Portable fluidic platform for rapid cell-free production of protein biologics
A portable fluidic platform for rapid and flexible end-to-end production of recombinant protein biologics includes a bioreactor system hosting stable and robust cell-free translation systems that is fluidically integrated with modular protein separation functionalities (e.g., size exclusion, ion exchange or affinity chromatography systems) for purification of the cell-free expressed product and which are configurable for process-specific isolation of different proteins, as well as for formulation. The bioreactor utilizes lysates from engineered eukaryotic (e.g., yeast) or prokaryotic (e.g., bacterial) strains that contain factors for protein folding and posttranslational modifications. Combination of various purification modules on the same fluidic platform allows flexibility of re-routing for purification of different proteins depending on specific target requirements. Protein synthesis and purification modules are integrated into self-contained disposable fluidic cartridge that eliminates cross-contamination between runs. The platform allows for flexible production of protein biologics within 24 hours (from DNA to purified product).
1. A portable, modular platform for cell-free production, purification and formulation of a protein, wherein the platform comprises:
a loading module for loading a reaction solution including one or more reaction reagents to a reactor module;
a feeder module for loading a feeding solution to the reactor module;
the reactor module having a membrane therein forming a first chamber and a second chamber, wherein the first chamber receives the reaction solution from the loading module and the second chamber receives the feeding solution from the feeder module, and wherein said reactor module facilitates production of a target protein via a process selected from the group consisting of: (i) continuous exchange cell-free (CECF) and (ii) batch protein synthesis;
a chromatography module for separating the target protein from one or more components of the reaction solution; and
a finishing module for polishing and formulation of the target protein;
wherein the production, separation and formulation of the target protein are completed within a period of 24 hours.
2. The platform of claim 1 , wherein:
the production, separation and formulation of the target protein are completed within a period of 12 hours.
3. The platform of claim 1 , wherein:
the production, separation and formulation of the target protein are completed within a period of 10 hours.
4. The platform of claim 1 , wherein:
the reactor module comprises a 2 ml chamber divided by the membrane into a 1 ml first chamber and a 1 ml second chamber.
5. The platform of claim 1 , wherein:
the reactor module holds a reaction volume between 0.1 and 5 ml.
6. The platform of claim 1 , wherein:
the first chamber and the second chamber are formed of polydimethylsiloxane (PDMS).
7. The platform of claim 1 , wherein:
the membrane is a 3.5 kDa dialysis membrane.
8. The platform of claim 1 , wherein:
production of the target protein is in an amount between about 40 μg/ml and about 8 mg/ml protein.
9. The platform of claim 1 , wherein:
the reaction solution further includes a lysate.
10. The platform of claim 9 , wherein:
the lysate comprises a eukaryotic cell-free protein synthesis system.
11. The platform of claim 9 , wherein:
the lysate comprises a prokaryotic cell-free protein synthesis system.
12. The platform of claim 1 , further comprising:
a control system for facilitating purification of different proteins on the same platform via switching among various chromatography matrices.
13. The platform of claim 1 , further comprising:
one or more sampling ports for removing sample aliquots from the reaction chamber for assessment of protein synthesis and formulation processes.
14. The platform of claim 1 , further comprising:
one or more syringe pumps with integrated rotor valves for directing fluidics between one or more of the loader, feeder, reactor, chromatography and finishing modules and facilitating addition and removal of a solution, including the reaction solution and the feeding solution.
15. The platform of claim 1 , wherein the reactor, chromatography and finishing modules are fluidically integrated into a self-contained disposable fluidic cartridge.
16. The platform of claim 1 , wherein:
the finishing module includes formulation via a size exclusion chromatography matrix.
17. The platform of claim 1 , wherein:
the chromatography module for separating the target protein employs at least one of (a) FLAG™-tag-based affinity chromatography, (b) protease affinity chromatography, and (c) size exclusion chromatography steps, or a combination thereof.
18. The platform of claim 1 , wherein:
the chromatography module for separating the target protein employs at least one of (a) ion-exchange chromatography, (b) protein concentration, and (c) size exclusion chromatography steps, or a combination thereof.