HK1194296A - Methods and compositions for production of vaccina virus - Google Patents

Description

Methods and compositions for producing vaccinia virus

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 61/515,724 filed on 5/8/2011, the entire contents of which are incorporated herein by reference.

Background

I. Field of the invention

Embodiments of the present invention generally relate to virology, medicine and viral therapeutics. Certain embodiments relate to methods of producing viruses.

II. background

Oncolytic Viruses (OV) are replicating therapeutic agents that have been designed and selected to specifically grow in and kill tumor cells. Several groups are in the early stages of commercialization based on preclinical animal models and early clinical data in humans. However, one challenge still facing the art is to produce large scale pharmaceutical grade viruses for delivery to patients. Viruses are biological entities rather than synthetic drugs, and thus the production method involves the production of the virus within the cell and the subsequent removal of contaminating cellular debris while maintaining the infectious potency. To date, most, if not all, commercial production of vaccinia virus products for human use has become a vaccine in non-human cell culture. Generally, for vaccine applications, patients receive low doses of virus at a local site (often intramuscularly), where contaminating cellular proteins may actually act as adjuvants to increase the immunogenicity of the formulation. In contrast, for oncolytic viral therapeutics, the dose of virus required is up to millions of times that of vaccine applications, and can be administered intravenously, intracranially, intraperitoneally, or by direct tumor injection. In the case of OV, it is critical to be able to produce large quantities of virus free of contaminating non-human cellular debris. In addition, vaccinia viruses are enveloped viruses that incorporate host cell proteins into the viral membrane. Incorporation of human proteins into the viral envelope increases its ability to move without being detected by the human host immune system.

There remains a need for additional methods and compositions for large-scale oncolytic virus production.

Disclosure of Invention

Vaccinia virus has been produced in HeLa cells cultured in suspension culture. However, suspension culture conditions are currently not suitable for large scale production of viruses. Thus, attempts to produce vaccinia virus on a large or commercial scale using HeLa cells have not been successful. Furthermore, the adherent HeLa cell line is not expected to produce sufficient numbers of cells to ensure the production of the required amount of vaccinia virus on a large scale using adherent HeLa cells. Methods for large scale production of vaccinia virus using adherent HeLa cells are described.

Certain embodiments relate to methods of producing vaccinia virus comprising one or more of the following steps.

In certain aspects, the method comprises: the surface-adherent HeLa cells were infected with vaccinia virus by contacting the adherent HeLa cells with vaccinia virus. Preferably, the vaccinia virus is a recombinant vaccinia virus, such as those described in the paragraph of U.S. patent application publication No. 2009/0004723 (incorporated herein by reference in its entirety). In certain embodiments, HeLa cells attached to a culture dish are specifically excluded from the scope of the claims. In several embodiments, the present invention provides a method of producing vaccinia virus comprising: (a) infecting HeLa cells adhered to a surface with vaccinia virus, (b) culturing the infected cells under conditions that allow production of progeny virus, and (c) harvesting vaccinia virus from the culture.

In certain embodiments, methods of producing vaccinia virus are provided, comprising: (a) by making at least 1x10⁷1x10⁸5x10 pieces⁸1x10⁹5x10 pieces⁹1x10¹⁰5x10 pieces¹⁰1x10¹¹5x10 pieces¹¹1x10¹²5x10 pieces¹²1x10¹³5x10 pieces¹³Contacting one or more (including all values and ranges therebetween) adherent HeLa cells with a vaccinia virus composition, infecting HeLa cells adherent to a surface with a recombinant vaccinia virus; and (b) culturing the infected cells under conditions that allow the production of 50, 60, 70, 80, 90, 100 or more (including all values and ranges therebetween) recombinant vaccinia viruses/cell. Preferably, at least 50 plaque forming units (pfu) vaccinia virus/HeLa cells, more preferably at least 75pfu vaccinia virus/HeLa cells, are produced according to the method.

In other embodiments, HeLa cells adhered to a surface are infected with vaccinia virus at a multiplicity of infection (m.o.i.) of 0.001 to 1.0, preferably 0.005 to 0.5, more preferably 0.01 to 0.1. In a particularly preferred embodiment, the m.o.i. is from 0.01 to 0.05, including all values and ranges therebetween. In other preferred embodiments, the m.o.i. is from 0.015 to 0.03. The term "multiplicity of infection" or "m.o.i." means the ratio of plaque forming units (pfu)/HeLa cells of the virus added during infection.

In a related embodiment, according to the method, at about 10⁴To about 10⁶HeLa cells/cm²Preferably about 10⁵HeLa cells/cm²Infecting adherent HeLa cells with vaccinia virus, wherein the concentration of said vaccinia virus is 10³-10⁵pfu/ml, preferably about 10⁴pfu/ml。

Culture of adherent HeLa cellsAdherent HeLa cells infected with vaccinia virus according to the method can be cultured by any suitable method, including (but not limited to) culturing in glass and plastic containers, such as culture flasks, bioreactors, including but not limited to: cell factories such as NunclonCell Factories^TMA unit cell such as CELLCUBE from Corning Life sciencesSystem, Soft Plastic bags (e.g., infusion bags) containing cell support matrix such as GibcoOr LampireA cell culture bag; bottle rolling; rotating bottles such as Magna-FlexSpinner flashes; a fermentation tank; or hollow fibers of different materials, such as the cell culture system from Cellex Biosciences (AcuSyst)Hollow fiber reactor), BioVest (Cell-Pharm)System100 or System2500) or Fibercell^TM. Culturing in a bioreactor is preferred because bioreactors allow for precise monitoring and control of variables such as temperature and pH. In certain aspects, CellBind may be used^TMA plastic article (www.sigmaaldrich.com). The cell culture may be in the form of a cluster of cells, such as spheroids on a microcarrier bead, or cells, tissues or tissue organoids on a scaffold (e.g., a biodegradable scaffold). In certain aspects, adherent HeLa cells are cultured in microcarriers, cells, cell factories, T-flasks or roller bottle vessels. In a preferred embodiment, adherent HeLa cells are cultured in one or more roller bottles, for example using a RollerCell40 instrument (cell s.a., Luxembourg). The container used may contain, but is not limited to, about, at least, or at most 1,700cm²、5,000cm²、10,000cm²、25,000cm²、50,000cm²、100,000cm²、150,000cm²、200,000cm²、500,000cm²(including all ranges and values therebetween). In certain aspects, the container has at least 168,000cm²The cumulative culture area of (3).

