Large-Scale Flavivirus Vaccine Production and Manufacturing

Abstract

The present invention provides methods for large scale production and manufacturing of flavivirus vaccines. The methods provided herein are particularly contemplated for large scale production and manufacturing of live attenuated flavivirus vaccines, such as live attenuated dengue virus vaccines. Additionally, the methods provided herein relate to the formulation of live attenuated, monovalent, bivalent, trivalent, or quadrivalent viral vaccine products.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 302,910, filed January 25, 2022, U.S. Provisional Application No. 63 / 302,920, filed January 25, 2022, U.S. Provisional Application No. 63 / 385,274, filed November 29, 2022, and U.S. Provisional Application No. 63 / 385,309, filed November 29, 2022, the entire contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a sequence listing, which has been submitted herewith in electronically readable XML ST.26 format via the Patent Center and is incorporated herein by reference in its entirety. The electronic sequence listing file was created on Jan. 24, 2023, is named "T08530WO.xml", and is 65KB in size.

[0003] The present invention provides methods for large scale production and manufacturing of flavivirus vaccines. The methods provided herein are particularly contemplated for large scale production and manufacturing of live attenuated flavivirus vaccines, such as live attenuated dengue virus vaccines. Additionally, the methods provided herein relate to the formulation of live attenuated, monovalent, bivalent, trivalent, or quadrivalent viral vaccine products. [Background technology]

[0004] Scaling up vaccine manufacturing capacity is essential to interrupt global transmission of the disease and halt the emergence of new variants of the virus. One of the biggest challenges in vaccine production and manufacturing is the economical use of resources to meet the ever-increasing challenge of delivery to billions of people around the world.

[0005] One group of viruses that continues to pose a growing threat to public health are the flaviviruses. These are vector-borne RNA viruses that can emerge unexpectedly in human populations and cause a spectrum of potentially severe diseases, including hepatitis, vascular shock syndrome, encephalitis, acute flaccid paralysis, congenital anomalies, and fetal death. This epidemiological pattern has occurred multiple times over the past 70 years, including epidemics of dengue and West Nile viruses, as well as the recent explosive Zika virus epidemic in the Americas. Flaviviruses are now distributed globally, infecting up to 400 million people annually. Of major concern, outbreaks of other less well-characterized flaviviruses have been reported in humans and animals in different parts of the world. The potential for these viruses to sustain epidemic transmission among humans remains poorly understood. Thus, there is an urgent need for more economical, efficient, and robust methods for large-scale production and manufacturing of flaviviruses that can reliably increase manufacturing capacity and meet high demand.

[0006] Early signs of supply pressures are observed across all vaccine production steps, e.g., the working virus seed used to infect the first production culture, or the bioreactors that produce the vaccine components, or the chromatography and filtration steps that purify the virus, or the filters and vials that contain the vaccine components. These individual challenges can have a compounding effect where the lack of a single input disrupts the entire manufacturing process. Thus, there is an urgent need to reinvent large-scale vaccine production and manufacturing at all steps that provides an economically coordinated process synchronized with the increasing demand for vaccines. Summary of the Invention

[0007] The present invention provides a method for large-scale flavivirus vaccine production and manufacturing, comprising the following consecutive steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing a flavivirus at a low MOI; (iii) harvesting to obtain a harvest; (iv) processing the harvest of step (iii) to obtain a processed harvest; and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step.

[0008] The present invention provides a method for large scale flavivirus vaccine production and manufacturing, comprising the following successive steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with an infection medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step. In this context, the infection method in step (ii) is static and the cells are infected in a monolayer.

[0009] The present invention provides a method for large scale flavivirus vaccine production and manufacturing comprising the following consecutive steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium comprising flavivirus at a low MOI; (iii) harvesting to obtain a harvest, wherein harvesting comprises a first harvesting step and at least one further harvesting step, wherein a medium change is performed at least 12 to 30 hours before the first harvesting step; (iv) processing the harvest of step (iii) to obtain a processed harvest; and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step.

[0010] The present invention provides a method for large scale flavivirus vaccine production and manufacturing comprising the following successive steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with an infection medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, where harvesting comprises a first harvesting step and at least one further harvesting step, where a medium change is performed at least 12-30 hours before the first harvesting step, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), where step (v) comprises at least one chromatography step. In this context, the infection method in step (ii) is static and the cells are infected in a monolayer.

[0011] The present invention provides a method for large-scale flavivirus vaccine production and manufacturing comprising the following consecutive steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing flavivirus at a low MOI; (iii) harvesting to obtain a harvest; (iv) processing the harvest of step (iii) to obtain a processed harvest; and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step, and step (v) comprises the consecutive steps of (va) ion exchange chromatography to obtain a purified harvest, and (vb) ultrafiltration.

[0012] The present invention provides a method for large scale flavivirus vaccine production and manufacturing comprising the sequential steps of: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium comprising a flavivirus; (iii) harvesting to obtain a harvest; (iv) processing the harvest of step (iii) to obtain a processed harvest; and (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step, wherein the pH is maintained in the range of 7.6 to 8.1 throughout steps (iii) to (v), and the difference between (a) the maximum pH occurring at any time throughout steps (iii) to (v) and (b) the minimum pH occurring at any time throughout steps (iii) to (v) is no more than 0.4 or 0.3 units.

[0013] According to the invention, preferably the total surface area of ​​the production culture is greater than, for example, 50,000 cm 2 ~1,000,000cm 2 , 100,000cm 2 ~1,000,000cm 2 , 50,000cm 2 ~450,000cm 2 , 100,000cm 2 ~450,000cm 2 35,000cm 2 This is achievable at a magnitude of 100 or more.

[0014] According to the present invention, preferably the flavivirus is a dengue virus, and preferably the dengue virus is selected from the group consisting of a live attenuated dengue-2 virus serotype, a dengue 2 / 1 chimera, a dengue 2 / 3 chimera, and a dengue 2 / 4 chimera.

[0015] The present invention also provides methods for large scale production and manufacturing of flavivirus vaccines, including pharmaceutical formulations, preferably tetravalent dengue virus vaccines including live attenuated dengue-2 virus serotypes, dengue 2 / 1 chimera, dengue 2 / 3 chimera, and dengue 2 / 4 chimera. [Brief description of the drawings]

[0016] [Figure 1] Illustrative overview of the production and manufacturing process of flavivirus vaccines. In this figure, an example of a manufacturing process according to the invention for a tetravalent dengue virus vaccine is shown. Reference to an individual serotype as Den-1, Den-2, Den-3, or Den-4 can be interpreted as either non-chimeric dengue serotypes 1, 2, 3, and 4, respectively, or one or more chimeric dengue serotypes, e.g., four chimeric dengue serotypes or three chimeric dengue serotypes, e.g., dengue 2 / 1 chimera or TDV-1 represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue 2 serotype or TDV-2 represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue 2 / 3 chimera or TDV-3 represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue 2 / 4 chimera or TDV-4 represented by SEQ ID NO:7 and / or SEQ ID NO:8, respectively. [Diagram 2]Illustrative overview of the production and manufacturing process of flavivirus vaccines. In this figure, an example of a manufacturing process according to the invention for a tetravalent dengue virus vaccine is shown. In the "processing the BDS" step, filtration may be performed before and / or after freezing and storage of the BDS. Reference to an individual serotype as Den-1, Den-2, Den-3, or Den-4 can be interpreted as either non-chimeric dengue serotypes 1, 2, 3, and 4, respectively, or one or more chimeric dengue serotypes, e.g., four chimeric dengue serotypes or three chimeric dengue serotypes, e.g., dengue 2 / 1 chimera or TDV-1 represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue 2 serotype or TDV-2 represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue 2 / 3 chimera or TDV-3 represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue 2 / 4 chimera or TDV-4 represented by SEQ ID NO:7 and / or SEQ ID NO:8, respectively. In one embodiment, TDV-1 and TDV-2 are manufactured in the same manner. In one embodiment, TDV-3 and TDV-4 are manufactured in the same manner as TDV-1 and TDV-2, but at a different scale of bulk drug substance manufacturing and / or processing. In one embodiment, TDV-4 is manufactured on a larger scale than TDV-1, TDV-2, and TDV-3. In one embodiment, TDV-3 and TDV-4 are manufactured on a larger scale than TDV-1 and TDV-2. [Diagram 3] Graph of virus titers obtained after infection with a low MOI of 0.005 (group 1) and a high MOI of 0.05 (group 2). Individual virus titers on days 5, 6, and 9 are shown (bars from left to right). [Figure 4] Graph of virus titers obtained after infection with a low MOI of 0.001 (group 1) and a high MOI of 0.05 (group 2). Individual virus titers are shown on days 5, 6, and 9 (bars from left to right). [Diagram 5] Graph showing the dependence of virus yield (grams) on pH variation - the higher the virus yield the lower the variation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] References to the word "can" (e.g., the phrase "can be") throughout this specification are not intended to be construed as limiting the invention to specific embodiments, but rather are illustrative and non-limiting examples.

[0018] Throughout this specification, reference to multiple steps being performed "sequentially" means that the steps are necessarily performed in that order. However, when the word "sequentially" is not referenced, the steps may or may not be performed in that order.

[0019] The methods of the invention relate to large-scale production and manufacturing of flavivirus vaccines. As used herein, "large-scale," also referred to as "production scale" or "manufacturing scale," refers to the isolation of virus achievable from a starting sample volume on the order of thousands of liters or thousands of square centimeters. Alternatively, large-scale refers to a volume of at least 1000 liters or more, or at least 35,000 cm. 2 In the context of the present invention, large-scale production refers to starting a production culture in a larger number of tissue culture vessels with a larger surface area for cell expansion and growth. For large-scale production and manufacturing of flavivirus vaccines according to the present invention, culture vessels that can be used are, for example, Corning® CellSTACK® culture chambers or Nunc TM EasyFill TM Cell Factory TM It is a system.

[0020] For example, a Corning® CellSTACK® culture chamber that may be used has a volume of 636 cm 2 1 stack (CF1) with a cell growth area of ​​1,272 cm 2 2 stacks (CF2) with a cell growth area of ​​3,180 cm 2 5 stacks (CF5) with a cell growth area of ​​6,360 cm2 10 stacks (CF10) with a cell growth area of ​​25,440 cm 2 The 40 stack (CF40) has a cell growth area of ​​1000 x 1000 mm. The recommended medium volumes required for each of these vessels are 130-200 mL for CF1, 260-400 mL for CF2, 650-1,000 mL for CF5, 1,300-2,000 mL for CF10, and 5,200-8,000 mL for 40 stack.

[0021] Thus, for large scale production and manufacturing of flavivirus vaccines, a "larger" number of culture vessels may be achieved, either with the culture vessels specified above, or with a total volume of the production culture of at least 1000 liters, or with a total surface area of ​​the production culture of at least 50,000 cm, for example. 2 ~1,000,000cm 2 , 100,000cm 2 ~1,000,000cm 2 , 50,000cm 2 ~450,000cm 2 , 100,000cm 2 ~450,000cm 2 35,000cm 2 The above dimensions may be achieved with any of the other commercially available culture vessel combinations.

[0022] Thus, for example, for large scale production and manufacturing of a flavivirus vaccine, 5-20 CF10 chambers, 10-20 CF10 chambers, 15-20 CF10 chambers may be used, or 1-20 CF40, 4 CF40, 5-20 CF40, 10-20 CF40, 15-20 CF40 chambers, or any combination thereof may be used. It is understood that each individual possibility can be combined with other possibilities to arrive at a combination of features, and that each possibility, and each combination, represents a separate embodiment of the invention.

[0023] Those skilled in the art will appreciate that large-scale production is specifically designed to meet the demands of viral vaccine production and manufacturing while improving the economics of process resources. Thus, the method of the present invention specifically relates to large-scale production and manufacturing of viral vaccines, which are subject to entirely different scientific and economic considerations than small-scale processes. Thus, the present invention preferably excludes small-scale processes of viral vaccine production.

[0024] Those skilled in the art will also understand that the methods of the present invention relate to the "production" of a flavivirus vaccine using a "seed" stock. The terms "production culture" and "seed culture" are well known in the art. Seed culture generally refers to a pure culture of a desired strain, i.e., a primary strain, and inoculum refers to a seed culture that is inoculated into a medium for growth, which is then considered the production culture. Thus, those skilled in the art will understand that methods used to develop seed stocks, or methods commonly used for seed cultures, are not applicable or relevant and cannot be used for large-scale production and manufacturing of viral vaccines.

[0025] The method of the present invention also relates to the large-scale production and manufacture of "attenuated" virus vaccines. Large-scale production and manufacture of attenuated virus vaccines is a challenge because attenuated viruses may have impaired replication rates. As used herein, the term "replication" refers to the process by which complementary strands of a nucleic acid molecule are synthesized by a polymerase enzyme. In the context of the present invention, the term "replication" with respect to a virus refers to the completion of the flavivirus life cycle, in which an infectious virus particle or virion binds to the surface of a host cell (usually to a specific cell surface molecule that is responsible for the specificity of infection). Once inside the cell, the virion is uncoated and flavivirus genes begin to be expressed, leading to the synthesis of proteins required for genome replication and the synthesis of new proteins, creating new capsids and cores leading to the assembly of progeny infectious virus particles that can themselves infect and replicate in new host cells. Thus, the virus life cycle is complete only when, within a single cell, infection by one or more virus particles or virions proceeds to the production of fully infectious progeny virus particles. The term "replication rate" refers to the factor at which a flavivirus population grows. Thus, as used herein, the term "attenuation" means that the replication rate of a virus is impaired.Several methods for evaluating the attenuation of a particular virus are known to those skilled in the art.For example, the attenuation of dengue virus can be measured using methods including temperature sensitivity, small plaque size, reduced replication in mosquito C6136 cell culture, reduced replication in intact mosquitoes, and reduced occurrence of viremia in monkeys.

[0026] The method of the present invention can be used for large-scale production and manufacturing of flaviviruses. The flavivirus genus includes enveloped positive-stranded RNA viruses such as West Nile (WN) virus, Japanese encephalitis virus (JEV), Zika virus, Dengue virus, Yellow fever virus (YF), Kyasanur Forest disease virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Tick-borne encephalitis virus, West Nile encephalitis virus, Central European encephalitis (TBE-W) virus, Far Eastern encephalitis (TBE-FE) virus, Kunjin virus, Tyureni virus, Ntaya virus, Uganda S virus, Modoc virus, BVDV (e.g., strains NADL and 890), CSFV Alfort 187, BDV BD31 virus, and / or GB virus-A, -B, and / or -C.

[0027] The genus Flavivirus includes highly pathogenic and potentially hemorrhagic fever viruses such as Yellow Fever Virus and Dengue Virus, encephalitis viruses such as Zika Virus, Japanese Encephalitis Virus, Murray Valley Encephalitis Virus and West Nile Virus, as well as several low pathogenic viruses. The Flavivirus genome includes, from 5' to 3', a 5' non-coding region (5'-NCR), a capsid protein (C) coding region, a pre-membrane protein (prM) coding region, an envelope protein (E) coding region, a region coding for nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) and a 3' non-coding region (3'-NCR). Flavivirus structural proteins are C, prM and E, and nonstructural proteins are NS1 to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and flavivirus proteases.

[0028] Preferred flavivirus vaccines for production according to the present invention are Zika and dengue vaccines. Thus, the method of the present invention can be used for the large-scale production and manufacturing of dengue viruses, i.e., each of the dengue serotypes. As used herein, dengue viruses are single-stranded, positive-sense RNA viruses of the Flaviviridae family. The Flaviviridae family includes three genera: Flavivirus, Hepacivirus, and Plaguevirus. As used herein, the term "dengue serotype" refers to a species of dengue virus that is defined by its cell surface antigens and thus can be distinguished from other dengue serotypes by serological methods known in the art. Currently, four serotypes of dengue virus are known, namely, dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), and dengue serotype 4 (DENV-4). Thus, the method of the present invention can also be used for the large-scale production and manufacturing of the four live attenuated dengue virus strains separately.

[0029] As used herein, the term "live attenuated dengue virus" refers to a viable, infectious dengue virus that is mutated to provide reduced virulence. A live attenuated dengue virus can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus with portions from two or more dengue serotypes. A "virus strain," particularly a "dengue virus strain," is a genetic subtype of a virus, particularly a dengue virus, characterized by a specific nucleic acid sequence. A dengue serotype can include different strains with different nucleic acid sequences that have the same cell surface antigens and thus are recognized by the same antibodies. A dengue virus strain can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus with portions from two or more dengue serotypes.

[0030] The live attenuated dengue virus strain can be, for example, (i) a chimeric dengue serotype 2 / 1 strain (TDV-1), (ii) a dengue serotype 2 strain (TDV-2), (iii) a chimeric dengue serotype 2 / 3 strain (TDV-3), and (iv) a chimeric dengue serotype 2 / 4 strain (TDV-4).

