# US 9,908,064 B2 · Portable fluidic platform for rapid cell-free production of protein biologics

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- Number: US 9,908,064 B2
- Document ID: US9908064B2
- Status: Granted
- Kind: B2
- Application number: 15/010,528
- Filed: 2016-01-29
- Published: 2016-08-11
- Granted: 2018-03-06
- Earliest claimed: 2015-02-06
- Assignees: Leidos, Inc.
- Applicants: Leidos, Inc.
- Inventors: John Dresios; Richard Holmes Griffey; Challise J. Sullivan; Erik D. Pendleton; Henri M. Sasmor; William L. Hicks; Eric M. Amendt
- Web page: https://patentgrep.com/patent/US9908064B2

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- B01D 15/10: characterised by constructional or operational features
- B01D 15/12: relating to the preparation of the feed
- B01D 15/14: relating to the introduction of the feed to the apparatus
- B01D 15/22: relating to the construction of the column
- B01D 15/34: Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
- B01D 15/361: {Ion-exchange}
- B01D 15/3804: {Affinity chromatography}
- C07K 14/505: Erythropoietin [EPO]
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C12M 21/18: {Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes}
- C12M 23/28: {disposable or single use}
- C12M 23/44: {Multiple separable units; Modules}
- C12M 29/00: {Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B)}
- C12M 41/48: {Automatic or computerized control (automatic analysis G01N35/00)}
- C12M 47/10: {Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02)}
- C12N 1/06: Lysis of microorganisms
- C12N 9/1223: {Phosphotransferases with a nitrogenous group as acceptor (2.7.3)}
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12Y 207/03002: Creatine kinase (2.7.3.2)
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

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).

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The platform of claim 1, wherein:
the production, separation and formulation of the target protein are completed within a period of 12 hours.

### Claim 3 (depends on 1)

The platform of claim 1, wherein:
the production, separation and formulation of the target protein are completed within a period of 10 hours.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

The platform of claim 1, wherein:
the reactor module holds a reaction volume between 0.1 and 5 ml.

### Claim 6 (depends on 1)

The platform of claim 1, wherein:
the first chamber and the second chamber are formed of polydimethylsiloxane (PDMS).

### Claim 7 (depends on 1)

The platform of claim 1, wherein:
the membrane is a 3.5 kDa dialysis membrane.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

The platform of claim 1, wherein:
the reaction solution further includes a lysate.

### Claim 10 (depends on 9)

The platform of claim 9, wherein:
the lysate comprises a eukaryotic cell-free protein synthesis system.

### Claim 11 (depends on 9)

The platform of claim 9, wherein:
the lysate comprises a prokaryotic cell-free protein synthesis system.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 1)

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.

### Claim 15 (depends on 1)

The platform of claim 1, wherein the reactor, chromatography and finishing modules are fluidically integrated into a self-contained disposable fluidic cartridge.

### Claim 16 (depends on 1)

The platform of claim 1, wherein:
the finishing module includes formulation via a size exclusion chromatography matrix.

### Claim 17 (depends on 1)

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.

### Claim 18 (depends on 1)

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.