Process for the fermentative production of antibodies
The present invention relates to a process for producing a correctly folded and assembled full-length antibody using an E. coli strain, which comprises fermenting an E. coli strain which leaks periplasmic proteins into the medium, comprising a gene coding for the heavy chain of an antibody functionally linked to a signal sequence coding for a signal peptide, and a second gene coding for the light chain of an antibody, functionally linked to a signal sequence coding for a signal peptide, in a culture medium, where the E. coli strain secretes a full-length antibody into the culture medium, and the full-length antibody is removed from the culture medium.
1. A method for producing a correctly folded and assembled full-length antibody using an E. coli strain, the method comprising: a) fermenting in a culture medium an E. coli strain which leaks periplasmic proteins into a medium, the E. coli strain comprising: a gene coding for a heavy chain of an antibody functionally linked to a signal sequence coding for a signal peptide; and a second gene coding for the light chain of an antibody, functionally linked to a signal sequence coding for a signal peptide, wherein the E. coli strain secretes a full-length antibody into a culture medium and wherein the leaky mutant is an E. coli strain selected from the group consisting of JF733, A592, A593, A586, CAG12184, G11e1, JE5511, E610, E623, JE5505, PM61, 6628, PM61R, JP1228, 207, and AE84064; and b) removing the full-length antibody from the culture medium.
2. The method of claim 1 , wherein the antibody is obtained in a yield of greater than 160 mg/l without coexpression of a periplasmic chaperone.
3. The method of claim 1 , wherein the signal sequence coding for a signal peptide is selected from the group consisting of the signal sequences of the phoA or ompA gene of E. coli or the signal sequence having sequence SEQ ID No: 1.
4. The method of claim 1 , wherein the 5′ ends of the genes of the light and heavy chain are linked in frame to different signal sequences coding for a signal peptide.
5. The method of claim 4 , wherein the 5′ end of the gene coding for the one chain of the antibody is functionally linked in frame to the signal sequence of the phoA or ompA gene of E. coli , and the 5′ end of the gene coding for the other chain of the antibody is linked in frame to the signal sequence SEQ ID No: 1.
6. The method of claim 1 , wherein the gene coding for the heavy chain of an antibody functionally linked to a signal sequence coding for a signal peptide, and the second gene coding for the light chain of an antibody, functionally linked to a signal sequence coding for a signal peptide, are combined in one operon.
7. The method of claim 1 , wherein fermentation takes place over a period of 16 to 150 hours.
8. The method of claim 1 , wherein the fermentation takes place with an oxygen partial pressure (pO 2 ) of between 10 and 70% saturation.
9. The method of claim 1 , wherein the fermentation takes place with an oxygen partial pressure (pO 2 ) of between 30 and 60% saturation.
10. The method of claim 1 , wherein the fermentation takes place with an oxygen partial pressure (pO 2 ) of between 45 and 55% saturation.
11. The method of claim 1 , wherein the pH in the culture medium is between pH 6 and pH 8.
12. The method of claim 1 , wherein the pH in the culture medium is between pH 6.5 and 7.5.
13. The method of claim 1 , wherein the temperature before induction of gene expression is reduced from 30° C. to 25° C. to prevent formation of inclusion bodies.
14. The method of claim 1 , wherein the removal of the full-length antibodies from the culture medium comprises:
separating the antibodies by centrifugation or filtration,
concentrating the antibodies by ultrafiltration; and
purifying the antibodies by precipitation, chromatography or ultrafiltration or affinity chromatography.