Methods for producing recombinant proteins

Abstract

The present invention provides a method for controlling the partitioning of a recombinant protein between the supernatant and the periplasm in E. coli host cell cultures wherein expression of the recombinant protein by said cells is under the control of an inducible system, which method comprises: a) providing an E. coli host cell culture b) changing the growth rate of the E. coli host cells c) inducing expression of the recombinant protein wherein steps (b) and (c) can be performed in any order or simultaneously; and subsequently d) determining the yield of recombinant protein in the culture supernatant and the E. coli host cell periplasm e) comparing the yield determined in step (d) with the yield determined when at least one other growth rate has been used in step (b) f) selecting a growth rate from the comparison made in step (e) in which the partitioning of the recombinant protein between the supernatant and the periplasm is most suited to the primary recovery of the recombinant protein.

The invention claimed is: 
     
       1. A method for determining the growth rate of E. coli host cells that results in partitioning of a recombinant protein produced by the host cells between the periplasm of the host cells and the supernatant of the culture medium in which the cells are grown that is most suited to the primary recovery of the recombinant protein, wherein expression of the recombinant protein by said cells is under the control of an inducible system, which method comprises: facilitates primary recovery of recombinant protein from the supernatant, the method comprising,
 a) providing an E. coli host cell culture; 
 b) changing the growth rate of the E. coli host cells such that the growth rate is in the range of 0.0005 to 0.04/h; 
 c) inducing expression of the recombinant protein under the control of an inducible system wherein steps (b) and (c) are performed in any order or simultaneously; and subsequently 
 d) determining the yield of recombinant protein in the supernatant of the culture medium and the E. coli host cell periplasm; 
 e) performing steps (b) and (d), and optionally steps (a) and (c), using a growth rate different from the growth rate initially used in step (b); 
 f) comparing the yield determined in step (d) with the yield determined in step (e); and 
 g) selecting a growth rate from the comparison made in step (f) , the selected growth rate being in the range of 0.0005 to 0.0092/h, the selected growth rate being most suited to the primary recovery of the recombinant protein from the supernatant.; 
 wherein the growth rate is in the range of 0.0005 to 0.0092/h. 
 
     
     
       2. The method according to  claim 1  wherein the yields compared in step (f) are from at least two cultures grown simultaneously. 
     
     
       3. The method according to  claim 1  wherein the yields compared in step (f) are from at least two cultures grown sequentially. 
     
     
       4. The method according to  claim 1  wherein the yields compared in step (f) are from one culture grown at at least two different growth rates by carrying out steps (a) to (d) using one growth rate in step (b) and then carrying out steps (b) and (d) at least once more varying the growth rate in step (b) each time. 
     
     
       5. The method according to  claim 1  wherein the growth rate in step (b) is reduced. 
     
     
       6. The method according to  claim 1  wherein changing the growth rate of the E. coli host cells in step (b) comprises adjusting the level of carbon available to the cells. 
     
     
       7. The method according to  claim 6  wherein the carbon source is selected from glucose, lactose, sucrose, fructose, glycerol, succinate and lactate. 
     
     
       8. The method according to  claim 7  wherein the carbon source is glycerol. 
     
     
       9. The method according to  claim 1  wherein changing the growth rate of the E. coli host cells in step (b) comprises adjusting the level of phosphate available to the cells. 
     
     
       10. The method according to  claim 1  wherein changing the growth rate of the E. coli host cells in step (b) comprises adjusting the level of oxygen available to the cells. 
     
     
       11. The method according to  claim 1  wherein the recombinant protein is targeted to the periplasm. 
     
     
       12. The method according to  claim 11  wherein the growth rate selected in step (g) is a growth rate at which greater than 80% of the recombinant protein produced by the host cells is expressed in the periplasm. 
     
     
       13. The method according to  claim 1  wherein the recombinant protein is targeted to the supernatant. 
     
     
       14. The method according to  claim 13  where wherein the growth rate selected in step (g) is a growth rate at which greater than 80% of the recombinant protein produced by the host cells is produced in the supernatant. 
     
     
       15. The method according to  claim 1  wherein the inducible system comprises a lac derived promoter. 
     
     
       16. The method according to  claim 15  wherein the lac derived promoter is lac, tac or trc. 
     
     
       17. The method according to  claim 15  wherein the promoter is induced with lactose or IPTG. 
     
     
       18. The method according to  claim 1  wherein the recombinant protein is an antibody or fragment thereof. 
     
     
       19. The method according to  claim 18  wherein the recombinant protein is an antibody that is an IgG. 
     
     
       20. The method according to  claim 18  wherein the recombinant protein is an antibody fragment that is a Fab, Fab′, F(ab′) 2  or scFv. 
     
     
       21. A method of producing a recombinant protein in E. coli host cells wherein expression of the recombinant protein by said cells is under the control of an inducible system and the partitioning of the recombinant protein between the supernatant of the culture medium in which the cells are grown and the periplasm of the host cells is most suited to the primary recovery of the recombinant protein said method primary recovery of the protein is from the supernatant, the method comprising,
 a) providing an E. coli host cell culture; 
 b) changing the growth rate of the E. coli host cells such that the growth rate is in the range of 0.0005 to 0.04/h; 
 c) inducing expression of the recombinant protein under the control of an inducible system wherein steps (b) and (c) are performed in any order or simultaneously; and subsequently 
 d) determining the yield of recombinant protein in the supernatant of the culture medium and the E. coli host cell periplasm; 
 e) performing steps (b) and (d), and optionally steps (a) and (c), using a growth rate different from the growth rate initially used in step (b); 
 f) comparing the yield determined in step (d) with the yield determined in step (e); 
 g) selecting a growth rate in the range of 0.0005 to 0.0092/h from the comparison made in step (f) the selected growth rate being most suited to the primary recovery of the recombinant protein from the supernatant; and 
 h) growing the E. coli host cells at the growth rate selected in step (g). 
 
