Preparation of substances with encapsulated cells

• US 4,409,331 A
• Filed: 04/28/1982
• Issued: 10/11/1983
• Est. Priority Date: 03/28/1979
• Status: Expired due to Term

• Current Assignee: Repligen Corporation
• Original Assignee: Damon Corporation (Thor Industries Incorporated)
• Inventors: Lim, Franklin
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/372,835
• Time in Patent Office: 531 Days
• Field of Search: 435/174, 435/177, 435/178, 435/180, 435/182, 435/240, 435/241, 424/DIG. 7, 424/93
• US Class Current: 435/178
• CPC Class Codes: A61K 2035/128 capsules, e.g. microcapsules
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: A61K 9/5031 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/5036 Polysaccharides, e.g. gums, alginate; Cyclodextrin
• CPC Class Codes: A61K 9/5073 having two or more different coatings optionally including drug-containing subcoatings
• CPC Class Codes: B01J 13/14 Polymerisation; cross-linking
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0012 Cell encapsulation
• CPC Class Codes: Y10S 424/07 Microporous membranes
• CPC Class Codes: Y10S 623/915 Method or apparatus for preparing biological material

Abstract

Disclosed is a system and process for producing substances produced in cells such as antibodies and biological response modifiers. Cells which produce the substance of interest are encapsulated within semipermeable membranes having an upper limit of permeability sufficient to allow traverse of ions, amino acids and other cell nutrients and then suspended in a culture medium. Serum components or other high molecular weight materials needed for ongoing viability and normal in vitro metabolism of certain types of cells may be included within the intracapsular volume and may be excluded from the extracapsular medium by limiting the permeability of the membranes. The substance of interest collects either in the intracapsular volume or the extracapsular medium, depending on the degree of permeability of the membranes and on the molecular weight of the substance.

Claims

1. A process for producing a substance which is produced by living cells, said process comprising the steps of:

A. encapsulating said cells within semipermeable membranes having a selected upper limit of permeability, said encapsulating being effected by forming said semipermeable membranes by reacting cationic groups on polymer chains having a molecular weight greater than about 3000 with anionic groups on a polysaccharide gel encapsulating said cells to crosslink surface layers of said polysaccharide gel to form said semipermeable membranes;

B. suspending said encapsulated cells in an aqueous culture medium;

C. allowing said cells to undergo metabolism in vitro and to produce said substance; and

D. harvesting said substance either from said aqueous medium or from within said membranes.

2. The process of claim 1 wherein said cells comprise hybridoma cells.

3. The process of claim 1 wherein said encapsulation step (A) is effected by the steps of:

(1) suspending the cells in an aqueous medium physiologically compatible therewith and containing a water-soluble gum having plural anionic moieties;

(2) forming the suspension into droplets containing the cells;

(3) subjecting the droplets to a solution of multivalent, physiologically compatible cations to gel the droplets to form discrete, shape-retaining, water-insoluble temporary capsules as said polysaccharide gel encapsulating said cells; and

(4) cross-linking surface layers of said temporary capsules to produce semipermeable membranes about said gelled droplets by subjecting them to said polymer containing cationic groups reactive with said anionic groups.

4. The process of claim 3 comprising the additional step of resolubilizing the gel within the membrane produced in step (4).

5. The process of claim 1 wherein said substance has a molecular weight below said selected upper permeability limit, said process comprising the step of allowing the substance to diffuse through said membranes into said aqueous medium and harvesting said substance therefrom.

6. The process of claim 1 or 5 wherein said cells are encapsulated together with a complete cell culture medium sufficient to maintain said cells and to allow biosynthesis of said substance in vitro.

7. The process of claim 1 or 5 wherein the aqueous medium used in step (B) is a complete cell culture medium sufficient to maintain said cells and to allow biosynthesis of said substance in vitro.

8. The process of claim 6 wherein a component having a molecular weight in excess of the upper permeability limit of said membranes is needed by said cells in order to allow in vitro biosynthesis of said substance, said process comprising the additional step of encapsulating said component together with said cells.

9. The process of claim 1 or 5 wherein said cells are mammalian cells.

10. The process of claim 1 or 5 comprising the additional step of allowing said cells to undergo mitosis within said capsule.

11. The process of claim 1 or 5 wherein said cells are cells which have been genetically modified.

12. The process of claim 1 or 5 wherein during said encapsulation step (A), spheroidal microcapsules having a diameter from about 0.4 to 1.0 mm are produced.

13. The process of claim 1 or 5 wherein the substance harvested in step (E) is selected from the group consisting of insulin, glucagon, prolactin, somatostatin, thyroxin, steroid hormones, pituitary hormones, interferons, FSH, and PTH.

14. The process of claim 1 or 5 wherein the substance harvested in step (E) is selected from the group consisting of hormones, interferons, lymphokines, and antibodies.

15. The process of claim 1 or 5 wherein the cells encapsulated in step (A) require contact with a component having a molecular weight in excess of the upper permeability limit of said membranes in order to maintain production of said substance, said component being encapsulated together with said cells in step (A) and the aqueous culture medium used in step (B) being substantially free of said component.

16. The process of claim 1 wherein said selected upper limit of permeability is below about 1.5×

10⁵ daltons.

17. The process of claim 1 wherein said cells comprise hybridoma cells, said substance comprises monoclonal antibodies having a molecular weight in excess of said selected upper limit of permeability, and said antibodies are harvested from within said membranes.

18. A system for producing a substance which is produced by living cells comprising viable cells encapsulated within semipermeable membranes and suspended in an aqueous culture medium for said cells,

the semipermeable membranes of said encapsulated viable cells having an upper limit of permeability sufficient to allow traverse of nutrients required by said cells, said semipermeable membranes comprising a polysaccharide gel having plural anionic groups cross-linked with a polymer having a molecular weight greater than about 3000 and having plural cationic groups, said semipermeable membranes encapsulating suspended viable, healthy, physiologically active cells disposed in a medium which includes all components (A) needed to maintain viability of said cells and which components are of a size in excess of the upper permeability limit of said membranes,

said aqueous culture medium comprising all components (B) needed to maintain viability of said cells having a molecular weight below the upper permeability limit of said membranes.

19. The system of claim 18 wherein said components (A) comprise serum components.

20. The system of claim 18 wherein said cells comprise mammalian cells.

21. The system of claim 18 wherein said cells comprise microorganisms.

22. The system of claim 18 wherein said cells comprise genetically modified cells.

23. The system of claim 18 wherein said cells comprise hybridoma cells.

24. The system of claim 18 wherein said cells comprise cells capable of producing in vitro a substance selected from the group consisting of hormones, interferons, lymphokines, and antibodies.

25. The system of claim 18 wherein said cells comprise cells capable of producing in vitro a substance selected from the group consisting of insulin, glycogen, growth hormones, pituitary hormones, steroid hormones, prolactin, somatostatin, PTH, and FSH.