Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-11-17
2004-06-01
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S002000, C435S283100, C435S306100, C435S372000
Reexamination Certificate
active
06743624
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a new process and apparatus for continuous purification and concentration of leukocytes from blood, preferably buffy coats. The leukocytes are used in the production of interferon.
PRIOR ART
Interferons constitute an endogenously produced immunologically active group of small proteins, which act as a natural defence against viral infections. They are synthesised and secreted by vertebrate cells following a virus infection. Interferons bind to the plasma membrane of other cells in the organism and induce an antiviral state in them by enhancing the production of three enzymes: an oligonucleotide synthetase, an endonuclease, and a kinase. In modern medical care, pharmaceutical compositions containing interferons are administered as a regimen against infections, specially viral infections, but also to generally boost the patient's immunological defence systems.
Interferons are presently manufactured via three different routes: recombinant, cell-line derived and human leukocyte derived. The human leukocyte derived interferon products can further be divided in partially purified and highly purified products. The present application concerns in particular highly purified human leukocyte interferon.
The large scale production of human leukocyte derived interferon is generally performed according to the process outlined by Kari Cantell et al. 1981 (Cantell, K., Hirvonen, S., Kauppinen, H-L. and Myllyla, G., Production of interferon in human leukocytes from normal donors with the use of Sendai virus, in
Methods in Enzymology
vol 78, p. 29-38, and Cantell, K., Hirvonen, S. and Koistinen, V., Partial purification of human leucocyte interferon on a large scale, in
Methods in Enzymology
, vol 78, p.499-505.) The process according to Cantell can be summarised as follows: Pooled buffy coats from healthy donors are suspended in cold 0.83% NH
4
Cl and centrifuged. In this step the leukocytes are purified and separated from other blood cells. Approximately 30% of the leukocytes are lost. The leukocytes are collected and incubated in modified Eagle's minimum essential medium (MEM). Further, the suspension is primed with priming interferon and then inoculated with Sendai virus, to initiate the production of interferon. The harvested crude interferon is then pooled and the interferon precipitated and purified further.
In production with blood an anticoagulant can be added to the blood to prevent clot formation, thereby maintaining the blood in a fluid state. When blood treated in this way is undisturbed, the cells gradually settle because they are denser than the plasma; the red cells go to the bottom, the white cells and platelets form a thin white layer (buffy coat) overlying the red cells, and the plasma appears in the upper portion of the container.
The leukocyte preparation steps are still mostly performed batch wise, using manually handled laboratory flasks and suitable equipment. Scale up has up to now been achieved by adding more flasks and centrifuges and naturally more personel, handling these flasks. The production of interferon according to the state of the art is thus plagued by the drawbacks, typical for labour intensive processes: high labour costs, low reproducibility, variations in yield etc. Nevertheless, most of the present processes have been focused on how to best utilise available laboratory equipment and methodology.
The present invention aims to overcome these drawbacks and to enable higher yield, better reproducibility and lower labour costs. Additionally, the invention aims to enable easier scale-up and GMP-verifiability of the process.
SUMMARY OF THE INVENTION
The present invention offers a solution to the above mentioned problems and shortcomings of conventional processes by introducing a process according to the attached claims. The inventive process has e.g. the advantages of being suitable for automation, thus improving the reproducibility, lowering the operator input needed and reducing labour costs. Further, the inventive process is easy to scale up and adapt to larger production volumes.
REFERENCES:
patent: 4294824 (1981-10-01), Jones et al.
patent: 4938876 (1990-07-01), Ohsol
patent: 5785869 (1998-07-01), Martinson et al.
“Methods in Enzymology”, Kari Cantell et al., 1981, vol. 78, pp. 29 to 38.
“Methods in Enzymology”, Kari Cantell et al., 1981, vol. 78, pp. 499 to 505.
Bionative AB
Burns Doane Swecker & Mathis L.L.P.
Saunders David
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