US4582799A
Expired / Lapsed
US
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Process for recovering nonsecreted substances produced by cells
Assignee: Abbott Biotech Inc
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Filing: 1983-04-15
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Granted: 1986-04-15
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Expires: 2003-04-15
Abstract
Abstract Disclosed is a process for recovering nonsecreted substances produced by cells. The process eliminates some of the high molecular weight contaminants thereby simplifying the purification process. The cells are encapsulated within a semipermeable membrane having properties which permit rapid passage of the relatively low molecular weight substances of interest but retard or prevent passage of higher molecular weight contaminants. The encapsulated cells are suspended in a culture medium and undergo normal cell growth and mitosis. The encapsulated cell culture grows to substantially fill the capsules but not rupture them. The cell membrane is then lysed without disrupting the capsule membrane. The permeability of the capsule membrane is such that the substances of interest diffuse rapidly through the capsule membrane into the extracapsular fluid while the higher molecular weight contaminants and cell fragments are retained within the capsule. The process is particularly useful for obtaining low to moderate molecular weight substances produced by pyrogen-producing prokaryotic, genetically modified organisms because the crude product can be collected relatively free of high molecular weight pyrogenic contaminants.
Inventors
- 👤 Allan P. Jarvis, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 17:42
CPC Classifications
C; C12; C12N; C12N11/00; C12N11/02; C12N11/04; C12N1/00; C12N1/06; C12P; C12P1/00; Y; Y10; Y10S; Y10S424/00; Y10S424/07; Y10S435/00; Y10S435/803
Claims Analysis
Total Claims: 31
Claim 1
Independent Claim
118 words
A process for recovering a non-secreted substance produced by living cells and for recovering said substance as a crude product having a reduced concentration of high molecular weight contaminants, said process comprising the steps of: A. encapsulating cells within a capsule membrane have permeability properties which permit passage of a non-secreted substance but retard passage of high molecular weight contaminants; B. suspending said encapsulated cells in an aqueous culture medium; C. allowing said cells to undergo metabolism within said capsule membrane; D. lysing the cell membranes of said cells without disrupting said capsule membrane; E. allowing said substance to diffuse preferentially through said capsule membrane into an extracaspular fluid, and F. harvesting said substance from said extracapsular fluid.
Claim 2
Dependent Claim
118 words
A process for recovering a non-secreted substance produced by living cells and for recovering said substance as a crude product having a reduced concentration of high molecular weight contaminants, said process comprising the steps of: A. encapsulating cells within a capsule membrane have permeability properties which permit passage of a non-secreted substance but retard passage of high molecular weight contaminants; B. suspending said encapsulated cells in an aqueous culture medium; C. allowing said cells to undergo metabolism within said capsule membrane; D. lysing the cell membranes of said cells without disrupting said capsule membrane; E. allowing said substance to diffuse preferentially through said capsule membrane into an extracaspular fluid, and F. harvesting said substance from said extracapsular fluid.
Claim 3
Dependent Claim
41 words
The process of claim 1 wherein said encapsulation step (A) is effected by forming said capsule membrane by reaction between plural cationic groups on polymer chains and plural anionic groups on a water-soluble polymer to form a water-insoluble salt bonded matrix.
Claim 4
Dependent Claim
102 words
The process of claim 1 wherein said encapsulation step (A) is effected by the steps of: (a) suspending the cells in an aqueous medium physiologically compatible therewith and containing a water-soluble polymer having plural anionic moieties; (b) forming the suspension into a droplet containing the cell; (c) subjecting the droplet to a solution of multivalent, physiologically compatible cations to gel the droplet as a discrete, shape-retaining, water-insoluble temporary matrix; and (d) cross-linking surface layers of said temporary matrix to produce a semipermeable capsule membrane about said droplet by subjecting it to a polymer comprising plural cationic groups reactive with said anionic moieties.
Claim 5
Dependent Claim
20 words
The process of claim 3 comprising the additional step of resolubilizing the gel within said capsule membrane after step (d).
Claim 6
Dependent Claim
22 words
The process of claim 1 wherein said aqueous culture medium is a cell growth medium sufficient to support biosynthesis of said substance.
Claim 7
Dependent Claim
19 words
The process of claim 5 wherein said cell growth medium is sufficient to support mitosis of said encapsulated cells.
Claim 8
Dependent Claim
43 words
The process of claim 6 comprising the additional step of allowing said cells to undergo mitosis within said capsule membrane between steps C and D to produce a cell colony substantially filling the volume within said capsule membrane without rupturing said capsule membrane.
Claim 9
Dependent Claim
22 words
The process of claim 1 wherein the lysing of said cell membranes is effected by suspending said encapsulated cells in a detergent.
Claim 10
Dependent Claim
33 words
The process of claim 8 wherein said detergent is an aqueous solution of a detergent selected from a group consisting of nonionic surfactants, guanidine hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and mixtures thereof.
Claim 11
Dependent Claim
11 words
The process of claim 1 wherein said cells are prokaryotic cells.
Claim 12
Dependent Claim
11 words
The process of claim 10 wherein said contaminant comprises a pyrogen.
