein's stability typically require the production of hundreds of variants and characterization of their biophysical properties in vitro in order to choose the best variant. The two new techniques are expected to be applicable to a range of proteins and could stabilize antibodies and therapeutic cytokines more quickly and efficiently than current methods (see "Two approaches for improving protein stability").

A team at The Scripps Research Institute uncovered an amino acid sequence that, when placed near glycosylation sites, helps stiffen the protein's backbone.1 This stabilizing amino acid sequence proved transplantable into nonglycosylated proteins, allowing them to become glycosylated and stabilized.

Pfizer Inc.already has obtained a license to the approach.

Meanwhile, a group at the Massachusetts Institute of Technology devised an enzymaticscheme to join thetwo ends of small cytokines like interferon-a2 (IFNA2; IFN-a2), G-CSF (CSF3) and erythropoietin (EPO) and tag them with the stabilizing molecule polyethylene glycol (PEG). The resulting pegylated circular proteinsretain all of their biological activity but are more stable than their parent molecules.2

Spicing with sugar

Jeffery Kelly, professor of chemistry and chairman of the Department of Molecular and Experimental Medicine at Scripps, said that increasing the stability of therapeutic proteins could lead to easier manufacturing and dosing.

"One key challenge for making biologic drugs is to have a long half-life, which leads to good patient compliance," said Kelly. "The other key challenge is manufacturing stable proteins. There's very strong evidence in the literature that the stability of protein-based drugs correlates with their half-life" on the shelf and in vivo.

Taking a rational approach, Kelly and Evan Powers, an associate professor of chemistry at Scripps, set out to crack the protein sequence code that would explain the stability caused by glycosylation, a post-translational modification naturally found in many useful proteins, including mAbs.

Glycosylation involves the addition of a sugar-rich glycan chain to specific sites containing anasparagine residue next to glycine andthreonine residues. Previous studies have shown that blocking glycosylation destabilizes naturally glycosylated proteins, so it is widely believed that glycosylation improves stability.

Indeed, researchers have long attempted to increase the stability of

nonglycosylated proteins by engineering glycosylation sites into them....

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