[0031] Together, TDV-1, TDV-2, TDV-3, and TDV-4 form a tetravalent dengue virus composition, designated "TDV" or "TAK-003," which is Takeda's dengue vaccine candidate for which it has recently submitted regulatory submissions.

[0032] In one embodiment, the tetravalent dengue virus composition "TAK-003" comprises: live attenuated dengue virus serotype 1, live attenuated dengue virus serotype 2, live attenuated dengue virus serotype 3, and comprising a live attenuated dengue virus serotype 4, Dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, dengue serotype 2 is a non-chimeric dengue serotype 2 strain, dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; Dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 and differ from the wild-type by at least three nucleotides, as follows: a) 5'-noncoding region (NCR)-57 (nt-57) b) NS1-53 Gly-to-Asp (nt-2579) c)NS3-250 Glu-to-Val(nt-5270), The three chimeric dengue strains were derived from dengue serotype 2 strains by replacing the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins from other dengue serotypes, resulting in the following chimeric dengue strains: DENV-2 / 1 chimera, DENV-2 / 3 chimeras, and DENV-2 / 4 chimera.

[0033] A "chimeric virus" or "chimeric strain" or "chimeric virus strain" generally comprises portions from at least two different viruses. For example, a chimeric virus can comprise prM and E proteins of dengue virus and other proteins from another flavivirus. A chimeric virus can comprise prM and E proteins of dengue virus and other proteins from another flavivirus, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, and tick-borne encephalitis virus. A chimeric virus can comprise prM and E proteins of dengue virus and other proteins from yellow fever virus strain YF-17D. Such a chimeric virus is present in the commercial product Dengvaxia® and is described, for example, in WO 98 / 37911, WO 03 / 101397, WO 2007 / 021672, WO 2008 / 007021, WO 2008 / 047023, and WO 2008 / 065315. Accordingly, certain embodiments of the invention include the production of a tetravalent composition of four chimeric dengue strains and / or four live attenuated dengue strains, for example a tetravalent composition of four live attenuated chimeric dengue strains.

[0034] A "chimeric dengue virus" or "chimeric dengue serotype strain" or "chimeric dengue strain" preferably comprises portions from at least two different dengue serotypes, i.e., a dengue-dengue chimera. Such chimeric dengue viruses are described in WO01 / 060847A2, WO2014 / 150939A2, and WO2017 / 179017A1.

[0035] In a preferred embodiment, the live attenuated Dengue-2 virus serotype or DENV-2 or TDV-2 is in the form of a DEN-2 PDK-53 variant derived from DENV-2 16681, which has a triple mutation in NS1-53, 5'NCR-57, and NS3-250 such that amino acid position 250 of the NS3 protein contains a valine residue, and a chimera has the DEN-2 PDK-53 genome as the viral backbone and one or more structural protein genes encoding the capsid, pre-membrane / membrane, or envelope of the DEN-2 PDK-53 genome replaced with one or more corresponding structural protein genes from DEN-1, DEN-3, or DEN-4.

[0036] In a preferred embodiment, the dengue 2 / 1 chimera or DENV-2 / 1 chimera or TDV-1 has two further mutations such that amino acid position 116 of the NS2A protein contains a leucine residue and amino acid position 92 of the NS2B protein contains an aspartic acid residue, the live attenuated dengue-2 virus serotype or DENV-2 or TDV-2 has two further mutations such that amino acid position 52 of the prM protein contains a glutamic acid residue and amino acid position 412 of the NS5 protein contains a valine residue, and the dengue-2 / 3 chimera or DENV-2 / 3 chimera or TDV-3 has the DEN-2 PDK-53 genome as the viral backbone and the DEN-2 PDK-53 genome replaced with the corresponding prM-E genes from wild type DEN-3 16562. The dengue-2 / 3 chimera or DENV-2 / 3 chimera or TDV-3 has a prM-E gene (nt -457 to -2373) of the PDK-53 genome, and the dengue-2 / 4 chimera or DENV-2 / 4 chimera or TDV-4 has one additional mutation in the corresponding prM-E gene from wild type DEN-3 16562 such that amino acid position 223 of the E protein contains a serine residue, the dengue-2 / 4 chimera or DENV-2 / 4 chimera or TDV-4 has two additional mutations such that amino acid position 66 of the NS2A protein contains a glycine residue and amino acid position 21 of the NS4A protein contains a valine residue, and the dengue-2 / 4 chimera or DENV-2 / 4 chimera or TDV-4 is of mixed genotype for amino acid position 99 of the NS2A protein which contains an arginine or lysine residue.

[0037] Thus, a dengue-2 / 1 chimera, or DENV-2 / 1 chimera, or TDV-1, has as its viral backbone the DEN-2 PDK-53 genome with the prM-E gene (nt -457 to -2379) of the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild type DEN-1 16007; a dengue-2 / 3 chimera, or DENV-2 / 3 chimera, or TDV-3, has as its viral backbone the DEN-2 PDK-53 genome with the prM-E gene (nt -457 to -2373) of the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild type DEN-3 16562; and a dengue-2 / 4 chimera, or DENV-2 / 4 chimera, or TDV-4, has as its viral backbone the DEN-2 PDK-53 genome with the prM-E gene (nt -457 to -2373) of the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild type DEN-3 16562. PDK-53 genome, with the prM-E gene of DEN-2 PDK-53 (nt -457 to -2379) replaced with the corresponding prM-E gene from wild-type DEN-4 1036.

[0038] Thus, a live attenuated dengue-2 virus serotype or DENV-2 or TDV-2 is represented by the polynucleotide of SEQ ID NO:3 or the polypeptide of SEQ ID NO:4, a dengue 2 / 1 chimera, or DENV-2 / 1 chimera, or TDV-1 has nonstructural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-1 virus serotype represented by the polynucleotide of SEQ ID NO:1 or the polypeptide of SEQ ID NO:2, a dengue 2 / 3 chimera, or DENV-2 / 3 chimera, or TDV-3 has nonstructural proteins derived from a modified live attenuated dengue-2 virus serotype and structural proteins derived from a dengue-3 virus serotype represented by the polynucleotide of SEQ ID NO:5 or the polypeptide of SEQ ID NO:6, and the dengue-2 / 4 chimera or DENV-2 / 4 chimera or TDV-4 has nonstructural proteins derived from a modified live attenuated dengue-2 virus serotype and structural proteins derived from a dengue-4 virus serotype represented by the polynucleotide of SEQ ID NO:7 or the polypeptide of SEQ ID NO:8.

[0039] RNA viruses included in TDV can be characterized by their RNA sequence. Alternatively or additionally, it is also common practice to characterize the genome of RNA viruses by corresponding DNA sequence, which is easily understood to reflect the RNA genome in the virus. Therefore, when referring to genomic RNA virus sequence, references to "t" or "thymine" throughout this disclosure should be understood as references to "u" or "uracil", respectively.

[0040] Providing cells in growth medium According to the present invention, the method for large scale production and manufacturing of flavivirus vaccines preferably comprises the step of providing cells in a growth medium. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1, represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8. The method described in this section is carried out separately for each of the four serotypes.

[0041] This step further involves expanding the cells to sufficient numbers in growth medium. For large scale flavivirus vaccine production and manufacturing purposes, a number of different cell lines can be used, including Madin-Darby canine kidney cells, monkey cell lines pMK, Vero, and human cell lines HEK293, MRC5, Per.C6, PMK, LLCMK2, BHK, and WI-38. All of these cell lines are well characterized with demonstrated safety profiles. Furthermore, these cell lines are suitable to support the replication of attenuated vaccine strains. Each of these cell lines represents a separate embodiment of the present invention.

[0042] Thus, for the purposes of the present invention, two cell banks can be developed using cell lines selected from Madin-Darby canine kidney cells, monkey cell lines pMK, Vero, and human cell lines HEK293, MRC5, Per.C6, PMK, LLCMK2, BHK, and WI-38: Master Cell Bank (MCB) and Working Cell Bank (WCB). As used herein, the term "master cell bank" refers to a culture of cells (e.g., fully characterized cells) that have been propagated from a single clone and stored under cryopreservation conditions. Cells from the MCB are used to develop the working cell bank. As used herein, the term "working cell bank" refers to a culture of cells (e.g., fully characterized cells) that have been propagated from a single vial of the MCB or from two pooled vials of the master cell bank and stored under cryopreservation conditions. Cells from the WCB are later used in production cell cultures.

[0043] The methods of the present invention also include the use of a growth medium and additive formulation for the expansion of cells, which may or may not be the same as the medium in which the WCB and MCB are developed. According to the present invention, the growth medium used for the expansion of cells is Growth Medium, Glutamine or GlutaMAX. TM , and serum.

[0044] The term "growth medium" refers to a medium for culturing cells that contains nutrients that maintain cell viability and support growth. Growth medium may contain any of the following nutrients in appropriate amounts and combinations: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, and other components such as peptide growth factors. "Growth medium" is known in the art and can be classified as natural or artificial cell culture media. Examples of cell culture media that can be used in the methods of the invention include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), DMEM / F-12, and Roswell Park Memorial Institute Medium (RPMI). Different culture media with different ranges of pH, glucose concentration, growth factors, and other supplements can be used for different cell types or different applications. In some embodiments, custom cell culture media or commercially available cell culture media such as Dulbecco's Modified Eagle's Medium, Minimum Essential Medium, RPMI medium, HA or HAT medium, or other media available from other commercial sources can be used. The growth medium may include one or more antibiotics (e.g., actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, penicillin, penicillin-streptomycin, polymyxin B, streptomycin, tetracycline, or any other suitable antibiotic, or any combination of two or more thereof). The growth medium may include one or more salts (e.g., balanced salts, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, etc.). The growth medium may also include one or more buffers (e.g., HEPES or other suitable buffers). The growth medium may also include differentiation factors. Growth or differentiation factors (e.g., WNT family proteins, BMP family proteins, IGF family proteins, etc.) may be added individually or in combination, for example, as a differentiation cocktail containing different factors that result in differentiation into a specific lineage. It is understood that each individual possibility can be combined with another possibility to arrive at a combination of features, and each possibility, and each combination, represents a separate embodiment of the invention.

[0045] The concentration of each of the individual components in the growth medium can be adapted to the needs of the cell line used and the flavivirus vaccine to be produced. For example, the concentration of glucose can be 3 g / L to 4 g / L glucose, 3.1 to 3.2 g / L, 3.2 to 3.3 g / L, 3.3 to 3.4 g / L, 3.4 to 3.5 g / L, 3.5 to 3.6 g / L, 3.6 to 3.7 g / L, 3.7 to 3.8 g / L, 3.8 to 3.9 g / L, or 3.9 to 4.0 g / L. The concentration of glucose can be, for example, 3.12 g / L, 3.22 g / L, 3.32 g / L, 3.42 g / L, 3.52 g / L, 3.62 g / L, 3.72 g / L, 3.82 g / L, or 3.92 g / L. Each of these possibilities represents a separate embodiment of the invention.

[0046] Glutamine or GlutaMAX TM The concentration of glutamine, if added, is 3.5 mM to 4.5 mM. TM , 3.5mM-3.6mM, 3.6mM-3.7mM, 3.7mM-3.8mM, 3.8mM-3.9mM, 4.0mM-4.1mM, 4.1-4.2mM, 4.2-4.3mM, 4.3-4.4mM, or 4.4-4.5mM. Glutamine or GlutaMAX TM can be, for example, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4.0 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, or 4.5 mM. Each of these possibilities represents a separate embodiment of the present invention.

[0047] "Serum" as used herein refers to mammalian serum and can be selected from human serum, horse serum, bovine serum, or sheep serum. Serum is widely used as a supplement to cell growth media for adherent mammalian cell cultures because it provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water-insoluble components, and other compounds necessary for the in vitro growth of cells, such as hormones and attachment factors. The term "serum" further encompasses serum-based solutions containing buffers. The serum used in the present invention can include the addition of substances such as salts, buffers, sugars, chelating agents, preservatives, and protease inhibitors. As used herein, the term "horse serum" refers to serum obtained from horses. As used herein, the term "bovine serum" refers to serum obtained from bovine, and commonly used bovine serum is referred to in the art as "fetal bovine serum (FBS)". As used herein, the term "sheep serum" refers to serum obtained from sheep. As used herein, the term "human serum" refers to serum obtained from humans. In some embodiments, the cell culture medium comprises serum (e.g., fetal bovine serum, calf serum, horse serum, porcine serum, or other serum). In some embodiments, the cell culture medium is serum-free. In some embodiments, the cell culture medium comprises human platelet lysate (hPL).

[0048] When serum is added, the concentration of serum may be in the range of 1-10%, for example, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10%. The concentration of serum may be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. Thus, the concentration of FBS in the medium may be in the range of 1-10%, for example, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10%. Thus, the concentration of horse serum in the medium may be in the range of 1-10%, for example, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10%. Thus, the concentration of sheep serum in the medium can be in the range of 1-10%, e.g., 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10%. Thus, the concentration of pig serum in the medium can be in the range of 1-10%, e.g., 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10%. Each of these possibilities represents a separate embodiment of the invention.

[0049] Further optional components of the growth medium for the purposes of the present invention include a pH indicator. The pH indicator that can be used in the method of the present invention can be phenol red. In the presence of phenol red, microbial contamination of the medium can be indicated by a shift to a lower pH value, which causes a color change of the pH indicator. Instead of or in addition to the pH indicator, a visual microscopic check for microbial contamination can be performed. Each possibility has been tested and represents a separate embodiment of the present invention.

[0050] The growth medium may or may not include a non-ionic surfactant such as a poloxamer. Preferably, the growth medium does not include a poloxamer. Thus, according to one embodiment of the invention, a method for large scale flavivirus vaccine production and manufacturing includes providing cells in a growth medium, the growth medium does not include a non-ionic surfactant such as a poloxamer.

[0051] In the context of the present invention, the growth medium preparation strategy is also a factor for controlling the growth of microorganisms. For the purposes of the present invention, the growth medium can be prepared either by "aseptically mixing" and homogenizing all the components, or by mixing the components and then "filtering" them depending on the virus being produced. The term "sterile" is understood to mean a state that is substantially free of growth of undesirable or pathogenic organisms and substantially free of the accumulation of debris or other media in which such organisms are likely to thrive.

[0052] The present invention includes preparing the growth medium in either of the two ways described above. Furthermore, the present invention includes using one way of preparing the growth medium for one flavivirus and another way for another flavivirus. Furthermore, when flaviviruses having different serotypes are being produced, the present invention includes producing one serotype in a growth medium prepared in one way and producing another serotype in a growth medium prepared in another way. For example, for the purposes of the present invention, when dengue serotypes are being produced separately, serotype 4 may be produced in a filtered growth medium while serotype 1 may be produced in a sterile mix, or vice versa. Each possibility represents a separate embodiment of the present invention. When filtration is used, preferably, a filter smaller than 1 μm is used, more preferably, a filter smaller than 0.5 μm is used.

[0053] Another optional additive that may be included is a dissociation reagent for adherent cell passaging to remove cells from the surface. As used herein, the term "dissociation reagent" refers to a solution or fluid that is contacted with anchorage-dependent cells and dissociates (can loosen and detach) the cells from the surface to which they are attached. Thus, when using adherent cells for the purposes of the present invention, the growth medium may also include a dissociation reagent, such as one or more chelating agents (ethylenediaminetetraacetic acid, "EDTA", ethylene glycol-bisbeta-aminoethyl ether N,N,N',N'-tetraacetic acid, "EGTA", versen, etc.). The growth medium may alternatively include one or more proteolytic enzymes (e.g., ficin, pepsin, trypsin, chymotrypsin, papain, etc., trypsin being the preferred enzyme). The proteolytic enzymes used in the present invention may be recombinant, e.g., commercially available TrypLE Select, or mammalian, e.g., porcine, trypsin. The growth medium may also include a combination of a chelating agent and a proteolytic enzyme. Each possibility represents a separate embodiment of the present invention.

[0054] The target volume of the dissociation reagent is determined by the surface area of ​​the culture vessel. The volume-to-surface area ratio of the dissociation reagent should be between 0.01 and 0.02 mL / cm. 2 , or 0.011 to 0.016 mL / cm 2 Thus, for example, the volume to surface area ratio of the protease may be 0.01 to 0.02 mL / cm. 2 , or 0.011 to 0.016 mL / cm 2 Thus, for example, the volume to surface area ratio of pepsin may be 0.01 to 0.02 mL / cm. 2 , or 0.011 to 0.016 mL / cm 2 Thus, for example, the volume to surface area ratio of TrypLE Select can be 0.01 to 0.02 mL / cm 2 , or 0.011 to 0.016 mL / cm 2 Thus, for example, the volume to surface area ratio of porcine trypsin can be 0.01-0.02 mL / cm. 2, or 0.011 to 0.016 mL / cm 2 It could be.

[0055] The duration of treatment with a dissociating agent may be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes. Thus, for example, the duration of treatment with a proteolytic enzyme may be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes. Thus, for example, the duration of treatment with TrypLE select may be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes. Thus, for example, the duration of treatment with pepsin may be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes. Thus, for example, the duration of treatment with porcine trypsin may be up to 100 minutes, up to 90 minutes, up to 80 minutes, up to 70 minutes, up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes.

