Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-11-30
2001-04-03
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070300, C435S366000, C435S369000, C435S455000, C435S463000
Reexamination Certificate
active
06210922
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a method of serum-free production of recombinant proteins and adenoviral vectors; cell lines viable in a serum-free medium and method of obtaining same.
(b) Description of Prior Art
The need for the production of recombinant proteins as well as viral particles able to mediate gene transfer and expression in vivo for gene therapy is constantly increasing. The use of recombinant adenovirus (AdV) in gene therapy as well as live viral vaccines has been extensively reported (Trapnell, B. C. and Gorzilia, M. 1994 Curr. Opinion Biotechnol. 5: 617-625). More recently, reported the use of AdV as vaccines for inducing specific T-cell immunity for cancer therapy. The transformed human embryonic kidney cell line 293 which constitutively express Ad5 E1A and E1B genes is used for its ability to support the replication of an E1A+E1B-defective adenoviral particle. These vectors are basically wild type human adenoviruses in which the E1A and E1B, the essential genes, have been deleted. These deletions, together with other less crucial ones, provide room in the capsid for recombinant genes and also prohibit the vector growth in normal cell lines.
The complementing human embryonic kidney 293 cell line and its derivatives which have been adapted to suspension culture, the 293S cells, will sustain the recombinant adenoviral vector replication. This cell line can be used for AdV production as well as for high level expression of r-proteins especially those for which activity is modulated by complex posttranslational modifications. Both types of product would benefit from the development of an effective serum-free medium (SFM) that would allow high cell growth rate and cell density as well as high yield of recombinant proteins and AdVs. The only serum-free formulations reported to support growth of 293 cells are proprietary in nature. Furthermore, doubling times ranging from 36 to 40 hours were much higher than doubling times of 18 to 22 hours measured in serum-containing media for single-cell suspension culture. There are, at present, no reports of adenoviral particle production by 293 cells in serum-free suspension culture.
The numerous problems associated with the presence of serum in culture medium are high cost, batch-to-batch variability, regulatory considerations and the difficulty in removing serum proteins when purifying the product of interest. These concerns are heightened when the final product is to be used as an injectable therapeutic. Considering this, one would expect that by now the adaptation of a cell line to SFM would be a routine laboratory work. However, since each cell line have it's very own growth and production characteristics, a universal SFM that would be efficient for all cell types does not yet exist. The multitude of commercial SFM formulations available assess the diversity of the needs. Furthermore, the literature often oversimplifies the development process of a SFM. Although, certain cell lines can be adapted spontaneously from serum-containing medium to SFM, a number of other cell lines need a sophisticated mixture of adaptation and screening steps in order to be weaned from serum-containing medium. This is especially true when more than one performance criteria has to be met. In practice, the development process usually involves: a) screening of media formulations, b) cell line adaptation following a blend of gradual or abrupt weaning steps, c) cell line cloning or sub-cloning in order to isolate better performing individual cells, and d) clone screening based on multiple performance criteria, namely cell growth rate, maximum cell density, resistance to agitation stress, lack of aggregate formation, and product yield. Since all the steps described above can rarely be performed in a simultaneous manner, empirical choices must be made during the development process that depend as much as on objective criteria as on a laboratory personnel experience and skills. To our knowledge, no universal method has been devised to address the SFM development process.
The large amount of viral particles required in gene therapy emphasizes the need for scale-up when possible. Suspension culture is often preferred to micro-carriers because of scale up and cost concerns. Testing of serum-free media is not trivial when the bioprocess involves infection of cells. The absence of serum in suspension culture leaves the shear-sensitive infected cells unprotected. The limited duration of the AdV production phase, 48 to 72 hours post infection when infected cells become fragile and might be subject to important cell lysis, may complicate the data analysis.
It would be highly desirable to be provided with a method of serum-free production of recombinant proteins and adenoviral vectors; cell lines viable in a serum-free medium and method of obtaining same.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a method of serum-free production of recombinant proteins and adenoviral vectors.
Another aim of the present invention is to provide cell lines viable in a serum-free medium.
Another aim of the present invention is to provide a method of obtaining cell lines viable in a serum-free medium.
In accordance with the present invention there is provided a serum-free medium viable cell line, which comprises an immortalized animal cell line for the expression of recombinant proteins and/or adenoviral vectors in a serum-free medium.
A preferred cell line of the present invention is a mammal cell line, or a human cell line, most preferably a human embryonic kidney cell line.
A preferred serum-free human embryonic kidney cell line in accordance with the present invention, which allows for the expression of recombinant adenoviral vectors, referred to as 293SF-3F6, has been deposited at the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.) on Sep. 25, 1998 under deposit number ATCC CRL-12585. This deposit is available to be public upon the grant of a patent to the assignee, National Research Council Canada, disclosing same. The deposit is also available as required by Foreign Patent laws in countries wherein counterpart applications are filed.
In accordance with the present invention there is provided with the use of the cell lines of the invention for establishing stable transfected cell line or for production of adenoviral vectors required for gene therapy.
In accordance with the present invention there is provided a method of serum-free production of recombinant proteins, which comprises the steps of:
a) transfecting a serum-free cell line of the invention with a cDNA coding for a recombinant protein to obtain transformants;
b) isolating a stable transformant of step a);
c) cultivating the isolated transformant of step b) in suspension in a serum-free medium to produce the recombinant proteins; and
d) isolating the recombinant proteins of step c).
The preferred recombinant protein may be selected from the group consisting of cytokines, G-protein coupled receptors, hormones and enzymes.
The step b) may be effected using a selection marker.
In accordance with the present invention there is provided a method of serum-free production of recombinant adenoviral vectors, which comprises the steps of:
a) transforming a serum-free cell line of the present invention, which is complementing defective of an adenoviral vector, with a recombinant adenoviral vector comprising a promoter operatively linked upstream to a marker gene and/or a therapeutic gene sequence relative to the direction of transcription in an adenoviral vector, wherein the promoter is controlling transcription of the marker gene and/or the therapeutic gene, wherein the cell line is complementing the recombinant adenoviral vector;
b) isolating a stable cell line of step a) using the marker; and
c) cultivating the isolated cells of step b) in suspension in a serum-free medium to produce the recombinant adenoviral vector.
The marker may be selected from the group consisting of green fluorescent pro
Côté M. T. Johanne
Kamen Amine A.
Massie Bernard
C{overscore (o)}té France
National Research Council of Canada
Swabey Ogilvy Renault
Witz Jean C.
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