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
1998-01-26
2002-01-22
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S375000, C435S069700, C435S070100, C435S091400, C435S325000, C435S320100, C435S358000, C435S363000
Reexamination Certificate
active
06340574
ABSTRACT:
This invention relates to methods of production of recombinant biopharmaceuticals and other desirable proteins, polypeptides and peptides using mammalian cell cultures. More particularly, the methods of the invention involve the use of specifically bioengineered mammalian cell lines for the production of complex proteins in low cost media. These cell lines have the acquired ability for autonomous and regulated growth in cheap, reproducible, fully-defined protein-free medium, with the cells supplying all of their own growth factor requirements.
Mammalian cells have become the host cells of choice for the production of many of the new biopharmaceuticals, specifically those recombinant proteins requiring complex post-translational modifications and folding which bacterial cells cannot carry out. The production costs of these cells are far higher than with bacterial cells, with the fermentation cost accounting for an estimated 30% of the total production costs. The need to use a serum source in the growth and often production phases of the fermentation, is one of the contributing factors to the high costs of the fermentation.
Removing the need for added serum or growth factors in the fermentation medium will reduce media costs considerably. In addition, media free of added serum or growth factors should also be free of any vital or other contaminants. Further, the purity of the expressed protein will be maximized thus minimizing the steps in purification stages and maximising the recovery yield. This will allow production of complex processed protein in mammalian cell hosts but with many of the cost advantages associated with bacterial cell hosts. Also, in future years, commercial and regulatory pressures may well dictate a requirement for such purity in the production of mammalian cell derived recombinant protein.
Furthermore, with the rising cost of serum due to more stringent testing, there is a great demand for the development of cell lines that can grow on fully defined media. Indeed, several methods have been developed for growth of cultured cells in serum-free medium (Branes & Sato, 1980), including CHO-K1 (Mendiaz, E., et al., 1986). Media claiming to sustain growth in the absence of serum are available commercially. These are based on the fact that the serum requirements by cells in culture can be replaced by a combination of growth factors which is unique for each cell type. It is still unclear whether any of these media can sustain growth indefinitely. More recently, CHO-K1 cells have been used for high level expression of protein in serum-free medium (Ogata, M., et al., 1993), but these cells were maintained with serum during the growth phase and growth factors were added to the serum-free medium during the production phase. Although this system offers increased ease of product recovery, other factors cited above as making protein/serum-free growth media desirable, including cost, are not satisfied by this approach.
Australian patent specification No. 22120/88 (Genentech, Inc.) describes an attempt at engineering CHO cells to grow autonomously in protein-free medium. It is unclear whether these cells, which carry genes encoding insulin, transferrin and a desired protein product, are capable of continuously growing in the absence of serum. It is also unclear whether these cells are capable of expressing the desired protein product at satisfactory levels. Further, if cells such as CHO cells are engineered to produce growth factors in a constitutive fashion, then the mitogenic agents causing cell division would be present all the time and uncontrolled growth of the cells would result. Thus, in fermentation situations, it would be expected that cell numbers would increase in an uncontrolled fashion, causing in the case of attached cell cultures, multilayering of the cells and in the case of cells self-immobilised or growing as flocs, containing division resulting in cells in the centre of the flocs becoming anaeroic and necrotic. In the case of suspension cultures, it would be expected that cell densities would increase as would toxic metabolic byproducts having a negative effect on the viability and metabolism of the cells and the culture.
Thus the present inventors have now identified and developed a method of producing recombinant proteins utilising mammalian cell lines engineered for autonomous and regulated growth in low cost, protein/serum-free media.
In a first aspect, the present invention provides a method for producing a desired recombinant protein, polypeptide or peptide comprising the step of:
culturing a mammalian host cell in culture medium, wherein said host cell includes:
(i) at least one introduced DNA sequence encoding the desired protein polypeptide or peptide expressibly linked to a first inducible promoter sequence,
(ii) at least one introduced DNA sequence encoding a protein, polypeptide and/or peptide factor(s) required for growth of the host cell in said culture medium expressibly linked to a promoter sequence, the expression of which is regulated by a repressor binding region, and
(iii) at least one DNA sequence encoding a repressor molecule which binds to the repressor binding region, expressibly linked to a second inducible promoter sequence, wherein the first and second inducible promoter sequence(s) may be the same or different.
The invention enables the use of low cost, protein/serum-free medium by utilising a host cell which is able to produce the protein, polypeptide and/or peptide growth factor(s) required for its growth in such medium. The culture medium used in the method of the method of the invention is, therefore, preferably serum-free or otherwise free of protein, polypeptide and/or peptide growth factor(s) necessary for the growth of the particular host cell type. However, methods wherein the culture medium includes one or more of the required growth factor(s) and the host cell itself expresses one or more of the same and/or other required growth factor(s), is also to be regarded as falling within the scope of the invention.
By expressibly linking the DNA sequence(s) encoding the protein, polypeptide and/or peptide factor(s) required for growth of the host cell of a regulated promoter, the expression of the protein, polypeptide and/or peptide growth factor(s) can be controllably regulated so that production of the factor(s
0
may be limited only to the stage of culturing where growth is required. Controllable regulation may be achieved by including a transcription-regulatory sequence (e.g., a repressor binding region such as from the lac repressor/operator system as modified for mammals. Hu and Davidson, 1987, and Kozak, 1986). Such transcription regulatory sequence(s) may be located at any location where it can exert a regulatory effect on expression from the second promoter. For example, the transcription regulatory sequence(s) may be located between the TATAA bus and the transcription start site or, alternatively, between the transcription start site and the AUG start codon.
Accordingly, the method of culturing may comprise a firs stage of culturing to a desired cell confluence and a second stage of culturing in the presence of the repressor. However, since the host cell further includes a DNA sequence encoding the repressor, the expression of which is regulated by an inducible promoter sequence, it is preferred that the method of culturing comprises a first stage of culturing to a desired cell confluence and a second stage of culturing in the presence of an inducer of second inducible promoter. Thus, the inducer may be added or applied to the host cell culture, thereby causing the expression of repressor and subsequent down regulation of growth factor(s) production.
It is also especially preferred to control the expression of the desired recombinant protein or peptide by the use of the same inducible promoter at that used to control the expression of the repressor. In this way, adding or applying inducer will cause the downregulation of growth factor(s) production and simultaneous expression of the desired protein or peptide. Th
Gray Peter Philip
Hunt Sybille Marie
Sleigh Merilyn Joy
Lazar-Wesley Eliane
Spector Lorraine
Sughrue & Mion, PLLC
Unisearch Limited
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