Cell line

Details Cell line Cell line

1996-06-27

1998-10-13

Ketter, James

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 6951, 4351723, 4352351, 435372, C12P 2102, C12N 510

Patent

active

058210806

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to Namalwa cell lines and their use.
Human alpha-interferons (.alpha.-IFNs) belong to a multigene family located on a 400 kb segment of human chromosome 9. The family comprises at least 13 genes encoding .alpha.-IFN subtypes. Each .alpha.-IFN protein consists of 165 or 166 amino acid residues with molecular weights of between 18 and 26 kD (depending on the degree of glycosylation). Although chemically distinct, the subtypes are closely related with regions of highly conserved amino acid sequences.
One commercially available source of .alpha.-IFN is human lymphoblastoid alpha nl IFN (.alpha. n1 IFN). Which is a natural .alpha.-IFN. It is produced by stimulating a Namalwa human lymphoblastoid cell line with Sendai virus to produce a mixture of at least 13 subtypes of .alpha.-IFN, which are then purified by chromatography (U.S. Pat. No. 4,216,203; EP-A-0000520; EP-A-0097353). The number and quantity of particular subtypes is kept within defined limits during the manufacturing process.
This technique for producing .alpha.-IFN was in fact developed in the early 1970's. It was during that period that the cell-line now used to produce all currently commercially available human lyphoblastoid interferon was generated from a Burkitt's lymphoma tumour. This tumour was removed from a single individual named Namalwa, thus generating the eponymously titled cell line (Strander H. et al, J. Clin. Microbiol. 1, 116-117, 1975 and Christofinis G. J. et al, J. Gen. Virol. 52, 169-171, 1981). Namalwa cells are publicly available under ATCC Deposit No. 00001432-CRL.
Namalwa cells have also been used for the preparation of recombinant proteins (Yanagi H. et al, Gene 76, 19-26, 1989 and Okamoto M. et al, Bio/Technology 550-553, 1990). Namalwa cells are attractive for the production of recombinant proteins of human origin. Correct post-translational processing of the recombinant human proteins can be expected. Further, industrial-scale production of Namalwa cell lines is well-established because of their use for producing .alpha.-IFN.
The possibility that there may be contaminants associated with products obtained from a Namalwa cell line has been extensively researched. Namalwa cells have been tested for contamination with bacteria, viruses and mycoplasma. Some level of Epstein-Barr virus (EBV) have been detected. Infectious virus is not formed, though. Further, EBV early antigen cannot be detected even when cells are treated with chemicals such as bromodeoxyuridine which can induce EBV replication.
However, the presence of genome of the squirrel monkey retrovirus (SMRV, a type D retrovirus) has been detected (Middleton et al, Int. J. Cancer 52, 451-454, 1992). SMRV was first isolated from a squirrel monkey lung culture and displays an extended host range in vitro including canine, human, chimpanzee, rhesus monkey and mink cells. The virus is of particular interest since it has been identified as a contaminant of a number of cell lines. To date, two strains of the virus, SMRV and SMRV-H, have been identified on the basis of differences in restriction endonuclease patterns and sequence variation.
It is possible to ensure that no viable SMRV particles survive the production protocols that are employed to obtain products using Namalwa cell lines. However, it is clearly preferable that no SMRV genome should be present in Namalwa cells. SMRV genomes were detected by Middleton et al (supra) in all eight of a random selection of eight Namalwa cell-lines sampled from laboratories world-wide. Hence it is considered likely that all prior, publicly available, Namalwa cells contain the SMRV genome.
Conclusively proving that particular cells are lacking a viral contaminant is clearly dependent upon the sensitivity of the particular assay system employed. Direct assay for the presence of viral DNA by techniques such as the polymerase chain reaction (PCR) may allow sensitivities of detection of up to one viral genome per 100 to 1000 cells, even up to one viral genome per 1000 cells. If however a new cell-line is cl

REFERENCES:
Middleton et al., Int. J. Cancer, vol. 52, 1992, 451-454 1992.

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