[0056] When a dissociation reagent is used and the growth medium contains serum, preferably the cells are washed before using the dissociation reagent. Preferably, washing is performed one, two, or three times before adding the dissociation reagent. Washing can be performed using any of the washing buffers, such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS) or Tris buffered saline (TBS). As used herein, "PBS" is an aqueous salt solution containing disodium hydrogen phosphate, sodium chloride, and in some formulations, potassium chloride and potassium dihydrogen phosphate. As used herein, "Dulbecco's phosphate buffered saline (DPBS)" is a balanced salt solution used for a variety of cell culture applications, such as washing cells before dissociation, transporting cell or tissue samples, diluting cells for counting, and preparing reagents.

[0057] When all cells are removed from the surface of the culture vessel, the cells are washed with the growth medium used for cell expansion. The targeted volume of growth medium used for one wash is determined by the surface area of ​​the culture vessel. The volume-to-surface area ratio of the wash with growth medium should be between 0.04 and 0.06 mL / cm. 2 or 0.046-0.053mL / cm 2 It could be.

[0058] It is to be understood that in the context of the present invention, each of the individual components of the medium described hereinabove is contemplated alone or in combination with each other, based on the cell line used and the virus produced. These components provide all the nutrients, growth factors and other proteins necessary for the growth of adherent cells on the surface of the culture vessel.

[0059] For purposes of the present invention, an operating range must be used that supports robust cell growth for large-scale production and manufacturing of flavivirus vaccines.

[0060] One of the parameters is the cell thawing process. The term "thawing" refers to raising the temperature of the cryopreserved composition or biological material, in this case the cells, to 0°C or higher, preferably 4°C or higher. The term "thawing" may also refer to raising the temperature of the cryopreserved composition or biological material to a temperature where all or part of the cryopreserved composition or biological material is free of ice crystals or substantially free of ice crystals. Thus, the term "thawing" includes full thawing and partial thawing. The duration of the cell thawing process may range from 1 to 5 minutes, or from 3 to 5 minutes. Thus, for example, if the cells used are Vero cells, the duration of thawing may range from 1 to 5 minutes, or from 3 to 5 minutes. After the thawing process, the thawed cells can be stored at ambient temperature. As used herein, "ambient temperature" refers to room temperature between 20 and 22°C. The storage period of the thawed cells can be extended up to 5 minutes, 6 minutes, 7 minutes, or 8 minutes. Thus, for example, if the cells used are Vero cells, the storage period of the thawed cells can be extended up to 5 minutes, 6 minutes, 7 minutes, or 8 minutes.

[0061] Other parameters are the percentage of carbon dioxide (CO2) and the temperature during incubation of the cells in the expansion process. For the purposes of the present invention, the CO2 percentage can be, for example, 2-8%, 4%-6%, or less than 6%, such as 5% or 6%, and a temperature range of 36°C to 39°C, or less than 39°C, such as, for example, 37°C or 38°C.

[0062] Another parameter is to determine the allowable time for cells to remain "dry" during passaging (i.e., if adherent cells are used, the cell monolayer will be without coverage of solution such as growth medium or DPBS removal). The time can be up to 2 hours, preferably less than 2 hours, e.g., 30 minutes, 45 minutes, 60 minutes. As used herein, the term "passaging" refers to the transfer of a cell or cells from a first growth environment to a second growth environment, where the second cell density is less than the first cell density.

[0063] Another parameter is the maximum process duration for passaging, i.e. the period starting from when the cells are outside the incubator and are passed and kept in growth medium. The maximum process time for passaging can be up to 20 hours, preferably less than 20 hours, or less than 15 hours, or less than 10 hours, for example, 5 hours, 6 hours, 7 hours, 8 hours or 9 hours.

[0064] Another parameter is the maximum cell division ratio, which can be set up to 1:8 regardless of the culture vessel used. This parameter depends heavily on the cell density and confluency to which the cells are expanded before infection. In the method of the invention, the minimum confluency of the cells before infection is 80-90%, preferably more than 80%. Thus, in the method of the invention, the cell seeding density is, for example, 1.75×10 4 or 2×10 4 cells / cm 2 1.5×10 etc. 4 ~3×10 4 cells / cm 2 and the preinfection cell density was in the range of 9 × 10 4 ~2×10 5 cells / cm 2 The range is.

[0065] Thus, for large scale flavivirus vaccine production and manufacturing, the duration of the cell thawing process may be in the range of 3-10 minutes, the incubation CO2 percentage may be 2-8%, the cell drying period may be 2 hours, the maximum process period for passaging may be up to 20 hours, and the maximum cell division ratio may be up to 1:8. Thus, for large scale flavivirus vaccine production and manufacturing, the duration of the cell thawing process may be in the range of 3-8 minutes, the incubation CO2 percentage may be 4%-6%, the cell drying period may be less than 2 hours, the maximum process period for passaging may be up to 20 hours, and the maximum cell division ratio may be up to 1:8. Thus, for large-scale flavivirus vaccine production and manufacturing, the duration of the cell thawing process may be in the range of 3-5 minutes, the incubation CO2 percentage may be less than 6%, the cell drying period may be less than 2 hours, the maximum process period for passaging may be up to 20 hours, and the maximum cell division ratio may be up to 1:8.

[0066] Thus, one embodiment of a method for large-scale production and manufacturing of a flavivirus vaccine comprises providing cells in a poloxamer-free growth medium, which comprises expanding the cells to sufficient numbers. It should be understood that in the context of the present invention, each of the individual method steps and / or components described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0067] Infection of cells According to one embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the consecutive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus at a low MOI. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1, represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8. The methods described in this section are performed separately for each of the four serotypes.

[0068] After the cells are expanded to sufficient numbers in the production vessel as described in the previous section, the cells are infected with a medium containing a working virus seed (WVS) containing the flavivirus. This medium is called the infection medium. The infection medium may or may not be the same as the growth medium. The purpose of this step is to ensure that the virus infects the cells and replicates to sufficient potency for collection and purification. Furthermore, if an attenuated virus is used, an additional purpose is that the virus retains its attenuation locus. The WCB is used to produce the master virus seed and working virus seed, MVS and WVS, respectively. As used herein, the term "master virus seed (MVS)" in the sense of this disclosure is the virus intended to be used for the production of the vaccine. The MVS is first used to inoculate the WVS, and then the WVS is used to inoculate the production culture.

[0069] As used herein, "production vessel" refers to a "culture vessel" that is inoculated with an infection medium containing WVS for the purpose of large-scale production and manufacturing of flavivirus vaccines. The term "culture vessel" may refer to any vessel in which cells may be cultured. Culture vessels include, but are not limited to, tissue culture flasks, 96-well plates, culture dishes, culture slides, and rotating wall vessels. For purposes of the present invention, tissue culture flasks that may be used include Corning® CellSTACK® culture chambers or Nunc® CellSTACK® culture chambers. TM EasyFill TM Cell Factory TM For example, the Corning® CellSTACK® culture chamber, which can be used alone or in combination, has a 636 cm 2 1 stack (CF1) with a cell growth area of ​​1,272 cm 2 2 stacks (CF2) with a cell growth area of ​​3,180 cm 2 5 stacks (CF5) with a cell growth area of ​​6,360 cm 2 10 stacks (CF10) with a cell growth area of ​​25,440 cm 2 The stack is 40 stack (CF40) with a cell growth area of ​​1000 x 1000 mm.

[0070] For the purposes of the present invention, maintaining cell viability while facilitating intracellular viral replication and the stability of the produced virus are particularly guaranteed. Therefore, all method steps of the present invention are carried out specifically to achieve these advantages. The method of the present invention also includes the use of an infection medium comprising a WVS containing a flavivirus for the infection of cells. Thus, the present invention includes the use of an infection medium comprising a flavivirus for the infection of cells. In the context of the method of the present invention, the infection medium can be the same as or different from the growth medium used for the expansion of cells.

[0071] Infection medium contains growth medium plus glutamine or GlutaMAX TM and non-ionic surfactants.

[0072] The term "nonionic surfactant" refers to a surfactant that does not contain positively or negatively charged functional groups. In contrast to anionic and cationic surfactants, nonionic surfactants do not ionize in solution. The nonionic surfactant may be selected from block copolymers, sorbitan esters, ethoxylated or propoxylated sorbitan esters, alkyl-polyglycosides (APGs), alkoxylated mono- or di-alkylamines, fatty acid monoethanolamides (FAMAs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GAs, or fatty acid glucamides, FAGAs), and combinations thereof.

[0073] The nonionic surfactant can be a high molecular weight nonionic surfactant. By "high molecular weight" is meant a molecular weight of 1500 or greater. The nonionic surfactant can be a nonionic triblock copolymer. The surfactant can be a nonionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (or EO-PO block copolymer). The EO-PO block copolymer can include blocks of polyethylene oxide (-CH2CHO-designated EO) and polypropylene oxide (-CH2CHCHO-designated PO). The PO block can be adjacent to two EO blocks in an EOx-POy-Eox configuration. Because the PO component is hydrophilic and the EO component is hydrophobic, the overall hydrophilicity, molecular weight, and surfactant properties of the copolymer can be tuned by varying x and y in the EOx-POy-Eox block structure. In aqueous solution, the EO-PO block copolymers self-assemble into micelles with a PO core and a corona of hydrophilic EO groups.

[0074] The non-ionic surfactant can be a poloxamer. A poloxamer is a non-ionic triblock copolymer composed of a central hydrophobic chain of poly(propylene oxide) flanked by two hydrophilic chains of poly(ethylene oxide). The length of the polymer block can be customized, resulting in different poloxamers with slightly different properties.

[0075] As used herein, "EO-PO block copolymer" may refer to a copolymer consisting of blocks of poly(ethylene oxide) and poly(propylene) oxide. Additionally, as used herein, "Pluronic" may refer to an EO-PO block copolymer in EOx-POy-EOx. This configuration of EO-PO block copolymer is also referred to as "poloxamer".

[0076] Thus, the nonionic surfactant may be Pluronic F127 (poloxamer 407), Pluronic F123 (poloxamer 403), Pluronic F-68 (poloxamer 188), Pluronic P123, Pluronic P85, other polyethylene oxide-polypropylene oxide (EO-PO) block copolymers of greater than 3,000-4,000 MW, or combinations thereof. As used herein, "poloxamer 407" is a hydrophilic nonionic surfactant consisting of a triblock copolymer consisting of a central propylene glycol block having about 56 repeat units and two adjacent hydrophilic polyethylene glycol blocks each containing about 101 repeat units. "Poloxamer 407" is also known by its trade names Pluronic F127 and Synperonic PE / F127. As used herein, "Poloxamer 188" (P188) is a non-ionic linear copolymer having an average molecular weight of 8400 Daltons, also referred to as Pluronic F68, FLOCOR and RheothRx. Pluronic P123 is a symmetric triblock copolymer, PEO-PPO-PEO, containing poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) in an alternating linear fashion. Pluronic P85 is a difunctional block copolymer surfactant terminated with primary hydroxyl groups.

[0077] Thus, according to one embodiment of the present invention, the preparation of the infection medium is carried out using glutamine or GlutaMAX TM and non-ionic detergents, and the addition of specific concentrations of glucose, glutamine or GlutaMAX in the infection medium. TMand non-ionic surfactants. The concentration of glucose in the infection medium can be 3 g / L to 4 g / L glucose, 3.1 to 3.2 g / L, 3.2 to 3.3 g / L, 3.3 to 3.4 g / L, 3.4 to 3.5 g / L, 3.5 to 3.6 g / L, 3.6 to 3.7 g / L, 3.7 to 3.8 g / L, 3.8 to 3.9 g / L, or 3.9 to 4.0 g / L. The concentration of glucose can be, for example, 3.11 g / L, 3.22 g / L, 3.33 g / L, 3.44 g / L, 3.55 g / L, 3.66 g / L, 3.77 g / L, 3.88 g / L, or 3.99 g / L. Each of these possibilities represents a separate embodiment of the invention.

[0078] Glutamine or GlutaMAX in infection medium TM The concentration of glutamine or GlutaMAX can be 3.5 mM to 4.5 mM, 3.5 mM to 3.6 mM, 3.6 mM to 3.7 mM, 3.7 mM to 3.8 mM, 3.8 mM to 3.9 mM, 4.0 mM to 4.1 mM, 4.1 mM to 4.2 mM, 4.2 mM to 4.3 mM, 4.3 mM to 4.4 mM, or 4.4 mM to 4.5 mM. TM can be, for example, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4.0 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, or 4.5 mM. Each of these possibilities represents a separate embodiment of the present invention.

[0079] To carry out certain embodiments of the method of the present invention, the addition of a non-ionic surfactant at a certain concentration enhances virus production compared to an infection medium without the addition of a non-ionic surfactant. For example, the concentration of the non-ionic surfactant can be 0.05% to 2.0% (w / v) in the medium. For example, 0.05%, 0.1%, 0.15%, 0.20%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% (w / v). Each possibility represents a separate embodiment of the present invention. For example, the concentration of poloxamer 407 and / or F127 can be 0.05% to 2.0% (w / v) in the medium. For example, 0.05%, 0.1%, 0.15%, 0.20%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% (w / v). Each possibility represents a separate embodiment of the present invention. For example, the concentration of poloxamer 403 and / or F68 can be 0.05% to 2.0% (w / v) in the medium. For example, 0.05%, 0.1%, 0.15%, 0.20%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% (w / v). Each possibility represents a separate embodiment of the present invention. For example, the concentration of P123 can be 0.05% to 2.0% (w / v) in the medium. For example, 0.05%, 0.1%, 0.15%, 0.20%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% (w / v). Each possibility represents a separate embodiment of the present invention. For example, the concentration of P85 can be 0.05% to 2.0% (w / v) in the medium, e.g., 0.05%, 0.1%, 0.15%, 0.20%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% (w / v), with each possibility representing a separate embodiment of the present invention.

[0080] Infection media with and without a pH indicator can be used. The pH indicator of the infection medium can be phenol red. If a pH indicator is not used, for example if the medium does not contain phenol red, visual microbial evaluation can be performed.

[0081] In the context of the present invention, the infection medium preparation strategy is also a factor for controlling the growth of the microorganism. The mode of preparing the infection medium may affect the growth or potency of the virus. For the purposes of the present invention, the infection medium can be prepared either by "aseptically mixing" and homogenizing all the components, or by mixing the components and then "filtering" them depending on the flavivirus being produced. The present invention includes preparing the infection medium in either of the above two modes. Furthermore, the present invention includes using one mode of preparing the infection medium for one flavivirus and another mode of preparing the infection medium for another flavivirus. Furthermore, if viruses with different serotypes are used, the present invention includes producing one serotype in an infection medium prepared in one mode and producing another serotype in an infection medium prepared in another mode. For example, for the purposes of the present invention, if dengue serotypes are produced separately, serotype 4 may be produced in a filtered growth medium, while serotype 1 may be produced in an aseptic mix, or vice versa. Each possibility represents a separate embodiment of the present invention. If filtration is used, preferably a filter smaller than 1 μm is used, more preferably a filter smaller than 0.5 μm.

[0082] The cells provided with growth medium are infected with medium containing the WVS of the flavivirus. This means that each flavivirus and each serotype of each flavivirus has its own WVS from which separate production cultures are initiated for large-scale production and manufacturing of flavivirus vaccines. Infection is a complex biological process involving various operational parameters that affect infection efficiency / yield, i.e., the virus attaching, penetrating, and infecting the cells.

[0083] The infection parameters used in the methods of the present invention have surprisingly been found to be reproducible and allow for large-scale production of flaviviruses in general, and large-scale production of attenuated flaviviruses.

[0084] For the purpose of achieving high infection efficiency / high yield / and / or high virus titer, the method of the present invention involves specific infection parameters.

[0085] One infection parameter is the multiplicity of infection ("MOI"), which refers to the ratio of the number of infectious viral particles deposited in a vessel and / or vessel divided by the number of target cells present in that vessel and / or vessel.

[0086] The probability that a given infectious viral particle will infect a target cell is given by the Poisson distribution.

number

[0087] It is possible to calculate the probability that a target cell is not infected when n = 0. Therefore, the probability that a target cell is infected by a viral particle is 1-P(0).

number

[0088] Therefore, the average percentage of cells that will be infected as a result of inoculation with a given MOI is given by the above formula. For example, when MOI=1, 63.2% of cells are infected. Therefore, those skilled in the art understand that MOI also affects the efficiency of virus production, which is calculated in terms of virus titer.

