Identification and quantification of conjugated peptides in antibody drug conjugates by mass spectrometry

Patent metadata

Field	Value
Publication number	US-11802879-B2
Application number	US-201716080903-A
Country	US
Kind code	B2
Filing date	Mar 2, 2017
Priority date	Mar 2, 2016
Publication date	Oct 31, 2023
Grant date	Oct 31, 2023

Abstract

The present disclosure relates to a streamlined, complete workflow for the qualitative and the quantitative analysis of conjugated peptides from antibody-drug conjugate (ADC) compounds.

First claim

We claim:

1. A method for analyzing site occupancy ratios in antibody-drug conjugates, said method comprising:
(i) providing a sample comprising an antibody-drug conjugate compound;
(ii) exposing the sample to de-salting;
(iii) exposing the sample to multienzyme digestion to form a digested peptide mixture, wherein the digested peptide mixture comprises conjugated peptide and unconjugated peptide;
(iv) ionizing the sample to form radical ion fragments;
(v) detecting the mass-to-charge ratios associated with the radical ion fragments;
(vi) analyzing the mass-to-charge ratios of the radical ion fragments; and
(vii) quantifying the site occupancy ratio in the sample by determining the ratio of conjugated peptide to conjugated peptide and unconjugated peptide, thereby analyzing site occupancy ratios in antibody-drug conjugates.

2. The method of claim 1 , further comprising identifying the conjugated peptide and unconjugated peptide in the sample.

3. The method of claim 1 , further comprising selecting two separate groups of mass-to-charge ratios of the radical ion fragments.

4. The method of claim 3 , wherein the first group of mass-to-charge ratios is associated with the radical ion fragments of the conjugated peptide.

5. The method of claim 3 , wherein the second group of mass-to-charge ratios is associated with the radical ion fragments of the unconjugated peptide.

6. The method of claim 3 , wherein the selection of the two separate groups of mass-to-charge ratios is based on known ion fragmentation patterns.

7. The method of claim 6 , wherein the known ion fragmentation patterns are stored in a library.

8. The method of claim 1 , wherein the multienzyme digestion comprises a first enzyme and a second enzyme.

9. The method of claim 8 , wherein the first enzyme is trypsin/Lys C.

10. The method of claim 9 , wherein the ratio of the trypsin/Lys C to sample is about 1:25 (w/w).

11. The method of claim 8 , wherein the second enzyme is Asp N.

12. The method of claim 11 , wherein the ratio of the Asp N to sample is about 1:100 (w/w).

Assignees

• Waters Technologies Corp

Inventors

• Chen Liuxi
• Chen Weibin
• Cubbedge Steven

Classifications

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N30/72

  Mass spectrometers {(mass spectrometers per se H01J49/00)} · CPC title

• G01N30/7233

  interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04) · CPC title

• G01N2030/027

  Liquid chromatography · CPC title

• H01J49/004

  Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn · CPC title