Method for assessing protein identity and stability

• US 9,823,255 B2
• Filed: 06/13/2014
• Issued: 11/21/2017
• Est. Priority Date: 06/17/2013
• Status: Active Grant

• Current Assignee: Armo BioSciences Inc.
• Original Assignee: Armo BioSciences Inc.
• Inventors: Mumm, John Brian, Van Vlasselaer, Peter
• Primary Examiner(s): Cordero Garcia, Marcela M
• Application Number:
  US14/895,880
• Publication Number:
  US 20160123991A1
• Time in Patent Office: 1,257 Days
• CPC Class Codes: G01N 2030/8831 involving peptides or proteins
• CPC Class Codes: G01N 2333/5428 IL-10
• CPC Class Codes: G01N 2496/00 Reference solutions for assays of biological material
• CPC Class Codes: G01N 2560/00 Chemical aspects of mass spectrometric analysis of biological material
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6851 Methods of protein analysis involving laser desorption ionisation mass spectrometry
• CPC Class Codes: G01N 33/6869 Interleukin

Abstract

The present invention relates to methods and other technologies that may be used to determine whether compositions (e.g., pharmaceutical compositions) comprising interleukin-10 molecules (e.g., pegylated interleukin-10) meet particular product-related specifications prior to being administered to a subject for the treatment and/or prevention of the diseases, disorders and conditions, and/or the symptoms thereof, described herein.

Claims

1. A method of confirming the amino acid sequence of a pegylated interleukin-10 (PEG-IL-10), comprising:

fragmenting a test PEG-IL-10 with a glutamyl endopeptidase (Endo Glu-C), to yield a test plurality of peptides;

separating peptide members of the test plurality of peptides by chromatography;

analyzing said separated peptide members using ultraviolet absorption spectroscopy to provide a test PEG-IL-10 chromatogram;

comparing the test PEG-IL-10 chromatogram to a reference standard chromatogram, said reference standard chromatogram generated by Endo Glu-C digestion of a reference standard PEG-IL-10, separating peptide members of the reference standard by chromatography, and analyzing said separated peptide members using ultraviolet absorption spectroscopy to provide the reference standard chromatogram;

wherein the amino acid sequence of the test PEG-IL-10 is confirmed by the substantial equivalency of the retention time of the peaks of the test PEG-IL-10 chromatogram and the reference standard chromatogram.

2. The method of claim 1, wherein both the test and reference standard PEG-IL-10 is PEG-hIL-10.

3. The method of claim 1, wherein the chromatography is reverse phase high performance liquid chromatography.

4. The method of claim 1, wherein the test and reference standard PEG-IL-10 comprises a mixture of mono-pegylated and di-pegylated IL-10.

5. The method of claim 4, wherein the PEG component of the test PEG-IL-10 has a molecular mass from about 5kDa to about 20kDa.