Adherent HeLa cells to be infected with vaccinia virus can be cultured in any suitable culture medium that supports growth, maintenance and attachment to the surface of a cell support, including, but not limited to, BME (basal eagle medium), MEM (minimal eagle medium), 199 medium, DMEM (Dulbecco's modified eagle medium), GMEM (Glasgow modified eagle medium), DMEM-HamF12 and Ham-F10, Isocove's modified Dulbecco's medium, MacCoy's 5A medium, or RPMI 1640. Preferably, the culture medium is a defined culture medium. In one aspect, the culture medium is substantially free of serum. Examples of serum-free media optimized for use with HeLa cells include, but are not limited to, VP-SFM (Gibco BRL/Life Technologies),Serum Free Medium (SFM) (Sigma Aldrich) and Quantum101 (GEHealthcare). In other embodiments, the culture medium may also comprise animal serum such as Fetal Bovine Serum (FBS). For example, the culture medium can comprise 5% to 10% serum (e.g., FBS), or can contain less than 5% serum (e.g., FBS), or any range or value therebetween. In one embodiment, the culture medium is DMEM supplemented with 5% to 10% FBS (such as 10% FBS).

The term "microcarrier" refers to small discrete particles that are used to culture cells and to which cells can attach. The microcarrier may be in any suitable shape, such as a rod, ball, etc. In many embodiments, the microcarrier comprises a microcarrier matrix that is coated to provide a surface suitable for cell culture. The polypeptide may be bonded, grafted, or otherwise attached to the surface coating. Microcarriers have been used in cell culture to provide high yields of attachment-dependent cells. Microcarriers are typically stirred or agitated in the cell culture medium, which provides a very large surface area to volume ratio for attachment and growth, compared to more traditional culture equipment. For detailed examples of microcarriers, see U.S. patent publication 2011/0027889, which is incorporated herein by reference in its entirety.

In certain embodiments, the vaccinia virus is IHD-J, Wyeth, Western Reserve, or Copenhagen strain of vaccinia virus. In certain aspects, the vaccinia virus is a recombinant vaccinia virus. The recombinant vaccinia virus may comprise a heterologous coding region. Heterologous coding regions, as used herein, are coding regions that do not naturally occur in the vaccinia virus genome. In certain aspects, the heterologous coding region may encode an immunostimulatory polypeptide. The immunostimulatory polypeptide may be a cytokine such as, but not limited to, granulocyte macrophage colony stimulating factor (GM-CSF).

In certain aspects, the recombinant vaccinia virus selectively replicates in tumor cells. The recombinant vaccinia virus may comprise a mutation in an endogenous gene. The mutation may be a deletion of a nucleic acid sequence, a substitution of a nucleic acid residue, or an insertion of one or more nucleic acid residues that affect the function or expression of the vaccinia virus. The mutation may result in selective growth of a tumor cell, or reduced growth of a non-tumor cell, or enhanced growth of a tumor cell, or a combination thereof. The recombinant vaccinia virus comprising a mutation in an endogenous gene may be thymidine kinase deficient (TK)^-) Vaccinia virus (i.e., a vaccinia virus having a mutation in the gene encoding thymidine kinase that renders the encoded polypeptide non-functional). In certain aspects, the recombinant vaccinia virus comprising a mutation in an endogenous gene is IHD-J, Wyeth, Western Reserve, or Copenhagen strain of vaccinia virus; and/or comprise a heterologous coding region. In another embodiment, vaccinia virus comprising a mutation in an endogenous gene also comprises a heterologous coding region, such as, but not limited to, granulocyte macrophage colonyA stimulator polypeptide coding region.

Culturing infected HeLa cellsCulturing adherent HeLa cells that have been contacted with a vaccinia virus under conditions that allow production of progeny vaccinia virus. Culture conditions may include, but are not limited to: defined infection medium, defined pH, defined culture vessel, defined temperature or temperature range, defined cell number, defined cell confluence, defined physical manipulations (e.g., enzymatic or chemical treatments, rotation frequency, shaking speed, etc.), defined gas phase conditions, defined culture time, defined cell seeding density, and defined cell passage number. In one aspect, the virus may simply be added to the medium for HeLa cell culture used to prepare adherent HeLa cells, in which case the medium for culture and the infection medium are the same. In other aspects, the culture medium used to prepare adherent HeLa cells is removed in the infection step, and HeLa cells that have been contacted with vaccinia virus are cultured with an infection medium (which may comprise serum or may be substantially serum free) including, but not limited to, BME, MEM, 199 medium, DMEM, GMEM, DMEM-HamF12 and Ham-F10, Isocove's modified Dulbecco's medium, MacCoy's 5A medium, or RPMI1640, any of which may optionally be supplemented with serum, until progeny virus is produced in a suitable infection medium. Serum-free media such as VP-SFM,Serum Free Medium (SFM) and Quantum101 are also suitable as infection medium.

