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Purified interleukin-15/fc fusion protein and preparation thereof

A technology of fusion protein and protein, which is applied in the direction of interleukin, peptide preparation method, animal/human protein, etc., and can solve the problems such as the difficulty of rejection

Inactive Publication Date: 2007-04-11
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, difficulties often arise with regard to rejection of the recipient organ, which is caused by an immune response to foreign cell surface antigens of the graft

Method used

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  • Purified interleukin-15/fc fusion protein and preparation thereof
  • Purified interleukin-15/fc fusion protein and preparation thereof
  • Purified interleukin-15/fc fusion protein and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Preparation of IL-15 / Fc in CHO-K1 cells

[0101] In order to prepare IL-15 / Fc-producing CHO-K1 cell lines, an IL-15 / Fc expression construct should be formed and tested in terms of its secretion properties, identity / integrity of the fragments it contains and suitable resistance genes optimize

[0102] a) Raw material

[0103] Human IL-15 / Fc expression construct (mutant IL-15 / human Fc) was provided by the Department of Immunology of the "Beth Israel Deaconness Medical Center" (Harvard Medical School, Boston, USA).

[0104] Oligonucleotides were obtained from NMG-Biotech (Ebersberg, Germany). Sequences of relevant signal peptides were obtained from GenBank.

[0105] Restriction enzymes (BgIII, XbaI, BamHT, SmaT, BstXI, ApaI), Lipfectamin2000, other molecular biology reagents (T4-DNA ligase, T4-polynucleotide kinase) and plasmids pSecTagA, pcDNA3.1 were obtained from Invitrogen (Karlsruhe, Germany ) or Amersham-Pharmacia (NheI, Protein A Sepharose Uppsala, Sw...

Embodiment 2

[0124] Transfection of eukaryotic cell lines (eg, CHO-K1 cells) with a plasmid containing the DNA of the desired product is a standard method for producing therapeutic proteins. However, the low productivity level of the stable cell clones thus produced is a known problem. Therefore, various methods exist for increasing the productivity of existing cell lines. In addition to attempts to increase the number of copies of the plasmid in the cell (eg via the methotrexate / DHFR system), it is also possible to modify the expression construct itself. Incorporation of introns in addition to strong promoters (such as the CMV promoter) can lead to better RNA stability and better RNA export from the nucleus, which can be carried out by the cell's splicing machinery. However, experimentation must be performed to determine which intron / transgene combination is suitable. To this end, various introns were combined with human IL-15-Fc to find out the combinatorial way to increase IL-15-Fc pr...

Embodiment 3

[0140] Embodiment 3: Purification of IL-15 / Fc fusion protein

[0141] a) purification and concentration

[0142] About 3100 liters of the supernatant from Example 2 (pMG10Ala7 plasmid) were clarified, concentrated and sterile filtered in 6 operations. This included clarification of the supernatant using a Profile Star filter (3 micron, 20 inches, Pall Corporation, East Hills, NY, USA). Subsequently, the supernatant was concentrated 10 to 15 times in total by a tangential flow filtration system (Millipore, Billerica, MA, USA) using a 2.0 square meter Biomax-30 membrane. The inlet pressure is 2-2.5 bar and the outlet pressure is 1.5 bar. After concentration, filter through a filter comprising a pre-filter (Polysep II (0.2 micron, 10 inches, Millipore, Billerica, MA, USA)) and a final filter (Durapore (0.22 micron, 10 inches, Millipore, Billerica, MA, USA)). The filter system sterile-filters the concentrate. Six different operations took 4.5 to 8 hours.

[0143] b) rProtein-...

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Abstract

The present invention relates to a process for purifying an interleukin-15 / Fc fusion protein from a composition, which process comprises a) applying the composition to an affinity chromatography column and eluting a first IL-15 / Fc eluate from the column and b) applying the eluate of step a) to an ion exchange chromatography column and eluting a second IL-15 / Fc eluate from the column; and to a purified interleukin-15 / Fc fusion protein and a composition, in particular a pharmaceutical composition, comprising such a fusion protein.

Description

technical field [0001] The present invention relates to a method of purifying an interleukin-15 / Fc fusion protein from a composition comprising a) applying the composition to an affinity chromatography column and eluting the first IL-15 / Fc elution from the column and b) applying the eluate from step a) to an ion-exchange chromatography column and eluting a second IL-15 / Fc eluate from the column; also to purified interleukin-15 / Fc fusion protein and comprising Compositions of such fusion proteins, especially pharmaceutical compositions. Background of the invention [0002] Organ or tissue transplantation has become the standard method and, in many cases, the only life-saving treatment for many life-threatening diseases. However, difficulties often arise with respect to rejection of the recipient organ, which is caused by an immune response to foreign cell surface antigens of the graft. One therapeutic approach to inhibit rejection is the suppression of humoral or cellular ...

Claims

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Application Information

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IPC IPC(8): C07K14/54C07K1/22C07K1/18
CPCC07K14/5443A61P35/00A61P43/00
Inventor A·埃尔曼
Owner F HOFFMANN LA ROCHE & CO AG
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