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Cell culture process

A cell and culture medium technology, applied in the field of production of recombinant therapeutic glycoproteins such as human erythropoietin

Inactive Publication Date: 2005-03-16
SANDOZ AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the medium still contains expensive functional recombinant proteins like insulin and transferrin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1-Construction of Plasmid Epo / neo

[0169] 1. Construction of p2-neo

[0170] 1.1 Preparation of vector fragments from pSV2neo containing the SV40 early promoter

[0171] The basis of vector construction is the pBR322 plasmid backbone contained in pSV2neo. The smaller EcoRI-PvuII restriction fragment contains the pBR322 backbone, and the adjacent PvuII-HindIII fragment from SV40 has a related fragment of the SV40 early promoter.

[0172] The plasmid pSV2neo (ATCC 37149) was cut with restriction enzymes EcoRI and HindIII. The resulting fragment sizes are 3092bp and 2637bp. The 2637bp fragment consists of an EcoRI-PvuII restriction fragment containing the pBR322 backbone and an adjacent PvuII-HindIII fragment containing the SV40 early promoter fragment. The 2637bp fragment was prepared and purified by gel electrophoresis.

[0173] 1.2 Preparation of neomycin resistance gene

[0174] The neo gene was extracted from the transposon Tn5 of pSV2neo. The gene is amplified a...

Embodiment 2

[0352] Example 2-Construction of Plasmid Epo / dhfr

[0353] 1. Construction of p2-dhfr-CDS

[0354] 1.1 Preparation of dhfr gene

[0355] The dhfr gene used for vector construction was obtained from the mouse cDNA present in the plasmid pLTRdhfr26 (ATCC 37295). The mouse dhfrcDNA nucleotide sequence (MUSDHFR) can be obtained under GenBank record number: L26316.

[0356] The dhfr was amplified from pLTRdhfr26 using the designed primers, which produced a fragment containing the coding region from the ATG at position 56 to the stop codon TAA at position 619. Taking into account the above-mentioned neomycin resistance gene amplification, HindIII and SpeI sites were introduced into the upstream and downstream amplification primers, respectively. In addition to the SpeI site, the EcoRI site was also introduced in the reverse primer. The sequence of the oligonucleotide is as follows:

[0357] Oligo 2004-13: Length: 39mer

[0358] 5′-ggg g aa gct t at ggt tcg acc att gaa ctg cat cgt cgc-...

Embodiment 3

[0386] Example 3-Production of recombinant CHO cells from pEpo / neo and pEpo / dhfr

[0387] On the day before lipofection, at 25cm 3 Inoculate 1-5×10 in T-flask or 96-well plate 4 Cells / cm 2 . The two plasmids were mixed at a ratio of 50:1=Epo / neo:Epo / dhfr, and the plasmid was adsorbed to the lipofection reagent (GIBCO / BRL) according to the manufacturer's procedure.

[0388] In short, we use 0.25μg DNA / cm 2 And 1.5μl lipofection reagent / cm 2 And adjust the DNA / lipid mixture to 200μl / cm2 Cell layer. The cells were plated with the transfection mixture in serum-free DMEM for 4 hours, and then the medium containing DNA was replaced with medium. After 24 hours of culture in serum-containing medium, the cells were transferred to selective medium. Start by culturing the transfected cell pool in selective medium to confluence, and then in expansion medium (4.8×10 -8 MMTX) culture, after which the cell culture supernatant was screened by ELISA against Epo. Determine the highest producer cell...

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PUM

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Abstract

The invention provides a method for producing a recombinant polypeptide of interest which method comprises: (a) providing a host cell which comprises a nucleotide sequence which encodes the recombinant polypeptide of interest and which directs expression of the recombinant polypeptide of interest in the host cell; (b) providing a serum-free culture medium which comprises (i) water, a plant-derived peptone, an osmolatity regulator, a buffer, an energy source, at least one amino acid, a lipid source or precursor, a source of iron, non-ferrous metal ions and optionally one or more vitamins and cofactors; and (ii) does not contain any full-length polypeptides; and (c) culturing the host cell in the culture medium under conditions that allow for expression of the recombinant polypeptide of interest.

Description

Invention field [0001] The present invention relates to a method for cost-effective production of recombinant therapeutic glycoproteins such as human erythropoietin (Epo) using recombinant cell lines. Background of the invention [0002] Erythropoietin (Epo) is the main hormone that regulates the proliferation and differentiation of erythroid progenitor cells and maintains the physiological level of circulating red blood cells. In the fetus, Epo is mainly produced in the liver, and about 90% of the production of Epo after the fetus is born is transferred to the kidney. When Epo levels decrease due to chronic or acute renal failure (for example, in cancer patients), Epo must be administered externally to prevent the development of anemia. Since the discovery of the Epo gene and its expression in rodent cells, human erythropoietin with therapeutic activity has been available. [0003] The human Epo gene encodes a signal peptide of 27 amino acids and a protein of 166 amino acids wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/505C12N5/10C12P21/02C12N15/09
CPCC12N2500/34C12N2500/99C07K14/505C12N2500/76C12N2510/02C12P21/02C12N2500/92C12N5/10C12N15/11C12N5/00
Inventor S·曾F-M·勃格纳R·库纳D·米勒F·温特路戈尔
Owner SANDOZ AG
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