Performance and dynamic range for oligonucleotide bioanalysis through reduction of non specific binding

Patent metadata

Field	Value
Publication number	US-2024189740-A1
Application number	US-202418437482-A
Country	US
Kind code	A1
Filing date	Feb 9, 2024
Priority date	Jan 17, 2020
Publication date	Jun 13, 2024
Grant date	—

Abstract

The present disclosure discusses a method of separating a sample including oligonucleotides including coating a flow path of a chromatographic system; injecting the sample comprising oligonucleotides into the chromatographic system; flowing the sample through the chromatographic system; and separating the oligonucleotides. In some examples, the coating of the flow path is non-binding with respect to the analyte, such as oligonucleotides. Consequently, the analyte does not bind to the coating of the flow path. The non-binding coating eliminates the need for passivation, which can eliminate the formerly needed time to passivate as well. In addition, analyte can be recovered with a first injection in a system, such as chromatographic system.

First claim

What is claimed is:

1 . A method of separating a sample comprising a low concentration of analyte:
providing to a chromatography system a chromatographic column having a metal flow path with a coating applied to wetted surfaces of the metal flow path by vapor deposition of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane to form a coated flow path; injecting the sample into the coated flow path of the chromatographic column; flowing the sample through the coated flow path of the chromatographic column; separating the sample; detecting at least an amount of the low concentration of the analyte above a minimum expected value when the low concentration of the analyte extends from about 1 ng/mL to about 5 μg/mL.

2 . The method of claim 1 , wherein injecting the sample is a first injection, and
wherein detecting comprises detecting the low concentration of the analyte after the first injection.

3 . The method of claim 1 , wherein a minimum of a linear dynamic range of the chromatographic system remains substantially similar over the first 50 injections.

4 . The method of claim 1 , wherein a chromatographic peak shape of the analyte of the separated sample is substantially similar to a reference chromatographic peak shape.

5 . The method of claim 1 , wherein the coating is uniformly distributed, such that the wetted surfaces are entirely coated.

6 . The method of claim 1 , wherein the coating on the wetted surfaces has a thickness of about 100 Å to about 1600 Å.

7 . The method of claim 1 , further comprising injecting another 50 samples,
wherein a concentration of the analyte of the 50 samples is substantially similar to the low concentration of the analyte of the first sample, and wherein the percent recovered of the analyte is substantially similar for all the samples.

8 . The method of claim 1 , further comprising injecting another 50 samples,
wherein a concentration of the analyte of the 50 samples is substantially similar to the concentration of the analyte of the first sample, and wherein an accuracy of the analyte detected is substantially similar for all the analyte samples.

9 . A method of separating a sample comprising:
flowing a first sample comprising oligonucleotides through a chromatography column having a fluid-contacting coating on metal surfaces defining a flow path, wherein the fluid-contacting coating is formed by vapor deposition of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; separating the first sample; detecting an analyte in the first sample; washing the flow path; flowing a second sample comprising oligonucleotides through the system; separating the second sample; and detecting the second sample, wherein a detected signal from the second sample is within 5% of a detected signal from the first sample and is within 10% of an expected value.

10 . The method of claim 9 , wherein flowing the second sample is immediately preceded by washing the flow path.

11 . The method of claim 9 , wherein separating the first sample and the second sample produce a substantially similar analyte signal.

12 . The method of claim 9 , wherein the chromatography column is in a chromatographic system having a detector.

13 . A method of separating a sample comprising:
injecting a first sample comprising oligonucleotides into a chromatography column having a fluid-contacting coating on metal surfaces defining a flow path, wherein the fluid-contacting coating is formed by vapor deposition of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; flowing the first sample through the chromatography column; and detecting oligonucleotides of the first sample at a concentration extending from about 1 ng/mL to about 10 μg/mL when injecting the first sample through the chromatography column.

14 . The method of claim 13 , further comprising:
injecting a second sample comprising oligonucleotides with a substantially similar concentration as the first sample; flowing the second sample through the chromatography column; and detecting an analyte of the second sample, wherein the amount of analyte detected with first sample is substantially similar to the amount of analyte detected with the second sample.

15 . The method of claim 13 , wherein the fluid-contacting coating maintains performance after flowing over 1,000 samples.

16 . The method of claim 13 , wherein the oligonucleotides do not bind to the fluid-contacting coating on the metallic surfaces of the flow path.

17 . The method of claim 13 , wherein the fluid-contacting coating is hydrophobic.

Assignees

• Waters Technologies Corp

Inventors

• Donegan Michael
• Gilar Martin
• Ngounou Wetie Armand Gatien

Classifications

• G01N30/34

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title

• G01N30/16

  Injection (G01N30/24 takes precedence) · CPC title

• G01N2030/8827

  involving nucleic acids · CPC title

• G01N30/56

  Packing methods or coating methods · CPC title

• B01D15/3828 Primary

  Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography · CPC title