# US 12,179,128 B2 · HPLC-based detection of flocculation agents in a protein sample

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- Number: US 12,179,128 B2
- Document ID: US12179128B2
- Status: Granted
- Kind: B2
- Application number: 17/620,137
- Filed: 2020-06-18
- Published: 2022-08-04
- Granted: 2024-12-31
- Earliest claimed: 2019-06-20
- Assignees: Ucb Biopharma Srl
- Applicants: Ucb Biopharma Srl
- Inventors: Annick Gervais; Joel Dubisy
- Web page: https://patentgrep.com/patent/US12179128B2

## CPC

- B01D 15/327: {with hydrophobic interaction}
- B01D 15/166: {Fluid composition conditioning, e.g. gradient}
- G01N 30/34: of fluid composition, e.g. gradient (G01N30/36 takes precedence)
- G01N 2030/8831: {involving peptides or proteins}
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The current application provides a rapid and simple method for measuring residual pDADMAC in a sample containing a recombinant protein of interest based on the combination of use of reversed-phase hydrophobic interaction HPLC and charged aerosol detection.

## Claims

### Claim 1

A method for measuring poly (diallyldimethylammonium chloride) (pDADMAC) in a sample containing a recombinant protein of interest, the method comprising the following steps:
a) applying the sample to a reversed-phase hydrophobic interaction HPLC column,
b) eluting bound pDADMAC from the column using hydrophobic gradient elution,
c) measuring pDADMAC recovered in step b) using a charged aerosol detector (CAD),
wherein the sample applied in a) comprises 0.2-3% (v/v) of a CAD-compatible acid.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the sample applied in step a) comprises a protein concentration of less than 200 mg/ml.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the sample applied in step a) comprises a protein concentration of 20-100 mg/ml.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the sample applied in step a) comprises a protein concentration of 20-60 mg/ml.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the sample applied in step a) has a pH from 2 to 4.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the sample applied in step a) comprises 0.5-1.5% (v/v) of a CAD-compatible acid.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said CAD-compatible acid is selected from trifluoro-acetic acid, acetic acid, heptafluorobutyric acid and formic acid.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the reversed-phase hydrophobic interaction HPLC is PLRP-S HPLC.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the method further comprises a step of determining the concentration of pDADMAC present in the sample by reference to a control.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the recombinant protein of interest is an antibody, an antibody fragment, or a derivative thereof.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the sample applied in step a) is a sample obtained directly from a step in purification of said recombinant protein to which the CAD-compatible acid has been added.