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Chromatographic purification of recombinant human erythropoietin

A technology for recombinant human erythropoietin, cell components, applied in erythropoietin, recombinant DNA technology, organic chemistry, etc., can solve the problems of leakage, unstable NaOH disinfection treatment, weak host cell protein separation, etc.

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

Disadvantages of this approach are the possible leakage of the Cibacron Blue 3G dye-ligand of the Blue-sepharose capture matrix, the rather weak separation of host cell proteins by the HIC step, and the instability of the silica-based RPC resin to NaOH sterilizing treatments

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Construction of embodiment 1-plasmid Epo / neo

[0157] 1. Construction of p2-neo

[0158] 1.1 Preparation of vector fragments from pSV2neo containing SV40 early promoter

[0159] The basis for vector construction is the pBR322 plasmid backbone contained in pSV2neo. A smaller EcoRI-PvuII restriction fragment encompasses the pBR322 backbone, and the adjacent PvuII-HindIII fragment from SV40 has the associated fragment of the SV40 early promoter.

[0160] Plasmid pSV2neo (ATCC 37149) was cut with restriction enzymes EcoRI and HindIII. The resulting fragment sizes were 3092bp and 2637bp. The 2637 bp fragment consisted of an EcoRI-PvuII restriction fragment containing the pBR322 backbone and an adjacent PvuII-HindIII fragment containing the SV40 early promoter fragment. A 2637 bp fragment was prepared and purified by gel electrophoresis.

[0161] 1.2 Preparation of neomycin resistance gene

[0162] The neo gene was extracted from the transposon Tn5 of pSV2neo. The gene...

Embodiment 2

[0340] Construction of embodiment 2-plasmid Epo / dhfr

[0341] 1. Construction of p2-dhfr-CDS

[0342] 1.1 Preparation of dhfr gene

[0343] The dhfr gene used for vector construction was obtained from mouse cDNA present in plasmid pLTRdhfr26 (ATCC 37295). The mouse dhfrcDNA nucleotide sequence (MUSDHFR) is available under GenBank Accession No.: L26316.

[0344] dhfr was amplified from pLTRdhfr26 using primers designed to generate a fragment containing the coding region from the start ATG at position 56 to the stop codon TAA at position 619. Considering the amplification of the neomycin resistance gene mentioned above, HindIII and SpeI sites were introduced in the upstream and downstream amplification primers, respectively. In the reverse primer an EcoRI site was also introduced in addition to the SpeI site. The sequence of the oligonucleotide is as follows:

[0345]Oddity 2004-13: Length: 39mer

[0346] 5′-ggg g aa gct t at ggt tcg acc att gaa ctg cat cgt cgc-3′ SEQ...

Embodiment 3

[0374] Example 3 - Generation of recombinant CHO cells from pEpo / neo and pEpo / dhfr

[0375] The day before lipofection, at 25cm 3 Inoculate 1-5 x 10 in T-flasks or 96-well plates 4 cells / cm 2 . The two plasmids were mixed at a ratio of 50:1 = Epo / neo:Epo / dhfr and the plasmids were adsorbed to lipofection reagent (GIBCO / BRL) according to the manufacturer's procedure.

[0376] Briefly, we utilized 0.25 μg DNA / cm 2 and 1.5 μl lipofection reagent / cm 2 , and adjust the DNA / lipid mixture to 200 μl / cm 2 cell layer. Cells were plated with the transfection mix in serum-free DMEM for 4 hours, after which time the medium was replaced with the medium containing the DNA. After 24 hours in serum-containing medium, cells were switched to selective medium. Initially culture the transfected pool of cells in selection medium to confluence, then in expansion medium (4.8 × 10 -8 MMTX) after which cell culture supernatants were screened by ELISA for Epo production. To determine the high...

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PUM

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Abstract

The invention provides a method for recovering and purifying recombinant human erythropoietin (rhEpo) from a cell culture medium comprising host cells, which method comprises the steps of: (a) removing host cells, cellular constituents and debris from the cell culture medium by centrifugation using a disc stack separator followed by a depeth filtration step to obtain a clarified culture medium supernatant; (b) adjusting the conductivity of the supernatant to 5 mS / cm or less, and a pH of between about 7.0 and 8.0; (c) applying the supernatant from step (b) to a column comprising an anion exchange chromatographic medium, washing the column, eluting the rhEpo from the column, and collecting the peak fraction (s) that contain rhEpo; (d) subjecting the combined peak fractions from step (c) to a reverse phase chromatography step using a polystyrene resin that can be run under medium pressure (< 10 bar) and is resistance to high concentrations of NaOH, such as Source 30RPC, the rhEpo being eluted using a linear gradient of an organic solvent; (e) applying one or more fractions eluted in step (d) which contain rhEpo to a column comprising Q-Seph HP anion exchange chromatographic media, washing the column, and eluting the rhEpo using a linear salt gradient; (f) selecting one or more fractions eluted in step (e) which contain rhEpo based on degree of sialylation of the rhEpo; and (g) subjecting one or more fractions eluted in step (f) which contain rhEpo by one or more size exclusion chromatographic steps using Superdex 75 prep grade to remove potential dimers and higher aggregates; and collecting the eluate containing rhEpo.

Description

technical field [0001] The present invention relates to a method for the production of recombinant human erythropoietin (Epo) with defined glycoform composition in a highly purified form, ie with low amounts of host cell proteins. This is achieved by a specific sequence of purification steps combined with analytical tools for the quantification of the isolated isoforms. Background technique [0002] Erythropoietin is the main hormone that regulates the proliferation and differentiation of erythroid progenitor cells and maintains the physiological level of circulating erythrocytes. In the fetus, erythropoietin (Epo) is mainly produced in the liver, and after birth, about 90% of its products are transferred to the kidney for production. When erythropoietin (Epo) declines due to chronic or acute renal failure, erythropoietin (Epo) must be administered externally to prevent the development of anemia that is developing. Since the discovery of the erythropoietin (Epo) gene and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/505C12P21/02
CPCC07K14/505C07K1/18C07K1/20B82Y5/00
Inventor P·埃利格尔N·帕尔马
Owner SANDOZ AG
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