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
2001-11-28
2004-05-25
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069200, C435S069400, C435S069500, C435S069600, C435S070100
Reexamination Certificate
active
06740505
ABSTRACT:
The present invention concerns the production of proteins by cell culture techniques and in particular concerns new and improved processes for the production of proteins useful in therapeutic and diagnostic applications.
The commercial production of proteins by cell culture particularly for use in medical applications remains a costly exercise principally due to the relatively low levels of proteins (particularly so-called “rare proteins”) produced by many cell types. One approach to addressing this problem is the use of agents to induce cells to produce higher than normal amounts of the desired protein. An example of such an agent is butyric acid or, more typically, a salt thereof such as sodium butyrate, which is believed to modify gene expression at a molecular level through its effect on the methylation state of DNA, which in turn affects transcriptional gene activation. The inducing agent supplements the culture media during the culturing process and is typically following a period of cell culturing. During the process the culture media including the inducing agent may be changed by either a continuous process in which new media is continually added as old medium is removed or in a batch type process in which some of the medium is removed from the cells and replaced.
In EP 0 239 292 B1, NB1/19 cells were incubated with a variety of sodium butyrate concentrations ranging from 0.1 mM to 1.0 mM for a period of only approximately 8 days. In WO 89/06686, a process of enhancing protein production by cultured eukaryotic cells through the addition of butyric acid or salt thereof in concentrations of 0.1 mM to 10.0 mM is suggested. In this disclosure, cells are first grown to confluence before the addition of the acid or salt. This is generally in line with accepted thinking that butyric acid, whilst effective in promoting protein production, also has a potentially deleterious effect on the viability of cells, making it prudent to bring cells to confluence or to a relatively high cell population, before exposing them to the butyric acid.
In U.S. Pat. No. 5,705,364, the use of an alkanoic acid or salt thereof at concentrations of between 0.1 mM and 20 mM in optional conjunction with osmotic control to specifically increase silac acid content of glycoproteins is discussed. In Nucleic Acids Res., 1983, 11, no.21 the effects of sodium butyrate on DNA-mediated gene transfer is investigated at concentrations ranging from 2 to 10 mM. In Can.Res, Vol.46, February 1986, 713-716, an investigation into the effects of sodium butyrate on the synthesis and methylation of DNA in normal cells and their transformed counterparts is disclosed. In this investigation, concentrations ranging from 5 to 100 mM are used. In U.S. Pat. No. 5,378,612, a culture medium for culturing transformed cells is disclosed. The culture media may contain butyric acid at an exemplified concentration of 1 mM.
The present inventors have found, contrary to conventional expectations, that the use and maintenance of unusually low concentrations of an alkanoic acid in culture media comprising culturing cells, leads to enhanced protein production without, over an extended period of time, significantly affecting cell viability. Furthermore, the low alkanoic acid concentration permits the presence of alkanoic acid in the culturing media at an earlier stage in the culturing process than hitherto described.
In accordance with the present invention, we provide a process for the production of a protein by cell culture which comprises the step of culturing eukaryotic cells which constitutively produce e.g. secrete, said protein in a culture media which media comprises an alkanoic acid and/or salt thereof at a maintained concentration of less than 0.1 mM.
In accordance with the present invention, we provide a process for the production of a protein by cell culture which comprises the steps of (a) culturing eukaryotic cells which constitutively produce e.g. secrete said protein in a culture media which media comprises an alkanoic acid and/or salt thereof at a concentration of less than 0.1 mM (b) subculturing the cell culture (c) supplementing the subculture media with additional alkanoic acid and/or salt thereof to maintain the concentration therein during the culturing process at less than 0.1 mM.
Novel proteins obtained by the process of the present invention as hereinbefore described also forms an aspect of the present invention.
It will be understood by those skilled in the art that the terms “maintained concentration” and “to maintain the concentration” does not necessarily imply that the concentration in the culturing media must be kept constant since a certain degree of variation in concentration is permissible whilst still achieving the same or similar results. Of course, Zero mM is excluded from the term “less than 0.1 mM”.
In accordance with the present invention we provide a process for the production of a protein by cell culture which comprises the steps of (a) culturing eukaryotic cells which constitutively produce e.g. secrete said protein in a culture media which comprises an alkanoic acid and/or salt thereof at a specified concentration which concentration is less than 0.1 mM.
Eukaryotic cells useful in the practice of the present invention may be e.g. yeast or animal and be anchorage dependent or independent. Preferably, the cells are mammalian e.g. rat, mouse and hamster. Cells of the present invention may be immortalised cells. Immortalised cells may be transformed or transfected with exogenous DNA e.g. via a plasmid, coding for the desired protein according to techniques standard and well known to those skilled in the art. Cells for use in the present invention include hybridoma cells e.g. hybrid cells produced by fusion of antibody producing cells with myeloma cells. The process of the present invention maybe used in the culturing of hybridoma cells for the production of immunoglobulins e.g. antibodies.
Cells useful in the present invention include NS0 cells (non-immunoglobulin secreting mouse myeloma B cells), and CHO cells (Chinese hamster ovaries). Preferably, cells of the present invention are derived from stable cell lines, the production of which is well known to those skilled in the art. For example, stable cell line expression may be produced using a glutaminine synthetase gene amplification system (such as commercially available from Celltech). Briefly, linearized expression vectors containing cDNA encoding e.g. hamster glutamine synthetase, under the control of an Early promoter such as SV40 and splicing and polyadenylation signals and cDNA of the desired protein, e.g. antibody heavy and light chains are introduced into mammalian cells by electroporation. Transfected cells are then selected for the ability to grow in, for example, a glutamine free medium. See Bebbington et al, 1992, Biotechnology 10, 169-175 to which the reader is specifically referred.
Proteins which maybe produced by the present invention include therapeutically and/or diagnostically useful eukaryotic proteins which may be naturally occuring or artificial (e.g. fusion proteins). Examples of proteins whose production may benefit from the present invention include hormones, for example growth hormone e.g. human growth hormone, enzymes, enzyme inhibitors or lymphokines. The process of the present invention is particularly useful in the production of immunoglobulins, e.g. naturally occurring and artificial (chimeric and humanised) antibodies or analogues or fragments thereof
Suitable fragments thereof include Fab, Fv fragments and single chain antibodies. Antibodies maybe polyclonal or more preferably monoclonal from any suitable class or subclass. It is preferred that the antibody is a IgG, particularly IgG
1
antibody or fragment derived therefrom. Preferably such proteins are suitable for use in the therapeutic (including prophylactic) or diagnostic treatment of human diseases, e.g. pro-inflammatory disorders such as rheumatoid arthritis, osteoarthritis, or other disorders. Thus in the case of antibodies, the antibody may be produced in
Islam Seema
Sharp Nigel Alan
Bennett Virqinia C.
Saunders David
SmithKline Beecham Corporation
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