Patent Application
20240043813
The present invention relates to a cation exchange chromatography process for the purification of enteroviruses.
The Enterovirus genus of the Picornaviridae family are small, non-enveloped, single stranded positive sense RNA viruses that contain several species of human pathogens including polioviruses, coxsackieviruses, echoviruses, numbered enteroviruses, and rhinoviruses [1]. Aside from the well-studied poliovirus, there has been an influx of research into the development of vaccines and therapeutics for diseases caused by non-polio enteroviruses such as EV-A71 (hand foot and mouth disease) [2], EV-D68 (respiratory disease) and Coxsackievirus A24 (acute hemorrhagic conjunctivitis) [3]. Enteroviruses have also been evaluated for use as oncolytic viral immunotherapies [4]. Coxsackievirus A21 (CVA21), derived from the wild-type strain, is currently being evaluated in phase 1b/2 clinical trials as a treatment for multiple types of cancer due to its selective infection and oncolysis of tumors overexpressing cell surface receptors ICAM-1[5].
The increasing demand for enterovirus viral vaccines and immunotherapies could challenge the conventional production platform. Gradient ultracentrifugation is commonly employed for the enrichment of full, genome containing capsids and impurity clearance, but may be a potential bottleneck in the purification process due to its low-throughput and labor-intensive protocols [6]. As evidenced by the recombinant adeno-associated viral gene therapy purification platform, a shift from gradient ultracentrifugation towards chromatography-based methods may improve scalability and productivity [7]. No chromatographic technique has been demonstrated for empty (lacking genome; product impurity) and full (genome containing; target product) enterovirus particle separation. There remains a need for a chromatography-based alternative to gradient ultracentrifugation that is capable of removing empty capsids and contaminating impurities to produce a purified composition of infectious, mature virions. This would enable an enterovirus purification process that is more suitable for large-scale commercial manufacturing.
The present invention comprises use of cation exchange chromatography to purify enterovirus from one or more impurities. In another aspect, the present invention provides use of glutathione affinity chromatography prior to the cation exchange purification. In one embodiment, the method selectively captures and enriches genome-containing full mature enterovirus virions from infected host-cell culture harvests, thereby removing one or more impurities such as non-infectious genome-lacking enterovirus procapsids, host-cell proteins (HCPs), host-cell DNA (HC-DNA), and media-related impurities such as bovine serum albumin (BSA).
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of various embodiments of the invention, as illustrated in the accompanying drawings.
The invention described here relates to a scalable cation exchange chromatography process for the purification of enteroviruses (i.e., Coxsackievirus A21, CVA21), including full mature virus particles, empty procapsids, and host cell proteins from a downstream process intermediate. The cation exchange chromatography (CEX) step can be run in bind and elute, or flow-through mode. The CEX purification process can be preceded by a glutathione-based affinity chromatography step followed by an anion exchange flowthrough step.
So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.
The term “about”, when modifying the quantity (e.g., mM, or M), potency (genome/pfu, particle/pfu), purity (ng/ml), ratio of a substance or composition, the pH of a solution, or the value of a parameter characterizing a step in a method, or the like refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition; through instrumental error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like. In certain embodiments, “about” can mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%. In one embodiment, “about” can mean a variation of ±10%.
As used herein, “x % (w/v)” is equivalent to x g/100 ml (for example, 5% w/v equals 50 mg/ml).
“CVA21” refers to Coxsackievirus A 21. One skilled in the art would understand that viruses may undergo mutation when cultured, passaged or propagated. The CVA21 may contain these mutations. Examples of CVA21 include but are not limited to the Kuykendall strain (GenBank accessions nos. AF546702 and AF465515), and Coe strain [9] with or without mutations. The CVA21 may be a homogenous or heterogeneous population with none, or one or more of these mutations.
When referring to the genus or species of enteroviruses, one skilled in the art would understand that viruses may undergo mutation when cultured, passaged or propagated. The enterovirus may contain these mutations. Examples of the specific enteroviruses include but are not limited to the those listed in GenBank or UnitPro data bases with or without mutations. The enterovirus may be a homogenous or heterogeneous population with none, or one or more of these mutations.
“Stationary phase” is meant any surface to which one or more ligands can immobilize to. The stationary phase may be a suspension, purification column, a discontinuous phase of discrete particles, plate, sensor, chip, capsule, cartridge, resin, beads, monolith, gel, a membrane, or filter etc. Examples of materials for forming the stationary phase include mechanically stable matrices such as porous or non-porous beads, inorganic materials (e.g., porous silica, controlled pore glass (CPG) and hydroxyapatite), synthetic organic polymers (e.g., polyacrylamide, polymethylmethacrylate, polystyrene-divinylbenzene, poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of the above) and polysaccharides (e.g., cellulose, agarose and dextran). See for examples.
By “binding” an enterovirus to a stationary phase is meant exposing the enterovirus of interest to the stationary phase under appropriate conditions (pH and/or conductivity) such that the enterovirus is reversibly associated with the stationary phase by interactions between the enterovirus and ligand immobilized on the stationary phase.
The term “equilibration solution” refers to a solution to equilibrate the stationary phase prior to loading the enterovirus on the stationary phase. The equilibration solution can comprise one or more of a salt and buffer, and optionally a surfactant. In one embodiment, the equilibration solution is the same condition as the loading solution comprising the enterovirus.
The term “loading solution” is the solution which is used to load the composition comprising the enterovirus of interest and one or more impurities onto the stationary phase. The loading solution may optionally further comprise one or more of a buffer, salt and surfactant.
The term “wash solution” when used herein refers to a solution used to wash or re-equilibrate the stationary phase, prior to eluting the enterovirus of interest. For washing, the conductivity and/or pH of the wash solution is/are such that the impurities (such as empty enterovirus pro-capsid, BSA, or HCP etc.) are removed from the stationary phase. For re-equilibration, the wash solution and elution solution may be the same, but this is not required. The wash solution can comprise one or more of a salt and buffer, and optionally a surfactant such as PS-80.
The “elution solution” is the solution used to elute the enterovirus of interest from the stationary phase. The elution solution can comprise one or more of a salt, or buffer, optionally a surfactant. The presence of one or more of free reduced glutathione (GSH), salt, buffer of the elution solution is/are such that the enterovirus of interest is eluted from the stationary phase.
A “strip solution” is a solution used to dissociate strongly bound components from the stationary phase prior to regenerating a column for re-use. The strip solution has a conductivity and/or pH as required to remove substantially all impurities and the enterovirus from the stationary phase. The strip solution can comprise one or more of a salt, buffer and GSH, and optionally a surfactant and/or reducing agent.
The term “conductivity” refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity. The unit of measurement for conductivity is mS/cm, and can be measured using a conductivity meter sold, e.g., within the GE Healthcare Äkta™ System. The conductivity of a solution may be altered by changing the concentration of ions therein. For example, the concentration of a buffering agent and/or concentration of a salt (e.g. NaCl or KCl) in the solution may be altered in order to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is modified to achieve the desired conductivity as in the Examples below.
By “purifying” an enterovirus of interest or “purified composition” is meant increasing the degree of purity of the enterovirus in the composition by removing (completely or partially) at least one impurity from the composition. The impurity can be empty procapsids, BSA, host cell components such as serum, proteins or nucleic acids, cellular debris, growth medium etc. The term is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the enterovirus.