[0089] The terms "infection efficiency" or "transduction efficiency" or "viral potency" or "viral titer" are used interchangeably and refer to the ability of flavivirus particles to bind to, penetrate and deliver their genome to the cytoplasm of host cells, thereby allowing expression of structural proteins in the host cells. Infection efficiency can be measured using analytical techniques known in the art, such as the immunofocus assay ("IFA assay"). The IFA assay is a well-known analytical technique used in the art. The principle of the assay is based on the classical viral plaque assay, in which serial dilutions of virus are placed on a monolayer of adherent cells from a suitable host. After a period allowing infectious virions to bind and be taken up by the cells, an overlay medium containing a thickening agent is added to prevent the spread of virions. Thus, progeny virions can only infect cells adjacent to the original infected cell. This results in an approximately circular focus of infection for every infectious unit of virus. The immunofocus assay differs from the classical plaque assay in that the site of infection is detected by immunostaining instead of visual observation of cytopathic effects. In particular, the term "viral titer" refers to the number of infectious viral particles, or "transducing units", that result in the infection of target cells. Viral titer in the context of the present invention can be measured using the IFA assay discussed herein above. Viral titer determined by IFA assay results in plaque-forming units (PFU). In one example, immune focus assay can be performed as described in detail in section 2.5 of Brewoo et al. (Vaccine. 2012 February 14; 30(8): 1513-1520. doi: 10.1016 / j.vaccine.2011.11.072). Alternatively, any other analytical method can be used to measure viral titer, for example, viral titer can be measured by a functional assay such as that described in Xiao et al., Exp. Neurobiol. 144:113-124, 1997, or Fisher et al., J. Virol. 70:520-532, 1996, the disclosures of both of which are incorporated herein by reference.

[0090] As used herein, a "high viral titer" is defined as a viral titer greater than or equal to 7.0 log 10 More than 7.5 log PFU / mL, preferably 10 Refers to a viral titer greater than PFU / mL.

[0091] For large-scale vaccine production and manufacturing, one of the biggest challenges is to obtain high virus titers to conserve MVS and WVS stocks from an economic standpoint and at the same time meet the population requirements in a timely manner. This challenge is further exacerbated in large-scale production and manufacturing of attenuated virus vaccines because attenuated viruses may have impaired replication kinetics.

[0092] It has been surprisingly found that due to the method of the present invention, an infection medium containing a flavivirus at a low MOI can be used for large-scale production and manufacture of a flavivirus vaccine. Even more surprisingly, it has been found that a low MOI can be used for large-scale production and manufacture of an attenuated flavivirus vaccine. In this context, the flavivirus can be a dengue virus. In one embodiment, by using the method of the present invention, a low MOI can be used for large-scale production and manufacture of a flavivirus vaccine that provides a higher average virus titer compared to the same method that includes the same process steps but uses a high MOI. Thus, by using the method of the present invention, a low MOI can be used for large-scale production and manufacture of an attenuated flavivirus vaccine that provides a higher average virus titer compared to the same method that includes the same process steps but uses a high MOI. In this context, a high MOI refers to an MOI of more than 0.008. In this context, the flavivirus can be a dengue virus. As used herein, "average virus titer" refers to the average virus titer of at least two harvesting steps.

[0093] As used herein, a "low MOI" refers to a MOI of 0.008 or less, such as less than 0.008, or 0.005 or less, such as less than 0.005, preferably 0.0001 to 0.008, or 0.0001 to 0.005, such as 0.001 to 0.005, for example, 0.0011, 0.0012, 0.0013, 0.0014. , 0.0015, 0.002, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.003, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.004, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, or 0.005. Therefore, for the purposes of the present invention, a low MOI in the range of 0.008 or less can be used. Therefore, for the purposes of the present invention, a low MOI in the range of 0.005 or less can be used. For the methods of the present invention, if flaviviruses with different serotypes are used, the infection of each of the serotypes can be carried out in the low MOI range. It is possible that one serotype infects a medium containing a flavivirus at a particular low MOI within the range of 0.008 or less and 0.005 or less, and another serotype infects a medium containing a flavivirus at another low MOI within the range of 0.008 or less and 0.005 or less, with each possibility representing a separate embodiment of the present invention.

[0094] Thus, by using the method of the present invention, a low MOI can be used for large-scale production and manufacture of flavivirus vaccines that provide high virus titers. Thus, by using the method of the present invention, a low MOI can be used for large-scale production and manufacture of attenuated flavivirus vaccines at high virus titers. In this context, the flavivirus can be a dengue virus. In one embodiment, by using the method of the present invention, a low MOI can be used for large-scale production and manufacture of flavivirus vaccines that provide higher average virus titers compared to the same method that includes the same process steps but uses a high MOI. Thus, by using the method of the present invention, a low MOI can be used for large-scale production and manufacture of attenuated flavivirus vaccines that provide higher average virus titers compared to the same method that includes the same process steps but uses a high MOI. In this context, a high MOI refers to an MOI of more than 0.008. In this context, the flavivirus can be a dengue virus. As used herein, "average virus titer" refers to the average virus titer of at least two harvesting steps.

[0095] In one embodiment, vero cells in more than ten CF-10 flasks are infected at a low MOI with dengue serotype 1, such as dengue 2 / 1 chimera represented by SEQ ID NO:1 and / or SEQ ID NO:2 or TDV-1.

[0096] In one embodiment, vero cells in more than ten CF-10 flasks are infected at a low MOI with dengue serotype 2 or TDV-2 represented by SEQ ID NO:3 and / or SEQ ID NO:4.

[0097] In one embodiment, vero cells in more than ten CF-10 or CF-40 flasks are infected at a low MOI with dengue serotype 3, such as dengue 3 / 1 chimera or TDV-3 represented by SEQ ID NO:5 and / or SEQ ID NO:6.

[0098] In one embodiment, vero cells in more than ten CF-40 flasks are infected at a low MOI with dengue serotype 4, such as dengue 2 / 4 chimera represented by SEQ ID NO:7 and / or SEQ ID NO:8 or TDV-4.

[0099] According to a particular embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium and (ii) infecting the cells of step (i) with the medium containing the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0100] In one embodiment, the large scale production and manufacturing of a flavivirus vaccine with high viral titer involves the use of a cell line that contains an abundant flavivirus receptor. In another embodiment, the large scale production and manufacturing of an attenuated flavivirus vaccine with high viral titer involves the use of a cell line that contains an abundant flavivirus receptor. In this context, the cell may be a Vero cell. In this context, the flavivirus may be a dengue virus and the flavivirus receptor may be a dengue receptor.

[0101] As used herein, "flavivirus receptor" refers to a cell surface receptor. A wide range of cell surface receptors are involved in flavivirus entry into different cell types, such as αvβ3 integrin C-type lectin receptor (CLR), phosphatidylserine receptor TIM (T cell immunoglobulin and mucin domain), and TYRO3, AXL and MER (TAM). As used herein, a cell line that is "enriched" in flavivirus receptor refers to a mosquito cell line, such as C636, that has some overlap with flavivirus receptors, for example, at least one or two common receptors.

[0102] As used herein, "dengue virus vaccine" means either a monovalent vaccine, i.e., one of the four dengue serotypes, a bivalent vaccine, i.e., two of the four serotypes, a trivalent vaccine, i.e., three of the four serotypes, or a quadrivalent vaccine, i.e., all four serotypes.

[0103] As used herein, "dengue virus receptor" refers to a cell surface receptor. These include carbohydrate molecules, lectins, and claudin-1 cell receptors. Carbohydrate molecules such as glycosaminoglycans (GAGs), sulfated polysaccharides, and glycosphingolipids (GSLs) are widely expressed cell surface coreceptors for DENV entry and are believed to enhance virus entry efficiency. As used herein, a cell line that is "enriched" in dengue virus receptors refers to a mosquito cell line such as C636, e.g., a cell line that has some overlap between dengue virus receptors and dengue virus receptors in at least one or two common receptors.

[0104] According to an aspect of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the consecutive steps of (i) providing cells containing abundant flavivirus receptors in a growth medium, and (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0105] Another infection parameter is the volume of infection medium per surface area of ​​the production vessel. For the purposes of the present invention, when using a low MOI range, the volume of infection medium per surface area of ​​the production vessel is such that it covers the monolayer of cells in the tissue culture vessel. However, whether the infection medium covers the cell monolayer or not may depend on the physical method of infection, i.e., either static or rocking.

[0106] Typically, it is understood that for the purpose of large-scale vaccine production, rocking / shaking infection methods should be used to ensure infection efficiency and virus potency. As used herein, "rocking / shaking" refers to either manually rocking the tissue culture vessel or using a rocking platform. The term "static" infection means that the tissue culture vessel is not moving during the infection process, and therefore the infection medium is not mechanically dispersed throughout the cell monolayer. On the other hand, the term "rocking" infection means that the tissue culture vessel is held on a platform that moves gently along a horizontal axis, or the tissue culture vessel is moved manually, resulting in mechanical dispersion of the infection medium throughout the monolayer.

[0107] It has surprisingly been found that the static infection method can be used for large scale production and manufacturing of flavivirus vaccines. Even more surprisingly, it has been found that the static infection method can be used for large scale production and manufacturing of attenuated flavivirus vaccines. In this context, the flavivirus can be a dengue virus.

[0108] Thus, for large-scale flavivirus vaccine production and manufacturing, static infection provides the same infection efficiency and / or virus titer, i.e., higher virus titer, compared to the rocking infection method. Thus, for large-scale flavivirus vaccine production and manufacturing, static infection provides the same infection efficiency and / or virus titer, i.e., higher virus titer, compared to the rocking infection method, when the cell monolayer is covered with infection medium. The term "covered with infection medium" refers to a state in which the cell monolayer is completely or almost completely immersed in infection medium. This will depend greatly on the volume of infection medium used and the tissue culture vessel used. Thus, whenever "static infection" is mentioned, it is understood that the volume of infection medium is such that it covers the cell monolayer.

[0109] Moreover, it has surprisingly been found that static infection in combination with a low MOI range provides high infection efficiency and / or high virus titers for large scale flavivirus vaccine production, and even more surprisingly, for large scale attenuated flavivirus vaccine production. In this context, the flavivirus may be a dengue virus.

[0110] According to an aspect of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium and (ii) statically infecting the cells of step (i) with a medium containing a flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0111] According to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells containing abundant flavivirus receptors in a growth medium, and (ii) statically infecting the cells of step (i) with a medium containing flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0112] Another infection parameter in the sense of the infection method is to infect cells in suspension or in a monolayer. The term "suspension" refers to cells dispersed in a medium. In this context, the cells may be adherent cells that have been dissociated from the culture vessel using a dissociation reagent. Typically, for the purpose of large-scale vaccine production, it is understood that the cells should be infected immediately after using the dissociation agent (when they are still in suspension) to ensure infection efficiency.

[0113] It has surprisingly been found that infection of cells in monolayers can be used for large scale production and manufacture of flavivirus vaccines. Even more surprisingly, it has been found that infection of cells in monolayers can be used for large scale production and manufacture of attenuated flavivirus vaccines. In this context, the cells can be Vero cells. In this context, the flavivirus can be Dengue virus.

[0114] Moreover, it has been surprisingly found that infection of cells in monolayers in combination with a low MOI provides high infection efficiency for large scale flavivirus vaccine production, and even more surprisingly, provides high infection efficiency for large scale attenuated flavivirus vaccine production. In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus.

[0115] Moreover, it has surprisingly been found that infection of cells in monolayers combined with low MOI and static infection provides high infection efficiency for large scale flavivirus vaccine production, and even more surprisingly, provides high infection efficiency for large scale attenuated flavivirus vaccine production. In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus.

[0116] As used herein, the term "monolayer" refers to a layer of cells in which the cells are not, or are not substantially, growing on top of one another, but are all growing side by side, often in contact with each other on the same growth surface.

[0117] According to an embodiment of the present invention, a method for large scale production and manufacturing of a flavivirus vaccine comprises the successive steps of (i) providing cells in a growth medium and (ii) statically infecting the cells of step (i) in a monolayer with a medium containing a flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0118] Thus, infection of cells in monolayers can be used in large scale production and manufacturing of flavivirus vaccines in cell lines containing abundant flavivirus receptors. Furthermore, infection of cells in monolayers combined with low MOI can be used in large scale production and manufacturing of flavivirus vaccines with high infection efficiency and / or high virus titer in cell lines containing abundant flavivirus receptors. Furthermore, infection of cells in monolayers combined with low MOI and static infection can be used in large scale production and manufacturing of flavivirus vaccines for high infection efficiency and / or high virus titer in cell lines containing abundant flavivirus receptors.

[0119] According to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells containing abundant flavivirus receptors in a growth medium, and (ii) statically infecting the cells of step (i) in a monolayer with a medium containing flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0120] Another infection parameter is the duration of infection and the temperature of infection. The duration of infection can be in the range of 50-250 minutes, 60-200 minutes, 70-150 minutes, 70, 80, 90, 100, 110, 120, 130, 140, or 150 minutes. Incubation of the cells during infection can be in the temperature range of 36-39°C, preferably below 39°C.

[0121] After infection, the cells may or may not be washed. If the cells are washed, washing can be performed one, two, three or four times. Washing can be performed using any of the washing buffers, such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS) or Tris buffered saline (TBS).

[0122] Thus, an aspect of the method for large-scale production and manufacturing of flavivirus vaccines includes infecting cells with a medium containing flavivirus at a low MOI. It should be understood that in the context of the present invention, each of the individual method steps and / or component elements described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0123] Collecting to get collectibles After infection of the cells, a harvesting step can be performed to obtain a harvest. Thus, according to an embodiment of the present invention, a method for large-scale production and manufacturing of a flavivirus vaccine comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the virus at a low MOI, and (iii) harvesting to obtain a harvest. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1, represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8. The methods described in this section are performed separately for each of the four serotypes.

[0124] As used herein, the term "harvest" refers to a composition obtained from harvesting, e.g., recovering the supernatant. Harvest refers to any intermediate composition from the time the supernatant is recovered through the first processing step.

[0125] According to the present invention, "harvesting" refers to the act of recovering at least a portion of the medium from the production culture in which the virus is produced and released. As used herein, a "portion" of the medium refers to the supernatant. Thus, "harvesting" refers to recovering the supernatant from the infected cells. In the context of the present invention, "harvesting the supernatant" or "harvesting step" is immediately followed by the addition of fresh medium, such as, for example, growth medium as described herein above. In a preferred embodiment, fresh medium refers to growth medium as described herein above.

[0126] In the context of the present invention, collecting may comprise a first collecting step and at least one further collecting step.

[0127] According to certain embodiments of the present invention, a medium exchange is carried out 12-30 hours, 24 hours, 48 ​​hours, 96 hours or 120 hours prior to the "first harvesting step", comprising harvesting the supernatant, discarding the harvested supernatant and adding fresh medium. Preferably, the medium exchange is carried out 12-30 hours prior to the first harvesting step. Thus, according to an embodiment of the present invention, harvesting comprises a first harvesting step and at least one further harvesting step. In the context of the present invention, the interval between each harvesting step is preferably at least 20-30 hours, 21-27 hours, 24 hours, 48 ​​hours or 96 hours.

[0128] Thus, according to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the virus at a low MOI, and (iii) harvesting to obtain a harvest, the harvesting comprising a first harvesting step and at least one further harvesting step, wherein a medium change is carried out at least 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0129] According to an embodiment of the method of the present invention, the medium change is performed 12 to 30 hours, 24 hours, 48 ​​hours, 96 hours, or 120 hours before the first harvesting step, i.e., the medium change is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after infection, and the first harvesting step is performed 1, 2, 3, or 4 days after the medium change, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after infection, followed by 1, 2, 3, 4, 5, 6, 7, 8, or 9 further harvesting steps. Fresh medium is added to the cells immediately after each harvesting step. The interval between each collection step is at least 20-30 h, 21-27 h, 24 h, 48 h, or 96 h.

[0130] Thus, according to an embodiment of the method of the present invention, the first harvesting step is performed on the second day after infection followed by one further harvesting step on the third day after infection, or the first harvesting step is performed on the third day after infection followed by one further harvesting step on the fourth day after infection, or the first harvesting step is performed on the fourth day after infection followed by one further harvesting step on the fifth day after infection, or the first harvesting step is performed on the fifth day after infection followed by one further harvesting step on the sixth day after infection, or the first harvesting step is performed on the sixth day after infection followed by one further harvesting step on the seventh day after infection. In this context, a medium change is performed 12 to 30 hours before the first harvesting step. Fresh medium is added to the cells immediately after the first harvesting step.

[0131] Thus, according to an embodiment of the method of the present invention, the first harvesting step is carried out on the second day after infection followed by two further harvesting steps on the third and fourth days after infection, or the first harvesting step is carried out on the third day after infection followed by two further harvesting steps on the fourth and fifth days after infection, or the first harvesting step is carried out on the fourth day after infection followed by two further harvesting steps on the fifth and sixth days after infection, or the first harvesting step is carried out on the fifth day after infection followed by two further harvesting steps on the sixth and seventh days after infection, or the first harvesting step is carried out on the sixth day after infection followed by two further harvesting steps on the seventh and eighth days after infection. In this context, a medium change is carried out 12 to 30 hours before the first harvesting step. Fresh medium is added to the cells immediately after each harvesting step.