In certain aspects, the infected cells are cultured in an infection medium comprising 5-10% serum (e.g., Fetal Bovine Serum (FBS)) at a pH of about 7, 7.2, 7.3, 7.4, 7.5.7.6, 7.7, 7.8, 7.9, or 8, preferably at a pH above 7.1, more preferably at a pH above 7.2, most preferably at a pH of about 7.3. In other aspects, the cells are cultured in an infection medium comprising less than 5% serum (e.g., FBS), such as 0% serum (e.g., FBS), at a pH of about 7, 7.2, 7.3, 7.4, 7.5.7.6, 7.7, 7.8, 7.9, or 8, preferably at a pH above 7.1, more preferably at a pH above 7.2, most preferably at a pH of about 7.3. In a related aspect, cells are cultured in infection medium comprising 0% to 10% serum (e.g., FBS) at a pH of about 7.3. In other embodiments, the cells are cultured in an infection medium comprising 0% to 10% serum (e.g., FBS) at a pH above 7.2 and below 7.6. In a preferred embodiment, the infection medium comprises DMEM and optionally 100-300mM glutamine. The cells may be cultured at a temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ℃. In a preferred embodiment, the cells are cultured at a temperature of 36 ℃ to 37.5 ℃, preferably at 37 ℃. In certain aspects, the infection medium specifically excludes one or more of dextran sulfate, Pluronic F-68, tween-80, and/or soy protein hydrolysate. In a preferred embodiment, the infection medium specifically excludes dextran sulfate and at least one of the following components: pluronic F-68, Tween-80 and soy protein hydrolysate. In other preferred embodiments, the infection medium is substantially free of all of these components.

The method may further comprise: before, during or after infection, HeLa cell cultures were expanded (expanded). HeLa cells can be passaged in at least 1, 2, 3, 4, 5, or more culture vessels (e.g., roller bottle culture vessels). In certain aspects, by using a composition comprising a protease and/or a collagenolytic enzyme (e.g., HyQtase)) The release solution of (3) to release the cells from the surface. Each vessel may have an increased culture area compared to previous culture vessels. In certain aspects, the culture vessel has at least 84,000cm²To 1,000,000cm²Total culture area of (a). In some aspects, the final passage is to at least 168,000cm²The culture container of (1).

In certain embodiments, the inoculation and sensing of the culture vesselThe time intervals between stains are 12, 24, 48, 72, 96, 120, 144, or more hours, including all values and ranges therebetween. In certain aspects, adherent HeLa cells are contacted with 1x10¹pfu/mL、1x10²pfu/mL、1x10³pfu/mL、1x10⁴pfu/mL、1x10⁵pfu/mL、1x10⁶pfu/mL、1x10⁷pfu/mL、1x10⁸pfu/mL、1x10⁹pfu/mL (including all values and ranges therebetween) of vaccinia virus. In a preferred embodiment, the density is made to be about 10⁴To about 10⁶HeLa cells/cm²(preferably about 10)⁵HeLa cells/cm²) Adhesion of HeLa cells and 10³-10⁵pfu/ml vaccinia virus, more preferably about 10⁴pfu/ml vaccinia virus.

In certain aspects, the method comprises: vaccinia virus produced by adherent HeLa cells was isolated. The harvested vaccinia virus may be concentrated and purified according to methods known to those skilled in the art. Other embodiments of the invention relate to vaccinia virus compositions produced by the methods described herein, such as those described in the paragraph of U.S. patent application publication No. 2009/0004723 (incorporated herein by reference in its entirety).

Other embodiments of the invention are discussed in the present application. Any embodiment discussed in relation to one aspect of the invention may also be applied to other aspects of the invention and vice versa. The embodiments in the examples section are to be understood as embodiments of the invention applicable to all aspects of the invention.

In the claims and/or the specification, the terms "a" or "an" when used in conjunction with the term "comprising" may mean "one" but it is also consistent with the meaning of "one or more," at least one, "and" one or more.

It is contemplated that any embodiment discussed herein may be practiced with any method or composition of the present invention and vice versa. In addition, the compositions and kits of the invention can be used to practice the methods of the invention.

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of the device or method used to determine the value.

The term "or" as used in the claims is intended to mean "and/or" unless explicitly indicated to indicate only that alternatives or alternatives are mutually exclusive, although the present disclosure supports definitions indicating only alternatives and "and/or". It is also contemplated that any object listed using the term "or" may also be specifically excluded.

As used in this specification and claims, the words "comprise" (and any form of comprise, such as "comprises" and "comprising"), "have" (and any form of have, such as "has" and "with"), "include" (and any form of include, such as "includes" and "containing") or "contain" (and any form of contain, such as "comprises" and "including") are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 illustrates the production of recombinant vaccinia virus strain JX-594 (pfu/cell) in different human cell lines. Cells were infected at a multiplicity of infection of 0.1 and at 72 hours post infection, lysates were collected and titers were determined on U-2OS cells.

FIG. 2 illustrates the effect of pH of the infection medium on the productivity of the recombinant vaccinia virus strain JX-594 in adherent HeLa cells. Cells were infected at a multiplicity of infection of 0.1 in medium of the indicated pH and at 72 hours post infection, lysates were collected and viral titers were determined on U-2OS cells.

Detailed Description

Vaccinia virus is an enveloped virus that produces 4 infectious forms that differ in their outer membrane: intracellular Mature Virions (IMVs), Intracellular Enveloped Virions (IEVs), cell-associated enveloped virions (CEVs) and Extracellular Enveloped Virions (EEVs). The EEV form is complement resistant due to the incorporation of complement-activated host regulators into host cells in the outer EEV membrane (Vanderplasschen et al, (1997) PNAS95: 7544-7549). The ability to escape complement inactivation and the general ability to escape recognition by the host immune system are important features of the efficacy of systemic delivery in patients.

The plasma membrane of IMV incorporates numerous host cell proteins during its assembly. Since IMVs represent the majority of infectious progeny, producer cells from the same species as the target patient for oncolytic virus therapy may be advantageous in terms of systemic spread within the patient, as the virus will be less immunogenic. These host cell proteins on the plasma membrane of IMV may also provide certain advantages during infection.

