US4517293A
Expired / Lapsed
US
⏱️ 0.0 years remaining
High yield process for in vitro production of serum-free and mitogen-free interleukin-2 using a roller bottle culture system
Assignee: Hooper Trading Co
•
Filing: 1984-02-03
•
Granted: 1985-05-14
•
Expires: 2004-02-03
Abstract
Abstract An improved in vitro cell culture process for producing in high yield and purity a serum-free and mitogen-free interleukin-2-containing conditioned supernatant. The incubation steps during stimulation and conditioning of the interleukin-2 producing cells is carried out with a rapidly rotating roller bottle culture system. Yields of interleukin-2 are on the order of 10-fold and higher than obtained by similar cultivations carried out in flat dishes or tubes or in a roller bottle culture system rotated at conventional speeds. Improvement in yields in both the static and roller culture systems are also achieved by incubating the IL-2 producing cells under high oxygen concentrations, for example atmospheres of at least 70% O 2 and at least 5% CO 2 . In addition to using peripheral mononuclear blood cells (PBL) as the source of leukocytes containing the IL-2 producer cells, it is also possible to use buffy coat cells which are readily available as a waste by-product from blood banks.
Inventors
- 👤 Hans-Åke Fabricius
- 👤 Roland Stahn
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 04:09
CPC Classifications
C; C12; C12P; C12P21/00; A; A61; A61K; A61K38/00; A61K38/16; A61K38/17; A61K38/19; A61K38/20; A61K38/2013; C12N; C12N1/00; C12N5/00; C12N5/06; C12N5/0602; C12N5/0634; C12N5/0646; C12P1/00; C12N2502/00; C12N2502/11; C12R; C12R2001/00; C12R2001/91
Claims Analysis
Total Claims: 5
Claim 1
Independent Claim
213 words
A process for producing a serum-free and mitogen-free interleukin-2 containing supernatant comprising: (1) stimulating IL-2 producing primary cells which have been washed to remove substantially all proteins bound to the external cell membrane surface by incubating the washed cells in a liquid tissue culture medium supplemented with serum protein and a mitogen in a roller bottle cultivation system which is caused to rotate at a speed sufficient to rotate said roller bottle at from about 5 to 20 revolutions per minute to thereby provide a homogeneous culture medium, disperse metabolic products and synthesized proteins throughout the culture medium, and maintain a uniform cell suspension in the culture medium; (2) separating and washing the stimulated cells to remove substantially all of the serum and mitogen; and (3) conditioning the cells obtained in step (2) by incubating the cells in the presence of a serum-free and mitogen-free liquid tissue culture medium in a roller bottle cultivation system which is caused to rotate at a speed sufficient to rotate said roller bottle at from about 5 to 20 revolutions per minute to thereby provide a homogeneous culture medium, disperse metabolic products and synthesized proteins throughout the culture medium, and maintain a uniform cell suspension in the culture medium to thereby transfer interleukin-2 into the liquid phase.
Claim 2
Dependent Claim
213 words
A process for producing a serum-free and mitogen-free interleukin-2 containing supernatant comprising: (1) stimulating IL-2 producing primary cells which have been washed to remove substantially all proteins bound to the external cell membrane surface by incubating the washed cells in a liquid tissue culture medium supplemented with serum protein and a mitogen in a roller bottle cultivation system which is caused to rotate at a speed sufficient to rotate said roller bottle at from about 5 to 20 revolutions per minute to thereby provide a homogeneous culture medium, disperse metabolic products and synthesized proteins throughout the culture medium, and maintain a uniform cell suspension in the culture medium; (2) separating and washing the stimulated cells to remove substantially all of the serum and mitogen; and (3) conditioning the cells obtained in step (2) by incubating the cells in the presence of a serum-free and mitogen-free liquid tissue culture medium in a roller bottle cultivation system which is caused to rotate at a speed sufficient to rotate said roller bottle at from about 5 to 20 revolutions per minute to thereby provide a homogeneous culture medium, disperse metabolic products and synthesized proteins throughout the culture medium, and maintain a uniform cell suspension in the culture medium to thereby transfer interleukin-2 into the liquid phase.
Claim 3
Dependent Claim
34 words
The process of claim 1 wherein the incubation in the stimulating and the conditioning steps is performed in an atmosphere comprising from 70 to 95% oxygen and from 5 to 15% CO 2 .
Claim 4
Dependent Claim
22 words
The process of claim 1 or 2 wherein the starting IL-2 producing primary cells are buffy-coat cells obtained from a human donor.
Claim 5
Dependent Claim
69 words
The process of claim 1 wherein the roller bottles have a diameter of from about 4 to about 5 inches and are filled with from about 50 to 200 milliliters of the liquid tissue culture medium, and wherein the roller bottles in the stimulation and conditioning incubation steps are rotated by the rollers of the roller bottle culture system at a speed of at least 6 revolutions per minute.
References Cited (Backward Citations)
US3812016A
Cited
US4337104A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-05-13 | 1988-11-12 | 1989-05-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-05-13 | 1992-11-12 | 1993-05-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-05-13 | 1996-11-12 | 1997-05-14 | $7700.00 | Window Closed / Lapsed |