# US 6,537,813 B1 · Concurrent flow mixing methods and apparatuses for the preparation of gene therapy vectors and compositions prepared thereby

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- Number: US 6,537,813 B1
- Document ID: US6537813B1
- Status: Granted
- Kind: B1
- Application number: 09/250,839
- Filed: 1999-02-16
- Granted: 2003-03-25
- Earliest claimed: 1998-02-13
- Assignees: Selective Genetics, Inc.
- Inventors: Xian Chen; Mark J. D'Andrea
- Web page: https://patentgrep.com/patent/US6537813B1

## CPC

- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 47/645: {Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The invention relates generally to methods and apparatuses that are adapted for the preparation of gene therapeutic compositions, as well as the compositions formed thereby. The invention also relates to methods adapted for making mixtures and condensate compositions. In the various embodiments, the present invention provides controlled and uniform mixing of gene therapy vectors and gene therapy vector vehicles for improved reproducibility, scaleability, stability, and pharmaceutical efficacy. Such compositions are suitable for use in mediation of disease.

## Claims

### Claim 1

A method for the preparation of a homogenous mixture comprising two or more molecular entities, comprising:
concurrently and separately introducing at least a first molecular entity-containing solution and a second molecular entity-containing solution, each in a controlled and independent manner, into at least a first flow-through mixer such that the two solutions contact, mix, and form the homogenous mixture, such that said homogenous mixture exits from the flow-through mixer at a controlled rate, and wherein said first and second molecular entity-containing solutions collectively comprise at least one vector and at least one vehicle; wherein said vehicle is selected from the group consisting of a ligand, a polycation, and a matrix formulation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mixer is a static mixer.

### Claim 3 (depends on 1)

The method of claim 1, wherein the mixer is a dynamic mixer.

### Claim 4 (depends on 1)

The method of claim 1, further comprising concurrently introducing a third molecular entity-containing solution.

### Claim 5 (depends on 1)

The method of claim 1, further comprising a second flow-through mixer, wherein the second flow-through mixture allows for the introduction of a third and/or fourth molecular entity-containing solution into the uniform mixture.

### Claim 6 (depends on 1)

The method of claim 1, wherein at least one molecular entity-containing solution comprises a medium that consists essentially of a non-aqueous medium.

### Claim 7 (depends on 6)

The method of claim 6, wherein the non-aqueous medium is selected from the group consisting of dimethylsulfoxide, tetramethyl urea, and N,N-dimethylacetamide.

### Claim 8 (depends on 6)

The method of claim 6, wherein the non-aqueous medium is mixed with an aqueous medium to form a uniformly mixed product.

### Claim 9 (depends on 1)

The method of claim 1, wherein the vehicle is a matrix formulation.

### Claim 10 (depends on 1)

The method of claim 1, wherein the vehicle is a polycation.

### Claim 11 (depends on 1)

The method of claim 1, wherein said flow-through mixer comprises:
a) a controllable first molecular entity-containing solution dispenser;
b) a controllable second molecular entity-containing solution dispenser;
c) a removal outlet; and
d) a chamber to receive delivery of solution from both dispensers a) and b) such that both solutions delivered by said solution dispensers will co-mix and flow through the removal outlet c).

### Claim 12 (depends on 1)

The method of claim 1, wherein the vector is a nucleic acid and the vehicle is a condensing agent.

### Claim 13

A method of preparing uniform size particles comprising a DNA:condensing agent condensation complex of a predetermined size, comprising the steps of:
a) providing i) a DNA-containing solution, ii) a condensing agent-containing solution, and iii) a flow-through mixer; and
b) concurrently and separately introducing said DNA-containing solution and said condensing agent-containing solution, each in a controlled and independent manner, into said mixer such that the two solutions contact, mix, and form a mixed solution containing a DNA:condensing agent condensation complex of predetermined size thereby, and such that said condensation complex exits from the flow-through mixer at a controlled rate such that further reaction of said condensation complex with additional DNA- and/or condensing agent-containing solution is avoided.

### Claim 14 (depends on 13)

The method of claim 13, wherein the mixer is a static mixer.

### Claim 15 (depends on 13)

The method of claim 13, further comprising the step of:
c) isolating the condensation complex from the mixed solution.

### Claim 16 (depends on 13)

The method of claim 13, wherein said flow-through mixer comprises:
a) a controllable DNA-containing solution dispenser;
b) a controllable condensing agent-containing solution dispenser;
c) a removal outlet; and
d) a chamber to receive delivery of solution from both dispensers a) and b) such that both solutions delivered by said solution dispensers will co-mix and flow through the removal outlet c).

### Claim 17 (depends on 13)

The method of claim 13, wherein said condensing agent comprises a polycationic molecule.

### Claim 18 (depends on 17)

The method of claim 17, wherein said polycationic molecules are selected from the group consisting of polycationic peptides, polycationic carbohydrates, polycationic synthetic polymers, inorganic multivalent cations, and synthetic viral particles.

### Claim 19 (depends on 13)

The method of claim 13, wherein the amounts of condensing agent and DNA used are such that the condensing agent/DNA charge ratio is from about 1 to about 5.

### Claim 20 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 2000 nm or smaller.

### Claim 21 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 1000 nm or smaller.

### Claim 22 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 500 nm or smaller.

### Claim 23 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 200 nm or smaller.

### Claim 24 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 100 nm or smaller.

### Claim 25 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 30-200 nm.

### Claim 26 (depends on 13)

The method of claim 13, wherein the condensation complex has a particle size of about 30-100 nm.

### Claim 27

A method for the preparation of a homogenous mixture comprising two or more molecular entities, comprising:
concurrently and separately introducing at least a first molecular entity-containing solution and a second molecular entity-containing solution, each in a controlled and independent manner, into at least a first flow-through mixer such that the two solutions contact, mix, and form the homogenous mixture, such that said homogenous mixture exits from the flow-through mixer at a controlled rate; and
isolating a resulting first molecular entity and second molecular entity composition from the resulting homogenous mixture,
wherein said first and second molecular entity-containing solutions collectively comprise at least one vector and at least one vehicle; wherein said vehicle is selected from the group consisting of a ligand, a polycation, and a matrix formulation.

### Claim 28

A method for the preparation of a homogenous mixture comprising two or more molecular entities, comprising:
concurrently and separately introducing at least a first molecular entity-containing solution and a second molecular entity-containing solution, each in a controlled and independent manner, into at least a first flow-through mixer such that the two solutions contact, mix, and form the homogenous mixture, such that said homogenous mixture exits from the flow-through mixer at a controlled rate, and wherein said first and second molecular entity-containing solutions collectively comprise at least one vector and at least one vehicle; wherein said vector is a virus and said vehicle is a matrix formulation.