Although IMV is the majority of infectious progeny, it has not been finalized that the EEV form of the virus is absent from cell culture preparations. Complement activation regulatory proteins from the same species as the target patient may play an important role in complement activation inhibition and thus escape immune clearance by the host immune system. Thus, the inventors developed a system for producing large amounts of vaccinia virus with human cellular components.

Historically, vaccinia virus used as a vaccine was produced from human scarring, however, it was subsequently cultured on the skin of calves, sheep and buffalos, in the chorioallantoic membrane of chicken embryos, or on primary bovine embryonic fibroblasts (Ellner, 1998). For a global cooperative work in the root elimination of smallpox, reserves are produced by lacerating the skin of calves to cause infection (Fenner, 1977). Viral stocks were prepared from calf lymph and this preparation was the only licensed and available vaccine called DRYVAX(Wyeth). The virus preparation is concentrated and lyophilized, which facilitates its stability (Fenner, 1977). At least 10⁸pfu/ml concentration of lyophilized material. It was reconstituted in 50% glycerol and 0.25% phenol (in sterile water for injection) and delivered intradermally by using a bifurcated needle (Fenner,1977) to up to 400 vaccinees at a dose of approximately 2.5x10⁵pfu-this ratio was used as a standard 10 for oncolytic viral vaccinia virus⁹pfu (or higher) dose is at least 10 lower⁴And (4) doubling.

The lead clinical candidate for oncolytic vaccinia virus was GM-CSF-expressing thymidine kinase inactivated vaccinia virus (Jennerex, JX-594). However, other vaccinia virus variants and strains can also be produced by the methods described herein. For example, JX-963 is a recombinant vaccinia virus that has, in addition to the removal of the TK gene, an additional deletion of the Vaccinia Growth Factor (VGF) gene constructed on the Western Reserve vaccinia backbone. This double deletion vaccinia virus (vvDD) has been shown to be tumor specific and to have anti-tumor activity in animal models (McCart et al, 2001; Thorne et al, 2007; Naik et al, 2006). The second vvDD variant is JX-929, which carries and expresses the human somatostatin receptor (SSTR2) in infected cells, which facilitates use¹¹¹Molecular imaging following systemic delivery of In-pentraxin (McCart et al, 2004).

I. Cell culture

Cell-based viral production has been examined for DRYVAXIn the alternative to vaccination and for the production of vaccinia virus for use in the fields of viral immunotherapy and gene therapy. Second generation vaccinia virus vaccine strains have been developed that were cultured in the monkey kidney cell line Vero (called ACAM 2000). In these studies, viruses were efficiently purified from cell cultures. In addition, cancer vaccines have also been produced in cells using vaccinia virus in combination with tumor-specific antigens (i.e., PSA, CEA) and immunostimulatory molecules (i.e., B7.1, ICAM-1, and LFA-1) and administered to patients in phase I and phase II clinical trials (Li, et al, 2005; Guo and Bartlett, 2004). However, in both of these cases, patients receive local, rather than systemic, administration of the virus preparation and often receive a fraction of the total virus dose required for in vivo oncolytic efficiency.

Poxviruses such as vaccinia represent some unique challenges in large-scale production and purification compared to other viruses that have been recommended as oncolytic virus candidates. As some of the largest viruses in nature, poxviruses can be seen by light microscopy and have a measured length of 200-. Due to its large size, vaccinia virus cannot be sterilized by filtration, thus any production method is usually shut down.

Since its entire life cycle occurs within the host cell cytoplasm, most infectious particles are not released into the cell culture medium, but are not retained within the infected cells, and thus purification requires lysis of the infected cells and purification of viral particles from cell debris.

In the morphogenesis of poxviruses, they obtain membranes from the neogenetic membrane synthesis in the early stages of morphogenesis and the extra-membrane from the host Golgi in the later stages (Moss, in Fields' virology B.N.fields, D.M.Knipe, P.M.Howley, D.E.Griffin, eds. (Lippincott Williams & Wilkins, Philadelphia,2001) pages 2849-2883).

The present method relates to an adherent cell method developed to produce GMP-grade viruses for cancer testing. The adherent cell method may be performed under serum-free conditions or in the presence of serum. The productivity goals of the production platforms described herein are at least 30, 100, 125, 150, 200 or more pfu/cell, with an estimated viral concentration per dose of about 10⁹pfu/ml。

One embodiment of the present invention relates to the practice of producing high titer JX-594 in cell culture. Various human cell lines were tested and it was found that HeLa (a human cervical cancer cell line) would support robust viral replication in adherent culture. The present method is not limited to HeLa cells and other adherent cells may be used in the production method, but currently HeLa cells would provide the best model cell line for the production of vaccinia virus.

A typical protocol for assessing viral productivity is to perform a standard plaque assay, which includes: serial dilutions were followed by a period of 2-3 days waiting for plaque formation, followed by titer calculation. As an alternative, quantitative PCR protocols (Q-PCR) have been standardized, which allow the number of viral genomes produced after infection to be determined. Amplification primers have been optimized which allow us to quantify the copy number of the viral E9L gene in infected cells and these results have been correlated with our standard plaque assay data. The advantage of this protocol is that it can be adapted to 96-well format and data is available in hours (rather than days). Thus, standard preliminary screens for the effects of media and supplements, time to harvest, and concentration of input virus can be performed rapidly in 96-well plates and analyzed. After this primary screening, all positive "hits" were confirmed using a classical plaque assay.