     
     
       22. The method according to  claim 21  wherein the recombinant protein produced is subsequently purified. 
     
     
       23. The method of  claim 21  wherein E. coli host cells are grown in a phosphate concentration in the range of 26.9 to 29.8 Mm. 
     
     
       24. A method for determining the growth rate of E. coli host cells that results in partitioning of a recombinant protein produced by the host cells between the periplasm of the host cells and the supernatant of the culture medium in which the cells are grown that is most suited to the primary recovery of the recombinant protein, wherein expression of the recombinant protein by said cells is under the control of an inducible system, which method comprises: facilitates primary recovery of recombinant protein from the periplasm, the method comprising,
 a) providing an E. coli host cell culture; 
 b) changing the growth rate of the E. coli host cells such that the growth rate is in the range of 0.0005 to 0.04/h; 
 c) inducing expression of the recombinant protein under the control of an inducible system wherein steps (b) and (c) are performed in any order or simultaneously; and subsequently 
 d) determining the yield of recombinant protein in the supernatant of the culture medium and the E. coli host cell periplasm; 
 e) performing steps (b) and (d), and optionally steps (a) and (c), using a growth rate different from the growth rate initially used in step (b); and 
 f) comparing the yield determined in step (d) with the yield determined in step (e); and 
 g) selecting a growth rate from the comparison made in step (f), the selected growth rate being in the range of 0.0005 to 0.0092/h, the selected growth rate being most suited to the primary recovery of the recombinant protein from the periplasm; 
 wherein the growth rate is in the range of 0.0005 to 0.0092/h. 
 
     
     
       25. The method according to  claim 24  wherein the yields compared in step (f) are from at least two cultures grown simultaneously. 
     
     
       26. The method according to  claim 24  wherein the yields compared in step (f) are from at least two cultures grown sequentially. 
     
     
       27. The method according to  claim 24  wherein the yields compared in step (f) are from one culture grown at at least two different growth rates by carrying out steps (a) to (d) using one growth rate in step (b) and then carrying out steps (b) and (d) at least once more varying the growth rate in step (b) each time. 
     
     
       28. The method according to  claim 24  wherein the growth rate in step (b) is reduced. 
     
     
       29. The method according to  claim 24  wherein changing the growth rate of the E. coli host cells in step (b) comprises adjusting the level of carbon available to the cells. 
     
     
       30. The method according to  claim 29  wherein the carbon source is selected from glucose, lactose, sucrose, fructose, glycerol, succinate and lactate. 
     
     
       31. The method according to  claim 30  wherein the carbon source is glycerol. 
     
     
       32. The method according to  claim 24  wherein changing the growth rate of the E. coli host cells in step (b) comprises adjusting the level of phosphate available to the cells. 
     
     
       33. The method according to  claim 24  wherein changing the growth rate of the E. coli host cells in step (b) comprises adjusting the level of oxygen available to the cells. 
     
     
       34. The method according to  claim 24  wherein the recombinant protein is targeted to the periplasm. 
     
     
       35. The method according to  claim 34  wherein the growth rate selected in step (g) is a growth rate at which greater than 80% of the recombinant protein produced by the host cells is expressed in the periplasm. 
     
     
       36. The method according to  claim 24  wherein the recombinant protein is targeted to the supernatant. 
     
     
       37. The method according to  claim 36  where wherein the growth rate selected in step (g) is a growth rate at which greater than 80% of the recombinant protein produced by the host cells is produced in the supernatant. 
     
     
       38. The method according to  claim 24  wherein the inducible system comprises a lac derived promoter. 
     
     
       39. The method according to  claim 38  wherein the lac derived promoter is lac, tac or trc. 
     
     
       40. The method according to  claim 38  wherein the promoter is induced with lactose or IPTG. 
     
     
       41. The method according to  claim 24  wherein the recombinant protein is an antibody or fragment thereof.  
     
     
       42. The method according to  claim 41  wherein the recombinant protein is an antibody that is an IgG. 
     
     
       43. The method according to  claim 41  wherein the recombinant protein is an antibody fragment that is a Fab, Fab′, F(ab′) 2  or scFv. 
     
     
       44. A method of producing a recombinant protein in E. coli host cells wherein expression of the recombinant protein by said cells is under the control of an inducible system and the partitioning of the recombinant protein between the supernatant of the culture medium in which the cells are grown and the periplasm of the host cells is most suited to the primary recovery of the recombinant protein said method primary recovery of the protein is from the periplasm, the method, comprising,
 a) providing an E. coli host cell culture; 
 b) changing the growth rate of the E. coli host cells such that the growth rate is in the range of 0.0005 to 0.04/h; 
 c) inducing expression of the recombinant protein under the control of an inducible system wherein steps (b) and (c) are performed in any order or simultaneously; and subsequently 
 d) determining the yield of recombinant protein in the supernatant of the culture medium and the E. coli host cell periplasm; 
 e) performing steps (b) and (d), and optionally steps (a) and (c), using a growth rate different from the growth rate initially used in step (b); 
 f) comparing the yield determined in step (d) with the yield determined in step (e); 
 g) selecting a growth rate in the range of 0.0005 to 0.0092/h from the comparison made in step (f) the selected growth rate being most suited to the primary recovery of the recombinant protein from the periplasm; and 
 h) growing the E. coli host cells at the growth rate selected in step (g). 
 
     
     
       45. The method according to  claim 44  wherein the recombinant protein produced is subsequently purified. 
     
     
       46. The method of  claim 44  wherein E. coli host cells are grown in a phosphate concentration in the range of 26.9 to 29.8 Mm.