As used herein, “glutathione is immobilized to a stationary phase” refers to a glutathione covalently attached to a stationary phase through conjugation of one or more reactive groups. In one embodiment, the glutathione stationary phase is a glutathione conjugated to the stationary phase through the thiol group of the glutathione.
“Surfactant” is a surface active agent that is amphipathic in nature.
“Mature virion” “full mature virion”, “full mature virus” or “full mature virus particle”, “full mature enterovirus”, “mature enterovirus”, “mature virus particle” refers to the mature enterovirus virion [(VP4-VP2-VP3-VP1)5]12+RNA as described in
“Empty capsid” refers to procapsid [(VP0-VP3-VP1)5]12, or degraded A-particle [(VP2-VP3-VP1)5]12 according to
“Full capsid” refers to mature virion or provirion [(VP0-VP3-VP1)5]12+RNA as described in
“Impurity” refers to a material different from the desired enterovirus. The impurity can be a serum (i.e. BSA), Host Cell Protein (HCP), Host Cell DNA (HC-DNA), non-infectious virus-related particles including VP0-containing enterovirus (protomers, pentamers, provirions, procapsids), VP2-containing enterovirus (A-particles, or degraded A-particles). In one embodiment, the desired enterovirus is full mature enterovirus (e.g. full mature CVA21).
The invention provides a method of purifying an enterovirus comprising the steps of:
In one embodiment, prior to step (a), equilibrating the stationary phase with an equilibration solution is performed.
In another aspect of the method, after step (a) but prior to step (b), it further comprises step (i) of washing the stationary phase with one or more wash solutions. In one embodiment, one or more impurities are removed from the wash step. In another embodiment, step (i) comprises a wash step with a wash solution having a conductivity higher than the equilibration solution or loading solution. In a further embodiment, the conductivity of the loading or equilibration solution is the same as the wash solution in the wash step.
Various commercially available cation ion exchange stationary phases may be used in the invention. Examples include but are not limited to Poros™ 50 HS (ThermoFisher Scientific, MA, USA), Capto™ S ImpAct (Cytiva Life Sciences, Uppsala, Sweden), Capto™ SP ImpRes (Cytiva Life Sciences), or Nuvia™ HR-S (Bio-Rad, CA, USA). In one embodiment, the stationary phase is Poros™ 50 HS. In another embodiment, the cation ion exchange ligand is a sulfonic acid (SO3−) functional group. The functional group can be C1-C6alkylSO3− (Poros 50 HS, Capto S, Capto S ImpAct, Capto SP ImpRes) or a sulfonic acid (SO3−) attached to a polymeric surface extender (Nuvia S and Nuvia HR-S). In one embodiment, the resin bead diameter is 30-70 μm. In another embodiment, the resin bead diameter is 30-60 μm. In another embodiment, the resin bead diameter is 40-50 μm.
In one embodiment, the loading solution, equilibration solution, wash solution or elution solution comprises a salt, preferably a monovalent metal ion salt, such as NaCl or KCl. In another embodiment, the loading solution or equilibration solution comprises about 50-500 mM NaCl or KCl. In another embodiment, the loading solution or equilibration solution comprises up to about 350 mM or 400 mM NaCl or KCl. In another embodiment, the loading solution or equilibration solution comprises about 400 mM NaCl or KCl.
In one embodiment, the wash solution comprises about 50-600 mM NaCl or KCl. In one embodiment, the wash solution comprises about 100-600 mM NaCl or KCl. In another embodiment, the wash solution comprises about 350-450 mM NaCl or KCl. In another embodiment, the wash solution comprises about 400-500 mM NaCl or KCl. In a further embodiment, the wash solution comprises about 500 mM NaCl or KCl.
The elution step may be performed with a solution with high ionic strength or high conductivity, and low pH (for example pH about 3.5-4.8). In one embodiment, the elution solution comprises about 350-1200 mM of monovalent salt. In one embodiment, the elution solution comprises about 300-900 mM of monovalent salt. In one embodiment, the elution solution comprises about 200-1000 mM of monovalent salt. In one embodiment, the elution solution comprises about 550-850 mM of NaCl or KCl. In another embodiment, the elution solution comprises about 800 mM NaCl, and optionally about 0.001-1% w/v PS-80. In yet a further embodiment, the elution solution comprises about 800 mM NaCl, and about 0.005% w/v PS-80.
In one embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 3.5-4.8. In another embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 3.8-4.5. In another embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 3.5-4.5. In another embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 4.2-4.8. In a another embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 4. In a further embodiment, one or more of the loading solution, equilibration solution and wash solutions has a pH of about 3.5-6.0, and the elution solution has a pH of about 3.5-4.8. In a further embodiment, one or more of the loading solution, equilibration solution and wash solutions has a pH of about 3.5-6.0, and the elution solution has a pH of about 3.8-4.5. In a further embodiment, one or more of the loading solution, equilibration solution and wash solutions has a pH of about 3.5-6.0, and about 50-500 mM monovalent salt, and the elution solution has a pH of about 3.8-4.5, and about 350-1200 mM monovalent salt. In a further embodiment, one or more of the loading solution, equilibration solution and wash solutions has a pH of about 3.5-6.0, and about 50-500 mM monovalent salt, and the elution solution has a pH of about 3.8-4.5, and about 200-1000 mM monovalent salt.
In one embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution further comprises a surfactant. In another embodiment, the surfactant is PS-80 or PS-20. In another embodiment, the surfactant is about 0.001-1% w/v PS-80. In another embodiment, the surfactant is about 0.001-0.1% w/v PS-80. In another embodiment, the surfactant is about 0.005% w/v PS-80.
In a another embodiment, the loading and equilibration solution has a pH of about 3.8-4.5, comprises about 350-450 mM NaCl or KCl, optionally about 0.001-0.1% w/v PS-80; the wash solution has a pH of about 3.8-4.5, comprises about 450-550 mM NaCl or KCl, optionally about 0.001-0.1% w/v PS-80; and the elution solution has a pH of about 3.8-4.5, comprises about 700-900 mM NaCl or KCl, and optionally about 0.001-0.1% w/v PS-80. In a preferred embodiment, the loading and equilibration solution comprises 50 mM citrate, pH 4.0, 400 mM NaCl, 0.005% w/v PS-80; the wash solution comprises 25 mM citrate, pH 4.0, 500 mM NaCl, w/v PS-80; and the elution solution comprises 25 mM citrate, pH 4.0, 800 mM NaCl, and 0.005% w/v PS-80.
The invention provides a method of purifying an enterovirus comprising the steps of:
In one embodiment, prior to step (a), the stationary phase is equilibrated with an equilibration solution.
In another aspect of the method, it further comprises step (i) of washing the stationary phase with one or more wash solutions after step (b) and further collecting the flow-through of the wash solutions. In one embodiment, one or more of the loading solution, equilibration solution, and wash solution has a pH of about 3.5-4.5. In another embodiment, one or more of the loading solution, equilibration solution, and wash solution has a pH of about 3.8-4.0. In a further embodiment, one or more of the loading solution, equilibration solution, and wash solution has a pH of about 3.8.
