Systems and methods for identifying viral contaminants

Patent metadata

Field	Value
Publication number	US-12460270-B2
Application number	US-201916284133-A
Country	US
Kind code	B2
Filing date	Feb 25, 2019
Priority date	Feb 28, 2018
Publication date	Nov 4, 2025
Grant date	Nov 4, 2025

Abstract

The present disclosure is directed to a streamlined sample preparation process, VERA ( V iral E nrichment by R educing A rtifacts), to tilt total genomic material in favor of DNA/RNA viral genomes. This reduction of host genomic artifacts can be completed in <8 hours from sample acquisition. Using a rapid library preparation protocol (˜1.5 minutes) and real-time nanopore sequencing, potential viral contamination, for example RNA viral contamination, can be identified in less than one workday from sample acquisition.

First claim

We claim:

1 . An amplification-free method for identifying a virus in a sample of a cell culture by reduction of host genomic material, comprising:
lysing eukaryotic cells in the sample of a cell culture; removing cellular debris from the sample; concentrating the sample via diafiltration to produce a retentate; treating the retentate with more than one nuclease to degrade eukaryotic nucleic acids, wherein the more than one nuclease is one or more endonucleases and one or more RNases; extracting viral nucleic acids from the nuclease-treated retentate; preparing a library of the extracted viral nucleic acids for sequencing; and sequencing the extracted viral nucleic acids from the prepared library without amplifying the extracted viral nucleic acids; and identifying the virus in the sample.

2 . The method of claim 1 , wherein the eukaryotic cells comprise Chinese Hamster Ovary cells.

3 . The method of claim 1 , wherein the eukaryotic cells secrete a protein drug product.

4 . The method of claim 3 , wherein the protein drug product is selected from the group consisting of an antibody, antigen binding antibody fragment, a fusion protein, and a recombinant protein.

5 . The method of claim 1 , further comprising the step of treating the cell culture to remove the identified virus.

6 . The method of claim 1 , wherein the cells are lysed using a freeze-thaw technique.

7 . The method of claim 1 , wherein the step of removing cellular debris comprises centrifuging the lysed sample to produce a supernatant and filtering the supernatant to remove cellular debris from the sample.

8 . The method of claim 1 , wherein the virus is an RNA virus.

9 . The method of claim 1 , wherein the virus is selected from the group consisting of Minute virus of mice (MVM), K virus, Mouse Encephalomyelitis virus, Mouse Adenovirus, MAV1, murine leukemia virus, Encephalomyocarditis (EMC), lactate dehydrogenase virus (LDV), Polyoma virus, mouse hepatitis virus (MHV) Sendai virus, lymphocytic choriomeningitis virus (LCM), Reo-virus type 3, Kilham rat virus, and Toolan's H-1 virus.

10 . An amplification-free method for detecting viral nucleic acids in a cell culture sample by reduction of host genomic material, comprising:
lysing cells in the cell culture sample to produce cellular debris; separating the cellular debris to produce a supernatant; concentrating the supernatant via diafiltration to produce a retentate; treating the retentate with more than one nuclease, wherein the more than one nuclease is one or more endonucleases and one or more RNases, thereby degrading the eukaryotic nucleic acids; extracting viral nucleic acids from the nuclease-treated retentate; preparing a library of the extracted viral nucleic acids for sequencing; and sequencing the viral nucleic acids from the prepared library without amplifying the extracted viral nucleic acids, wherein viral nucleic acid reads obtained from the sequencing are greater than the cellular nucleic acid reads obtained from the sequencing; and identifying the virus in the sample.

11 . The method of claim 10 , wherein the viral nucleic acid reads are at least 51% of the total reads obtained from the sequencing.

12 . The method of claim 10 , wherein the viral nucleic acid reads are between 50 to 99% of the total reads obtained from the sequencing.

13 . The method of claim 10 , wherein the viral nucleic acid reads are at least 80% of the total reads obtained from the sequencing.

14 . The method of claim 10 , wherein the viral nucleic acid reads are at least 85% of the total reads obtained from the sequencing.

15 . The method of claim 10 , wherein the viral nucleic acid reads are at least 90% of total reads obtained from the sequencing.

16 . The method of claim 10 , wherein the virus is an RNA virus.

17 . The method of claim 1 , wherein the sequencing of the extracted viral nucleic acids from the library is completed within 8 hours.

18 . The method of claim 1 , wherein the sequencing is real-time nanopore sequencing.

19 . The method of claim 1 , wherein viral nucleic acid reads obtained from the sequencing are at least 51% of the total reads obtained from the sequencing.

20 . The method of claim 1 , wherein viral nucleic acid reads obtained from the sequencing are at least 90% of the total reads obtained from the sequencing.

21 . The method of claim 1 , wherein the library of the viral nucleic acids is prepared within 15 minutes.

22 . The method of claim 1 , wherein the more than one nuclease comprises two to five nucleases or nuclease mixtures.

23 . The method of claim 10 , wherein the more than one nuclease comprises two to five nucleases or nuclease mixtures.

Assignees

• Regeneron Pharma

Inventors

• Vanhoute David
• Davis Steven

Classifications

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title