As more and more biopharmaceuticals move along the torturous process of development, clinical evaluation, and approval, dealing with changes to these complex protein molecules becomes increasingly paramount. A number of meetings and symposia have examined this question lately, and it’s clear that the technology is ramping up to successfully add or delete whole classes of changes when necessary.
Gary Walsh, Ph.D., associate professor at the University of Limerick in Ireland, foresees a wave of advances in fundamental post-translational modification (PTM) engineering technologies as new protein therapeutics work their way through the research lab and into the marketplace. With more than 2,000 biopharmaceuticals in clinical and preclinical development, there will be a pressing need for platforms that can successfully address the upscaling of highly modified protein therapeutics.
Dr. Walsh believes that biosimilars will become increasingly important and the issue of achieving their appropriate post-translational modification profile will constitute a major challenge in coming years. The EMA has already approved six biosimilars, and many more are being evaluated.
Whereas glycosylation is the major concern of post-translational engineers, there are many other alterations in protein molecules that may be required for appropriate performance. These include acetylation, ADP-ribosylation, acylation, phosphorylation, disulfide bond formation, proteolytic processing, γ-carboxylation, β-hydroxylation, amidation, and sulphation.
A major component of research in this area is concerned with the redesign of production systems to move post-translational modifications forward on an industrial scale, according to Dr. Walsh. As an example, cell lines are being engineered for improved glycosylation production by inactivating glycosylation enzymes that are problematic. One of these is the Potelligent cell line, in which fucosyltransferase is knocked out from the CHO parent cell. This allows the stable production of fucose-free antibodies, which show dramatically enhanced ADCC in vitro and ex vivo, and improved in vivo activity.
Another engineering accomplishment eliminates the problem of hypermannosylation in yeast. GlycoFi has radically engineered Pichia pastoris, knocking out four genes while at the same time introducing 14 others.
Yet another protein-production platform that has received relatively little notice is plant systems, which may generate hyperglycosylated products containing α-1,3-fucose and β-1,2-xylose. Greenovation Biotech working with moss, and Biolex which is based on the exploitation of duckweed, have engineered around this problem.
“Choosing an expression system capable of generating an appropriate post-translational product profile remains one of the most crucial decisions drug developers must make,” says Dr. Walsh.