In another aspect of the method, one or more of the loading solution, equilibration solution, and wash solution comprises about 400-1500 mM monovalent salt. In one embodiment, one or more of the loading solution, equilibration solution, and wash solution comprises about 350-800 mM monovalent salt (e.g. NaCl or KCl). In one embodiment, one or more of the loading solution, equilibration solution, and wash solution comprises about 900-1100 mM monovalent salt (e.g. NaCl or KCl), and has a pH of about 3.5-4.0 or 3.8-4.0. In one embodiment, one or more of the loading solution, equilibration solution, and wash solution comprises about 550-700 mM monovalent salt (e.g. NaCl or KCl), and has a pH of about 4.0-4.7 or 4.0-4.5. In one embodiment, one or more of the loading solution, equilibration solution, and wash solution comprises about 450-800 mM monovalent salt (e.g. NaCl or KCl), and has a pH of about 4.0-4.7 or 4.0-4.5. In one embodiment, the loading solution has the same conductivity as the equilibration solution or wash solution.
In another aspect of the method, one or more of the loading solution, equilibration solution, and wash solution further comprises a surfactant. In another embodiment, the surfactant is PS-80 or PS-20. In another embodiment, the surfactant is about 0.001-1% w/v PS-80. In another embodiment, the surfactant is about 0.001-0.1% w/v PS-80. In another embodiment, the surfactant is about 0.005% w/v PS-80.
In one embodiment, the desired enterovirus is full mature enterovirus. In one embodiment, the desired enterovirus is Coxsackievirus. In one embodiment, the desired enterovirus is full mature CVA21. In one embodiment, at least the full mature enterovirus binds to the stationary phase upon loading the solution. In one embodiment, the purification process removes one or more impurities such as serum (i.e. BSA), HCP, HC-DNA, non-infectious virus-related particles including but not limited to VP0-containing enterovirus (protomers, pentamers, provirions, procapsids), VP2-containing enterovirus (A-particles, or empty capsids from degraded A-particles). In a further embodiment, the purification process removes enterovirus empty procapsids (e.g., CVA21 empty procapsids).
The CEX purification method of the invention can be preceded by glutathione affinity (GSH) chromatography. After conducting GSH chromatography in bind and elute mode, the GSH elution product after solution adjustment, can be loaded to the CEX stationary phase. Alternatively, the GSH elution product (with or without solution adjustment) can be loaded to an anion exchange stationary phase, the flow-through collected; and after solution adjustment, applied to the CEX stationary phase. In one embodiment, the glutathione affinity chromatography stationary phase comprises a glutathione (GSH) immobilized to the surface of a stationary phase. Glutathione (also named L-glutathione, reduced glutathione, or GSH) is a biologically-active tri-peptide (glutamic acid-cysteine-glycine) in human cells used to control redox potential and is involved in many cellular functions [11]. GSH has the following chemical structure and name:
The glutathione can be immobilized to the stationary phase through conjugation of the SH group using maleimide, haloacetyl, pyridyl disulfide, epoxy or other similar sulthydryl-reactive based chemistries. See for examples. GSH resin is also commercially available through several vendors (Cytiva Life Sciences, ThermoFisher Scientific, Qiagen, Sigma).
In batch mode, the stationary phase is utilized free in solution. For utilization in flow mode, the stationary phase is packaged into a column, capsule, cartridge, filter or other support and a flowrate of about 1-500 cm/hr is used.
In one aspect, the invention provides a method of purifying an enterovirus comprising the steps of:
In one embodiment, prior to step (a), equilibrating the stationary phase with an equilibration solution is performed. In one embodiment, one or more impurities are in the flowthrough of step (a).
In another aspect of the method, after step a) but prior to step (b), it further comprises step i) of washing the stationary phase with one or more wash solutions. In one embodiment, one or more impurities are removed from the wash step. In another embodiment, step (i) comprises a first wash step with a wash solution having a conductivity higher than the equilibration solution or loading solution. In another embodiment, step (i) comprises a second wash step with a wash solution having a conductivity lower than the wash solution in the first wash step. In a further embodiment, the conductivity of the elution solution is the same as the wash solution in the second wash step.
In one embodiment of the GSH chromatography steps a) and b), the loading solution, equilibration solution, wash solution or elution solution comprises a salt, preferably a monovalent metal ion salt, such as NaCl or KCl. In another embodiment, the loading solution or equilibration solution comprises about 50-200 mM NaCl or KCl. In a another embodiment, the loading solution or equilibration solution comprises about 100 mM NaCl or KCl.
In one embodiment of the GSH chromatography steps a) and b), the wash solution comprises about 50-400 mM NaCl or KCl. In another embodiment, the wash solution comprises about 350-450 mM NaCl or KCl. In another embodiment, the wash solution comprises about 400-500 mM NaCl or KCl. In a further embodiment, the wash solution comprises about 400 mM NaCl or KCl. In a further embodiment, a first wash solution comprises about 100-500 mM NaCl or KCl and a second wash solution comprises about 50-500 mM NaCl or KCl. In a further embodiment, a first wash solution comprises about 350-500 mM NaCl or KCl and the second wash solution comprises about 50-150 mM NaCl or KCl. In a further embodiment, the first wash solution comprises about 400 mM NaCl or KCl and the second wash solution comprises about 75 mM NaCl or KCl. In a further embodiment, the second wash solution comprises about 50-150 mM NaCl or KCl. In a further embodiment, the second wash solution comprises about 100 mM NaCl or KCl.
The elution of the GSH chromatography step may be performed with a solution with high ionic strength or high conductivity, low pH (for example pH about 5-7), or in the presence of free GSH, or a combination thereof. In one embodiment, the elution solution comprises about 0.5-1 M of monovalent salt such as NaCl or KCl. In one embodiment, the elution solution comprises about 0.5 M of NaCl or KCl. In one embodiment, the elution solution comprises about 50-500 mM of NaCl or KCl. In another embodiment, the elution solution comprises about 0.1-100 mM glutathione. In another embodiment, the elution solution comprises about 0.1-50 mM glutathione. In another embodiment, the elution solution comprises about 0.1-25 mM glutathione. In another embodiment, the glutathione in the elution solution is about 1 mM. In one embodiment, the elution solution comprises about 0.5-5 mM glutathione and about 75-150 mM NaCl or KCl. In one embodiment, the elution solution comprises about 0.5-25 mM glutathione and about 50-500 mM NaCl or KCl. In another embodiment, the elution solution comprises about 0.1-100 mM glutathione and about 75-150 mM NaCl, and optionally about 0.001-1% w/v PS-80. In yet a further embodiment, the elution solution comprises about 100 mM NaCl, about 1 mM glutathione, and about 0.005% w/v PS-80.
In one embodiment of the GSH chromatography steps a) and b), one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 6.5-8.5. In a another embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 7-8. In a another embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 8. In a further embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 6-9. In yet a further embodiment, one or more of the loading solution, equilibration solution, wash solutions and elution solution has a pH of about 5-10.
