In theory, cell-free expression (CFE) systems offer several benefits with the major one being that they are much quicker to set up and optimize, notes Vincent Noireaux, PhD, from the University of Minnesota.

“In cell-free gene expression systems proteins are produced outside cells, typically in an extract energized with necessary nutrients like ribonucleosides and amino acids. CFE systems are more flexible at the level of the speed of production,” he says, adding that there are also potential advantages in terms of the quality of the final products. “CFE has some advantages at the level of biosafety and biosecurity.”

The current problem is the limited range of proteins CFE systems can produce.

“Current CFE systems cannot produce all types of proteins,” explains Noireaux. “There are limitations on the type and amount of proteins that can be produced,” although these issues look likely to be addressed thanks to advances in the field.

For example, until recently producing proteins with disulfide bonds using CFE was impossible because such bonds do not form in the reducing environment in which current systems operate. This shortcoming is a major challenge. According to Noireaux and colleagues, this is because “the majority of biopharmaceuticals are eukaryotic extracellular proteins requiring this post-translational modification.”

However, one research group overcame this issue by modifying an existing CFE system. Specifically, they deleted the glutathione reductase gene and inactivated other reductase enzymes using alkylating agents.

“This approach has yielded protein targets that contain multiple disulfide bonds with 95% correctly folded,” explains Noireaux. “Furthermore, conditions for large-scale production have been identified at the microtiter-plate scale…This contrasts with process optimization for biopharmaceutical production using mammalian cell lines, which faces multiple challenges in moving between equivalent scales.”

And there are signs the biopharmaceutical companies are willing to embrace CFE for commercial-scale protein drug production.

“I would say that CFE has become interesting as an alternative to in vivo approaches,” continues Noireaux. “There are already companies specialized in CFE for biomanufacturing, for example, Sutro Biopharma, based on the West Coast.”