[0132] Thus, according to an embodiment of the method of the present invention, the first harvesting step is performed on the second day after infection followed by three further harvesting steps on the third, fourth and fifth days after infection, or the first harvesting step is performed on the third day after infection followed by three further harvesting steps on the fourth, fifth and sixth days after infection, or the first harvesting step is performed on the fourth day after infection followed by three further harvesting steps on the fifth, sixth and seventh days after infection, or the first harvesting step is performed on the fifth day after infection followed by three further harvesting steps on the sixth, seventh and eighth days after infection, or the first harvesting step is performed on the sixth day after infection followed by three further harvesting steps on the seventh, eighth and nineth days after infection. In this context, a medium change is performed 12 to 30 hours before the first harvesting step. Fresh medium is added to the cells immediately after each harvesting step.

[0133] Thus, according to an embodiment of the method of the invention, the first harvesting step is carried out on the second day after infection followed by four further harvesting steps on the third, fourth, fifth and sixth days after infection, or the first harvesting step is carried out on the third day after infection followed by four further harvesting steps on the fourth, fifth, sixth and seventh days after infection, or the first harvesting step is carried out on the fourth day after infection followed by four further harvesting steps on the fifth, sixth, seventh and eighth days after infection, or the first harvesting step is carried out on the fifth day after infection followed by four further harvesting steps on the sixth, seventh, eighth and nineth days after infection, or the first harvesting step is carried out on the sixth day after infection followed by four further harvesting steps on the seventh, eighth, nineth and tenth days after infection. In this context, a medium change is carried out 12 to 30 hours before the first harvesting step. Immediately after each harvesting step, fresh medium is added to the cells.

[0134] Thus, according to an embodiment of the method of the present invention, the first harvesting step is performed on day 2 post-infection followed by five further harvesting steps on days 3, 4, 5, 6 and 7 post-infection, or the first harvesting step is performed on day 3 post-infection followed by five further harvesting steps on days 4, 5, 6, 7 and 8 post-infection, or the first harvesting step is performed on day 4 post-infection followed by five further harvesting steps on days 5, 6, 7, 8 and 9 post-infection. In a preferred embodiment, the first harvesting step is performed on day 5 post-infection followed by five further harvesting steps on days 6, 7, 8, 9 and 10 post-infection. In one embodiment, the first harvesting step is performed on day 6 post-infection followed by five further harvesting steps on days 7, 8, 9, 10 and 11 post-infection. In this context, a medium change is carried out 12 to 30 hours before the first harvesting step. Immediately after each harvesting step, fresh medium is added to the cells.

[0135] Thus, according to an embodiment of the method of the present invention, a first harvesting step is performed on day 2 post-infection followed by six further harvesting steps on days 3, 4, 5, 6, 7 and 8 post-infection, or a first harvesting step is performed on day 3 post-infection followed by six further harvesting steps on days 4, 5, 6, 7, 8 and 9 post-infection, or a first harvesting step is performed on day 4 post-infection followed by six further harvesting steps on days 5, 6, 7, 8 and 9 post-infection. Six further harvesting steps are performed on the 5th, 6th, 7th, 8th, 9th and 10th days after infection, or the first harvesting step is performed on the 5th day after infection followed by six further harvesting steps on the 6th, 7th, 8th, 9th, 10th and 11th days after infection, or the first harvesting step is performed on the 6th day after infection followed by six further harvesting steps on the 7th, 8th, 9th, 10th, 11th and 12th days after infection. In this context, a medium change is performed 12 to 30 hours before the first harvesting step. Fresh medium is added to the cells immediately after each harvesting step.

[0136] Thus, according to an embodiment of the method of the present invention, a first harvesting step is performed on day 2 post-infection followed by seven further harvesting steps on days 3, 4, 5, 6, 7, 8, and 9 post-infection, or a first harvesting step is performed on day 3 post-infection followed by seven further harvesting steps on days 4, 5, 6, 7, 8, 9, and 10 post-infection, or a first harvesting step is performed on day 4 post-infection followed by seven further harvesting steps on days 5 post-infection. Alternatively, the first harvesting step is performed on day 5 post-infection followed by seven further harvesting steps on days 6, 7, 8, 9, 10, 11 and 12 post-infection, or the first harvesting step is performed on day 6 post-infection followed by seven further harvesting steps on days 7, 8, 9, 10, 11, 12 and 13 post-infection. In this context, a medium change is performed 12-30 hours before the first harvesting step. Fresh medium is added to the cells immediately after each harvesting step.

[0137] Thus, according to an embodiment of the method of the present invention, a first harvesting step is performed on day 2 post-infection followed by eight further harvesting steps on days 3, 4, 5, 6, 7, 8, 9, 10 and 11 post-infection, or a first harvesting step is performed on day 3 post-infection followed by eight further harvesting steps on days 4, 5, 6, 7, 8, 9, 10 and 11 post-infection, or a first harvesting step is performed on day 4 post-infection followed by eight further harvesting steps on days 5, 6, 7, 8, 9, 10 and 11 post-infection. Alternatively, the first harvesting step is performed on day 5 post-infection followed by eight further harvesting steps on days 6, 7, 8, 9, 10, 11, 12 and 13 post-infection, or the first harvesting step is performed on day 6 post-infection followed by eight further harvesting steps on days 7, 8, 9, 10, 11, 12, 13 and 14 post-infection. In this context, a medium change is performed 12-30 hours before the first harvesting step. Fresh medium is added to the cells immediately after each harvesting step.

[0138] Thus, according to an embodiment of the method of the present invention, a first harvesting step is performed on day 2 post-infection followed by nine further harvesting steps on days 3, 4, 5, 6, 7, 8, 9, 10 and 11 post-infection, or a first harvesting step is performed on day 3 post-infection followed by nine further harvesting steps on days 4, 5, 6, 7, 8, 9, 10, 11 and 12 post-infection, or a first harvesting step is performed on day 4 post-infection followed by nine further harvesting steps on days 5, 6 and 13 post-infection. , 7, 8, 9, 10, 11, 12, 13 and 14 days after infection, or the first harvesting step is performed on day 5 post-infection followed by 9 further harvesting steps on days 6, 7, 8, 9, 10, 11, 12, 13 and 14 after infection, or the first harvesting step is performed on day 6 post-infection followed by 9 further harvesting steps on days 7, 8, 9, 10, 11, 12, 13, 14 and 15 after infection. In this context, a medium change is performed 12-30 hours before the first harvesting step. Fresh medium is added to the cells immediately after each harvesting step.

[0139] According to an embodiment of the present invention, large-scale production and manufacturing of flavivirus vaccines may include a step of medium exchange 12-30 hours before the first daily harvest. For large-scale vaccine production and manufacturing, another challenge is to maintain a balance between high virus titer and removing process-related impurities. This is because the step of removing process-related impurities may also have a negative effect on the virus titer. It has surprisingly been found that a medium exchange 12-30 hours, i.e. less than 48 hours before the first daily harvest, achieves an optimal balance of virus titer waste on the one hand, but reduced concentration of impurities on the other hand.

[0140] As used herein, the term "process-related impurities" refers to any undesirable components such as nucleotides, polynucleotides, non-target proteins (such as host cell proteins, HCPs), other cellular components (such as lipids and glycolipids), and any other contaminants that arise from or during the production, separation, and / or purification process. Other process-related impurities, especially in large-scale vaccine production, include defective interfering particles or DIPs. DIPs are natural by-products of viral replication. DIPs interfere with the growth and spread of infectious standard viruses (STVs), reducing viral yields by competing for viral and cellular resources, and inducing antiviral responses. It has been surprisingly found that the methods of the present invention remove process-related impurities, significantly enhancing viral production, as seen by significant improvements in viral titers.

[0141] It has been surprisingly found that a medium change 12-30 hours, i.e. less than 48 hours, before the first harvesting step in the method of production and manufacture of a flavivirus vaccine does not significantly affect the viral titer compared to no medium change. Furthermore, it has been surprisingly found that a medium change 12-30 hours before the first harvesting step in the method of production and manufacture of a flavivirus vaccine in combination with at least 2, at least 3, at least 4, or most preferably at least 5 further harvesting steps, e.g. 7 or more further harvesting steps, does not significantly affect the viral titer compared to no medium change. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0142] Thus, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step combined with infection of cells at a low MOI provides high virus titers. Further, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step followed by at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, combined with infection of cells at a low MOI range provides high virus titers. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0143] Thus, according to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the virus at a low MOI, and (iii) harvesting to obtain a harvest, the harvesting comprising a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, and a medium change is performed 12 to 30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0144] Thus, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step combined with static infection of cells at a low MOI range provides high virus titers. Furthermore, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step followed by at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, combined with infection of cells at a low MOI provides high virus titers. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0145] Thus, according to an embodiment of the present invention, large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) with a medium containing the flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, the harvesting comprising a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, with a medium change being carried out 12 to 30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0146] Thus, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours prior to the first harvesting step combined with static infection of cells in monolayers at a low MOI provides high virus titers. Furthermore, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours prior to the first harvesting step followed by at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, combined with static infection of cells in monolayers at a low MOI provides high virus titers. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0147] Thus, according to an embodiment of the present invention, large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) in a monolayer with a medium containing the flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, the harvesting comprising a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, with a medium change being carried out 12 to 30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0148] Thus, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step in combination with infection of cells containing abundant flavivirus receptors at a low MOI range provides high virus titers. Furthermore, according to the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step in combination with infection of cells containing abundant flavivirus receptors at a low MOI range provides high virus titers. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0149] Thus, according to an embodiment of the present invention, large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells containing abundant flavivirus receptors in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, the harvesting comprising a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, and a medium change is performed 12 to 30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0150] Thus, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step combined with a static infection of cells containing abundant flavivirus receptors at a low MOI range provides high flavivirus titers. Furthermore, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step followed by at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, combined with a static infection of cells containing abundant flavivirus receptors at a low MOI range provides high virus titers. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0151] Thus, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step combined with a static infection of cells in a monolayer containing abundant flavivirus receptors at a low MOI range provides high flavivirus titers. Furthermore, according to an embodiment of the method of the present invention, for large scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvesting step followed by at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, combined with a static infection of cells in a monolayer containing abundant flavivirus receptors at a low MOI range provides high flavivirus titers. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0152] Thus, according to an embodiment of the present invention, large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells containing abundant flavivirus receptors in a growth medium, (ii) statically infecting the cells of step (i) in a monolayer with a medium containing flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, the harvesting comprising a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven or more further harvesting steps, with a medium change being carried out 12 to 30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a non-ionic detergent. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0153] In certain embodiments, the pH is maintained in the range of 7.6 to 8.1 throughout the above steps.

[0154] Thus, an embodiment of a method for large scale production and manufacturing of a flavivirus vaccine includes a harvesting step, including a first harvesting step and at least one further harvesting step, where a medium change is performed at least 12-30 hours prior to the first harvesting step. In the context of the present invention, it is to be understood that each of the individual method steps and / or component elements described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0155] Processing collections According to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii). In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1, represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8. The methods described in this section are performed separately for each of the four serotypes.

[0156] As discussed above, as used herein, the term "harvest" refers to a composition obtained from a harvesting operation, e.g., collection of the supernatant. "Harvest" or "crude harvest" are used interchangeably and refer to any intermediate composition from the point at which the supernatant is collected through the first processing step.

[0157] The term "processing a harvest" refers to one or more manipulation steps performed on a obtained harvest. Processing a harvest may therefore include one or all of the steps including clarification, stabilization, freezing, thawing, and pooling. In a preferred embodiment, processing a harvest refers to clarification and stabilization, in that order. In a preferred embodiment, a processed harvest refers to a harvest that has been clarified and stabilized, in that order.

[0158] Collection processing: Collection clarification In the context of the present invention, processing the harvest may include clarification of the harvest. The harvest may be clarified to remove cells and cellular debris that may accumulate during the infection process. Clarification of the harvest may include one or more non-membrane filtration steps, such as centrifugation and / or depth filtration. These steps are performed at the time of collection.

[0159] Depth filtration uses porous filtration media to separate particles and solids from liquids. Filter media that can be used for depth filtration can be cellulose acetate, polypropylene, glass fiber fleece, and cellulose acetate protected by polyethersulfone. Filters used for depth filtration can have pore sizes of, for example, 0.1 μm to 1 μm, 0.2 μm to 0.8 μm, or 0.2 μm to 0.45 μm.

[0160] The filter used for the clarification of the harvest can be a heterogeneous bilayer of polyethersulfone with pore sizes of 0.2 μm and 0.45 μm. Thus, for the clarification of the harvest, several heterogeneous bilayer filters can be used, such as Sartoclean (cellulose acetate) 0.8+0.65 μm, Sartopure (polypolypyrene) 0.65 μm, Sartoclean (fiberglass fleece) 0.8+0.65 μm, Sartopore 2 (polyethersulfone) 0.8+0.45 μm, Sartopore 2 (polyethersulfone) 0.45+0.2 μm, or Sartopore 2 XLG (polyethersulfone) 0.8+0.2 μm. Each possibility represents a separate embodiment of the invention.

[0161] At the end of the clarification step, the resulting composition is referred to as the "clarified harvest," which refers to the harvest that has been subjected to the processing step of clarification.

[0162] Thus, according to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) including clarification of the harvest. In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0163] Processing of collections: stabilization of collections In the context of the present invention, processing the harvest may include stabilization. For the method of the present invention, a buffered excipient composition comprising one or more of a surfactant, a sugar, and a protein may be used. The buffered excipient composition may comprise a TRIS buffer, a phosphate buffer, a sodium citrate buffer, a 2-(N-morpholino)ethanesulfonic acid (MES) buffer, a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer, and a salt (e.g., sodium chloride, magnesium chloride, or calcium chloride). In some embodiments, the buffer may be protein-free. In one embodiment, the buffered excipient composition comprises a non-ionic surfactant, a sugar, and a protein in phosphate buffered saline (PBS). The buffered excipient composition is also referred to as a stabilizing buffer. Thus, the terms buffered excipient composition and stabilizing buffer are used interchangeably.

[0164] Thus, the method of the present invention may use a stabilization buffer that includes one or more excipients, including a surfactant, a sugar, and a protein. The stabilization buffer may include a phosphate buffer, a sodium citrate buffer, a 2-(N-morpholino)ethanesulfonic acid (MES) buffer, a 3-morpholinopropane-1-sulfonic acid (MOPS) buffer, and a salt (e.g., sodium chloride, magnesium chloride, or calcium chloride). In some embodiments, the stabilization buffer may be protein-free. In one embodiment, the stabilization buffer includes a non-ionic surfactant, a sugar, and a protein in phosphate buffered saline (PBS).

[0165] As used herein, the term "sugar" includes monosaccharides (e.g., glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabinose), disaccharides (e.g., trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, or fructose), trisaccharides (e.g., acarbose, raffinose, melizitose, panose, or cellotriose), and sugar polymers (e.g., dextran, xanthan, pullulan, cyclodextrin, amylose, amylopectin, starch, cello-oligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, or chitin). Additionally, as used herein, the term "sugar" also includes sugar alcohols such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol.

[0166] The sugar used in the stabilizing buffer may be a non-reducing sugar. A non-reducing sugar does not contain an aldehyde or ketone group that can be oxidized. Examples of non-reducing sugars include sucrose, trehalose, or its hydrates, such as trehalose dihydrate.

[0167] The concentration of the sugar in the stabilizing buffer solution may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, it is 35% to 50% (w / v). The concentration of the non-reducing sugar in the stabilizing buffer solution may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, it is 35% to 50% (w / v). The concentration of the sucrose in the stabilizing buffer solution may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, it is 35% to 50% (w / v). The concentration of trehalose in the stabilizing buffer solution may be 20% to 60% (w / v), 25% to 55% (w / v), 35% to 50%, or 45% (w / v). Preferably, it is 35% to 50% (w / v). The concentration of trehalose dihydrate in the stabilizing buffer solution may be 20% to 60% (w / v), 25% to 55% (w / v), 35% to 50%, or 45% (w / v). Preferably, it is 35% to 50% (w / v).

[0168] To carry out an embodiment of the method of the present invention, the surfactant in the stabilization buffer may be a non-ionic surfactant. The concentration of the non-ionic surfactant in the stabilization buffer may preferably be 0.25% to 5%. For example, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, or 3%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of poloxamer 407 and / or F127 may preferably be 0.25% to 5%. For example, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, or 3%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of poloxamer 403 and / or F123 may preferably be 0.25% to 5%. For example, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, or 3%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of P123 may be preferably 0.25% to 5%. For example, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, or 3%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of P85 may be preferably 0.25% to 5%. For example, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, or 3%. Each possibility represents a separate embodiment of the present invention.

[0169] The protein that may be present in the stabilizing buffer may be any protein that is essentially inert and does not react with the virus. In particular, the protein does not affect the structure or infectivity of the virus. Thus, the protein may be a structural protein or a serum protein. The protein may be selected from albumin, collagen, hydrolyzed collagen, gelatin, and hydrolyzed gelatin.