In one embodiment of the GSH chromatography steps a) and b), one or more of the loading solution, equilibration solution, wash solutions and elution solution further comprises a surfactant. In another embodiment, the surfactant is PS-80 or PS-20. In another embodiment, the surfactant is about 0.001-1% w/v PS-80. In another embodiment, the surfactant is about w/v PS-80. In another embodiment, the surfactant is about 0.005% w/v PS-80. In one embodiment, one or more of the loading solution, wash solutions and elution solution further comprises EDTA, or a reducing agent such as DTT or ß-mercaptoethanol. In another embodiment, the reducing agent is DTT. In another embodiment, the DTT is at about 0.1-10 mM. In another embodiment, the DTT is at about 0.1-5 mM. In another embodiment, the DTT is at about 1 mM.
Embodiments of the CEX chromatography steps in c) and d) were described in the CEX section above. The methods of the invention can be used in conjunction with other chromatography or purification steps to remove impurities. In one embodiment, after step b) but prior to step c) above, comprises the steps of
In one embodiment, the loading solution in step 1) comprises about 50-500 mM monovalent salt concentration at pH about 6-9.
In another aspect, the invention provides a method of purifying Coxsackievirus (e.g. CVA21) comprising the steps of:
In one embodiment, after step d) but prior to step e) above, comprises the steps of
In another aspect, the invention provides a purified composition of the enterovirus obtainable by or produced by the foregoing purification steps and/or embodiments of the invention.
In one embodiment, the desired enterovirus is full mature enterovirus. In one embodiment, the desired enterovirus is full mature Coxsackievirus. In one embodiment, the desired enterovirus is full mature CVA21. In one embodiment, at least the full mature enterovirus binds to the stationary phase upon loading the solution. In one embodiment, the purification process removes one or more impurities such as serum (i.e. BSA), HCP, HC-DNA, non-infectious virus-related particles including but not limited to VP0-containing enterovirus (protomers, pentamers, provirions, procapsids), VP2-containing enterovirus (A-particles, or empty capsids from degraded A-particles). In a further embodiment, the purification process removes enterovirus empty procapsids (e.g., CVA21 empty procapsids).
Any suitable source of enterovirus may be used in the methods of the invention [1]. The enterovirus particle can be poliovirus, Group A Coxsackievirus, Group B Coxsackievirus, echovirus, rhinovirus, and numbered enterovirus. In one embodiment, the enterovirus is a Group A, B or C enterovirus. In one embodiment, the enterovirus is a Group C enterovirus. In one embodiment, the enterovirus is a Group A or B Coxsackievirus. In another embodiment, the enterovirus is Group A Coxsackievirus. In one embodiment, the Group C enterovirus is a Group A Coxsackievirus selected from the group consisting of CVA1, CVA11, CVA13, CVA15, CVA17, CVA18, CVA19, CVA20a, CVA20b, CVA20c, CVA21, CVA22 and CVA24. In one embodiment, the Group A Coxsackievirus is selected from the group consisting of CVA13, CVA15, CVA18, CVA20, and CVA21. Various suitable strains of these viruses may be obtained from the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA, such as material deposited under the Budapest Treaty on the dates provided below, and is available according to the terms of the Budapest Treaty: Coxsackie group A virus, strain CVA13, ATCC No.: PTA-8854, deposited Dec. 10, 2007; Coxsackie group A virus, strain CVA15 (G9), ATCC No.: PTA-8616, deposited Aug. 15, 2007; Coxsackie group A virus, strain CVA18, ATCC No.: PTA-8853, deposited Dec. 20, 2007; and Coxsackie group A virus, strain CVA21 (Kuykendall), ATCC No.: PTA-8852, deposited Dec. 20, 2007. Other Group A Coxsackie virus under Group C enterovirus referenced in the literature include but are not limited to CVA1 (GenBank accession no. AF499635, [13]), CVA11 (GenBank accession no. AF499636), CVA17 (GenBank accession no. AF499639), CVA19 (GenBank accession no. AF499641), CVA20 (GenBank accession no. AF499642), CVA20a ([14]), CVA20b ([14]), CVA20c ([15]), CVA22 (GenBank accession no. AF499643; [14]), and CVA24 (GenBank accession no. EF026081; [16]). In a preferred embodiment, the enterovirus is a Coxsackievirus A21.
In another embodiment, the enterovirus is a Group B enterovirus. In another embodiment, the Group B enterovirus is echovirus. In another embodiment, the Group B enterovirus is echovirus-1 (EV-1). Examples of echovirus-1 include those with GenBank accession nos. AF029859, AF029859.2 and AF250874.
In another embodiment, the enterovirus is a Group B Coxsackievirus. In a further embodiment, the Group B Coxsackievirus is Coxsackievirus B3 (CVB3) or Coxsackievirus B4 (CVB4).
In a further embodiment, the enterovirus is a Rhinovirus A, B or C. In another embodiment, the enterovirus is Rhinovirus A or B. In yet a further embodiment, the enterovirus is Human Rhinovirus 14 (HRV14). In yet a further embodiment, the enterovirus is Human Rhinovirus 1B or 35. An example of Human Rhinovirus 1B is Genbank accession no. D00239.1. An example of human Rhinovirus 35 is Genbank accession no. EU870473. A summary of the current understanding of enterovirus morphogenesis is detailed in
The examples are presented in order to more fully illustrate the various embodiments of the invention. These examples should in no way be construed as limiting the scope of the invention recited in the appended claims.
High throughput chromatography experiments were performed using Opus® RoboColumns® (Repligen, MA, USA) on a Tecan EVO® 150 robotic station (base unit) operated by EVOware® v2.8 which was equipped with an 8-channel Liquid Handling (LiHa) arm and an eccentric Robot Manipulator (RoMa) arm (Tecan Group Ltd., Marmedorf, Switzerland). The LiHa arm was equipped with short stainless-steel tips and for the operation of the RoboColumns the robotic station was fitted with the Te-Chrom™ and Te-Shuttle™ modules (fraction collection system) and integrated with an Infinite® M1000 pro reader (Tecan Group Ltd.).
The described configuration of the robotic station allowed for up to 8 RoboColumn-based chromatographic separations to be run in parallel in a process described in [17]. A total of 12 separations were performed aiming to evaluate the separation of full mature virus particles and empty procapsids on a selection of ion exchange resins (Tables 1 and 2). The aforementioned separation was tested in a range of mobile phase conditions for cation exchange (CEX) resin Poros™ 50 HS (ThermoFisher Scientific). Additional CEX resins Capto™ S ImpAct, SP ImpRes, S (Cytiva Life Sciences) and Nuvia™ S and HR-S(Bio-Rad) were also evaluated along with the strong anion exchanger (AEX) Nuvia HP-Q (Bio-Rad). All separations (Tables 1 and 2) employed 200 μL RoboColumns and were run in bind and elute mode with a salt gradient and a residence time of 2 minutes for all phases. Each separation included an equilibration, load, wash, elution and strip phase with their durations, in terms of column volumes, shown in Tables 1 and 2. These tables also describe the composition of buffers employed in each phase of each separation. Here, the pH of these buffers across all phases, apart from the strip, remained constant and the same applied to the salt level during the equilibration, load and wash phase. Hence, each separation could be identified by the resin used and the combination of pH and salt level employed.
