Method for assessing protein identity and stability

• US 10,209,261 B2
• Filed: 10/13/2017
• Issued: 02/19/2019
• 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:
  US15/783,883
• Publication Number:
  US 20180031570A1
• Time in Patent Office: 494 Days
• Field of Search: None
• 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 the steps of:

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

b) adding a reducing agent in an amount sufficient to reduce the disulfide bonds of the test PEG-IL10;

(c) separating peptide members of the test plurality of peptides by chromatography;

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

(e) 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, adding a reducing agent in an amount sufficient to reduce the disulfide bonds of the reference standard PEG-IL-10, separating peptide members of the reference standard PEG-IL-10 by chromatography, and analyzing said separated peptide members using ultraviolet absorption spectroscopy to provide the reference standard PEG-IL10 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 PEG-IL10 chromatogram.

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

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

4. The method of claim 2, wherein both the test PEG-IL-10 and the reference standard PEG-IL-10 comprises mixtures 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 5 kDa to about 20 kDa.

6. The method of claim 4, wherein both the test and reference standard PEG-IL-10 comprises mixtures of mono-pegylated and di-pegylated recombinant human IL-10.

7. The method of claim 6, wherein the reducing agent is dithiothreitol.

8. The method of claim 7, wherein the PEG component of the test PEG-IL-10 has a molecular mass from about 5 kDa to about 20 kDa.

9. The method of claim 8, wherein the test PEG-IL-10 and reference standard PEG-IL-10 each further comprise a linker.

10. The method of claim 6, wherein the ratio of the mono-pegylated to di-pegylated recombinant human IL-10 of the test and reference PEG-IL-10 mixtures is approximately 1:

1.

11. The method of claim 10, wherein the reducing agent is dithiothreitol.

12. The method of claim 11, wherein the test PEG-IL-10 and reference standard PEG-IL-10 each further comprise a linker.

13. The method of claim 12, wherein the PEG component of the test PEG-IL-10 has a molecular mass of about 5 kDa.