(666e) Synthesis of Therapeutic Proteins Using a Continuous Exchange Microfluidic System

The development of cell-free protein expression technology enables creation of a potentially flexible tool for on-demand production of protein-based therapeutics. Although cell-based methods are the conventional technology for protein production, cell-free expression systems allow for greater control over the reaction conditions and may be optimized for the production of a specific product. Small scale production methods can lower production costs and can accelerate the manufacture of a variety of proteins, enabling protein screening assays and the validation and functional analysis of protein products. While there are many advantages in using cell-free protein expression systems, there are still several implementation challenges that further depend on the type of protein targeted and the expression system to be used (i.e. bacterial, eukaryotic, mammalian). Proper protein folding, glycosylation patterns, overall yields, and concentration must be considered. Along these lines, significant effort has gone into the development of robust cell extracts, including methods to control the formation of disulfide bonds during protein folding. Proper disulfide bond formation is critical for many therapeutic proteins. Yields and concentrations are difficult to optimize in micro-scale systems in which reactants are quickly exhausted and inhibitory molecules can become concentrated. To address this, cell-free micro-scale systems with membrane dialysis capabilities have been developed. Well engineered continuous exchange micro-reactors can feed in needed components such as ATP and amino acids, while also decreasing the concentrations of inhibitory species such as phosphates in order to extend translation reactions and increase protein yields.

Here, we describe the development of a continuous exchange micro-reactor, using a microfluidic device, in order to produce a single therapeutic dose of a protein such as human granulocyte-macrophage colony-stimulating factor (hGM-CSF) at a desired amount of ~1 mg. This platform is compatible with other micro-scale protein purification and processing modules. The microfluidic devices fit on either a standard glass slide (50 µl reactor) or a 2” x 3” glass slide (250 µl reactor) and allow mixing of up to three components that are fed into a common channel. The three components or inputs are an E. coli based cell extract including T7 polymerase, a master mix that is composed of all necessary nutrients and protein building components, and plasmid DNA encoding the desired protein. Rapid mixing of the components as they flow through the channel and the ability to operate in batch or continuous-flow mode provides significant flexibility in operation over conventional bulk reactors. Additionally, the microfluidic design allows pairing of a supporting feeder channel with the micro-reactor, separated by a membrane, to allow for exchange of components and the continuous addition of energy and nutrients. Here, we present our progress in optimizing the micro-reactor design and provide a scalability analysis, a comparison of batch and continuous flow modes, and a quantitative comparison of proteins yields from microfluidic reactors, microfluidic reactors with component exchange, and more commonly used static, bulk reactors.