The CEX resin-based separations employed a Citrate buffer system with varying pH between 3.8 and 6.0 and NaCl concentration to match the desired mobile phase conditions during the equilibration, wash, and elution phases (Tables 1 and 2). Conversely, for the AEX resin, a Tris buffer system was used with a pH of 9.0 and varying NaCl concentration for the equilibration, wash and elution phases (Tables 1 and 2). In all separations, the columns were stripped using a 100 mM Tris pH 7.0, 1000 mM NaCl buffer. All separations also employed the same duration for the equilibration, wash, and strip phases (i.e., 9, 5 and 5 column volumes, CVs, respectively) whereas the CVs varied during the elution and load phases. For the former, this was accrued due to maintaining a constant salt elution gradient slope of ˜60 mM CV−1. For example, for separations 1 and 2 (Table 1), the columns were eluted in 19 CVs (450 mM NaCl to 1500 mM NaCl) whereas separations 7-11 (Table 2) were run with a 24 CV gradient (50 mM NaCl to 1500 mM NaCl). Since RoboColumn experiments do not allow for an ‘on the fly’ mixing of mobile phases, the elution gradients were simulated by step gradients. Here, each step had a size of 1 CV and a salt level (Csalt) determined by the equation Csalt=Csalt,o+60×CVelution, where Csalt,o is the starting salt level in the gradient (e.g., 50 mM NaCl or 450 mM NaCl) and CVelution corresponds to the elution phase column volume number. Here, Csalt, is also the salt level of the buffer used in the equilibration, load and wash phases. The steps in the gradient were generated by mixing, for each buffer system, the low (50 mM NaCl or 450 mM NaCl) and high (1000 mM NaCl) salt buffers for a given pH at different ratios to obtain the desired salt concentrations. For the loading of the columns, 60 or 30 CVs were employed (Tables 1 and 2). Here, the product pool from a preceding Affinity Chromatography (AC) step was diluted 3-fold in concentrated buffers with a composition designed to match the composition (pH, NaCl concentration and buffer system concentration) of the equilibration mobile phase buffer post dilution. For the AEX resin separation, the Tris concentration was increased to 70 mM during the load compared to 50 mM Tris at the equilibration phase.
Finally, all separations were fractionated by collecting fractions every 200 μL, or one CV. These were collected in UV transparent 96 well microplates (Corning Inc., NY, USA) and were read on a plate reader at 260 nm, 280 nm, 900 nm and 990 nm. The made measurements were employed to construct chromatographic traces and to determine how the collected fractions should be pooled and which fractions required further analysis. Here, the fractions were pooled in a fashion yielding up to five pools containing flowthrough fractions (FT1-FT5), one pool containing the wash fractions (W), and one pool containing the strip fractions (S). The fractions collected during the elution of the columns were pooled in three different ways. Pools E1 and E2 contained the fractions in approximately the first and second half of the main elution peak respectively whereas pool E3 contained all fractions included in pools E1 and E2 in addition to a few fractions flowing the complete elution of the main peak in the gradient. All pooling was carried out on the described robotic station. The analysis of these pools and individual fractions took place via analytical methods including quantitative western blotting and SDS-PAGE.
The aforementioned robotic system and methodology were also employed to perform column challenge experiments. These were carried out by increasing the levels of impurities presented to the chromatography column and observing how well full mature virus particles could be separated from impurities such as host cell DNA and bovine serum albumin (BSA). For this purpose, 0.6 mL Poros™ 50 HS columns were equilibrated for 5 CVs before they were loaded for 20 CVs and washed for 5 CVs with equilibration buffer. The columns were then eluted for 13 CVs with a slope of 75 mM CV−1 and stripped for 5 CVs. Fractions were collected every 200 μL in UV transparent 96 well microplates (Corning Inc.) and the residence time was set to 2 min across all steps. The employed mobile phases during the equilibration and wash steps were comprised of a 50 mM citrate, 100 mM NaCl, 0.005% w/v PS-80 buffer system at different pH values. These spanned a pH range of 3.8-4.2 and remained constant across the entire separation. To generate buffers employed during the elution of the columns, the equilibration and wash buffers were mixed at desired ratios with a 50 mM citrate, 1000 mM NaCl, 0.005% PS-80 buffer prepared at the same pH. The latter was also used to strip the columns. Finally, here the load to the columns was the product from an early application of the preceding Affinity Chromatography step diluted 3-fold in concentrated mobile phase to match the equilibration buffer composition. Post dilution, the load was spiked with BSA (Sigma-Aldrich, MO, USA) and λ DNA (ThermoFisher Scientific) to concentrations of 0.1 g L−1 and 200 ng mL−1 respectively. These corresponded to loading to the columns amounts of 1.2 mg BSA and 2.4 μg λ DNA which represented a ˜>100-fold increase of such impurities in a typical Affinity Chromatography product. Fractions and their pools were analyzed via analytical methods including quantitative western blotting, SDS-PAGE, Quant-iT™ PicoGreen™ dsDNA (Invitrogen, CA, USA) and Pierce™ Coomassie Plus (Bradford) total protein assay (ThermoFisher Scientific).
Chromatography experiments in batch mode were performed using 96 well PreDictor™ chromatography plates pre-dispensed with 20 μL of resin Capto™ S ImpAct (both from Cytiva, MA, USA). The plates were operated manually and based on the manufacturer's instructions. When deviations from the suggested protocol were employed, these are detailed in the description of the derived results.
Before describing the analytical methods, it is important to first underline a key process in the morphogenesis of enteroviruses, CVA21, with a detailed review provided in [8] and depicted here schematically in
Starting material, fractions and elution pools were assayed for full mature virus particles (VP4) and empty procapsids (VP0) via quantitative western blotting using a Sally Sue™ system and a 12-230 kDa Sally Sue™ Separation Module kit (Protein Simple, CA, USA). Samples were prepared using an Anti-Rabbit Detection Module (Protein Simple), according to the manufacturer's protocol, and denatured in a Mastercycler® Gradient (Eppendorf, NY, USA) for 5 min at 95° C. For their analysis, an anti-VP4 rabbit pAb (Lifetein LLC, NJ, USA) was used which was diluted to 20 μg mL−1 in Antibody Diluent 2 (Protein Simple). Upon their preparation, the samples were loaded to the capillaries for 9 sec, separated for 40 min at 250 V, and immobilized for 250 sec. This was followed by their exposure to antibody diluent for 23 min, to anti-VP4 rabbit primary antibody for 30 min, and to the anti-rabbit secondary antibody for 30 min. The capillaries were then imaged with the chemiluminescence detection settings and the HDR detection profile. For data analysis purposes, the results were analyzed using the 8 sec exposure time setting with a dropped lines method for peak integration. All samples were diluted with a concentrated Tris, pH 7.5 buffer, 0.005% w/v PS-80 to a final composition of ˜150 mM Tris, pH 7.5, 0.005% w/v PS-80 prior to their analysis.