Claim 13
Dependent Claim
15 words
The process of claim 11 wherein said capsule membrane is substantially impermeable to said pyrogen.
Claim 14
Dependent Claim
11 words
The process of claim 11 wherein said pyrogen comprises an endotoxin.
Claim 15
Dependent Claim
18 words
The process of claim 11 wherein said pyrogen has a molecular weight greater than about 5×10 4 daltons.
Claim 16
Dependent Claim
12 words
The process of claim 1 wherein said cells comprise genetically modified cells.
Claim 17
Dependent Claim
11 words
The process of claim 15 wherein said contaminant comprises a pyrogen.
Claim 18
Dependent Claim
15 words
The process of claim 16 wherein said capsule membrane is substantially impermeable to said pyrogen.
Claim 19
Dependent Claim
11 words
The process of claim 16 wherein said pyrogen comprises an endotoxin.
Claim 20
Dependent Claim
18 words
The process of claim 16 wherein said pyrogen has a molecular weight greater than about 5×10 4 daltons.
Claim 21
Dependent Claim
24 words
The process of claim 1 wherein during said encapsulation step (A), spheroidal microcapsules having a diameter from about 0.1 to 1.0 millimeters are produced.
Claim 22
Dependent Claim
35 words
The process of claim 1 wherein said capsule membrane has an upper limit of permability greater than the molecular weight of said substance and lower than the molecular weight of said high molecular weight contaminant.
Claim 23
Dependent Claim
38 words
The process of claim 1 wherein said capsule membrane defines a multiplicity of tortuous-path pores, and the rate at which said pores allow passage of a molecule is an inverse function of the molecular weight of said molecule.
Claim 24
Dependent Claim
137 words
A process for recovering a nonsecreted substance produced by genetically modified prokaryotic cells, and for recovering the substance as a crude product having a reduced concentration of high molecular weight pyrogens, said process comprising the steps of: A. encapsulating genetically modified prokaryotic cells within an enclosed capsule defining an intracapsular volume, said capsule comprising a capsule membrane defining a multiplicity of tortuous-path pores which permit passage of a non-secreted substance while retarding the passage of a pyrogen; B. suspending said encapsulated cells in an aqueous culture medium; C. allowing said cells to undergo metabolism within said capsule membrane; D. lysing the cell membranes of said cells without disrupting said capsule membrane; E. allowing said substance to diffuse through the pores of said capsule membrane into an extracapsular fluid; and F. harvesting said substance from said extracapsular fluid.
Claim 25
Dependent Claim
137 words
A process for recovering a nonsecreted substance produced by genetically modified prokaryotic cells, and for recovering the substance as a crude product having a reduced concentration of high molecular weight pyrogens, said process comprising the steps of: A. encapsulating genetically modified prokaryotic cells within an enclosed capsule defining an intracapsular volume, said capsule comprising a capsule membrane defining a multiplicity of tortuous-path pores which permit passage of a non-secreted substance while retarding the passage of a pyrogen; B. suspending said encapsulated cells in an aqueous culture medium; C. allowing said cells to undergo metabolism within said capsule membrane; D. lysing the cell membranes of said cells without disrupting said capsule membrane; E. allowing said substance to diffuse through the pores of said capsule membrane into an extracapsular fluid; and F. harvesting said substance from said extracapsular fluid.
Claim 26
Dependent Claim
33 words
The process of claim 23 comprising the additional step of culturing said cells within said capsule membrane to produce a cell colony which substantially fills said intracapsular volume without rupturing said capsule membrane.
Claim 27
Dependent Claim
22 words
The process of claim 23 wherein the lysing of said cell membranes is effected by suspending said encapsulated cells in a detergent.
Claim 28
Dependent Claim
33 words
The process of claim 25 wherein said detergent is an aqueous solution of a detergent selected from a group consisting of non-ionic surfactants, guanidine hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and mixtures thereof.
Claim 29
Dependent Claim
41 words
The process of claim 23 wherein said encapsulation step (A) is effected by forming said capsule membrane by reaction between plural cationic groups on polymer chains and plural anionic groups on a water-soluble polymer to form a water-insoluble salt bounded matrix.
Claim 30
Dependent Claim
101 words
The process of claim 23 wherein said encapsulation step (A) is effected by the steps of: (a) suspending the cells in aqueous medium physiologically compatible therewith and containing a water-soluble polymer having plural anionic moieties; (b) forming the suspension into a droplet containing the cells; (c) subjecting the droplet to a solution of multivalent, physiologically compatible cations to gel the droplet as a discrete, shape-retaining, water-insoluble temporary matrix; and (d) cross-linking surface layers of said temporary matrix to produce a semipermeable capsule membrane about said droplet by subjecting it to a polymer comprising plural cationic groups reactive with said anionic moieties.
Claim 31
Dependent Claim
20 words
The process of claim 28 comprising the additional step of resolubilizing the gel within said capsule membrane after step (d).
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-04-14 | 1989-10-14 | 1990-04-15 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-04-14 | 1993-10-14 | 1994-04-15 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-04-14 | 1997-10-14 | 1998-04-15 | $7700.00 | Window Closed / Lapsed |