Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-30
2002-05-28
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S070100, C435S455000, C435S366000, C435S069100
Reexamination Certificate
active
06395484
ABSTRACT:
DESCRIPTION
The invention relates to a method for the selection of human cells for the preparation of human proteins by endogenous gene activation in order to produce human proteins in economical yields and in a form which is suitable for the production of a pharmaceutical preparation. The invention furthermore relates to a method for the production of human proteins in a cell line identified in this manner.
The production of human proteins by endogenous gene activation in a human cell line is known. For example, WO93/09222, WO94/12650, and WO95/31560 describe the production of human erythropoietin and other human proteins in human cell lines by endogenous gene activation.
In the documents cited, however, there is no mention of the fact that certain criteria must be observed in selecting the cell lines used for the production of human proteins. Therefore there can be no assurance that the desired human protein can be obtained in the desired yield and form, and free of contamination in the cell line chosen for its production. Accordingly, in the above-named documents only generally low yields of human proteins are achieved.
It was the objective of the present invention to eliminate the disadvantages of the state of the art and especially to offer criteria for the selection of human starting cell lines which are suitable for an endogenous activation of a predetermined target gene.
This objective is attained by a method for the selection of human cell lines for the production of human proteins by the activation of a target gene endogenously present in the cell line, characterized in that
(a) a human cell line is tested for the presence of the following features:
(i) a target gene with the desired nucleic acid sequence,
(ii) at least 5 population doublings within 14 days in a suspension culture, and
(iii) at least 5 population doublings within 14 days in a serum-free culture medium, and
(b) using a cell line having features (i), (ii), and (iii) as starting cell line for the endogenous activation of the target gene.
If one is facing the task of activating a human cell gene in a human cell line by gene targeting and obtaining a cell which is capable of the production of the target protein in satisfactory yield and in the desired form, according to the invention one will test several cell lines for the presence of a number of features which this cell line must possess in order to be a suitable candidate for the later large-scale production of the target protein. Preferably, immortalized cell lines, especially tumor cell lines, are tested since they have important advantages over non-immortalized cells regarding culturability.
According to feature (i) the human cell line is studied to see whether the target gene, i.e., the gene to be activated by endogenous gene activation, really has the desired nucleic acid sequence, generally the nucleic acid sequence of the natural target gene. Tumor cell lines and other cell lines in permanent culture often exhibit a series of mutations in their genome. Therefore it is an important aspect in the selection of a suitable cell line whether the cells have a correct gene for the desired product. The sequencing can be done by culturing the cells in the usual manner and sequencing the target gene. If necessary, the target gene can be amplified by PCR or other amplification methods prior to sequencing.
Another important feature for the selection of a cell line is culturability in suspension. Suspension cells are easier to ferment and the fermentation can be adapted more easily to larger dimensions, e.g., in a large fermenter with a capacity of, for example, 10 liters to 50,000 liters. Therefore the selected cells should be either suspension cells or they should easily adapt to a suspension culture. For this purpose the cells are cultured for 14 days with constant stirring. If the cells show at least five population doublings within this period they are considered suitable for suspension culture. Determining the number of population doublings can be done by periodically determining the cell count, e.g., by cell counting or by measuring the optical density of the cell suspension.
Another important feature in the selection of human cells is culturability in a serum-free medium. Since the purification of proteins from serum-free cell cultures is substantially easier and in serum-free culture there is no danger of contamination with animal pathogens, e.g., viruses, the selected cells should be able to grow in a serum-free culture. So the selected cells should be cultured for 14 days in a density of 1 to 10×10
5
cells per ml in culture vessels with a serum-free medium (e.g., RPMI 1640 with ITS by Boehringer Mannheim). If the cells during this culture show at least 5 doublings of population, which can be determined by cell counting, they are considered as suitable for serum-free culture.
Another important feature, and one preferred according to the invention is the generation time (iv). The selected cells in media such as, e.g., DMEM 10% fetal calf serum or RPMI 1640 with 10% fetal calf serum, have a high proliferation, i.e., they should have 10 to 256 population doublings, preferably 64 to 128 population doublings, within a week in culture. For this purpose the cells are seeded in culture dishes in a concentration of 0.1 to 10×10
5
cells per ml, preferably 0.5 to 2×10
5
cells per ml, and the cell count is made every two to three days by means of a cell chamber with or without trypsinization. Cells which show a sufficiently short generation time are especially suitable for large-scale production of human proteins by endogenous gene activation.
Another preferred feature is the absence of any detectable endogenous expression, i.e., transcription and translation, of the target gene (v). Preferably, for the endogenous gene activation, those cell lines are selected which have substantially no endogenous expression of the target gene. For this purpose the cells can be seeded in a cell density of 0.01 to 2×10
6
cells/ml, preferably 0.5 to 1×10
6
cells/ml of culture medium. After a predetermined time, e.g., 24 hours, the cell supernatant is removed, the cells are discarded, and the content of the target protein is determined in the cell supernatant by known test methods, e.g, ELISA. In the case of EPO the detection limit is, for example, 10 pg/EPO/ml. Cells seeded at 10
6
cells/ml, which synthesize less than 10 pg of protein are considered as nonproductive and are especially suitable.
Still another important and preferred feature is the polysomia of the target gene in the cell to be selected (vi). The presence of more than two chromosomal copies of the target gene in the cell significantly increases the yields in the homologous recombination. For the production of EPO whose gene is on chromosome 7, the cells Namalwa (Nadkarni et al., Cancer 23 (1969), 64-79) or HeLa S3 (Puck et al., J. Exp. Med. 103 (1956), 273-284), which have chromosome 7 in triplicate, have proven especially suitable. Additional examples of cell lines which contain chromosome 7 in a great number of copies are the colon adenocarcinoma cell line SW-480 (ATCC CCL-228; Leibovitz et al., Cancer Res. 36 (1976), 4562-4567), the malignantmelanoma cell line SK-MEL-3 (ATCC HTB 69; Fogh and Tremp in: Human Tumor Cells in vitro, pp.115-159, J. Fogh (ed.), Plenum Press, New York 1975), the colon adenocarcinoma cell Colo-320 (ATCC CCL-220; Quinn et al., Cancer Res. 39 (1979), 4914-4924), the melanoma cell line MEL-HO (DSM ACC 62; Holzmann et al., Int. J. Cancer 41 (1988), 542-547) and the kidney carcinoma cell line A-498 (DSM ACC 55; Giard et al., J. Natl. Cancer Inst. 51 (1973), 1417-1423).
The examination of the number of chromosomes in the genome of a cell line can be performed by using DNA probes which are specific for the particular chromosome and/or the locus of the target gene.
Still another preferred feature of a starting cell line used for an endogenous gene activation is a correct glycosylation of the desired target protein (vii). A human cell line is preferably used
Brandt Michael
Franze Reinhard
Pessara Ulrich
Fulbright & Jaworski LLP
Katcheves Konstantina
Roche Diagnostics GmbH
Yucel Remy
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