[0170] Albumins are a family of globular, non-glycosylated proteins that are present especially in the blood of vertebrates. They are water-soluble, moderately soluble in concentrated salt solutions, and undergo thermal denaturation. Albumins suitable for use in the method of the present invention include mammalian serum albumins, such as human serum albumin and bovine serum albumin or lactalbumin. Serum albumin is one of the most common proteins in vertebrate blood and has multiple functions. Human serum albumin is not glycosylated and has a single free thiol group. Human serum albumin can be recombinant human serum albumin or human serum albumin purified from human serum. Preferably, human serum albumin purified from human serum is used.

[0171] As used herein, the term "collagen" refers to an extracellular family of fibrous proteins characterized by their rigid triple-stranded helical structure. Three collagen polypeptide chains ("α chains") are wound around each other to form this helical molecule. The term is also intended to encompass various types of collagen, with the preferred form being type I collagen.

[0172] The term "gelatin" refers to a heterogeneous mixture of water-soluble proteins of high average molecular weight. Gelatin is not found in nature, but is derived from collagen by the action of hydrolysis. Gelatin is obtained by boiling skin, tendons, bones, and ligaments in water. Gelatin is a colorless or slightly yellowish transparent sheet, flake or coarse powder that absorbs in the range of about 5 to about 10 times its weight in water and forms a gel in solution.

[0173] The concentration of the protein in the stabilizing buffer may be 0.1% to 0.5% (w / v), 0.2% to 0.4% (w / v), 0.15% to 0.3% (w / v), or 0.3% (w / v), preferably 0.2% to 0.4% (w / v). The concentration of albumin in the stabilizing buffer may be 0.1% to 0.5% (w / v), 0.2% to 0.4% (w / v), 0.15% to 0.3% (w / v), or 0.3% (w / v), preferably 0.2% to 0.4% (w / v). The concentration of human serum albumin or human serum albumin purified from human serum in the stabilizing buffer can be 0.1% to 0.5% (w / v), 0.2% to 0.4% (w / v), 0.15% to 0.3% (w / v), or 0.3% (w / v), preferably 0.2% to 0.4% (w / v). The concentration of collagen in the stabilizing buffer can be 0.1% to 0.5% (w / v), 0.2% to 0.4% (w / v), 0.15% to 0.3% (w / v), or 0.3% (w / v), preferably 0.2% to 0.4% (w / v). The concentration of gelatin in the stabilizing buffer can be 0.1% to 0.5% (w / v), 0.2% to 0.4% (w / v), 0.15% to 0.3% (w / v), or 0.3% (w / v), preferably 0.2% to 0.4% (w / v).

[0174] The concentration of the excipient in the stabilization buffer in the stabilized harvest is 1 / 7 to 1 / 15 times, preferably 1 / 12 to 1 / 15 times, that in the stabilization buffer. For example, if the concentration of the excipient in the stabilization buffer in the stabilized harvest is 1 / 14 times that in the stabilization buffer, the following exemplary concentrations in the stabilized harvest will result:

[0175] The concentration of sugar in the stabilized harvest can be, for example, 1.4% to 4% (w / v), 1.8% to 3.9% (w / v), 2.5% to 3.6% (w / v), or 3% (w / v). The concentration of non-reducing sugar in the stabilized harvest can be 1.4% to 4% (w / v), 1.8% to 3.9% (w / v), 2.5% to 3.6% (w / v), or 3% (w / v). The concentration of sucrose in the stabilized harvest can be 1.4% to 4% (w / v), 1.8% to 3.9% (w / v), 2.5% to 3.6% (w / v), or 3% (w / v). The concentration of trehalose in the stabilized harvest may be 1.4%-4% (w / v), 1.8%-3.9% (w / v), 2.5%-3.6% (w / v) or 3% (w / v).The concentration of trehalose dihydrate in the stabilized harvest may be 1.4%-4% (w / v), 1.8%-3.9% (w / v), 2.5%-3.6% (w / v) or 3% (w / v).

[0176] To carry out an embodiment of the method of the present invention, the non-ionic surfactant in the stabilized harvest can be 0.01% to 0.4%. For example, 0.1%, 0.125%, 0.14%, 0.16%, 0.18%, or 0.2%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of poloxamer 407 and / or F127 can be 0.01% to 0.4%. For example, 0.1%, 0.125%, 0.14%, 0.16%, 0.18%, or 0.2%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of poloxamer 403 and / or F123 can be 0.01% to 0.4%. For example, 0.1%, 0.125%, 0.14%, 0.16%, 0.18%, or 0.2%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of P123 can be 0.01% to 0.4%. For example, 0.1%, 0.125%, 0.14%, 0.16%, 0.18%, or 0.2%. Each possibility represents a separate embodiment of the present invention. For example, the concentration of P85 can be 0.01% to 0.4%. For example, 0.1%, 0.125%, 0.14%, 0.16%, 0.18%, or 0.2%. Each possibility represents a separate embodiment of the present invention.

[0177] The concentration of albumin in the stabilized harvest may be 0.007%-0.04% (w / v), 0.01%-0.03% (w / v), 0.01%-0.075% (w / v), or 0.02% (w / v). The concentration of human serum albumin or human serum albumin purified from human serum in the stabilized harvest may be 0.007%-0.04% (w / v), 0.01%-0.03% (w / v), 0.01%-0.075% (w / v), or 0.02% (w / v). The concentration of collagen in the stabilized collection may be 0.007%-0.04% (w / v), 0.01%-0.03% (w / v), 0.01%-0.075% (w / v), or 0.02% (w / v). The concentration of gelatin in the stabilized collection may be 0.007%-0.04% (w / v), 0.01%-0.03% (w / v), 0.01%-0.075% (w / v), or 0.02% (w / v).

[0178] For other ratios, such as 1 / 7 to 1 / 15, or 1 / 12 to 1 / 15, one of skill in the art can readily calculate the resulting concentration of excipient in the stabilized harvest.

[0179] A composition referred to as a "stabilized harvest" refers to a crude harvest that has been subjected to a process step of stabilization or a crude harvest that has been subjected to the process steps of clarification and stabilization in any order. Thus, the term clarified stabilized harvest refers to a crude harvest that has been clarified and stabilized in that order. Thus, the term stabilized clarified harvest refers to a crude harvest that has been stabilized and clarified in that order.

[0180] Thus, according to an embodiment of the present invention, a method for large scale viral vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium comprising a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) including stabilization of the harvest. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated viral vaccine. Thus, according to an embodiment of the present invention, a method for large scale viral vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium comprising a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. Thus, according to an embodiment of the present invention, a method for large scale production and manufacturing of a virus vaccine comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) including stabilization and clarification of the harvest. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine.

[0181] Processing of collections: freezing, thawing and pooling Freezing is another processing modality that can be used to store and freeze the composition at any point during processing below -30°C.

[0182] Thawing is another processing mode and refers to adapting a frozen composition to room temperature.

[0183] Pooling is another processing regime and involves pooling one or more compositions at any point during processing in order to perform further processing steps on the pooled compositions or to perform purification steps on the pooled processed collection.

[0184] According to embodiments of the invention, each "harvest" or "crude harvest" may be frozen immediately after the harvesting step, subsequently thawed, and then subjected to one or several processing or purification steps on the processed harvest. Furthermore, according to embodiments of the method of the invention, the first harvest obtained from the first harvesting step may be frozen immediately after the harvesting step, subsequently thawed, pooled with at least one further harvest obtained from at least one further harvesting step, and then subjected to one or more further processing or purification steps.

[0185] Furthermore, according to embodiments of the methods of the present invention, the first harvest resulting from the first harvesting step may be immediately clarified or may then be immediately frozen, subsequently thawed and pooled with at least one further clarified harvest (which may or may not have been frozen and thawed) resulting from at least one further harvesting step, and then subjected to one or more further processing or purification steps.

[0186] Furthermore, according to embodiments of the methods of the present invention, the first harvest resulting from the first harvesting step may be immediately clarified and stabilized, or may then be immediately frozen, subsequently thawed and pooled with at least one further clarified and stabilized harvest (which may or may not have been frozen and thawed) resulting from at least one further harvesting step, and then subjected to one or more further processing or purification steps.

[0187] In a preferred embodiment, the first harvest resulting from the first harvesting step is immediately clarified and stabilized on the same day.

[0188] As used herein throughout this disclosure, "immediately on the same day" or "on the same day" refers to a time interval ranging from a few minutes to a few hours, but less than 20 hours, preferably less than 10 hours, or less than 12 hours, such as 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, or 5 hours or less, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less.

[0189] Purification of the processed harvest: at least one chromatography step In a preferred embodiment, the first harvest resulting from the first harvesting step is immediately clarified, stabilized and purified in at least one chromatography step on the same day.

[0190] Purifying the processed harvest may include a step of removing host cell DNA. The main purpose of this step is to remove host cell DNA (HC DNA) while ensuring maximum virus recovery. "Host cell DNA" originates from the cells in which the infectious viral particles were produced. Host cell DNA can be distinguished from viral nucleic acid by its nucleic acid sequence, which differs from the nucleic acid sequence of the viral nucleic acid. Several methods for removing HC DNA are known in the art. These are either enzymatic methods or enzymatic methods combined with chromatography-based methods.

[0191] In the context of the present invention, enzymatic methods can be used for the removal of host cell DNA, followed by one or more chromatography steps. One of the enzymatic methods is enzymatic degradation using endonucleases. These enzymes act on nucleic acids by specifically catalyzing the hydrolysis of internal phosphodiester bonds in DNA and RNA chains, breaking them down into smaller nucleotides. The smaller nucleotides / nucleic acid fragments and endonucleases can then be easily removed from the process during subsequent downstream processing.

[0192] In the context of the present invention, purification of the processed harvest may necessarily include a chromatography step. Regardless of the fact that an enzymatic method is used for HC DNA removal or not, a chromatography step is preferably necessarily included and may be ion exchange chromatography (i.e., either cation exchange chromatography or anion exchange chromatography).

[0193] In an embodiment of the method of the present invention, the chromatography step for host cell DNA removal can be performed in a bind and elute or flow-through mode. In the bind and elute mode, the substance to be purified binds to the anion exchange group, while the impurities do not. The substance to be purified can be eluted from the anion exchange group by changing one or more chromatographic conditions, such as the salt conditions in the buffer or the pH of the buffer. In the flow-through mode, the impurities bind to the anion exchange group, while the substance to be purified does not bind, but can be directly recovered from the flow-through.

[0194] Ion exchange chromatography (i.e., cation and anion exchange chromatography, respectively) relies on charge-charge interactions between components in the sample and the charges of the immobilized functional groups. In anion exchange chromatography, the bound ions present in the sample are negative and the immobilized functional groups are positive, whereas in cation exchange chromatography, the bound ions present in the sample are positive and the immobilized functional groups are negative.

[0195] Commonly used anion exchange functional groups are Q-resin, quaternary amine, and DEAE resin (diethylaminoethane). Commonly used cation exchange groups are CM, quaternary amine, S, methylsulfonate, and SP, sulfonyl group. In general, however, the anion or cation exchange chromatography step can be carried out using all common commercially available anion or cation exchange resins or membranes.

[0196] Typical strong anion exchange groups that can be used for the purposes of the present invention include functional groups such as quaternary aminoethyl (QAE) moieties, primary amines (PA), quaternary ammonium (Q) moieties, and trimethylammonium ethyl (TMAE) groups. Resins with quaternary aminoethyl (QAE) moieties include, for example, Toyopearl QAE (available from Tosoh Bioscience, Germany), Selectacel QAE (quaternary aminoethyl derivative of cellulose, available from Polysciences Inc., Pennsylvania USA), and others. Resins with quaternary ammonium (Q) moieties include, for example, Sartobind® Q (available from Sartorius, Germany), Mustang® Q Acrodisc (available from Pall, Germany), Q Sepharose XL, Q Sepharose FF, Q Sepharose HP, Resource Q (available from GE Healthcare, Germany), Macro Prep High Q (Bio-Rad, California, USA), Toyopearl Super Q (available from Tosoh Bioscience, Germany), and UNOsphere Q (available from Bio-Rad, California, USA). Resins with trimethylammonium ethyl (TMAE) groups include, for example, Fractogel EMD TMAE (available from Merck, Germany). Resins with primary amine groups include Sartobind STIC primary amine resin (available from Sartorius, Germany).

[0197] For example, the anion exchange chromatography step can be performed using an anion exchange chromatography membrane having quaternary ammonium groups. The membrane base material can be selected from stabilized reinforced cellulose and polyethersulfone. The membrane base material can be stabilized reinforced cellulose and the functional groups are quaternary ammonium groups. The anion exchange chromatography step can or can not involve the use of a monolithic support. The nominal pore size of the membrane can be between 0.5 μm and 5 μm, or greater than 3 μm. The membrane area can be between 20 and 50 cm 2 , 25~45cm 2 , 30~40cm 2 , for example, 32cm 2 , 34cm 2 , 36cm 2 , or 38cm 2 The flow rate can be 1-50 ml / min, or 10-40 ml / min, depending on the column used.

[0198] For example, the cation exchange chromatography step can be performed using a cation exchange chromatography membrane with quaternary amine groups. The membrane base material can be selected from stabilized reinforced cellulose and polyethersulfone. The cation exchange chromatography step may or may not involve the use of a monolithic support. The nominal pore size of the membrane can be between 0.5 μm and 5 μM, or greater than 3 μm. The membrane area can be between 20 and 50 cm. 2 , 25~45cm 2 , 30~40cm 2 The flow rate may be from 1 to 50 ml / min, or from 10 to 40 ml / min.

[0199] In a particular embodiment of the method of the invention, the host cell DNA is removed from the sample containing viral particles and host cell DNA. The term "removing host cell DNA" means that the content of host cell DNA after the method of the invention is performed is lower than the content of host cell DNA before the method of the invention is performed. In one embodiment, the method of the invention results in at least a 10-fold reduction in the host cell DNA content. Preferably, the method of the invention results in at least a 12-fold or 15-fold reduction in the host cell DNA content, more preferably at least an 18-fold or 20-fold reduction, most preferably at least a 22-fold reduction. The fold reduction can be calculated by dividing the host cell DNA content in the sample before the method is performed by the host cell DNA content in the sample after the method is performed. Preferably, the content of host cell DNA after the method of the invention is performed is lower than the detection limit of the assay used to determine the host cell DNA content. In one embodiment, the content of host cell DNA after the method of the invention is performed is 50 ng / ml or less.

[0200] The method for determining the host cell DNA content in the biological sample obtained from the host cell is known in the art, and includes quantitative PCR using primers that specifically bind to the host cell DNA but do not bind to the viral nucleic acid.Kits for determining the host cell DNA content are commercially available, for example, from ThermoFisher.Preferably, Picogreen® dye is used to determine the host cell DNA content.

[0201] Typically, for large-scale vaccine production, the harvest, or the clarified harvest, or the clarified and stabilized harvest, or the stabilized and clarified harvest, is first concentrated in an ultrafiltration step before loading it into a chromatography column, such as a hydrophobic interaction chromatography column, or an ion exchange chromatography column, i.e., an anion exchange chromatography column or a cation exchange chromatography column. This is mainly because concentrating the harvest before column chromatography allows for faster loading and increases the column capacity to improve productivity. Furthermore, it is understood that ultrafiltration before a chromatography step typically increases the efficiency of the chromatography. Furthermore, it is understood that ultrafiltration of the harvest, or the clarified harvest, or the clarified and stabilized harvest, or the stabilized and clarified harvest, before a chromatography step typically requires less dilution to adjust the pH and conductivity for the chromatography step, ultimately increasing column productivity, reducing the use of consumables, and contributing to improved process economics. As used herein, "concentration" means that the total volume of the harvest is reduced, and therefore the concentration of the components in the harvest is increased.

[0202] As used herein, ultrafiltration (UF) refers to a technique that relies on the use of polymeric membranes with highly defined pore sizes to separate molecules according to size. Simply put, the UF procedure relies on the use of fluid pressure to direct the movement of smaller molecules through the UF membrane with the simultaneous retention of larger molecules. Ultrafiltration can be performed in one of two modes of operation: direct flow filtration (DFF), or tangential flow filtration (TFF).

[0203] The chromatography step can be performed directly on the harvest, or on the clarified harvest, or on the clarified and stabilized harvest, or on the stabilized and clarified harvest, i.e. directly without any prior chromatography step and / or without a prior ultrafiltration step, which has surprisingly been found to not result in a substantial loss of virus titer compared to when the harvest is first concentrated with a membrane filtration step or by a prior chromatography step.

[0204] As used herein, the term "feed composition" refers to the solution that is introduced into a chromatography column or ultrafiltration membrane. The feed composition is directly related to how the harvest is treated before the chromatography step, for example by clarification, or stabilization, or clarification and stabilization. Thus, it has been surprisingly found that the clarification, stabilization, or clarification and stabilization of the harvest is followed directly by a chromatography step, and does not result in substantial loss of virus titer, compared to when the same feed composition is first concentrated in a membrane filtration step, such as a TFF step.

[0205] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: a direct chromatography step on the clarified and stabilized harvest, followed by ultrafiltration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the harvesting step, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by a chromatography step, followed by ultrafiltration are all performed on the same day.