Tested in commercial serum-free mediumThe effect of the components present on the productivity of the virus. The HeLaS3 CELL line has been adapted for suspension growth, and in our hands, these CELLs are in EX-CELL^TMHeLa serum-free suspension medium (SAFC Biosciences) grew as well as adherent cells in serum-containing medium. However, as described above, in spite of the excellent cell growth characteristics, the inventors observed very poor virus growth in this medium (less than 1 virus particle per infected cell). Although EX-CELL^TMThe exact formulation of HeLa serum-free medium is proprietary, a known component of which is sulfated polysaccharide (dextran sulfate), apparently added to help prevent cell aggregation. However, dextran sulfate is also known to inhibit replication of a variety of enveloped viruses including retroviruses, herpes viruses, rhabdoviruses and arenaviruses (De Clercq, 1993). Approximately 20mg/L dextran sulfate was present in EX-CELL medium. Various dextran sulfate concentrations were used and evaluated in the rapid assay described above. It was found that dextran sulfate does have a dose-dependent negative effect on virus productivity, which was subsequently confirmed using classical titer assays. Although removal of dextran sulfate did increase viral productivity, it was still lower than that conventionally achieved with adherent HeLa cells in serum-containing media. Other media components that affect viral productivity include: pluronic F-68, a bifunctional block copolymer surfactant that is generally non-toxic to cells; tween-80, a commonly used detergent; and soy hydrolysate. In certain aspects of the invention, the medium lacks one or more of dextran sulfate, Pluronic F-68, Tween-80 and soy protein hydrolysate. Examples of some of these media formulations and serum supplements are depicted in table 1.

TABLE 1 culture Medium formulation and supplements

Name of culture Medium	Suppliers of goods
		ExCell HeLa SFM	SAFC

ExCell 293 SFM	SAFC
		OPTIPRO SFM	Invitrogen
UltraCulture	Cambrex
		CHO-S-SFM II	Invitrogen
293 SFM II	Invitrogen
		VP-SFM	Invitrogen
IS-293-V	Irvine Scientific
		BD Nu Serum supplement	BD Biosciences

Since vaccinia virus prefers cell-to-cell contact for efficient diffusion, poor cell-to-cell delivery of the virus may limit suspension cell cultures. In certain aspects, aggregation of cells may be promoted, or cells may be cultured on microcarriers, or adherent culture may be sufficient for virus production.

In one non-limiting example, the following conditions were used for growth of JX-594, which consistently resulted in titers in excess of 100 pfu/cell. At 4x10⁴Individual cell/cm²Cells were seeded in roller bottles and grown in culture for 2 days. At this point, the growth medium was removed and the medium containing 10 was added⁴pfu/ml JX-594 fresh medium. Cells were maintained for an additional 60-70 hours, at which point cells were collected and virus harvested. This protocol can produce about 170 doses (10)⁹pfu/dose) of JX-594. In this process, the inventors found that factors that had a surprising effect on virus yield were not anticipated. For example, the pH of the medium can have an effect on JX-594 replication. Virus production was optimized in a well buffered medium at ph 7.3. Productivity from adherent cultures was significantly better than all attempts at suspension culture.

To ensure the production of high quality virus preparations that are substantially free of contaminating products, a number of assays can be used to analyze the virus purity and/or quality. These assays will ensure that the virus preparation is comparable to other validated virus preparation batches and that the method is successful.

Feature(s)	Method of producing a composite material
		Infectious titer	Plaque assay
Potency-cytotoxicity	ED on U2OS cells50

Genome titre	qPCR for E9L
		Total DNA	pico green
Amount of host cell DNA	qDNA-PCR
		Total protein (ug/mL)	BCA
Host cell protein (Overall)	ELISA(Cygnus)

In certain embodiments, vaccinia virus can be isolated and purified. Methods of purifying vaccinia virus may comprise: a. loading a solid phase substrate with vaccinia virus contained in a liquid phase; b. washing the substrate; eluting vaccinia virus.

In certain aspects, the matrix may comprise a ligand that binds vaccinia. The ligand may be attached to a solid phase matrix by binding or coupling to the matrix. The interaction between the ligand and the virus forms a reversible complex, and thus, the matrix reversibly retains the virus. In certain aspects, glycosaminoglycans (GAGs), particularly heparan sulfate or heparin, or GAG-like substances are used as ligands. As used herein, a "glycosaminoglycan" (GAG) is an unbranched long polysaccharide consisting of repeating disaccharide units.

The ligand may be a biomolecule, for example, a peptide and/or a lectin and/or an antibody and/or preferably a carbohydrate. The ligand may also comprise or consist of a sulphate ester. In another embodiment, the ligand comprises one or more negatively charged sulfate groups.

In certain aspects, the ligand is a hydrophobic molecule, for example, an aromatic phenyl group, PPG group, butyl, or hexyl.

In one aspect, the method comprises: hydrophobic Interaction Chromatography (HIC) was used to purify vaccinia virus. In another embodiment, the method comprises: vaccinia virus was purified by HIC and affinity chromatography. The use of HIC can provide high viral yield and a substantial reduction in DNA and protein contaminants. The DNA contamination level can be reduced to 0.01% of the original starting material and the protein contamination level can be reduced to less than 0.1%.

In certain aspects, sulfated cellulose can be used with HIC phenyl column chromatography to produce viral particles in high yield and purity levels.

In certain aspects, the DNA is degraded or removed before or after purification of the vaccinia virus. By treatment with nucleases, e.g. BenzonaseThe treatment can remove DNA. Nuclease treatment reduces vaccines or viruses containing intact oncogenes or other functional DNA sequencesThe possibility of a carrier.

In related aspects, the protein is degraded or removed before or after purification of the vaccinia virus. By protease treatment, e.g. TrypLE^TMAnd (4) removing proteins by virtue of selection treatment. Preferably, a combination of nuclease and protease treatment is employed to reduce or eliminate contaminating HeLa DNA and proteins.

The matrix may be a gel, bead, well, membrane, column, or the like. In a preferred embodiment of the invention, the solid phase is a membrane, in particular a cellulose membrane. A wide range of modified polymers capable of binding viruses may be used. Examples of such polymers are: cellulose derivatives (cellulose esters, cellulose hydrates, cellulose acetates, cellulose nitrates); agarose and derivatives thereof; polysaccharides such as chitin or chitosan; polyolefins (polypropylene); polysulfones; polyether sulfone; polystyrene; aromatic and aliphatic polyamides; polysulfonamides; halogenated polymers (polyvinyl chloride, polyvinyl fluoride, polyvinylidene fluoride); a polyester; homopolymers and copolymers of acrylonitrile.