Fractions, pools and starting materials were also analyzed via gel electrophoresis using NuPAGE™ 12% Bis-Tris 1.0 mm 10-well gels (Invitrogen, CA, USA) to track empty procapsids and full mature virus particles (VP0 and VP2 respectively—VP4 has a molecular weight close to the low limit of the gel and cannot be reliably tracked). For this purpose, 700 μL of denaturing buffer was prepared by mixing 200 μL of NuPAGE Sample Reducing Agent (10×) (Invitrogen) and 500 μL of NuPAGE LDS Sample Buffer (4×) (Invitrogen). 14 μL and 26 μL per well of denaturing buffer and sample, respectively, were mixed together in a 96 well PCR plate (ThermoFisher Scientific) which was then sealed with an adhesive aluminum foil and centrifuged for a few minutes at 3000 rpm on a Sorvall™ Legend™ XTR centrifuge (ThermoFisher Scientific). The PCR plate was then denatured in a Mastercycler Gradient (Eppendorf) for 10 min at 70° C. Following denaturation, 25 μL of sample per lane were loaded into separate lanes of a gel with the latter also including a lane loaded with 2 μL of Mark12 Unstained Standard (Invitrogen). The prepared gels were electrophoresed in a 1×MOPS running buffer, prepared from NuPAGE MOPS SDS Running Buffer (20×) (Invitrogen), for 50 min at 200 V. The gels were then stained with a Pierce™ Silver Stain Kit (ThermoFisher Scientific) according to the manufacturer's protocol, with a 2 min development time. Finally, the gels were imaged with a Gel Doc™ EZ System (Bio-Rad) with a Silver Stain autoexposure scan protocol.
Starting material and collected fractions used for and generated from the cation exchange chromatography experiments respectively were analyzed for verification of presence of empty procapsids and full mature virus particles via sucrose density gradient centrifugation. For this purpose, four linear gradients were prepared at 11 mL using buffers of 15 mM Tris, 150 mM NaCl, 0.005% w/v PS-80, pH 8.0 containing sucrose levels at concentrations of 45% (w/v) and 15% (w/v). Upon their preparation, 1 mL of samples were layered on their top and the gradients were centrifuged at 36000 rpm for 100 min at 4° C. using an Optima™-SE Ultracentrifuge (Beckman Coulter, CA, USA). Post centrifugation, twelve fractions of equal volumes were collected from the top of the gradients and stored at 4° C. until further processing.
Colorimetric assays Quant-iT™ PicoGreen™ dsDNA (Invitrogen, CA, USA) and Pierce™ Coomassie Plus (Bradford) (ThermoFisher Scientific) were deployed as per the manufacturer's instructions. The aforementioned SDS-PAGE protocol was also used to track BSA in the assayed fractions and pools. BSA tracking was also performed via quantitative western blotting as described in [18].
The chromatographic traces recorded across the performed separations showed the excellent repeatability of the RoboColumn method since the duplicated runs yielded traces overlapping with each other almost perfectly (
A single strong peak was observed in the elution gradient for all separations (
These results indicate that the empty procapsids are more retained than the full mature virus particles under low pH value conditions. This can be observed in the SDS-PAGE results since at low pH values (e.g.,
While the elution yields of the full mature virus particles undergo an increase from ˜75% to 100% by increasing the pH (
For the CEX step to be successful in separating full mature virus particles and empty procapsids it must also lead to high elution yields while minimizing any yield losses due to full mature virus particles flowing through during the loading of the column. As mentioned, across all tested conditions for Poros™ 50 HS, and for the rest of the CEX resins, the quantitative western results showed no full mature virus particles or empty procapsids in the flowthrough and wash fractions. Hence, all performed separations showed 100% binding yields even if the chromatography traces showed weak flowthrough signals at 260 nm (i.e., impurities flowing through and full mature virus particles or empty procapsids). The high binding yields for Poros™ HS were also accompanied by wide operating windows in terms of binding salt level as a function of the pH (i.e., salt level in equilibration, load and wash phases and also at the beginning of the salt gradient).
The elution yields for the full mature virus particles and empty procapsids in
Hence, taking into consideration the elution yields from elution pool E3 (
Finally, an interesting and unexpected result of the CEX application at pHs of 5.0 and above needs to also be highlighted. The lane images in
The retention trends in
The achieved purification of full mature virus particles from empty procapsids renders the CEX step in flow through mode at low pH conditions as a viable alternative to running it in bind and elute mode. This was further supported by the resulting full mature virus particle yields (
The CEX step in bind and elute mode is also capable of separating the full mature virus particles from impurities in the gradient and thus improving the purity of the elution product. The column challenge studies with BSA and DNA aimed to demonstrate this.
Five alternative cation exchangers were also tested, in addition to the cation exchange resin Poros™ 50 HS, to determine whether they could also deliver a good separation between empty procapsids and full mature virus particles at a pH of 4.0 (
Conversely, the AEX resin Nuvia HP-Q, run at a pH of 9.0 (
An anion exchange resin, such as Nuvia HP-Q, run at strong binding conditions (no full mature virus particles or empty procapsids were detected in the flowthrough and wash fractions), cannot separate full mature virus particles and empty procapsids and hence lead to product pools with high yield and purity. In contrast, cation exchangers, evaluated in bind and elute mode, were characterized across a range of conditions and were shown to be able to deliver a robust step for separating full mature virus particles and empty procapsids viral while returning high elution yields for full mature virus particles. At the same time, the CEX step serves to concentrate the product, which facilitates further processing activities, while removing process impurities, which either flow through or elute at higher salt levels than the full mature virus particles. The benefits of the CEX step were also demonstrated at scale where it delivered a concentrated product with high yields and free of empty procapsids and impurities.
To demonstrate the wide applicability of GSH affinity purification for enteroviruses, 8 different serotypes, encompassing several enterovirus species including Enterovirus B, Enterovirus C, Rhinovirus A, and Rhinovirus B, were evaluated. The strains were purchased from the American Type Culture Collection (ATCC) and amplified in two infections using two cell lines and upstream conditions (Table 4) based on infection protocols commonly used for producing enteroviruses. Cells were planted in tissue culture-treated vented flasks in growth media. Several days post plant, the growth media were decanted and 1 mL of enterovirus inoculum was added to the cell layer. The flasks were incubated for 2 hours before 39 mL of production media were added to each flask and incubated based on the upstream condition used. Upon confirmation of cytopathic effect, the flasks were harvested by collecting the supernatant. The harvests were then stored at −70° C. until they were purified via GSH affinity chromatography.
GSH affinity chromatography was performed using RoboColumns packed with 0.6 mL of Glutathione Sepharose® 4 Fast Flow resin, (GSH Sepharose 4 FF from Cytiva Life Sciences). For each purification, the columns were equilibrated with 5 CVs of Phosphate Buffered Saline (PBS), pH 7.4. Following equilibration, the columns were loaded with 50 CVs of thawed and clarified harvest. Post loading, the columns were washed sequentially with 5 CVs of wash 1 buffer (15 mM Tris, 400 mM NaCl, 1 mM DTT, 0.005% w/v PS-80, pH 8.0) and 5 CVs of wash 2 buffer (15 mM Tris, 150 mM NaCl, 1 mM DTT, 0.005% w/v PS-80, pH 8.0). The columns were eluted with 5 CVs of elution buffer (15 mM Tris, 150 mM NaCl, 1 mM DTT, 1 mM GSH, 0.005% w/v PS-80, pH 8.0) and stripped with 5 CVs of a buffer containing 15 mM Tris, 1000 mM NaCl, 1 mM DTT, 10 mM GSH, 0.005% w/v PS-80, pH 8.0. All steps were performed with a residence time of 4 min and fractions were collected every 200 μL in UV plates (Corning Inc.).