[0206] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: a direct chromatography step on the clarified and stabilized harvest, followed by tangential flow filtration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the harvesting step, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by a chromatography step, followed by tangential flow filtration are all performed on the same day.

[0207] Thus, according to an aspect of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following sequential steps: a direct ion exchange chromatography step on the clarified and stabilized harvest, followed by ultrafiltration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the harvesting steps, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by ion exchange chromatography, followed by ultrafiltration are all performed on the same day.

[0208] Thus, according to an aspect of the invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following sequential steps: a direct ion exchange chromatography step on the clarified and stabilized harvest, followed by tangential flow filtration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the steps of harvesting, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by ion exchange chromatography, followed by tangential flow filtration are all performed on the same day.

[0209] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following sequential steps: a direct anion exchange chromatography step on the clarified and stabilized harvest, followed by ultrafiltration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the harvesting steps, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by anion exchange chromatography, followed by ultrafiltration are all performed on the same day.

[0210] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: a direct anion exchange chromatography step on the clarified and stabilized harvest, followed by tangential flow filtration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the harvesting steps, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by anion exchange chromatography, followed by tangential flow filtration are all performed on the same day.

[0211] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following sequential steps: a direct cation exchange chromatography step on the clarified and stabilized harvest, followed by ultrafiltration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the harvesting steps, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by cation exchange chromatography, followed by ultrafiltration are all performed on the same day.

[0212] Thus, according to an aspect of the invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following sequential steps: a direct cation exchange chromatography step on the clarified and stabilized harvest, followed by tangential flow filtration. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, the steps of harvesting, harvest clarification, harvest stabilization, purification of the clarified and stabilized harvest by cation exchange chromatography, followed by tangential flow filtration are all performed on the same day.

[0213] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest, (v) purifying the processed harvest of step (iv) by the following consecutive steps: (va) direct ion exchange chromatography on the processed harvest and (vb) tangential flow filtration on the purified harvest obtained in step (va). In this context, the cells can be Vero cells. In this context, the flavivirus can be Dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, steps (iii), (iv) and (v) are all performed on the same day.

[0214] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest, (v) purifying the processed harvest of step (iv) by the following consecutive steps: (va) anion exchange chromatography directly on the processed harvest and (vb) tangential flow filtration on the purified harvest obtained in step (va). In this context, the cells can be Vero cells. In this context, the flavivirus can be Dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, steps (iii), (iv) and (v) are all performed on the same day.

[0215] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest, (v) purifying the processed harvest of step (iv) by the following consecutive steps: (va) direct cation exchange chromatography on the processed harvest and (vb) tangential flow filtration on the purified harvest obtained in step (va). In this context, the cells can be Vero cells. In this context, the flavivirus can be Dengue virus. In this context, the vaccine can be a live attenuated virus vaccine. Preferably, steps (iii), (iv) and (v) are all performed on the same day.

[0216] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following successive steps: (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) in a monolayer with medium containing the virus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest, (v) purifying the processed harvest of step (iv) by the successive steps of (va) direct ion exchange chromatography on the processed harvest and (vb) tangential flow filtration on the purified harvest obtained in step (va). In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. In this context, harvesting comprises a first harvesting step and at least three further harvesting steps. In this context, a medium change is performed 12 to 30 hours before the first harvesting step. Preferably, steps (iii), (iv) and (v) are all carried out on the same day.

[0217] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following successive steps: (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) in a monolayer with medium containing the virus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest, (v) purifying the processed harvest of step (iv) by the successive steps of (va) anion exchange chromatography directly on the processed harvest and (vb) tangential flow filtration on the purified harvest obtained in step (va). In this context, the cells may be Vero cells. In this context, the flavivirus may be Dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. In this context, harvesting comprises a first harvesting step and at least three further harvesting steps. In this context, a medium change is performed 12 to 30 hours before the first harvesting step. Preferably, steps (iii), (iv) and (v) are all carried out on the same day.

[0218] Thus, according to an embodiment of the present invention, high virus titers can be obtained when large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) in a monolayer with medium containing the virus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) including clarification and stabilization of the harvest, (v) purifying the processed harvest of step (iv) by the following consecutive steps: (va) direct cation exchange chromatography on the processed harvest and (vb) tangential flow filtration on the purified harvest obtained in step (va). In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated virus vaccine. In this context, the harvesting comprises a first harvesting step and at least three further harvesting steps. In this context, the medium change is carried out 12 to 30 hours prior to the first harvesting step, and preferably steps (iii), (iv) and (v) are all carried out on the same day.

[0219] At the end of one or more chromatography steps, the resulting composition is referred to as a "purified harvest," which refers to a processed harvest that has been subjected to at least one chromatography step.

[0220] The term "clarified, purified harvest" refers to a crude harvest that has been subjected to a clarification step and one or more chromatographic steps, in that order. The term "clarified, stabilized, purified harvest" refers to a crude harvest that has been subjected to a clarification step, a stabilization step, and one or more chromatographic steps, in that order. The term stabilized, clarified, purified harvest refers to a crude harvest that has been subjected to a clarification step, one or more chromatographic steps, and a stabilization step, in that order.

[0221] In one embodiment, by using the method of the present invention described hereinabove, a low MOI can be used for the large-scale production and manufacture of flavivirus vaccines, which provides a higher average virus titer than the same method with the same process steps but using a high MOI. Thus, by using the method of the present invention, a low MOI can be used for the large-scale production and manufacture of attenuated flavivirus vaccines, which provides a higher average virus titer than the same method with the same process steps but using a high MOI. In this context, a high MOI refers to an MOI of more than 0.008. In this context, the flavivirus can be a dengue virus. As used herein, "average virus titer" refers to the average virus titer of at least two harvesting steps.

[0222] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), wherein step (v) comprises at least one chromatography step; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided, wherein the difference is 0.4 units or less.

[0223] In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), wherein step (v) comprises at least one chromatography step; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided in which the difference is 0.3 units or less. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0224] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), wherein step (v) comprises at least one chromatography step; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided, wherein the difference is 0.4 units or less. In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method results in a higher virus yield compared to the same method with the same process steps, but allows the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0225] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), wherein step (v) comprises at least one chromatography step; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided in which the difference is 0.3 units or less. In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0226] mg / cm 2 Calculation of virus yield at 1000 s can be performed as follows when using one type of flask: (i) Total number of flasks × cm 2 Area of ​​each flask = total area of ​​unit (ii) Total virus yield in milligrams / cm obtained from step (i) above 2 Unit total area = virus yield / cm 2

[0227] When two or more types of flasks are used, mg / cm 2 Calculation of unit virus yield can be performed as follows: (i) (number of type 1 flasks) × (cm 2 (area of ​​each type 1 flask in units) + (number of type 2 flasks) × (cm 2 (area of ​​each flask) = total area (ii) Total virus yield in milligrams / cm obtained from step (i) above 2 Unit total area = virus yield / cm 2

[0228] Purification of the processed harvest: Ultrafiltration In the context of the present invention, the step of purifying the harvest may comprise a step of ultrafiltration to obtain the drug substance.

[0229] Thus, the feed composition, i.e., the input to the ultrafiltration step, is either a clarified, purified harvest, a clarified, stabilized, purified harvest, a stabilized, clarified, purified harvest, a stabilized, purified harvest, or a purified only harvest.

[0230] The purpose of implementing an ultrafiltration step in the method of the present invention is to minimize the harvest volume without affecting the potency / recovery of the virus. According to an embodiment of the present invention, the ultrafiltration step can be a direct flow filtration (DFF) or a tangential flow filtration step (TFF).

[0231] According to an embodiment of the present invention, a method for large scale viral vaccine production and manufacturing comprises the successive steps of: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv) comprising at least one chromatography step and an ultrafiltration step. Preferably, steps (iii), (iv), and (v) are all performed on the same day.

[0232] According to an embodiment of the present invention, a method for large scale viral vaccine production and manufacturing comprises the sequential steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv) comprising at least one chromatography step and a direct flow filtration step. Preferably, steps (iii), (iv) and (v) are all performed on the same day.

[0233] According to an embodiment of the present invention, a method for large scale viral vaccine production and manufacturing comprises the successive steps of: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with a medium containing a flavivirus at a low MOI; (iii) harvesting to obtain a harvest; (iv) processing the harvest of step (iii) to obtain a processed harvest; and (v) purifying the processed harvest of step (iv) comprising at least one chromatography step and a tangential flow filtration step. In this context, the cells may be Vero cells. In this context, the flavivirus may be a dengue virus. In this context, the vaccine may be a live attenuated viral vaccine. Preferably, steps (iii), (iv) and (v) are all performed on the same day.

[0234] Direct flow filtration (DFF) is applied when, due to the feed pressure, all the fluid to be filtered is directed in a direction perpendicular to the filtration surface. As contaminants are trapped in the filtration media or accumulate on the surface and blocked over the duration of the filtration process, the differential pressure across the filter increases. The filtrate leaves the filter downstream. When a certain differential pressure is reached, after which the fluid flow rate decreases and / or the filter reaches its end differential pressure, filtration is stopped and the filter may be discarded or regenerated for reuse. On the other hand, tangential flow filtration (TFF) is a process in which the feed stream flows parallel to the membrane surface. The applied pressure causes a portion of the flow stream to pass through the membrane (filtrate), while the remaining portion (retentate) is returned to the feed reservoir for recirculation. Thus, in TFF, the majority of the feed moves tangentially across the surface of the filter, rather than into the filter.

[0235] After at least one processing step, the resulting composition can be subjected to one or more tangential flow filtration (TFF) steps, one TFF step and one DFF step, or one TFF step, which serves to concentrate the sample to ensure sufficient potency and remove small molecular weight impurities.

[0236] Suitable filter materials for TFF can be crosslinked cellulose and polyethersulfone. Preferably, crosslinked cellulosic polymers available from Sartorius under the name Hydrosart are used. The cut-off size of the filtration material used for tangential flow filtration determines whether a particular compound is present in the filtrate or retentate. The cut-off size can be less than 1 / 3 to 1 / 5 of the size of the virus to be purified. For embodiments of the method of the present invention, the cut-off size can be 50 to 300 kDa. Within the range of 50 to 300 kDa, the cut-off size can be 100 kDa. Thus, for the purposes of the present invention, a crosslinked cellulosic polymer filter with a cut-off size of 100 kDa can be used for the TFF step. Typical TFF membranes used for this step include molecular weight cut-off (MWCO) sizes of 100 kDa (Hydrosart) and polyethersulfone (PES) of 300 kDa.

[0237] The TFF process can be characterized by the flow rate and the transmembrane pressure difference. The flow rate is proportional to the transmembrane pressure difference and inversely proportional to the resistance of the membrane and the filter cake. The flow rate is given as the feed flow rate per unit area of ​​the membrane, which is liters per square meter per hour (LMH). In the TFF step of the present invention, the flow rate can be 100-500 LMH. Within the range of 100-500 LMH, the flow rate can be 120-400 LMH, 150-300 LMH, or 300 LMH. The transmembrane pressure difference (TMP) is the pressure difference between the two sides of the membrane. The TMP can be 0.2-0.5 bar, 0.25-0.45 bar, 0.30-0.45 bar, 0.35-0.45 bar, or 0.35-0.40 bar, or 0.30-0.50 bar.

[0238] Two basic filter configurations are commonly used in TFF: cartridge filters and cassette filters. In cartridge filters (often called hollow fiber filters), the membranes form a set of parallel hollow fibers. The feed stream passes through the lumen of the fibers, and the permeate is collected from the outside of the fibers. In cassette filters, several flat sheets of membranes are held apart from each other and from the cassette housing by a support screen. The feed stream passes through the space between the two sheets, and the permeate is collected from the opposite side of the sheets. In embodiments of the TFF step in the method of the present invention, both cartridge or cassette filters can be used. In other embodiments of the TFF step in the method of the present invention, only cartridge filters can be used. Alternatively, in the TFF step in the method of the present invention, only cassette filters can be used.

[0239] In the TFF step, the solution containing the virus for flavivirus vaccine production is in the retentate. The obtained retentate is also a drug substance. After the TFF step, the feed volume can be concentrated, for example, 2-10x, 3-8x, 4-6x, or 5x. At the end of the TFF step, a drug substance is obtained. In one embodiment, the feed can be a clarified and purified harvest. In one embodiment, the feed can be a clarified, stabilized and purified harvest. In one embodiment, the feed can be a stabilized, clarified and purified harvest. In one embodiment, the feed can be a stabilized and purified harvest. In one embodiment, the feed can be a purified harvest.

[0240] In certain embodiments, the pH is maintained in the range of 7.6 to 8.1 throughout the above steps.

[0241] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided in which the difference is 0.4 units or less. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0242] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided in which the difference is 0.3 units or less. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0243] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided, wherein the difference is 0.4 units or less. In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method results in a higher virus yield compared to the same method with the same process steps, but allows the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0244] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is A method is provided in which the difference is 0.3 units or less. In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0245] Thus, an embodiment of the method for large-scale flavivirus vaccine production and flavivirus vaccine manufacturing includes a step of processing the harvest and a step of purifying the processed harvest, which includes at least one chromatography step followed by an ultrafiltration step. The purification step includes an ultrafiltration step after the chromatography step. At the end of the ultrafiltration step, a drug substance is obtained. If a bivalent, trivalent or tetravalent final drug product is intended, the drug substance at this step still includes each individual monovalent drug substance. It should be understood that in the context of the present invention, each of the individual method steps and / or component components described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove alone and in combination with each other represents a separate embodiment of the present invention.

[0246] Processing of API to obtain bulk API According to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the following consecutive steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) to obtain a processed harvest, (v) purifying the processed harvest of step (iv) comprising at least one chromatography step to obtain a purified harvest, (v) an ultrafiltration step to obtain a drug substance, (vi) processing the drug substance obtained in step (v) comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising the buffer flush and the drug substance, which is the bulk drug substance. Preferably, the ultrafiltration is a tangential flow filtration. Preferably, the chromatography step is an anion exchange chromatography. In this context, the cells may be Vero cells. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1, represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8. The methods described in this section are performed separately for each of the four serotypes.

[0247] The TFF step for the purposes of the present invention may or may not be followed by diafiltration. Diafiltration is a fractionation process that washes smaller molecules through the membrane, leaving larger molecules in the retentate without changing the final concentration. It can be used to remove salts or exchange buffer. In a preferred embodiment, the method of the present invention includes flushing the filtration membrane with buffer 1, 2, 3, 4, or 5 times to obtain a buffer flush and a composition containing drug substance that is bulk drug substance. Preferably, the steps of harvesting, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest by chromatography steps and tangential flow filtration, followed by flushing steps, are all performed on the same day.

[0248] The TFF step for the purposes of the present invention may or may not be followed by diafiltration. Diafiltration is a fractionation process that washes smaller molecules through the membrane and leaves larger molecules in the retentate without changing the final concentration. It can be used to remove salts or exchange buffer. In an exemplary embodiment, the method of the present invention includes a step of flushing the filter membrane with buffer 1, 2, 3, 4 or 5 times to obtain a buffer flush, and preferably, the steps of harvesting, harvest clarification, harvest stabilization, chromatography step and purification of the clarified and stabilized harvest by tangential flow filtration, followed by flushing step are all performed on the same day.

[0249] After the TFF step, the composition comprising the drug substance and a buffer flush is the bulk drug substance. Thus, according to an embodiment of the present invention, the bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least two buffer flushes of the TFF membrane. Thus, according to an embodiment of the present invention, the bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least three buffer flushes of the TFF membrane. Thus, according to an embodiment of the present invention, the bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least four buffer flushes of the TFF membrane.

[0250] After the TFF step, the drug substance and the buffer flushes can be mixed to obtain the bulk drug substance. Thus, according to an exemplary embodiment of the present invention, the bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least two buffer flushes of the TFF membrane. Thus, according to an embodiment of the present invention, the bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least three buffer flushes of the TFF membrane. Thus, according to an embodiment of the present invention, the bulk drug substance is a composition comprising the drug substance (TFF retentate) and at least four buffer flushes of the TFF membrane.

[0251] The flushing buffer comprises the same buffered excipient composition / stabilizing buffer that was added in the harvest processing step, more specifically in the harvest stabilization step, except that the excipients in the flushing buffer are at a lower concentration compared to the buffered excipient composition / stabilizing buffer.

[0252] The flushing buffer comprises one or more excipients including, for example, a non-ionic surfactant, a non-reducing sugar, and albumin, all of which are described in the section on stabilization of the harvest. The flushing buffer used for flushing may comprise F127, trehalose dihydrate, and human serum albumin. The buffer used for flushing preferably comprises 0.5-5% non-ionic surfactant, 20-40% non-reducing sugar, and 0.05-0.3% albumin. The buffer used for flushing preferably comprises 0.5-5% F127, 20-40% trehalose dihydrate, and 0.05-0.3% human serum albumin.

