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Cell lines and methods for producing proteins

a technology of cell lines and methods, applied in the field of transgenic avian generation, can solve the problems of primary cultures of normal oviduct cells unsuitable for large-scale in vitro production of heterologous proteins, and achieve the effect of facilitating selection of tumor cells

Inactive Publication Date: 2005-04-28
SYNAGEVA BIOPHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is based on the generation of avian cell lines that can produce heterologous polypeptides for multiple passages in cell culture. These cell lines can be derived from oviduct tumor cells, which are manipulated and propagated in cell culture. The cell lines can be sustained in culture for at least 3 passages, and can produce the heterologous protein for at least 5 passages. The cell lines can be isolated from various avian sources such as chickens, turkeys, ducks, and more. The invention also features a method of producing a heterologous protein by culturing oviduct tumor cells and isolating the protein. The heterologous proteins produced include antibodies, cytokines, fusion proteins, growth factors, enzymes, structural proteins, interferons, granulocyte-colony stimulating factor, and erythropoietins. The invention provides pharmaceutical compositions comprising the heterologous proteins and a pharmaceutically acceptable carrier."

Problems solved by technology

Cells collected and cultured in this manner typically die or differentiate into cells that do not produce egg white protein within three days making primary cultures of normal oviduct cells unsuitable for use in large scale in vitro production of heterologous proteins.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of an Immortal Cell Line from Avian Oviduct Tissue

[0080] Epithelial adenoma tumors are isolated from a chicken oviduct and are finely minced into 1 to 2 mm pieces which are incubated in F12 medium with 0.8 mg / ml collagenase. The tissue is shaken at 37° C. for 30 minutes and triturated to generate cell clumps consisting of approximately 100 to 1000 cells. The clumps of cells are separated from red blood cells and cellular debris which is discarded. The clumps are suspended in pre-warmed F12 media, supplemented with 0.8 mg / ml collagenase and 1.0 mg / ml dispase, and incubated at 37° C. with shaking. Cell clumps are triturated every ten minutes to generate clumps consisting of about 5 to 100 cells. The cells are pelleted with low speed centrifugation and the resultant pellet is suspended in ice cold F12 which is filtered through a single layer of sterile surgical gauze tape to remove larger cell clumps. The cells are washed twice with ice cold F12, pelleted and resuspended in...

example 2

Production of an Immortal Cell Line from Avian Oviduct Tissue of a Transgenic Avian and Production of Heterologous Protein

[0083] Transgenic chickens which produce eggs with interferon present in the egg white are disclosed in U.S. Pat. No. 6,730,822. An interferon producing transgenic chicken is matured to five years of age. Oviduct tissue is removed from the chicken and several tumors are isolated. An immortal cell line is obtained from the tumors as described in Example 1.

[0084] Growth medium in which the immortal ovalbumin producing cell lines are passaged tests positive for interferon.

example 3

Production of an Immortal Cell Line from Avian Tubular Gland Cells

[0085] Freshly harvested chicken oviduct tissue is isolated from a healthy 40 week old chicken. The tissue is finely minced into small pieces approximately 1 mm in diameter. The tissue is shaken at 42° C. for 20 min and triturated to generate clumps each consisting of about 100 to 1000 cells. The clumps of cells are separated from red blood cells and cellular debris which is discarded and are then suspended in pre-warmed McCoy's 5A medium, supplemented with 0.8 mg / ml collagenase and 1.0 mg / ml dispase, and incubated at 42° C. with shaking. The cell clumps are triturated every ten minutes to generate clumps consisting of about 5 to 100 cells. The cells are then collected by low speed centrifugation. The resultant pellet is suspended in ice cold McCoy's 5A medium and filtered through a single layer of sterile surgical gauze tape to remove large clumps. The cells are then washed 2 times in McCoy's 5A medium.

[0086] Cells...

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Abstract

The present invention relates to the isolation and enrichment of avian oviduct tumor cells to obtain sustainable cell lines that can be passaged multiple times in cell culture and can be used for long-term production of heterologous polypeptides at higher yields. The methods of the instant invention involve the manipulation and / or propagation of oviduct tumor cells derived from either wild-type or transgenic avians.

Description

RELATED APPLICATION INFORMATION [0001] This application claims the benefit of U.S. provisional application 60 / 509,353, filed Oct. 7, 2003.BACKGROUND OF THE INVENTION [0002] Recent advances have allowed for the generation of transgenic avians that express heterologous proteins in their oviduct cells. The avian oviduct contains an infundibulum, magnum, isthmus, shell gland, vagina and cloaca (Etches, Reproduction in Poultry. 1996, New York, N.Y.: CABI Publishing. 318). An ovulated ovum enters the tract through the infundibulum and continues to the magnum where the majority of egg white proteins, such as ovalbumin, ovomucoid and lysozyme, are produced by tubular gland cells and deposited on the ova. (Palmiter, J Biol Chem, 1972. 247: 6450-61; Yu and Marquardt, Biol Reprod, 1973. 8: 283-98). Expression of the major egg white proteins is controlled by hormone responsive elements of the respective promoters (Palmiter, J Biol Chem, 1972. 247: 6450-61; Schimke et al., Basic Life Sci, 1973. ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12N5/071C12N15/86C12P21/04
CPCC12N5/0682C12N2517/02C12N2510/02C12N5/0693
Inventor PARKER, STEPHEN H.DEO, YASHWANT M.
Owner SYNAGEVA BIOPHARMA CORP
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