Serum-free stable transfection and production of recombinant human proteins in human cell lines
The present invention relates to an improved method for the serum-free production of an immortalized human cell line stably transfected under serum-free conditions with a specific vector carrying the gene coding for the protein of interest. Furthermore the invention relates to a production cell line obtained by said method, a production method for said protein of interest utilizing said production cell line, and the specific vector carrying the gene of interest itself.
1. A method for preparing an immortalized human cell line stably transfected with a nucleic acid sequence comprising a gene encoding a human target protein or a derivative or mutant thereof, a promoter and a bovine growth hormone polyadenylation (polyA) signal, said promoter and polyA signal being linked to the 5′ and 3′ end of the gene encoding said human target protein, respectively, which method comprises transfecting an immortalized human host cell line under serum-free conditions with a transfection vector comprising said nucleic acid sequence and an origin of replication.
2. The method of claim 1 , wherein the human target protein is a human plasma protein selected from blood clotting factors such as selected from the group consisting of factor IX, factor VIII (wild-type and B-domain deleted), Factor VII/VIIa, and von Willebrand factor (vWF), growth factors, erythropoietin, colony-stimulating factors (CSFs), granulocyte stimulating factor (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), cytokines, interleukins, protease inhibitors, alpha-1-antitrypsin (A1AT), chymotrypsin, transport proteins, hormones, inhibitory or regulatory acting proteins, and derivatives and mutants thereof.
3. The method of claim 1 , wherein in the transfection vector
(i) the promoter is selected from viral promoters, housekeeping gene promoters and tissue specific promoters; and/or
(ii) the origin of replication allows the replication and amplification of the plasmid in bacteria;
(iii) the vector further carries at least one gene for a selection marker being under control of a promoter as defined in (i) above; and/or
(iv) the vector further carries one or more further regulatory elements, said regulatory elements selected from splice sites, recombination sites, polyA sites, enhancers, multicloning sites and prokaryotic plasmid sequences.
4. The method of claim 3 wherein the transfection vector carries a CMV promoter, a hygromycin gene, a polyA sequence and the gene of interest and is derived from the pcDNA3.1 vector having the sequence of SEQ ID NO:4 or 5.
5. The method according to claim 1 wherein the immortalized human cell line
(i) is capable of being transfected and being cultured under serum-free conditions; and/or
(ii) has integrated adenoviral sequences into its genome; and/or
(iii) is selected from the group of kidney, bladder, liver, lung, cardiac muscle, smooth muscle, ovary and gastrointestinal cells, preferably is a human kidney cell line; and/or
(iv) is not capable of expressing a human prion protein.
6. The method according to claim 5 wherein the kidney cells are human foetal kidney cells selected from 293 cells, 293T cells, or 293F cells.
7. The method of claim 6 , wherein the cell line is 293F and the transfection vector is derived from pcDNA3.1, wherein the vector encodes human blood clotting factor IX as shown by bps 939 to 2324 of SEQ ID NO:1, human A1AT as encoded by bps 973 to 2259 of SEQ ID NO:2, wt human factor VIII as shown in SEQ ID NO:9, or B-domain deleted human factor VIII as shown in SEQ ID NO:3, FVII/FVIIa, G-CSF including G-CSFb shown in SEQ ID NO:27, or von Willebrand factor.
8. The method according to claim 1 , wherein the serum-free transfection is effected in suspension culture without serum with a cationic transfection agent or calcium phosphate.
9. The method according to claim 1 , which further comprises selection for stably transfected cells by productivity, activity and product quality of the produced recombinant protein.
10. The method according to claim 5 where the kidney cells are human foetal kidney cells 293F3 cells.
11. The method according to claim 8 , wherein the serum-free transfection is effected in suspension culture without serum with lipofectamine 2000 CD reagent.
12. The method of claim 2 wherein the human target protein is selected from factor IX, factor VIII, wild-type factor VIII, B-domain deleted factor VIII, factor VII/VIIa, G-CSF, vWF and A1AT.
13. The method of claim 2 wherein the human target protein is blood clotting factor IX as encoded by bps 939 to 2324 of SEQ ID NO:1, human A1AT as encoded by bps 973 to 2259 of SEQ ID NO:2, wt factor VIII as shown in SEQ ID NO:9, B-domain deleted human factor VIII as encoded by bps 783 to 5162 of SEQ ID N03, factor VII/VIIa as encoded by SEQ ID NOs:13 and 14, G-CSF as encoded by SEQ ID NOs:15, 16 and 17, or vWF as encoded by SEQ ID NO:18.