# US 10,954,492 B2 · Processes for production and purification of nucleic acid-containing compositions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,954,492 B2 - Document ID: US10954492B2 - Status: Granted - Kind: B2 - Application number: 15/579,137 - Filed: 2016-06-10 - Published: 2018-06-14 - Granted: 2021-03-23 - Earliest claimed: 2015-06-10 - Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services - Applicants: THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Services - Inventors: Trevor Lane Broadt; Samir Hussein Shaban; Yueqing Xie; Jianwei Sean Zhu; George Mitra - Web page: https://patentgrep.com/patent/US10954492B2 ## CPC - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61K 39/125: Picornaviridae, e.g. calicivirus - B01D 15/363: {Anion-exchange} - C12N 7/02: Recovery or purification - C12N 2770/32651: Methods of production or purification of viral material - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - 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) ## Abstract Described are improved processes for production and purification of nucleic acid-containing compositions, such as non-naturally occurring viruses, for example, recombinant polioviruses that can be employed as oncolytic agents. Some of the described improved processes relate to improved processes for producing viral DNA template. Also described are improved processes for chromatography purification of nucleic acid-containing compositions, in which the nucleic acid is quantified in chromatography fractions using a rapid detection method of the one or more nucleic acid sequences of the nucleic acid-containing composition, such as detection by real time RT-qPCR. In addition, improved processes for production and purification of oncolytic poliovirus, such as PVS-RIPO, are described. Compositions generated using these methods are also provided. ## Claims ### Claim 1 A purification process for obtaining a composition comprising a live non-naturally occurring poliovirus, comprising: separating an aqueous fluid comprising the live non-naturally occurring poliovirus on a size separation chromatography column; measuring by quantitative polymerase chain reaction (qPCR) one or more nucleic acid sequences found in the live non-naturally occurring poliovirus in at least one fraction of the eluate from said size separation column; collecting at least one positive fraction of the eluate from said size separation column containing the one or more nucleic acid sequences found in the live non-naturally occurring poliovirus; pooling the at least one positive fraction; separating the pooled at least one positive fraction on an anion exchange chromatography column; and collecting in at least one positive fraction of the flow-through eluate from said anion exchange chromatography column containing the live non-naturally occurring poliovirus. ### Claim 2 A purification process for obtaining a composition comprising a live non-naturally occurring poliovirus, comprising: separating an aqueous fluid comprising the live non-naturally occurring poliovirus on a Sepharose 6 fast flow (FF) separation chromatography column, collecting at least one positive fraction of the eluate from said Sepharose 6 FF separation column containing the one or more nucleic acid sequences found in the live non-naturally occurring poliovirus; pooling the at least one positive fraction; separating the pooled at least one positive fraction on a Super Q 650M resin anion exchange chromatography column, and collecting in at least one positive fraction of the flow-through eluate from said Super Q 650M resin anion exchange chromatography column containing the live non-naturally occurring poliovirus. ### Claim 3 (depends on 1) The method of claim 1, further comprising: introducing plasmid DNA comprising a template sequence of the live non-naturally occurring poliovirus into one or more bacterial cells, thereby generating the one or more bacterial cells transformed with the plasmid DNA; growing a solid phase culture of the one or more transformed bacterial cells, thereby generating one or more bacterial colonies; detecting the presence of one or more nucleic acid sequences from the template sequence of the live non-naturally occurring poliovirus in at least one of the one or more bacterial colonies; propagating a culture of bacterial cells from the at least one bacterial colony in which the presence of the one or more nucleic acid sequences was detected; extracting the plasmid DNA comprising the template sequence of the live non-naturally occurring poliovirus from the propagated bacterial cells, wherein the bacterial cells are not frozen between the propagating and the extracting steps; infecting mammalian host cells with the plasmid DNA; culturing the mammalian host cells with the plasmid DNA; obtaining liquid cell culture medium from the mammalian host cells, debris of the mammalian host cells, or both, which comprise the live non-naturally occurring poliovirus; and incubating the liquid cell culture medium from the mammalian host cells, debris of the mammalian host cells or both, with a nuclease enzyme capable of digesting free nucleic acids in solution but not encapsulated viral nucleic acids, thereby generating an aqueous fluid comprising the live non-naturally occurring poliovirus. ### Claim 4 (depends on 1) The purification process of claim 1, wherein the process does not contain any further chromatography separation steps after the anion exchange chromatography separation step. ### Claim 