Vaccinia virus can be purified under sterile conditions to obtain an active, stable, and highly pure virus preparation. The vaccinia virus may be natural or recombinant.

As used herein, "contaminant" encompasses any undesirable substance that may be derived from a host cell used in virus culture (e.g., host cell DNA or protein) or from any additive used in production processes including upstream (e.g., gentamicin) and downstream (e.g., Benzonase).

"Industrial-scale" or "large-scale" production of vaccinia virus or recombinant vaccinia virus as used herein includes 1.0X10 capable of providing a minimum of 50,000 doses per batch (production run)⁸Virus particles (Total minimum 5.0X 10)¹²Individual viral particles).

The "purity" of the vaccinia virus preparation or vaccine used herein was investigated with respect to the content of contaminating DNA, protein, Benzonase and gentamicin. Purity is expressed as a specific impurity, which is the amount of each impurity per dose (e.g., ng DNA/dose).

As used herein, "stability" refers to a measure of how the quality of a viral preparation (bulk pharmaceutical material (BDS) or final pharmaceutical product (FDP)) changes over time under the influence of various environmental factors, such as temperature, humidity and light, and establishes a re-certification phase (for BDS) or shelf life (for FDP) under recommended storage conditions.

The ligands make it possible to elute bound vaccinia viruses under mild conditions where the vaccinia viruses retain their biological activity completely. This means that the virus is infectious. Infectivity of vaccinia virus can be preserved during purification, such that the original TCID is preserved during purification₅₀(half the tissue culture infectious dose) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Preferably, the original TCID is retained during the purification process₅₀At least 30% of the total weight of the composition.

In one embodiment, the solid phase matrix is a gel or membrane having a pore size of 0.25 μm, preferably more than 0.25 μm, more preferably 1.0-3.0 μm, so as to exhibit a linear flow rate of 10cm/min to 20cm/min under actual purification conditions. The pore size of the matrix may be 0.25-0.5 μm, 0.5-0.75 μm, 0.75-1.0 μm, 1.0-2.0 μm, 2.0-3.0 μm, or greater than 3.0 μm.

Loading the solid phase with ligand can be achieved by a partitioning scheme, a column scheme or a membrane scheme.

For purification of large viruses (e.g., vaccinia virus), membrane protocols may have some benefits. The large pore size and the availability of ligands on the membrane surface allow for high binding capacity of even large viral particles.

In one embodiment, the virus binds to the ligand in ammonium sulfate (e.g., at 0.2M, 0.4M, 0.6M, 0.8M, 1.0M, 1.2M, l.4m, 1.6M, 1.8M, or 2.0M).

When binding of vaccinia virus or recombinant virus to the ligand or matrix has progressed sufficiently, host cell contaminants (particularly host cell DNA and proteins) that remain in the liquid phase can be removed by washing the matrix to which vaccinia virus binds with an appropriate wash medium. In one aspect, the substrate is washed with 0.2M, 0.4M, 0.6M, 0.8M, 1.0M, 1.2M, l.4m, 1.6M, 1.8M or 2.0M ammonium sulfate.

Bound vaccinia virus or recombinant virus can be eluted from the matrix. Elution of captured vaccinia virus can be achieved with agents that specifically disrupt the specific interaction between the ligand or matrix and vaccinia virus, or with agents that non-specifically disrupt the electrostatic interaction between the ligand or matrix and surface proteins. In one aspect, the reagent is ammonium sulfate. In another aspect, the vaccinia virus can be eluted with a GAG or GAG-like ligand. In another aspect, the agent is sodium chloride, more preferably with increasing NaCl concentration gradients in the range of 0.15M to 2.0M.

The viral suspension may be pretreated prior to loading onto the substrate, in particular to remove contaminants from vaccinia virus in liquid phase culture. The pretreatment may be one or more (alone or in combination) of the following steps: homogenizing host cells, ultrasonic processing, freezing/thawing, hypotonic lysis, high pressure processing, centrifugation, filtration, nuclease treatment, protease treatment, cation exchange, and selective precipitation.

Depending on the agent used to elute the vaccinia virus or recombinant virus, post-treatment may be performed to increase the purity of the virus preparation. The post-treatment may be ultrafiltration/diafiltration to further remove impurities and/or specific or non-specific reagents for elution.

Typically, after elution of the virus, the pH is increased, in particular to a pH up to 9 or higher, in particular to pH7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5, 8.6, 8.8, 9.0, 9.2, 9.4, 9.5, 9.6, 9.8, 10.0, 10.2, 10.4 or 10.5.

The practice of the present invention employs techniques of molecular biology, protein analysis, and microbiology, which are within the skill of those skilled in the art. Such techniques are explained fully, for example, in Ausubel et al 1995, eds, Current Protocols in Molecular Biology, John Wiley & Sons, New York.

Example II

The following examples are given for the purpose of illustrating different embodiments of the present invention and are not intended to limit the invention in any way. It will be readily apparent to those skilled in the art that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent therein. The examples of the present invention, together with the methods described herein, presently represent preferred embodiments, are illustrative and are not intended as limitations on the scope of the invention. Variations and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Various human cell lines were tested for JX-594 production. Briefly, adherent HeLa cells, A549 cells, MCF-7 cells, M14 cells, U-2OS cells, HeLa S3 cells in suspension, and adherent HeLa S3 cells were infected with JX-594 at a multiplicity of infection of 0.1, and lysates were collected at 72 hours post-infection and the titers of the viruses were determined on U-2OS cells. The results are shown in FIG. 1. Surprisingly good results were obtained in adherent HeLa cells.

