Optimized host cells for protein production

• US 20080220432A1
• Filed: 11/30/2007
• Published: 09/11/2008
• Est. Priority Date: 11/30/2006
• Status: Abandoned Application

• Current Assignee: Chromocell Corporation
• Original Assignee: Chromocell Corporation
• Inventors: Shekdar, Kambiz, Sawchuk, Dennis
• Application Number:
  US11/998,646
• Publication Number:
  US 20080220432A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

The present invention relates to methods for isolating cells that express increased levels of an RNA or protein of interest, wherein the cells exhibit altered growth profiles, such as cells with increased or decreased rates of proliferation, increased or decreased rates of apoptosis, or cells with a biphasic growth profile.

Claims

1. A method for isolating a cell with an increased rate of cell proliferation, comprising the steps of:

contacting a population of cells with a fluorescent reagent for monitoring the rate of cell proliferation; and

isolating the cell that exhibits a level of fluorescence of the fluorescent reagent that correlates with increased cell proliferation.

2. The method of claim 1, wherein detection of fluorescence is carried out using flow cytometric cell sorting technology.

3. A method for isolating a cell with an increased rate of cell proliferation, wherein the cell also expresses high levels of an RNA of interest, comprising the steps of:

contacting a population of cells with a fluorogenic probe that fluoresces upon hybridization to said RNA of interest;

contacting said population with a fluorescent reagent for monitoring the rate of cell proliferation, wherein the reagent fluoresces at a wavelength different than that of the fluorogenic probe; and

isolating the cell that exhibits increased fluorescence of the fluorogenic probe and a level of fluorescence of the fluorescent reagent that correlates with increased cell proliferation.

4. The method of claim 3, wherein detection of the fluorescence of the fluorogenic probe is assayed simultaneously with detection of the fluorescence of the fluorescent reagent.

5. The method of claim 3, wherein detection of fluorescence is carried out using flow cytometric cell sorting technology.

6. The method of claim 3, wherein said fluorescent reagent for monitoring the rate of cell proliferation is selected from the group consisting of:

carboxyfluorescein diacetate succinimidyl ester, SNARF-1 carboxylic acid or acetate succinimidyl ester.

7. The method of claim 3, wherein said cell is a mammalian, bacterial, insect, plant, microbial, algal or fungal cell.

8. The method of claim 7, wherein said mammalian cell is selected from the group consisting of:

a Chinese Hamster Ovary (CHO) cell, a NS0 cell, a HEK 293 cell, and a Per.C6 cell.

9. The method of claim 7, wherein said bacterial cell is a BL21 cell.

10. The method of claim 7, wherein said fungal cell is selected from the group consisting of:

a Chrysosporium cell, an Aspergillus cell, a Trichoderma cell, a Dictyostelium cell, a Candida cell, a Saccharomyces cell, a Schizosaccharomyces cell and a Penicillium cell.

11. The method of claim 7, wherein said insect cell is a SF9 cell or a SF21 cell.

12. The method of claim 3, further comprising the step of contacting the cell that exhibits increased fluorescence of the fluorogenic probe and altered fluorescence of the fluorescent reagent with a reagent for monitoring an apoptotic or pre-apoptotic marker.

13. The method of claim 12, wherein said reagent is Annexin-V or propidium iodide.

14. The method of claim 3, wherein the RNA of interest is selected from the group consisting of:

a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA, and an snRNA.

15. The method of claim 14, wherein said messenger RNA encodes an immunoglobulin heavy chain, an immunoglobulin light chain, a single chain Fv, an Fab, Fab′

, or (Fab′

)₂ antibody fragment or an antigen binding fragment of an immunoglobulin.

16. The method of claim 3, wherein the RNA of interest is an endogenous RNA.

17. The method of claim 3, wherein the RNA of interest is a heterologous RNA.

18. The method of claim 3, further comprising the step of culturing the isolated cell to produce a cell culture.

19. The method of claim 18, further comprising the step of measuring the density of the cell culture.

20. A method of producing a cell culture with increased cell density, wherein cells in the cell culture express high levels of an RNA of interest, comprising the steps of:

contacting a population of cells with a fluorogenic probe that fluoresces upon hybridization to said RNA of interest;

isolating a cell from the population that exhibits increased fluorescence of the fluorogenic probe;

culturing the isolated cell to produce a first cell culture;

repeating the previous steps to isolate a second cell culture;

comparing the maximum-attained density of the first and second cell cultures; and

identifying the cell culture with increased cell density wherein cells in the cell culture expresses high levels of the RNA of interest.

21. The method of claim 20, wherein detection of fluorescence is carried out using flow cytometric cell sorting technology.

22. The method of claim 20, wherein said cell is a mammalian, bacterial insect, plant, algal or fungal cell.

23. The method of claim 22, wherein said mammalian cell is selected from the group consisting of:

a Chinese Hamster Ovary (CHO) cell, a NS0 cell, a HEK 293 cell, and a Per.C6 cell.

24. The method of claim 22, wherein said bacterial cell is a BL21 cell.

25. The method of claim 22, wherein said fungal cell is selected from the group consisting of:

a Chrysosporium cell, an Aspergillus cell, a Trichoderma cell, a Dictyostelium cell, a Candida cell, a Saccharomyces cell, a Schizosaccharomyces cell and a Penicillium cell.