5 (depends on 1) The purification process of claim 1, wherein the process contains two chromatography separation steps. ### Claim 6 (depends on 1) The purification process of claim 1, further comprising concentrating by diafilatration the live non-naturally occurring poliovirus eluted in the flow-through eluate. ### Claim 7 (depends on 1) The purification process of claim 1 wherein the purification process is conducted in less than 8 hours. ### Claim 8 (depends on 1) The purification process of claim 1, wherein the purification yield of the purification process is at least 50%, wherein the yield of live non-naturally occurring poliovirus from the process is at least 5×1011 pfu, wherein the infectivity of the live non-naturally occurring poliovirus eluted in the flow-through eluate is at least 1×1012 Tissue Culture Infectious Dose (TCID)50, or combinations thereof. ### Claim 9 (depends on 1) The purification process of claim 1, wherein the aqueous fluid comprising the live non-naturally occurring poliovirus is a liquid cell culture medium obtained by a process comprising culturing, in a one or more rounds of cell culture, host cells infected with the live non-naturally occurring poliovirus. ### Claim 10 (depends on 9) The purification process of claim 9, wherein the liquid cell culture medium is obtained by a process further comprising, after culturing, separating the liquid cell culture medium from the host cells, debris of the host cells or both. ### Claim 11 (depends on 9) The purification process of claim 9, wherein the liquid cell culture medium is obtained by a process further comprising incubating the liquid cell culture medium with a nuclease enzyme capable of digesting free nucleic acids in solution but not encapsulated viral nucleic acids. ### Claim 12 (depends on 9) The purification process of claim 9, wherein the live non-naturally occurring poliovirus is obtained by a process comprising: introducing plasmid DNA comprising a template sequence of the live non-naturally occurring poliovirus into one or more bacterial cells, thereby generating the one or more bacterial cells transformed with the plasmid DNA; growing a solid phase culture of the one or more transformed bacterial cells, thereby generating one or more bacterial colonies; detecting the presence of one or more nucleic acid sequences from the template sequence of the live non-naturally occurring poliovirus in at least one of the one or more bacterial colonies; propagating a culture of bacterial cells from the at least one bacterial colony in which the presence of the one or more nucleic acid sequences was detected; and extracting the plasmid DNA comprising the template sequence of the live non-naturally occurring poliovirus from the propagated bacterial cells, wherein the bacterial cells are not frozen between the propagating and the extracting steps. ### Claim 13 (depends on 9) The purification process of claim 9, wherein the host cells infected with the live non-naturally occurring poliovirus are obtained by a process comprising: introducing plasmid DNA comprising a template sequence of the live non-naturally occurring poliovirus into one or more bacterial cells, thereby generating the one or more bacterial cells transformed with the plasmid DNA; growing a solid phase culture of the one or more transformed bacterial cells, thereby generating one or more bacterial colonies; detecting the presence of one or more nucleic acid sequences from the template sequence of the live non-naturally occurring poliovirus in at least one of the one or more bacterial colonies; propagating a culture of bacterial cells from the at least one bacterial colony in which the presence of one or more nucleic acid sequence was detected; extracting the plasmid DNA comprising the template sequence of the live non-naturally occurring poliovirus from the propagated bacterial cells, wherein the bacterial cells are not frozen between the propagating and the extracting steps; generating naked RNA of the live non-naturally occurring poliovirus by in vitro translation of the template sequence; and introducing the naked RNA of the live non-naturally occurring poliovirus into host cells, thereby generating host cells infected with the live non-naturally occurring poliovirus. ### Claim 14 (depends on 3) The purification process of claim 3, wherein the plasmid is a bacterial plasmid comprising an E. coli origin of replication, and wherein the one or more bacterial cells are E. coli cells. ### Claim 15 (depends on 9) The purification process of claim 9, wherein the host cells are mammalian host cells. ### Claim 16 (depends on 3) The purification process of claim 3, wherein the mammalian host cells are Vero cells. ### Claim 17 (depends on 1) The process of claim 1, wherein the live non-naturally occurring poliovirus is an oncolytic poliovirus or a Sabin polio virus. ### Claim 18 (depends on 1) The process of claim 1, wherein the live non-naturally occurring poliovirus is PVS-RIPO. ### Claim 19 (depends on 1) The purification process of claim 1, wherein the purification yield of the purification process is at least 50%. ### Claim 20 (depends on 1) The purification process of claim 1, wherein the yield of live non-naturally occurring poliovirus from the process is at least 5×1011 pfu. ### Claim 21 (depends on 1) The purification process of claim 1, wherein the infectivity of the live non-naturally occurring poliovirus eluted in the flow-through eluate is at least 1×1012 Tissue Culture Infectious Dose (TCID)50.