Chromatograms were generated by measuring the optical absorbance of fractions at 260 nm and 280 nm. An elution peak, corresponding typically to a single fraction, was observed between 60-65 CVs. The clarified harvest (
GSH affinity chromatography was evaluated across 2 experiments with CVA21 clarified harvests produced using upstream cell culture conditions A-C (Table 5). 20 mL HiPrep columns packed with GSH Sepharose 4 Fast Flow resin were used on an Äkta Pure 150M FPLC system with UNICORN™ system control software (Cytiva Life Sciences). The CVA21 clarified cell culture harvests were loaded to the column at a flow rate of 100 cm hr−1 until a column loading of 200 CVs was reached. The GSH column was washed at a flow rate of 150 cm hr−1 with 8 CVs of a GSH Wash 1 buffer containing 15 mM Tris, 400 mM NaCl, 0.005% w/v PS-80, pH 8.0 and then 4 CVs of a GSH Wash 2 buffer containing 15 mM Tris, 75 mM NaCl, 0.005% w/v PS-80, 1 mM DTT, pH 8.0. The bound CVA21 particles were eluted with 4 CVs of a GSH Elution solution containing 15 mM Tris, 75 mM NaCl, 0.005% w/v PS-80, 1 mM DTT, 1 mM GSH, pH 8.0 at a flow rate of 150 cm hr−1. The GSH column was stripped with 4 CVs of a GSH Strip buffer containing 15 mM Tris, 1000 mM NaCl, 0.005% w/v PS-80, 1 mM DTT, 10 mM GSH, pH 8.0 and regenerated with a 0.1 N NaOH, 1 M NaCl solution at a flow rate of 150 cm hr−1.
The clarified harvest and GSH elution product samples were analyzed by SDS-PAGE with silver stain (
A scalable purification of enteroviruses was demonstrated using the process in
In some enterovirus cell cultures, the lytic activity of the virus is sufficient to lyse the cells and no lysis step is needed. In other enterovirus cell cultures, a lysis step such as detergent lysis with PS-80, PS-20, or other surfactant ranging from 0.01-2% w/v may be implemented prior to the clarification step to fully lyse the cells. In the current example with CVA21, no lysis step was performed.
The clarified harvest is loaded directly to a GSH affinity chromatography column. For the GSH chromatography operation, GSH immobilized resin is packed into manufacturing scale chromatography columns and operated with a chromatography skid such as Äkta Pilot or Äkta Ready (both from Cytiva Life Sciences). In the current example with CVA21, a 14 cm diameter column packed with GSH Sepharose 4 FF was used on the Äkta Pilot with UNICORN system control software (Cytiva Life Sciences). The CVA21 clarified cell culture harvest was loaded to the column at a flow rate of 100 cm hr−1 until a column loading of 150-200 CVs. The GSH column was washed at a flow rate of 150 cm hr−1 with 8 CVs of a GSH Wash 1 buffer containing 15 mM Tris, 400 mM NaCl, 0.005% w/v PS-80, pH 8.0 and then 4 CVs of a GSH Wash 2 buffer containing 15 mM Tris, 150 mM NaCl, 0.005% w/v PS-80, 1 mM DTT, pH 8.0. The bound CVA21 particles were eluted with 4 CVs of a GSH Elution solution containing 15 mM Tris, 150 mM NaCl, 0.005% w/v PS-80, 1 mM DTT, 1 mM GSH, pH 8.0 at a flow rate of 150 cm hr−1. The GSH column was stripped with 4 CVs of a GSH Strip buffer containing 15 mM Tris, 1000 mM NaCl, 0.005% w/v PS-80, 1 mM DTT, 10 mM GSH, pH 8.0 and regenerated with a 0.1 N NaOH, 1 M NaCl solution at a flow rate of 150 cm hr−1.
The GSH elution product is loaded directly to an optional polishing anion exchange (AEX) chromatography step operated in flow-through mode for additional residual impurity clearance. The AEX chromatography step may use common AEX chromatography media such as Poros™ 50 HQ (ThermoFisher Scientific), Capto Q (Cytiva Life Sciences), or Nuvia Q (Bio-Rad) or other AEX stationary phases. For large scale AEX chromatography operation, AEX resin is packed into manufacturing scale chromatography columns and run with a chromatography skid such as Äkta Pilot at a flow rate of 50-300 cm hr−1. The AEX column is equilibrated in 3-5 CVs AEX Equilibration buffer composed of a solution at pH 6-9 and a monovalent salt concentration of 50-500 mM. The GSH elution product in a solution at pH 6-9 and a monovalent salt concentration of 50-500 mM is loaded to the AEX column followed by a 1-3 CV chase with the AEX equilibration buffer. The enterovirus particles flow through while impurities including HC-DNA and impurity protein bind to the AEX resin. The column is stripped with 3-5 CVs of AEX Strip buffer composed of a solution at pH 6-9 and a monovalent salt concentration of 500-1500 mM and regenerated with a solution containing 0.1-0.5 N sodium hydroxide. The AEX buffer solutions may contain a surfactant such as PS-80, PS-20 or other similar surfactant at a concentration of 0.001-1% w/v. In the current example with CVA21, a 5 cm diameter column packed with Poros™ 50 HQ resin was run on an Äkta Pilot with UNICORN system control software at a flowrate of 200 cm hr−1. The AEX column was equilibrated with 4 CVs of an AEX equilibration buffer consisting of 15 mM Tris, 150 mM NaCl, 0.005% w/v PS-80, pH 8.0. The GSH elution product containing CVA21 particles was loaded to the column until a loading of 25-CVs and chased with 2 CVs of AEX equilibration buffer. The CVA21 particles flowed through while residual impurities bound to the column. The AEX column was stripped with 4 CVs of an AEX Strip buffer containing 15 mM Tris, 1000 mM NaCl, 0.005% w/v PS-80, pH 8.0 and regenerated with 4 CVs of a 0.5 N NaOH solution. The AEX chromatography step may be omitted if the desired residual impurity specifications in the final purified composition are met without AEX. In this situation, the GSH elution product is forwarded to the solution adjustment step.
In the solution adjustment step, the AEX FT or GSH elution (if AEX is not performed) product is adjusted to solution conditions compatible with binding to the CEX chromatography resin in the subsequent CEX chromatography step. The AEX FT or GSH elution product is initially in a solution at pH 6-9 and a monovalent salt concentration of 50-500 mM. If necessary, concentrated stock solutions of 0.5-1.5 M adjustment buffer solution, consisting of a buffer species such as citrate, at pH 3.5-6.0 and 2-5 M adjustment monovalent salt solution, such as NaCl, are spiked into the AEX FT to bring the solution pH down to pH 3.5-6.0 and increase the monovalent salt concentration to 50-500 mM. One or both adjustment solutions may not be required if the AEX FT is already at the target pH or monovalent salt concentration of the loading solution to the CEX step. In the current example with CVA21, a 1 M sodium citrate, pH 4.0 solution and a 5 M NaCl solution are spiked into the AEX FT, initially at pH 8.0 and 150 mM NaCl, to target a final sodium citrate concentration of 50 mM at pH ˜4.1 and a final NaCl concentration of 400 mM. The concentrated stock solutions were slowly added to the AEX FT product over 5-10 minutes with mixing. This solution adjusted sample was designated CEX feed and represented the target CEX loading solution.