26. The method of claim 22, wherein said insect cell is a SF9 cell or a SF21 cell.

27. The method of claim 20, further comprising the step of contacting the cell that exhibits increased fluorescence of the fluorogenic probe with a reagent for monitoring an apoptotic or pre-apoptotic marker.

28. The method of claim 27, wherein said reagent is Annexin-V or propidium iodide.

29. The method of claim 20, wherein the RNA of interest is selected from the group consisting of:

a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA, and an snRNA.

30. The method of claim 29, wherein said messenger RNA encodes an immunoglobulin heavy chain, an immunoglobulin light chain, a single chain Fv, an Fab, Fab′

, or (Fab′

)₂ antibody fragment or an antigen binding fragment of an immunoglobulin.

31. The method of claim 20, wherein the RNA of interest is an endogenous RNA.

32. The method of claim 20, wherein the RNA of interest is a heterologous RNA.

33. A method for isolating a cell with a biphasic growth profile, wherein the cell has an increased rate of proliferation in a first portion of the growth profile, and wherein the cell has a decreased rate of proliferation in a second portion of the growth profile, comprising the steps of:

contacting a first population of cells with a fluorescent reagent for monitoring the rate of cell proliferation;

culturing said population of cells at low cell density,

isolating a cell from said population of cells that exhibits altered fluorescence of the fluorescent reagent in the first portion of the growth profile;

culturing the isolated cell to produce a second population of cells;

contacting said second population of cells with a fluorescent reagent for monitoring the rate of cell proliferation;

culturing said second population of cells at high cell density; and

,

isolating a cell exhibiting an unaltered or slower rate of alteration of fluuorescence of the fluorescent reagent in the second portion of the growth profile as compared to the altered fluorescence of the fluorescenct reagent in the first portion of the growth profile, thereby isolating a cell with a biphasic growth profile, wherein the cell has an increased rate of proliferation in a first portion of the growth profile, and wherein the cell has a decreased rate of proliferation in a second portion of the growth profile.

34. A method for isolating a cell with a biphasic growth profile, wherein the cell has an increased rate of proliferation in a first portion of the growth profile, and wherein the cell has a decreased rate of proliferation in a second portion of the growth profile, and wherein the cell expresses high levels of an RNA of interest in the second portion of the growth profile, comprising the steps of:

contacting a population of cells with a fluorogenic probe that fluoresces upon hybridization to said RNA of interest;

contacting said population with a fluorescent reagent for monitoring the rate of cell proliferation, wherein the reagent fluoresces at a wavelength different than that of the fluorogenic probe; and

isolating a cell that exhibits altered fluorescence of the fluorescent reagent in the first portion of the growth profile, unaltered or decreased change of fluorescence of the fluorescent reagent in the second portion of the growth profile, and increased fluorescence of the fluorogenic probe in the second portion of the growth profile.

35. The method of claim 34, wherein the cell grows to increased density in the first portion of the growth profile.

36. The method of claim 34, wherein detection of the fluorescence of the fluorogenic probe is assayed simultaneously with detection of the fluorescence of the fluorescent reagent during the second portion of the growth profile.

37. The method of claim 34, wherein detection of fluorescence is carried out using flow cytometric cell sorting technology.

38. The method of claim 34, wherein said fluorescent reagent for monitoring the rate of cell proliferation is selected from the group consisting of:

carboxyfluorescein diacetate succinimidyl ester and SNARF-1 carboxylic acid, acetate succinimidyl ester.

39. The method of claim 34, wherein said cell is a mammalian, bacterial, insect, plant, microbial, algal or fungal cell.

40. The method of claim 39, wherein said mammalian cell is selected from the group consisting of:

a Chinese Hamster Ovary (CHO) cell, a NS0 cell, a HEK 293 cell, and a Per.C6 cell.

41. The method of claim 39, wherein said bacterial cell is a BL21 cell.

42. The method of claim 39, wherein said fungal cell is selected from the group consisting of:

a Chrysosporium cell, an Aspergillus cell, a Trichoderma cell, a Dictyostelium cell, a Candida cell, a Saccharomyces cell, a Schizosaccharomyces cell and a Penicillium cell.

43. The method of claim 39, wherein said insect cell is a SF9 cell or a SF21 cell.

44. The method of claim 34, further comprising the step of contacting the cell that exhibits increased fluorescence of the fluorogenic probe and unaltered fluorescence of the fluorescent reagent with a reagent for monitoring an apoptotic or pre-apoptotic marker.

45. The method of claim 44, wherein said reagent is Annexin-V or propidium iodide.

46. The method of claim 34, wherein the RNA of interest is selected from the group consisting of:

a messenger RNA that encodes a protein, an antisense RNA molecule, a structural RNA, a ribosomal RNA, an hnRNA, and an snRNA.

47. The method of claim 46, wherein said messenger RNA encodes an immunoglobulin heavy chain, an immunoglobulin light chain, a single chain Fv, an Fab, Fab′

, or (Fab′

)₂ antibody fragment, or an antigen binding fragment of an immunoglobulin.

48. The method of claim 34, wherein the RNA of interest is an endogenous RNA.

49. The method of claim 34, wherein the RNA of interest is a heterologous RNA.

50. The method of claim 34, further comprising the step of culturing the isolated cell to produce a cell culture.

51. The method of claim 50, further comprising the step of measuring the density of the cell culture.