In an effort to optimize virus production in adherent HeLa cells, different parameters were examined. The pH of the infection medium was found to have an unexpected and surprising effect on virus production (see figure 2). Under otherwise identical conditions, production of JX-594 increased from about 12 pfu/cell at an infection medium pH of 7.1 to about 60 pfu/cell at an infection medium pH of 7.3. When the pH of the infection medium is below 7.1, virus production is almost absent. Further increases in virus production are observed when the multiplicity of infection is in the range of 0.005 to 0.05 (especially 0.01 to 0.03), and when the temperature is maintained at 36 ℃ to 37.5 ℃ during and after infection. The use of plastic roller bottles as an attachment surface also promotes high virus yield.

It was also determined that the presence and amount of serum in the medium used to culture adherent HeLa cells prior to infection affected virus yield. The best yield was obtained when the medium contained 10% FBS: reducing the amount of serum in the medium results in a corresponding loss of virus yield. Nevertheless, optimization of other parameters (e.g. pH, multiplicity of infection, attachment surface) will to some extent offset yield loss caused by reduced serum concentrations.

However, the presence and amount of serum in the infection medium does not significantly affect virus yield. Thus, in order to obtain surprisingly good yields of vaccinia virus in adherent HeLa cells according to the invention, the infection medium may comprise serum, or may be substantially serum-free.

A flow chart describing a non-limiting embodiment of the optimized upstream process is as follows:

JX-594 upstream Process

Virus thawing and inoculum expansion. The upstream process was started by thawing a sufficient number of vials from the cell bank. In a polystyrene cell culture vessel (636 cm)²) (Nunc Nalgene) incubating the resulting cell suspension. Cells were cultured to some confluence (about 80-95%) as determined by visual evaluation using commonly used cell culture protocols.

At passage 1, the medium was removed, the cells were rinsed with PBS, and the cells were removed from the culture vessel. Using Vi-Cell XR (Beckman Coulter) or haemocytometerThe cells were counted and then 1 × 10⁴-6x10⁴Individual cell/cm²Was inoculated into 4 polystyrene culture vessels (total area 2544 cm)²) Each vessel contained 200mL of medium-100. The culture was incubated at 37 ℃.

If at 2x10⁴The cultures were inoculated, then the target date for the next passage was 4 days after inoculation. If at 4x10⁴The cultures were inoculated, then the target date for the next passage was 3 days after inoculation. This process was defined to provide approximately 1x10 at each passage⁵Individual cell/cm²。

Roller bottle cell culture expansion at the target date of passage 2, the medium was removed, the cells were rinsed with PBS, and the cells were detached from the polystyrene culture vessel.

At 1x10⁴-6x10⁴Individual cell/cm²(ii) cells were seeded into an RC-40 roller bottle apparatus (Synthecon, total surface area 8400 cm)²) 2 Extended Surface (ES) polystyrene roller bottles (RB, Cellon). Each bottle containing 900mL of medium 100 was cultured and the cell culture was incubated in an RC-40 incubator (Sanyo) at 37 ℃.

At the target date of passage 3, the cell culture medium was removed, the cells were washed with PBS, and the cells were detached. The cell suspension was collected from the ES flask, mixed 1:1 with the medium 100, and the viable cells were counted. Then 1x10⁴-6x10⁴Individual cell/cm²Appropriate amounts of cell suspension were seeded into 4 ES RBs (total surface area 16800 cm)²) In (1). In each bottle, the cell culture was incubated with 900mL of medium at 37 ℃.

During passage 4, the cell culture was transferred to 10 RBs (total surface area 42000 cm)²) And during passage 5, the cell culture was expanded to 20 RBs (total surface area 84000 cm)²). Passage 6 was the first key step in the JX-594 production process, as the cell culture was expanded to 40 RBs (total surface area 168000 cm)²). Inoculation Density of 40 RBs for JX-594 productionCritical and has 4x10⁴Individual cell/cm²To determine the target. During passage 6, samples of cell suspension and conditioned medium were collected for DNA fingerprinting and occasional reagent testing. During the seed production phase, the temperature of the cell culture was controlled at 37 ℃.

Infection and JX-594 production the interval from inoculation of 40 RBs to infection with JX-594 from several vials (either from the master virus bank or the working virus bank) was a 72 hour target. On the day of infection, medium was removed from the cell culture and used with a medium containing 1 × 10⁴pfu/mL of fresh medium-100 replacement of JX-594 virus. The titer of the infection medium is a key process parameter and has a value of 1X10⁴pfu/mL identified target. Cell density was not counted at infection, but was expected to be 1 × 10 based on passage history⁵Individual cell/cm². The duration of the infection is controlled in the range of 40-80 hours, and preferably about 44 hours. The temperature of the cell culture during infection is a critical process parameter and is controlled at 37 ℃.

Viral titers of up to and even greater than 100 pfu/cell are frequently obtained according to this method

The classical method for small scale preparation of poxviruses from cells has involved hypotonic lysis followed by several rounds of freeze-thawing and sonication. The inventors of the present invention have found that freezing/thawing and sonication are largely unnecessary steps, which often results in reduced virus titers. Thus, downstream processing of JX-594 used nuclease (Benzonase) treatment to digest HeLa DNA and protease (TrypLE)^TMSelect) to digest HeLa protein, followed by Tangential Flow Filtration (TFF). Treatment of crude cultures (containing virus and cellular debris in hypotonic lysis buffer) with Benzonase resulted in significantly less contaminating host (HeLa) DNA in the preparations (see figure 3). Thus, nuclease treatment in combination with TFF would provide a significantly purer preparation of virus, containing significantly less contaminating host DNA and proteins in the preparation, while retaining the infectious titer of the virus. In particular, nuclease/protease treatment is usedThe combination of irtff and TFF has reduced DNA contamination from 40 μ g DNA/dose to 3 μ g DNA/dose and protein contamination from 12mg protein/dose to 4mg protein/dose (dose =1x 10)⁹JX-594 of pfu). Further reductions can be achieved by employing one or more chromatographic steps. In one aspect, the one or more chromatography steps comprise ion exchange chromatography. In another aspect, the one or more chromatography steps comprise pseudo-affinity chromatography, preferably based on heparin (or heparin-like molecules) or sulfated cellulose, exploiting the heparin-binding capacity of the vaccinia virus a27L protein. In another aspect, the one or more chromatography steps comprise membrane adsorption chromatography, such as membrane affinity chromatography. Preferred membranes have a microporous structure with a pore size of at least 3 μ M.

