System and method for production of on-demand proteins in a portable unit for point of care delivery

Patent metadata

Field	Value
Publication number	US-10774304-B2
Application number	US-201916660993-A
Country	US
Kind code	B2
Filing date	Oct 23, 2019
Priority date	Mar 8, 2011
Publication date	Sep 15, 2020
Grant date	Sep 15, 2020

Abstract

A portable and mobile bioprocessing system and method for protein manufacturing that is compact, integrated and suited for on-demand production of any type of proteins and for delivery of the produced proteins to patients or for assay purposes. The portable system and method can also be used for efficient on-demand production of any type of protein with point-of-care delivery.

First claim

That which is claimed is:

1. A cell-free bioprocessing system for the production of on-demand synthesized desired target protein, the system comprising:
a production module for producing a protein; and
a purification module for receiving the protein from the production module and for purifying the protein from reagents, wherein the production module comprises a bioreactor for cell-free based protein expression, wherein the bioreactor comprises a resin column for binding of an RNA polymerase to catalyzes the polymerization of message ribonucleotides,
and wherein the purification module comprises a membrane chromatography component for receiving the protein from the production module and for outputting purified protein; and a diafiltration component for receiving the purified protein from the membrane chromatography component and for outputting further purified protein.

2. The cell-free bioprocessing system of claim 1 , wherein bioreactor comprises the inclusion of cell lysate of CHO or E. coli cells, reaction mixture, amino acids, nucleotides and a DNA template.

3. The cell-free bioprocessing system of claim 2 , wherein the DNA template is reactivated from an inactive state dry or frozen state.

4. The cell-free bioprocessing system of claim 1 , wherein the resin column for binding of an RNA polymerase is a Ni-NTA (nickel-nitrilotriacetic acid) column.

5. The cell-free bioprocessing system of claim 1 , wherein the RNA polymerase is selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase.

6. The cell-free bioprocessing system of claim 1 , wherein the RNA polymerase is T7 RNA polymerase used for transcription of the DNA template to synthesis a messenger RNA.

7. The cell-free bioprocessing system of claim 6 , wherein the DNA template comprises promoter nucleotide sequences for the T7 RNA polymerase to starts transcription of the DNA.

8. The cell-free bioprocessing system of claim 1 , further comprise a processor for controlling and/or monitoring at least the production module and/or the purification module.

9. The cell-free bioprocessing system of claim 8 , wherein the processor is communicatively connected to at least the production module and/or purification module to control the timing, temperature and other parameters necessary for optimizing the production and purification of the synthesized proteins to provide a sufficient amount of or a therapeutic dosage of the synthesized protein.

10. A cell-free bioprocessing system for production of a desired target protein comprising:
(a) a bioreactor for protein expression comprising:
a DNA template encoding the desired target protein;
a Ni-NTA resin column comprising an RNA polymerase for synthesizing mRNA by transcription of the DNA template;
(b) a membrane chromatography component for receiving and purifying protein output by the bioreactor; and
(c) a diafiltration component for receiving purified protein from the membrane chromatography and for further purifying the purified protein.

11. A method of preforming an in vitro protein synthesis reaction of a desired target protein, the method comprising the steps of
(a) providing a cell-free lysate in a bioreactor;
(b) adding to the bioreactor, before or after step (a) at least a buffer, salts, amino acids as part of a reaction mixture;
(c) adding a DNA template for the desired target protein to the reaction mixture, the DNA template encoding the expression of a protein and including a promoter recognized by an RNA polymerase in the reaction, wherein the RNA polymerase is bound to a resin column within the bioreactor; and
(d) incubating the reason mixture under conditions such that DNA template is transcribed to a messenger RNA (mRNA) molecule by transcription and then the mRNA is translated into the desired target protein.

12. The method of claim 11 , wherein the DNA template is fresh sample or from a preserved source and revitalized from a frozen or dry state.

13. The method of claim 11 , wherein the desired target protein is needed during a natural disaster.

Assignees

• Univ Maryland

Inventors

• Rao Govind
• Kostov Yordan
• Tolosa Leah
• Ge Xudong
• Frey Douglas
• Borhani Shayan Gholipour
• Burgenson David

Classifications

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12M47/12

  Purification (C12M47/04 takes precedence) · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12M41/12

  of temperature (controlling the temperature of chemical or physical processes B01J19/0013, heating or cooling apparatus for laboratory use B01L7/00) · CPC title

• C12M47/10 Primary

  Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02) · CPC title