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PROCESS FOR CORRECTION OF A DISULFIDE MISFOLD IN Fc MOLECULES

a technology of fc molecules and disulfide misfolds, which is applied in the direction of drug compositions, peptide/protein ingredients, metabolic disorders, etc., can solve the problems of aggregation or stability problems in production, limited use of fc fusion molecules,

Inactive Publication Date: 2010-10-21
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite their advantages, use of Fc fusion molecules may be limited by misfolding upon expression in a desired cell line.
Such misfolded Fc fusion molecules may generate an immune response in vivo or may cause aggregation or stability problems in production.

Method used

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  • PROCESS FOR CORRECTION OF A DISULFIDE MISFOLD IN Fc MOLECULES
  • PROCESS FOR CORRECTION OF A DISULFIDE MISFOLD IN Fc MOLECULES
  • PROCESS FOR CORRECTION OF A DISULFIDE MISFOLD IN Fc MOLECULES

Examples

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working examples

[0149]The following disclosure(s) are illustrative rather than limiting.

example 1

Identification of Cysteines in a Fc Construct of Osteoprotegerin by CDAP Labeling and Alkaline-Induced Cleavage with LC-MS Analysis

Abstract

[0150]Purpose. To assay for cysteines in a Fc construct of Osteoprotegerin stored in acidic media and to ascertain their location.

[0151]Methods. The reagent 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) was employed to selectively cyanylate stable free-sulfhydryl groups at pH 4. Cyanylation reactions were analyzed by HPLC coupled with mass-spectral analysis (LC-MS). Reversed-phase HPLC was used to separate component peaks from the native and CDAP-reacted samples. The reversed-phase samples were collected, introduced into a solution of aqueous ammonium hydroxide plus guanidine hydrochloride and subsequently analyzed by LC-MS. Results. Reversed-phase HPLC analysis of Fc-Osteoprotegerin gave two peaks. LC-MS of the primary CDAP reaction revealed selective cyanylation of two-sulfhydryl groups in a peak that represents approximately 10% ...

example 2

[0167]Purpose. Significant differences were observed in the stability of Fc-OPG as a function of copper treatment of the bulk protein, where Fc-OPG treated with copper was significantly more stable than Fc-OPG that was not. Specifically, the Fc-OPG that was not treated with copper was more prone to aggregation than the copper-treated Fc-OPG. The improved stability of Fc-OPG is thought to be due to the conversion of an unstable fraction of Fc-OPG with incomplete disulfide structure to a form with intact disulfide bonding upon treatment with copper ion.

[0168]Methods. Reversed-phase chromatography: Reversed-phase chromatography was performed on a Hewlett-Packard 1100 or 1090 HPLC system equipped with a diode-array detector and controlled by Chemstation Software. Fc-OPG samples were analyzed on a Zorbax (4.6 mm×25 cm) 300SB column. The column is initially equilibrated at 90% buffer A (HPLC grade water containing 0.1% TFA). The samples were eluted with a linear gradient of 4.2% buffer B / ...

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Abstract

The present invention concerns a process by which a misfold in an Fc fusion molecule can be prevented or corrected. In one embodiment, the process comprises (a) preparing a pharmacologically active compound comprising an Fc domain; (b) treating the fusion molecule with a copper (II) halide; and (c) isolating the treated fusion molecule. The pharmacologically active compound can be an antibody or a fusion molecule comprising a pharmacologically active domain and an Fc domain. The preferred copper (II) halide is CuCl2. The preferred concentration thereof is at least about 10 mM for fusion molecules prepared in E. coli; at least about 30 mM for fusion molecules prepared in CHO cells. The process can be employed with any number of pharmacologically active domains. Preferred pharmacologically active domains include OPG proteins, leptin proteins, soluble portions of TNF receptors (e.g., wherein the fusion molecule is etanercept), IL-1ra proteins, and TPO-mimetic peptides. The Fc domain preferably has a human sequence, with an Fc sequence derived from IgG1 most preferred. An exemplary Fc sequence is shown in FIG. 5 hereinafter.

Description

REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. patent application Ser. No. 10 / 925,183, now allowed, which is a divisional of U.S. patent application Ser. No. 09 / 709,704, filed Nov. 9, 2000, now U.S. Pat. No. 6,808,902, which claim the benefit of U.S. Provisional Application Ser. No. 60 / 165,188, filed Nov. 12, 1999. The above-identified applications are incorporated herein by reference.REFERENCE TO THE SEQUENCE LISTING[0002]The present application is being filed along with a Sequence Listing in electronic format via EFS-Web. The Sequence Listing is provided as a text file entitled A584USDIV2st25.txt, created Apr. 9, 2010, which is 24,863 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0003]Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/00C12N15/09A61K38/00A61K39/395A61P3/04A61P3/10A61P19/10A61P43/00C07K1/113C07K14/52C07K14/705C07K19/00C12P21/02
CPCC07K1/1133C07K14/70578C07K2319/00Y10S530/867Y10S530/866Y10S530/825C07K2319/30A61P19/10A61P3/04A61P43/00A61P3/10
Inventor TREUHEIT, MICHAEL J.O'CONNER, SHEILA R.KOSKY, ANDREW A.
Owner AMGEN INC
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