Reference to the literature

To the extent that they provide exemplary operational or other details to supplement those described herein, the following references are specifically incorporated by reference herein.

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Claims (39)

1. A method for producing vaccinia virus, comprising:

(a) infecting said HeLa cells attached to a surface with vaccinia virus by contacting said HeLa cells with vaccinia virus at 0.005-1.0 plaque forming units (pfu)/multiplicity of infection of cells (m.o.i.);

(b) culturing the infected cells in an infection medium having a pH above 7.1; and

(c) vaccinia virus was harvested from the culture.

2. The method of claim 1, wherein the density of adherent HeLa cells during step (a) is 10⁴-10⁶HeLa cells/cm²。

3. The method of claim 2, wherein the density of adherent HeLa cells during step (a) is about 10⁵HeLa cells/cm²。

4. The method of any one of claims 1-3, wherein the HeLa cells are attached to a unit cell, cell factory, T-flask, roller bottle, or microcarrier.

5. The method of claim 4, wherein the HeLa cells are attached to one or more plastic roller bottle containers.

6. The method of claim 5, wherein the one or more roller bottles comprise at least 168,000cm²The culture area of (a).

7. The method of any one of claims 1-6, wherein the concentration of vaccinia virus during step (a) is at 10³-10⁵pfu/ml。

8. The method of claim 7, wherein the concentration of vaccinia virus during step (a) is about 10⁴pfu/ml。

9. The method of any one of claims 1-8, wherein the pH of the infection medium is above 7.2, and preferably about 7.3.

10. The method according to any one of claims 1-9, wherein steps (a) and (b) are performed at a temperature of 36 ℃ to 37.5 ℃, preferably at 37 ℃.

11. The method according to any one of claims 1-10, wherein step (b) is carried out for a period of 40-80 hours, preferably about 44 hours.

12. The method according to any of claims 1 to 11, wherein at least 50pfu vaccinia virus/HeLa cells are produced, preferably at least 75pfu vaccinia virus/HeLa cells are produced.

13. The method of any one of claims 1-12, wherein the culture medium does not comprise one or more of: dextran sulfate, PluronicF-68, polysorbate 80 and soy hydrolysate.

14. The method of claim 13, wherein the culture medium does not comprise dextran sulfate.

15. The method of claim 14, wherein the medium lacks dextran sulfate, PluronicF-68, polysorbate 80 and soy hydrolysate.

16. The method of any one of claims 1-15, wherein the culture medium comprises fetal bovine serum.

17. The method of claim 16, wherein the culture medium comprises less than 10% fetal bovine serum.

18. The method of any one of claims 1-15, wherein the culture medium does not comprise serum.

19. The method according to any one of claims 1-18, wherein the m.o.i. of step (a) is about 0.01-0.5 pfu/cell, and preferably about 0.02 pfu/cell.

19. The method of any one of claims 1-18, wherein the harvested virus is subjected to one or more purification steps.

20. The method of claim 19, wherein the purifying comprises: treating the harvested virus with a nuclease to remove HeLa cell nucleic acids and/or a protease to remove HeLa cell proteins.

21. The method of claim 19 or 20, wherein the purification comprises a tangential flow filtration step.

22. The method according to any one of claims 19-21, wherein the purification comprises a heparin affinity chromatography step.

23. The method of any one of claims 19-22, wherein the purification comprises a membrane absorption chromatography step.

24. The method of any one of claims 1-23, wherein the vaccinia virus is IHD-J, Wyeth, Western Reserve, or copenhagen strain of vaccinia virus.

25. The method of claim 24, wherein the vaccinia virus is a recombinant vaccinia virus.

26. The method of claim 25, wherein the recombinant vaccinia virus comprises a heterologous coding region encoding an immunostimulatory polypeptide.

27. The method of claim 26, wherein the immunostimulatory polypeptide is a cytokine.

28. The method of claim 27, wherein the cytokine is granulocyte macrophage colony-stimulating factor (GM-CSF).

29. The method of claim 28, wherein the recombinant vaccinia virus selectively replicates in tumor cells.

30. The method of claim 29, wherein the recombinant vaccinia virus comprises a mutation in an endogenous gene.

31. The method of claim 30, wherein the recombinant vaccinia virus lacks a functional thymidine kinase gene.

32. The method of claim 31, wherein the recombinant vaccinia virus is IHD-J, Wyeth, Western Reserve, or copenhagen strain of vaccinia virus.

33. The method of claim 32, wherein the recombinant vaccinia virus encodes a granulocyte macrophage colony stimulating factor polypeptide.

34. The method according to any one of claims 1-33, the method further comprising: HeLa cultures were expanded prior to infection.

35. The method of claim 34, wherein the HeLa cells are passaged through at least one roller bottle culture vessel having an increased culture area compared to a previous culture vessel.

36. The method of claim 35, wherein the roller bottle culture vessel has at least 850cm²The culture area of (a).

37. The method of claim 35, wherein the final passage is to at least 168,000cm²The culture area of (3) is a roller bottle culture container.

38. The method of claim 37, wherein the interval between inoculating the roller bottle culture vessel and infection is 48-96 hours.

39. The method of claim 38, wherein the adherent HeLa cells are contacted with 2x10³-1x10⁵Individual recombinant replication competent vaccinia viruses per ml.