Canadian Patents Database

Claims page

---

Patent Document Number: 2446185

(54) English Title: ENHANCED PRODUCTION OF RECOMBINANT PROTEINS BY TRANSIENT TRANSFECTION OF SUSPENSION-GROWING MAMMALIAN CELLS

(54) French Title: PRODUCTION AMELIOREE DE PROTEINES DE RECOMBINAISON PAR TRANSFECTION TRANSITOIRE DE CELLULES MAMMALIENNES PROLIFERANT EN SUSPENSION

---

Claims:

Note: Claims are shown in the official language in which they were submitted.

CLAIMS:
1. An expression vector for expression of a recombinant protein in human
kidney
cells designated 293 which cells stably express EBNA 1 protein or a fragment
thereof retaining function of the EBNA 1 protein, said vector comprising:
(a) a first DNA sequence encoding the recombinant protein, the first DNA
sequence under control of a CMV5 promoter; and,
(b) a second DNA sequence enhancing transcriptional activity of the
promoter, the second DNA sequence comprising the oriP sequence of Epstein Barr

virus (EBV) or a fragment thereof to enhance transcriptional activity of the
promoter,
the fragment comprising EBNA1 binding sites and retaining function of oriP.
2. The expression vector according to claim 1, wherein the vector is a
plasmid.
3. The expression vector according to claim 1 or 2, wherein the fragment of
the
EBV oriP sequence comprises BstX I family of repeats (FR) fragment, BstX EcoR
I
FR fragment or the FR domain.
4. The expression vector according to any one of claims 1 to 3, wherein the
EBNA 1 binding sites comprise one or more of nucleotides 36-63 as set forth in
SEQ
ID NO: 1, nucleotides 66-83 as set forth in SEQ ID NO: 1, nucleotides 126-143
as
set forth in SEQ ID NO: 1, nucleotides 276-293 as set forth in SEQ ID NO: 1 or

nucleotides 302-319 as set forth in SEQ ID NO: 1.
5. A human kidney cell line designated 2935FE and deposited under IDAC
Accession No. 020502, wherein cells of said cell line are transfected with the

expression vector as defined in any one of claims 1 to 4.
6. A process for preparing transfected human kidney cells of the 293 cell
line,
which process comprises transfecting said human kidney cells with an
expression
vector as defined in any one of claims 1 to 4.
7. The process according to claim 6, wherein transfecting is carried out in
the
presence of polyethyleneimine (PEI) as a transfection reagent.
8. The process according to claim 7, wherein the polyethyleneimine is a
linear
polyethyleneimine or a branched polyethyleneimine.
24

9. The process according to claim 8, wherein the polyethyleneimine has an
average molecular weight of from 10 to 100 kDa.
10. The process according to claim 9, wherein the average molecular weight of
the polyethyleneimine is about 25 kDa.
11. A process for preparing a recombinant protein comprising culturing
transfected human kidney cells prepared by the process of any one of claims 6
to 10
to yield the protein.
12. The process according to claim 11, wherein the cells are cultured in serum-

free medium.
13. The process according to claim 11, wherein the cells are cultured in a
culture
medium comprising a serum or a subtraction thereof.
14. The process according to claim 11, wherein the cells are cultured in a
culture
medium comprising a peptone.
15. The process according to claim 14, wherein the peptone is gelatine peptone

N3.
16. The process according to any one of claims 11 to 15 carried out in a
culture
medium without changing the culture medium.
17. A process for preparing a recombinant protein comprising transfecting
human
kidney cells of the 293 cell line with an expression vector according to any
one of
claims 1 to 4 and then culturing the transfected human kidney cells to yield
said
protein.
18. A process for preparing a recombinant protein comprising culturing cells
of the
human kidney cell line as defined in claim 5 to yield the protein.



25