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Process For the Preparation of Glycosylated Interferon Beta

a glycosylated interferon and beta technology, applied in the field of glycosylated interferon beta preparation, can solve the problems of complex process, increased cost, and many limitations of serum, and achieve the effect of improving the stability of serum

Inactive Publication Date: 2008-09-11
ARES TRADING SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the greatest challenges is the development of cost effective and efficient processes for the production of recombinant proteins on a commercial scale.
Although widely used, serum has many limitations.
It contains high levels of numerous proteins interfering with the limited quantities of the desired protein of interest produced by the cells.
These proteins derived from the serum must be separated from the product during downstream processing such as purification of the protein of interest, which complicates the process and increases the cost.
While many methods are now available for large-scale preparation of proteins, crude products, such as cell culture harvest, contain not only the desired product but also impurities which are difficult to separate from the desired product.
As a further difficulty, many purification methods may contain steps requiring application of low or high pH, high salt concentrations or other extreme conditions that may jeopardize the biological activity of a given protein.
Thus, for any protein it is a challenge to establish a purification process allowing for sufficient purity while retaining the biological activity of the protein.
Glycosylation was shown to also be important for solubility, since the IFN-β precipitated after deglycosylation with glycopeptidase F. In addition, IFN-β produced by E. coli showed folding problems, due to lack of glycosylation in the bacterial expression system.
For example, non-glycosylated forms of recombinant interferon-β were shown to have significantly lower stability and also lower biological activity (Runkel et al., 1998).

Method used

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  • Process For the Preparation of Glycosylated Interferon Beta
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  • Process For the Preparation of Glycosylated Interferon Beta

Examples

Experimental program
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Effect test

example 1

Preparation of a Chinese Hamster Ovary (CHO) Clone Producing IFN-Beta at High Levels

[0164]This Example describes the generation of an interferon beta producing CHO clone.

[0165]The basic procedure, particularly with regard to the preparation of the expression plasmid, is described in Mory et al. (1981).

[0166]An overview over the process of generating the clone is depicted in FIG. 1. A DNA fragment comprising the human interferon-β coding region was isolated from a human peripheral blood cell genomic DNA library. A DHFR-deficient CHO cell line was transfected with a recombinant plasmid containing both the human IFN-β coding sequence and the mouse DHFR gene as a selectable and amplifiable marker. After selection in thymidine-free medium, gene amplification with methotrexate (MTX), and cloning, a cell producing IFN-β-1a at high levels was isolated. The cells were subjected to genotypic and phenotypic characterization.

[0167]Construction of the Expression Plasmid Carrying the hIFN-β and t...

example 2

Process for the Production of Interferon Beta

[0247]The overall goal of this experiment was to develop a process for producing IFN beta-1a from the clone described in example 1 under serum-free conditions.

[0248]The serum-free process was developed at 75 L bioreactor scale with internal packed bed Fibra-Cel® carriers.

[0249]The cells were thawed and expanded over 21 days in a commercially available serum-free medium having the following modifications:

TABLE 1Modification of serum-free mediumIngredientsComposition in % (w / w)HEPES 20 mMProline  1 mMPhenol Red 15 mg / LSodium chloride6150 mg / L

[0250]30×109 cells were seeded in the 75 L bioreactor (high seeding).

[0251]The runs were divided into the following phases:[0252]a growth phase I at 37° C. (until working day 2 or working day 4 or when the glucose consumption rate (GCR) was ≧2.0±1.0 g·L−1·d−1)[0253]a growth phase II at 35° C. (until working day 7 or when GCR≧8.0±0.5 g·L−1·d−1)[0254]a production phase at 33° C.

[0255]This temperature stra...

example 3

Purification of the Interferon Protein Product

[0260]The purification process of the IFN-β-1a from cell culture supernatant included four chromatographic and four filtration stages, as shown in FIG. 2. The purification stages were performed in the following order:[0261]Stage I: Harvest clarification by filtration[0262]Stage II: Affinity chromatography on a Blue Sepharose 6 fast flow (6 FF) column;[0263]Stage III: Ultrafiltration[0264]Stage IV: Cation exchange chromatography using preferably a CM Sepharose FF column;[0265]Stage V: Hydrophobic chromatography RP-HPLC;[0266]Stage VI: Ultrafiltration and dialysis;[0267]Stage VII: Size Exclusion (SE) chromatography;[0268]Stage VIII: Microfiltration.

[0269]The purification of the active ingredient started with dye affinity chromatography on Blue Sepharose 6 FF (BS 6 FF), which was the major purification stage. The amount of host cell derived proteins as well as DNA from ruptured CHO cells was reduced by several orders of magnitude and theref...

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Abstract

The present invention relates to a process for the production of interferon beta, and to an interferon beta composition having a unique glycosylation pattern.

Description

FIELD OF THE INVENTION[0001]The present invention relates to processes for producing recombinant human interferon beta under serum-free culture conditions, processes of purifying recombinant human interferon beta and to an interferon beta protein having a unique glycosylation pattern.BACKGROUND OF THE INVENTION[0002]Proteins have become commercially important as drugs that are also generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for the production of recombinant proteins on a commercial scale.[0003]The biotech industry makes an extensive use of mammalian cells for the manufacturing of recombinant glycoproteins for human therapy.[0004]Suitable cells that are widely used for production of polypeptides turned out to be Chinese Hamster Ovary (CHO) cells.[0005]CHO cells were first cultured by Puck (1958) from a biopsy of an ovary from a female Chinese hamster. From these original cells a number of sub-lines were ...

Claims

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Application Information

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IPC IPC(8): A61K38/21C12P21/04C07K1/00C12N15/00A61P25/00C12N5/06C12N15/11C07K14/00
CPCC07K14/565C12N5/0031C12N2500/12C12N2500/32C12N2500/99C12N5/06C12N15/00A61K38/215C12N2510/02C12N5/0037C12N2500/90C12N2500/92A61P25/00A61P25/28A61P31/12A61P35/00C07K1/18C07K1/20C12N5/00
Inventor FISCHER, DINABERNARD, ALAINDUCOMMUN, PAULROSSI, MARA
Owner ARES TRADING SA
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