The CEX chromatography step, operated in bind-elute mode, is implemented to improve process robustness as a secondary step for empty procapsid clearance, to clear residual impurities, and to provide additional volume reduction. The CEX step may use common chromatography media such as Poros™ 50 HS (ThermoFisher Scientific), Capto S (Cytiva Life Sciences), or Nuvia S (Bio-Rad) or other CEX stationary phases. For large scale CEX chromatography operation, CEX resin is packed into large scale chromatography columns and run with a chromatography skid such as Äkta Pilot at a flow rate of 50-300 cm hr−1. The CEX column is equilibrated in 3-5 CVs of CEX Equilibration buffer composed of a solution at pH 3.5-6.0 and a monovalent salt concentration of 50-500 mM. The CEX feed in a CEX loading solution at pH 3.5-6.0 and a monovalent salt concentration of 50-500 mM is loaded to the CEX column. The enterovirus particles bind to the CEX resin while some residual impurities may flow through. The CEX column is washed with 3-5 CVs of a CEX Wash buffer solution, composed of a solution at pH 3.5-6.0 and a monovalent salt concentration of 100-600 mM, to remove residual impurities. The full mature virions are selectively eluted from the CEX column using 3-5 CVs of a CEX elution buffer solution, composed of a solution at pH 3.5-4.8 and a monovalent salt concentration of 200-1000 mM NaCl, while the empty procapsids remain bound to the CEX resin. The empty procapsids and other residual impurities are eluted with 3-5 CVs of CEX Strip buffer, composed of a solution at pH 4.0-8.0 and a monovalent salt concentration of 500-1500 mM and the CEX column is regenerated with a solution containing 0.1-0.5 N sodium hydroxide. The CEX buffer solutions may contain a surfactant such as PS-80, PS-20 or other similar surfactant at a concentration of 0.001-1% w/v. In the current example with CVA21, a 5 cm diameter column packed with Poros™ 50 HS resin was run on an Äkta Pilot with UNICORN system control software at a flowrate of 200 cm hr−1. The CEX column was equilibrated with 4 CVs of an CEX equilibration buffer consisting of 50 mM sodium citrate, 400 mM NaCl, 0.005% w/v PS-80, pH 4.0. The CEX feed product containing CVA21 particles was loaded to the column until a loading of 25-30 CVs. The column was washed with 4 CVs of a CEX Wash buffer consisting of 25 mM sodium citrate, 500 mM NaCl, 0.005% w/v PS-80, pH 4.0. The full mature CVA21 virions were selectively eluted from the CEX column with 4 CVs of a CEX elution buffer consisting of 25 mM sodium citrate, 800 mM NaCl, 0.005% w/v PS-80, pH 4.0. The empty CVA21 procapsids were eluted with 4 CVs of a CEX strip buffer consisting of 25 mM sodium citrate, 1000 mM NaCl, 0.005% w/v PS-80, pH 7.0 and the column was regenerated with 4 CVs of a 0.5 N NaOH solution.
The CEX elution product, consisting of purified full mature enterovirus virions, is buffer exchanged into a stabilizing buffer by ultrafiltration/diafiltration (UF/DF) via tangential-flow filtration (TFF) or size-exclusion chromatography (SEC) in desalting mode. For TFF, the enterovirus particles are retained by a hollow fiber or a cassette with a molecular weight cut-off of about 50-500 kDa, while other small solution components permeate through the membrane. The TFF may be operated with a crossflow shear rate of about 1,000-8,000 s−1, a transmembrane pressure (TMP) of about 0.1-10 psig, and a permeate flux of about 5-60 L m−2 hr−1. The CEX elution product is diafiltered with 5-10 diavolumes into a 1× stabilizing buffer solution consisting of a buffering species at about pH 6-8. A UF step may be performed before or after DF. An optional neutralization step may be performed prior to TFF where the CEX elution product is diluted 2-5-fold into a 2-5× concentrated stabilizing buffer solution. An optional filtration step consisting of a filter with a pore size of about 0.1-1 μm may be used prior to TFF. For buffer exchange with SEC, the CEX elution product is loaded to SEC column packed with resin such as Sephadex (Cytiva Life Sciences) and operated in desalting mode using a chromatography skid such as Äkta Pilot. In the current example with CVA21, the CEX elution product was neutralized by diluting 3-fold into a 3× concentrated stabilizing buffer solution. The neutralized CEX elution product was filtered using a Durapore® 0.22 μm filter (Merck Millipore) to generate a TFF feed solution. The TFF feed solution was initially concentrated 2-3-fold and then buffer exchanged into the 1× stabilizing buffer solution using a Spectrum 300 kDa hollow fiber filter (Repligen) at a crossflow of 2000 s−1, TMP of 1-2 psig, and permeate flux of 20-40 LMH.
A final filtration step is performed with the buffer exchanged TFF or SEC elution product. A filter pore size of 0.1-0.5 μm is used. The final purified composition of enterovirus in the stabilizing buffer solution is frozen and stored at <−60° C. In the current example with CVA21, a Durapore 0.22 μm filter (Merck Millipore) was used.
The CVA21 purification process detailed above was demonstrated for 4 batches produced from upstream cell culture conditions A and B. As an example, the purification process intermediate samples for Batch 4 with cell culture condition B were characterized by SDS-PAGE with silver stain (
For Batch 4, the starting material was also analyzed through sucrose gradient analysis and it was shown to be rich in both empty procapsids and full mature virus particles (
The observations that multiple cation exchange resins resulted in a good separation between full mature virus particles and empty procapsids, along with the fact that residual HCPs could flow through while virus particles bound to the resins, led to the exploration of cation exchange chromatography as a purification step for additional enteroviruses to CVA21. Five enterovirus serotypes were tested to support this: (1) Coxsackievirus A13 (CVA13), (2) Coxsackievirus A15 (CVA15), (3) Coxsackievirus A18 (CVA18), (4) Human Rhinovirus 1B (RV1B), and (5) Human Rhinovirus 35 (RV35). For these tests, enterovirus stocks were purified using small scale columns packed with 200 μL of affinity chromatography resin and the elution products were adjusted to a pH of 4.0 and a salt level of 100 mM NaCl. These were then further purified using 96 well plate batch chromatography as described in Table 7. Here, the plates were pre-dispensed with 20 μL of resin Capto™ S ImpAct (Cytiva, MA, USA) since this resin was also found to provide good purification for CVA21 (
U.S. provisional application No. 63/126,743, filed Dec. 17, 2020, and U.S. provisional application No. 63/211,162, filed Jun. 16, 2020 are incorporated by reference in their entirety. All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. To the extent that the references provide a definition for a claimed term that conflicts with the definitions provided in the instant specification, the definitions provided in the instant specification shall be used to interpret the claimed invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/063647 | 12/16/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63211162 | Jun 2021 | US | |
| 63126743 | Dec 2020 | US |
