Methods of assessing antibody-drug conjugates

Patent metadata

Field	Value
Publication number	US-10976241-B2
Application number	US-201816121909-A
Country	US
Kind code	B2
Filing date	Sep 5, 2018
Priority date	Sep 8, 2017
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

The present disclosure provides methods of assessing DAR of ADC products that provide advantages over known methods. Specifically, methods of the disclosure can be used in high-throughput applications and/or without having to dilute ADC samples during the assessment.

First claim

What is claimed:

1. A method of determining a ratio of drug to antibody (DAR) in a sample comprising an antibody-drug conjugate, comprising:
applying the sample to a size exclusion chromatography matrix;
detecting absorbance response of the sample at a first light wavelength (λ 1 ), wherein the first light wavelength is a predetermined absorbance maxima of the antibody;
detecting absorbance response of the sample at a second light wavelength (λ 2 ), wherein the second light wavelength is a predetermined absorbance maxima of the drug;
determining a total absorbance of the sample at the first light wavelength and a total absorbance of the sample at the second light wavelength, each total absorbance calculated by integrating peaks of absorbance responses over a time interval by summing an area of high molecular weight (HMW) peaks, low molecular weight (LMW) peaks and a main peak; and,
calculating the DAR using the following Equation 1:

DAR
=

ɛ
λ2
mAb

*
Total
⁢

⁢

Area
λ1

-

ɛ
λ1
mAb

*
Total
⁢

⁢

Area
λ2

ɛ
λ1
drug

*
Total
⁢

⁢

Area
λ2

-

ɛ
λ2
drug

*
Total
⁢

⁢

Area
λ1

wherein ε λ1 mAb is an extinction coefficient of the antibody at the first light wavelength; ε λ2 mAb is an extinction coefficient of the antibody at the second light wavelength; ε λ1 drug is an extinction coefficient of the drug at the first light wavelength; ε λ2 drug is an extinction coefficient of the drug at the second light wavelength; Total Area λ1 is the total absorbance of the sample at the first light wavelength; and Total Area λ2 is the total absorbance of the sample at the second light wavelength.

2. A method of determining a ratio of drug to antibody (DAR) in a first sample comprising an antibody-drug conjugate, comprising:
measuring total absorbance of the first sample comprising the antibody-drug conjugate by:
applying the first sample comprising the antibody-drug conjugate to a size exclusion chromatography matrix;
detecting absorbance response of the first sample at a first light wavelength (λ 1 ), wherein the first wavelength is a predetermined absorbance maxima of the antibody;
detecting absorbance response of the first sample at a second light wavelength (λ 2 ), wherein the second wavelength is a predetermined absorbance maxima of the drug;
determining a total absorbance of the first sample at the first light wavelength and a total absorbance of the first sample at the second light wavelength, each total absorbance calculated by integrating peaks of absorbance responses over a time interval by summing an area of high molecular weight (HMW) peaks, low molecular weight (LMW) peaks and a main peak;

measuring total absorbance of a second sample comprising the antibody by:
applying the second sample comprising the antibody to a size exclusion chromatography matrix;
detecting absorbance response of the second sample comprising the antibody at the first light wavelength (λ 1 );
detecting absorbance response of the second sample comprising the antibody at the second light wavelength (λ 2 ); and
determining a total absorbance of the second sample comprising the antibody at the first light wavelength and a total absorbance of the second sample at the second light wavelength, each total absorbance calculated by integrating peaks of absorbance responses over a time interval by summing an area of high molecular weight (HMW) peaks, low molecular weight (LMW) peaks and a main peak; and

calculating the DAR using the following Equation 2:

DAR
=

ɛ
λ1
mAb

Total
⁢

⁢

Area
λ1
mAb

*

Total
⁢

⁢

Area
λ2
ADC

*
Total
⁢

⁢

Area
λ1
mAb

-

Total
⁢

⁢

Area
λ1
ADC

*
Total
⁢

⁢

Area
λ2
mAb

Assignees

• Regeneron Pharma
• Regeneran Pharmaceuticals Inc

Inventors

• Skinner Andria
• Kulyba Natallia

Classifications

• G01N21/31

  Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry {(G01N21/72 takes precedence)} · CPC title

• A61K47/6803

  Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates · CPC title

• G01N21/3103 Primary

  Atomic absorption analysis · CPC title

• A61K47/66 Primary

  the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells · CPC title

• A61K47/68033

  the drug being a maytansine · CPC title