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US4965199A
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Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
Assignee: Genentech Inc
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Filing: 1987-08-07
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Granted: 1990-10-23
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Expires: 2007-08-07
Abstract
Abstract A method for producing factor VIII in recombinant mammalian host cells utilizing an expression vector containing a selectable marker DNA and an amplifiable marker DNA. The initial selection is based upon the selectable marker and subsequent amplification of factor VIII DNA and amplifiable marker DNA is conducted in cells not deficient in the amplifiable marker.
Inventors
- 👤 Daniel J. Capon
- 👤 Richard M. Lawn
- 👤 Arthur D. Levinson
- 👤 Gordon A. Vehar
- 👤 William I. Wood
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 11:52
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/745; C07K14/755; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/11; C12N15/62; C12N15/63; C12N15/79; C12N15/85; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; A; A61; A61K; A61K38/00; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/80; C12N2800/00; C12N2800/10; C12N2800/108; C12N2830/00; C12N2830/55; C12N2840/00; C12N2840/10; C12N2840/105; C12N2840/44; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948; Y10S930/00; Y10S930/01; Y10S930/10
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
99 words
A process for producing factor VIII which comprises: (a) cotransfecting a mammalian host cell with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker, and a third DNA sequence encoding a selectable marker; (b) growing the transfected cell in a non-selective medium; (c) transferring the transfected cells to a selection medium and selecting for such selectable marker resistant cells; and (d) amplifying said amplifiable marker DNA sequence by culturing the selected cells of step (c) in media containing increasing amounts of selection agent, wherein the host cell is not deficient in the amplifiable marker.
Claim 2
Dependent Claim
99 words
A process for producing factor VIII which comprises: (a) cotransfecting a mammalian host cell with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker, and a third DNA sequence encoding a selectable marker; (b) growing the transfected cell in a non-selective medium; (c) transferring the transfected cells to a selection medium and selecting for such selectable marker resistant cells; and (d) amplifying said amplifiable marker DNA sequence by culturing the selected cells of step (c) in media containing increasing amounts of selection agent, wherein the host cell is not deficient in the amplifiable marker.
Claim 3
Dependent Claim
20 words
The process of claim 1 which additionally includes the step of purifying factor VIII from the media of step (d).
Claim 4
Dependent Claim
14 words
The process of claim 1 wherein the selectable marker is a neomycin resistance marker.
Claim 5
Dependent Claim
23 words
The process of claim 3 wherein the selectable marker conferring neomycin resistance is the neomycin phosphotransferase marker and the selection medium contains G418.
Claim 6
Dependent Claim
21 words
The process of claim 1 wherein the DNA sequences encoding Factor VIII and the amplifiable marker are contained within one vector.
Claim 7
Dependent Claim
32 words
The process of claim 1 wherein the selection agent is methotrexate and the amplifiable marker is DHFR protein that binds to methotrexate with an affinity comparable to that of wild-type DHFR protein.
Claim 8
Dependent Claim
40 words
A mammalian host cell cotransfected with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker and a third DNA sequence encoding a selectable marker, wherein the host cell is not deficient in the amplifiable marker.
Claim 9
Dependent Claim
40 words
A mammalian host cell cotransfected with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker and a third DNA sequence encoding a selectable marker, wherein the host cell is not deficient in the amplifiable marker.
Claim 10
Dependent Claim
27 words
The host cell of claim 2 wherein the amplifiable marker is DHFR protein that binds to methotrexate with an affinity comparable to that of wild-type DHFR protein.
Claim 11
Dependent Claim
13 words
The host cell of claim 1 that is BHK (ATCC No. CRL 8544).
Claim 12
Dependent Claim
22 words
The host cell of claim 7 wherein the DNA sequences encoding Factor VIII and the amplifiable marker are contained within one vector.
Claim 13
Dependent Claim
13 words
The process of claim 6 wherein the DHFR protein is wild-type DHFR protein.
Claim 14
Dependent Claim
15 words
The host cell of claim 1 wherein the selectable marker is a neomycin resistance marker.
Claim 15
Dependent Claim
14 words
The host cell cf claim 8 wherein the DHFR protein is wild-type DHFR protein.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1993-10-22 | 1994-04-23 | 1994-10-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-10-22 | 1998-04-23 | 1998-10-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-10-22 | 2002-04-23 | 2002-10-23 | $7700.00 | Window Closed / Lapsed |