Preparation of functional human factor VIII in mammalian cells using methotrexate based selection

{
  "publication_number": "US4965199A",
  "country": "US",
  "kind": "A",
  "title": "Preparation of functional human factor VIII in mammalian cells using methotrexate based selection",
  "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.",
  "claims": [
    "1. 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.",
    "2. The process of claim 1 which additionally includes the step of purifying factor VIII from the media of step (d).",
    "3. The process of claim 1 wherein the selectable marker is a neomycin resistance marker.",
    "4. The process of claim 3 wherein the selectable marker conferring neomycin resistance is the neomycin phosphotransferase marker and the selection medium contains G418.",
    "5. The process of claim 1 wherein the DNA sequences encoding Factor VIII and the amplifiable marker are contained within one vector.",
    "6. 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.",
    "7. 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.",
    "8. 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.",
    "9. The host cell of claim 1 that is BHK (ATCC No. CRL 8544).",
    "10. The host cell of claim 7 wherein the DNA sequences encoding Factor VIII and the amplifiable marker are contained within one vector.",
    "11. The process of claim 6 wherein the DHFR protein is wild-type DHFR protein.",
    "12. The host cell of claim 1 wherein the selectable marker is a neomycin resistance marker.",
    "13. The host cell cf claim 8 wherein the DHFR protein is wild-type DHFR protein."
  ],
  "cpc": [
    "C07K 14/755",
    "A61K 38/00",
    "C07K 2319/00",
    "C07K 2319/02",
    "C07K 2319/80",
    "C12N 15/1034",
    "C12N 15/62",
    "C12N 15/85",
    "C12N 2800/108",
    "C12N 2830/00",
    "C12N 2830/55",
    "C12N 2840/105",
    "C12N 2840/44",
    "C12Q 1/6832",
    "Y10S 435/948",
    "Y10S 930/10"
  ],
  "assignees": [
    "GENENTECH INC"
  ],
  "filing_date": "1987-08-07",
  "publication_date": "1990-10-23",
  "grant_date": "1990-10-23",
  "priority_date": "1984-04-20",
  "application_number": "US-8375887-A",
  "family_id": "26769699",
  "citations": [
    "EP0150735A2",
    "EP0157556A2",
    "US4027013A",
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    "US4104266A",
    "US4188318A",
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    "US4396601A",
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    "US4404131A",
    "US4757006A",
    "WO8400560A1",
    "WO8501961A1",
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  ]
}