[0253] The flushing buffer may contain the same excipients as the stabilizing buffer added in step (ii) of the method, including sugars, non-ionic surfactants, and proteins. Thus, in a preferred embodiment, the flushing buffer contains the same excipients as the stabilizing buffer added in step (ii) of the method, including sugars, non-ionic surfactants, and proteins, except that the excipients in the flushing buffer are at a lower concentration compared to the excipients in the stabilizing buffer.

[0254] The flushing buffer may contain the same excipients as the stabilized or purified harvest, including sugars, non-ionic surfactants, and proteins. Thus, in a preferred embodiment, the flushing buffer contains the same excipients as the stabilized or purified harvest, including sugars, non-ionic surfactants, and proteins, except that the excipients in the flushing buffer are at a higher concentration compared to the excipients in the stabilized or purified harvest.

[0255] The flushing buffer may contain the same excipients as the drug substance, including sugars, non-ionic surfactants, and proteins. In a preferred embodiment, the flushing buffer contains the same excipients as the drug substance, including sugars, non-ionic surfactants, and proteins, except that the sugars in the flushing buffer are at a higher concentration compared to the sugars in the drug substance.

[0256] Thus, an embodiment of the process for large scale production and manufacturing of a flavivirus vaccine comprises a further step (vi) of processing the drug substance to obtain a drug substance-containing composition that is the bulk drug substance, comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer, the flushing buffer comprising a sugar, a surfactant, and a protein. Preferably, steps (iii), (iv), (v), and (vi) are all performed on the same day.

[0257] In an exemplary embodiment, the method for large scale production and manufacturing of a flavivirus vaccine comprises a further step (vi) of processing the drug substance to obtain a buffer flush, comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer, the flushing buffer comprising a sugar, a surfactant, and a protein. Preferably, steps (iii), (iv), (v), and (vi) are all performed on the same day.

[0258] According to an embodiment of the process of the present invention, the drug substance and the bulk drug substance comprise the same excipients, including sugar, non-ionic surfactant, and protein, as those of the stabilizing buffer added in step (ii) of the process. In a preferred embodiment, the concentrations of excipients, including sugar, non-ionic surfactant, and protein, in the drug substance and the bulk drug substance are the same.

[0259] In one embodiment, the concentrations of excipients in the bulk drug substance from the buffered excipient composition / stabilizing buffer added in step (ii) are 2-5 times higher compared to the concentrations of these excipients in the stabilized or purified harvest. In a preferred embodiment, the concentrations of these excipients in the bulk drug substance are 5 times higher compared to the concentrations of these excipients in the stabilized or purified harvest.

[0260] In one embodiment, the concentrations of excipients in the bulk drug substance from the buffered excipient composition / stabilizing buffer added in step (ii), including sugars, proteins and / or non-ionic surfactants, are 2-5 times higher compared to the concentrations of these excipients in the stabilized or purified harvest. In a preferred embodiment, the concentrations of these excipients in the bulk drug substance are 5 times higher compared to the concentrations of these excipients in the stabilized or purified harvest.

[0261] In certain embodiments, the pH is maintained in the range of 7.6 to 8.1 throughout the above steps.

[0262] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; (vi) processing the drug substance obtained from step (vb) to obtain a bulk drug substance; Throughout steps (iii) to (vi), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) to (vi); and (b) The difference between the maximum pH at any time point through steps (iii) to (vi) is A method is provided, wherein the difference is 0.4 units or less. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0263] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; (vi) processing the drug substance obtained from step (vb) to obtain a bulk drug substance; Throughout steps (iii) to (vi), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) to (vi); and (b) The difference between the maximum pH at any time point through steps (iii) to (vi) is A method is provided in which the difference is 0.3 units or less. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0264] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), wherein step (vi) comprises the following successive steps: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; (vi) processing the drug substance obtained from step (vb) to obtain a bulk drug substance; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) to (vi); and (b) The difference between the maximum pH at any time point through steps (iii) to (vi) is A method is provided, wherein the difference is 0.4 units or less. In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method results in a higher virus yield compared to the same method with the same process steps, but allows the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0265] Thus, in certain embodiments, a method for large scale production and manufacturing of a flavivirus vaccine is provided, comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing the flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), comprising the steps of: (va) ion exchange chromatography, preferably anion exchange chromatography, to obtain a purified harvest; (vb) purifying, comprising ultrafiltration, preferably tangential flow filtration, to obtain the drug substance; (vi) processing the drug substance obtained from step (vb) to obtain a bulk drug substance; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) to (vi); and (b) The difference between the maximum pH at any time point through steps (iii) to (vi) is A method is provided in which the difference is 0.3 units or less. In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, the higher virus yield is at least 1 mg / cm. 2 , e.g., 1.5 mg / cm 2 Regarding high virus yields.

[0266] Thus, an embodiment of the method for large scale production and manufacturing of flavivirus vaccines includes a step of processing the drug substance, including mixing the drug substance with a buffer flush to obtain a bulk drug substance. The flushing step results in a composition comprising the buffer flush and the drug substance that is the bulk drug substance. If a bivalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still comprises each individual monovalent bulk drug substance. It should be understood that in the context of the present invention, each of the individual method steps and / or components or ingredients described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove alone and in combination with each other represents a separate embodiment of the present invention.

[0267] Bulk Drug Substance Processing According to an embodiment of the present invention, a method for large scale flavivirus vaccine production and manufacturing comprises the successive steps of (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii) to obtain a processed harvest, (v) purifying the processed harvest of step (iv) comprising at least one chromatography step to obtain a purified harvest, (v) an ultrafiltration step to obtain a drug substance, (vi) processing the drug substance obtained in step (v) to obtain a bulk drug substance, preferably comprising at least one flushing step of the ultrafiltration membrane with a flushing buffer, and (vii) processing the bulk drug substance. Preferably, the ultrafiltration is a tangential flow filtration. Preferably, the chromatography step is an anion exchange chromatography. In this context, the cells may be Vero cells. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1, represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8. The methods described in this section are performed separately for each of the four serotypes.

[0268] Processing the bulk drug substance may include a depth filtration step, which may be carried out in the same manner as the harvest clarification step.

[0269] Thus, depth filtration uses porous filtration media to separate particles and solids from liquids. Filter media that can be used for depth filtration can be cellulose acetate, polypropylene, glass fiber fleece and cellulose acetate protected by polyethersulfone. Filters used for depth filtration can have pore sizes of 0.1 μm to 1 μm, 0.2 μm to 0.8 μm, or 0.2 μm to 0.45 μm.

[0270] The filter used for the clarification of the harvest can be a heterogeneous bilayer of polyethersulfone with pore sizes of 0.2 μm and 0.45 μm. Thus, for the clarification of the harvest, several heterogeneous bilayer filters can be used, such as Sartoclean (cellulose acetate) 0.8+0.65 μm, Sartopure (polypolypyrene) 0.65 μm, Sartoclean (fiberglass fleece) 0.8+0.65 μm, Sartopore 2 (polyethersulfone) 0.8+0.45 μm, Sartopore 2 (polyethersulfone) 0.45+0.2 μm, or Sartopore 2 XLG (polyethersulfone) 0.8+0.2 μm. Each possibility represents a separate embodiment of the invention.

[0271] According to the method of the present invention, preferably the bulk drug substance after this step contains the same excipients as were present in the bulk drug substance prior to the processing step.

[0272] Freezing is another way of handling bulk drug substances and can be used to store and freeze bulk drug substances, preferably below -30°C.

[0273] Thawing is another way of processing bulk drug substances and refers to adapting frozen bulk drug substances to room temperature.

[0274] Pooling is another way of processing bulk drug substances and involves pooling two or more bulk drug substances.

[0275] In one embodiment of the method of the present invention, the first harvest obtained from the first collecting step is clarified and stabilized on the same day. The clarified and stabilized harvest is also purified on the same day using at least one chromatography step, preferably anion exchange chromatography. The purified harvest is also subjected to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain a first drug substance. The first drug substance is also mixed with a flushing buffer on the same day to obtain a composition comprising a buffer flush and the first drug substance, which is the first bulk drug substance. In one embodiment, 20-30 hours after the first collecting step, a second collecting step is performed and the steps are repeated to obtain a second bulk drug substance. In one embodiment, up to 10 collecting steps are performed and the steps are repeated to obtain up to 10 bulk drug substances, with an interval between each collecting step of 20-30 hours.

[0276] In a preferred embodiment, six collecting steps are carried out, and the above steps are repeated to obtain six bulk drug substances on six different days, with an interval of 20-30 hours between each collecting step.

[0277] In a preferred embodiment, the above steps are performed in the same manner for each of the four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1 represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2, represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3, represented by SEQ ID NO:5 and / or SEQ ID NO:6, and dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4, represented by SEQ ID NO:7 and / or SEQ ID NO:8.

[0278] Thus, in a preferred embodiment, for dengue serotype 1, six bulk drug substances are obtained from six collection steps, each bulk drug substance is obtained at the end of each collection day, and the interval between each collection step is 20-30 hours. Thus, in a preferred embodiment, for dengue serotype 2, six bulk drug substances are obtained from six collection steps, each bulk drug substance is obtained at the end of each collection day, and the interval between each collection step is 20-30 hours. Thus, in a preferred embodiment, for dengue serotype 3, six bulk drug substances are obtained from six collection steps, each bulk drug substance is obtained at the end of each collection day, and the interval between each collection step is 20-30 hours. Thus, in a preferred embodiment, for dengue serotype 4, six bulk drug substances are obtained from six collection steps, each bulk drug substance is obtained at the end of each collection day, and the interval between each collection step is 20-30 hours.

[0279] In one embodiment, each bulk drug substance may be frozen on the same day, or may be subjected to a depth filtration step on the same day and then frozen on the same day.

[0280] In a preferred embodiment, each bulk drug substance of dengue serotype 1 is immediately frozen on the same day. In a preferred embodiment, each of the frozen bulk drug substances of dengue serotype 1 is thawed, pooled, and then subjected to a step of depth filtration. In a preferred embodiment, six bulk drug substances of dengue serotype 1 are thawed, pooled, and then subjected to a step of depth filtration.

[0281] In a preferred embodiment, each bulk drug substance of dengue serotype 2 is immediately frozen on the same day. In a preferred embodiment, each of the frozen bulk drug substances of dengue serotype 2 is thawed, pooled, and then subjected to a step of depth filtration. In a preferred embodiment, six bulk drug substances of dengue serotype 2 are thawed, pooled, and then subjected to a step of depth filtration.

[0282] In a preferred embodiment, each bulk drug substance of dengue serotype 3 is immediately subjected to a step of depth filtration and then frozen on the same day. In a preferred embodiment, six bulk drug substances of dengue serotype 3 are individually subjected to a step of depth filtration and then frozen individually on the same day.

[0283] In a preferred embodiment, each bulk drug substance of dengue serotype 4 is immediately subjected to a step of depth filtration and then frozen on the same day. In a preferred embodiment, six bulk drug substances of dengue serotype 4 are individually subjected to a step of depth filtration and then frozen individually on the same day.

[0284] In a preferred embodiment, the method of the invention comprises the following steps for dengue serotype 1, such as dengue 2 / 1 chimera represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 or TDV-1: (i) providing cells in a growth medium as described in the previous section; (ii) infecting cells with a medium containing dengue serotype 1 at a low MOI as described in the previous section; (iii) obtaining a first harvest from the first harvesting step; (iv) processing the first harvest on the same day, including clarification and stabilization of the harvest; (v) (va) at least one chromatography step; preferably or anion exchange chromatography, and (vb) purifying the clarified and stabilized harvest on the same day, including subjecting the purified harvest to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain the first bulk drug substance, (vi) the first bulk drug substance is also processed on the same day, preferably mixed with a flushing buffer on the same day to obtain a buffer flush and a composition comprising the first bulk drug substance, which is the first bulk drug substance, (vii) (vii-a) processing the first bulk drug substance, including freezing the first bulk drug substance to obtain a frozen first bulk drug substance. In a preferred embodiment, six collecting steps are performed, and the above steps are repeated to obtain six bulk drug substances on six different days, with the interval between each collecting step being 20-30 hours. In one embodiment, the method comprises the steps of: (vii-b) thawing the six bulk drug substances; (vii-c) pooling the six bulk drug substances; (vii-d) subjecting the pooled six bulk drug substances to a depth filtration step; and (vii-e) freezing the filtered bulk drug substances.

[0285] In a preferred embodiment, the method of the invention comprises the following steps for dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and / or SEQ ID NO: 4: (i) providing cells in a growth medium as described in the previous section; (ii) infecting cells with a medium containing dengue serotype 2 at a low MOI as described in the previous section; (iii) obtaining a first harvest from the first harvesting step; (iv) processing the first harvest on the same day including clarification and stabilization of the harvest; (v) (va) at least one chromatography step, preferably anionization; and (vb) the purified harvest is also subjected to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain the first bulk drug substance, (vi) the first bulk drug substance is also processed on the same day, preferably mixed with a flushing buffer on the same day to obtain a buffer flush and a composition comprising the first bulk drug substance, which is the first bulk drug substance, (vii) (vii-a) processing the first bulk drug substance, including freezing the first bulk drug substance to obtain a frozen first bulk drug substance. In a preferred embodiment, six collecting steps are performed, and the above steps are repeated to obtain six bulk drug substances on six different days, with the interval between each collecting step being 20-30 hours. In one embodiment, the method comprises the steps of: (vii-b) thawing the six bulk drug substances; (vii-c) pooling the six bulk drug substances; (vii-d) subjecting the pooled six bulk drug substances to a depth filtration step; and (vii-e) freezing the filtered bulk drug substances.

[0286] In a preferred embodiment, the method of the invention comprises the following steps for dengue serotype 3 or TDV-3 represented by SEQ ID NO:5 and / or SEQ ID NO:6: (i) providing cells in a growth medium as described in the previous section; (ii) infecting the cells with a medium containing dengue serotype 3 at a low MOI as described in the previous section; (iii) obtaining a first harvest from the first harvesting step; (iv) processing the first harvest on the same day including clarification and stabilization of the harvest; (v) (va) at least one chromatography step, preferably anion exchange chromatography. and (vb) purifying the clarified and stabilized harvest on the same day, including subjecting the purified harvest to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain the first bulk drug substance; (vi) the first bulk drug substance is also processed on the same day, preferably mixed with a flushing buffer on the same day to obtain a buffer flush and a composition comprising the first bulk drug substance, the first bulk drug substance; (vii) processing the first bulk drug substance, including (vii-a) subjecting the first bulk drug substance to a depth filtration step, and (vii-b) freezing the filtered first bulk drug substance. In a preferred embodiment, six harvesting steps are performed, and the above steps are repeated to obtain six bulk drug substances on six different days, with the interval between each harvesting step being 20-30 hours.

[0287] In a preferred embodiment, the method of the invention comprises the following steps for dengue serotype 4 or TDV-4 represented by SEQ ID NO: 7 and / or SEQ ID NO: 8: (i) providing cells in a growth medium as described in the previous section; (ii) infecting the cells with a medium containing dengue serotype 4 at a low MOI as described in the previous section; (iii) obtaining a first harvest from the first harvesting step; (iv) processing the first harvest on the same day including clarification and stabilization of the harvest; (v) (va) at least one chromatography step, preferably anion exchange chromatography. and (vb) purifying the clarified and stabilized harvest on the same day, including subjecting the purified harvest to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain the first bulk drug substance; (vi) the first bulk drug substance is also processed on the same day, preferably mixed with a flushing buffer on the same day to obtain a buffer flush and a composition comprising the first bulk drug substance, the first bulk drug substance; (vii) processing the first bulk drug substance, including (vii-a) subjecting the first bulk drug substance to a depth filtration step, and (vii-b) freezing the filtered first bulk drug substance. In a preferred embodiment, six harvesting steps are performed, and the above steps are repeated to obtain six bulk drug substances on six different days, with the interval between each harvesting step being 20-30 hours.

[0288] Thus, an embodiment of the method for large-scale production and manufacturing of flavivirus vaccines includes a step of processing the bulk drug substance, including a depth filtration step. If a bivalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still includes each individual monovalent bulk drug substance. It should be understood that in the context of the present invention, each of the individual method steps and / or components or ingredients described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0289] Bulk Drug Substance Freezing and Storage The method of the present invention also provides a means and method for freezing and storing bulk drug substances.

[0290] The bulk drug substance can be frozen and stored at any time. One such time is after filtration of the BDS. After the filtration step, the bulk drug substance can be frozen and stored. The purpose of the freezing step is to ensure viral potency for the material to be used in the drug product formulation. At this step, the bulk drug substance can be frozen and stored using any of the commercially available bags.

[0291] In one embodiment, the bulk drug substance is frozen and stored after filtration of the BDS as discussed in the previous section, e.g., for TDV-3 and TDV-4. In one embodiment, the bulk drug substance can be frozen and stored immediately after obtaining the bulk drug substance from each collecting step, after which the bulk drug substance from each collecting step can be thawed, pooled, filtered as discussed above, and then frozen and stored, e.g., for TDV-1 and TDV-2.

[0292] Bags are used to freeze bulk drug substances and store them until thawed for use in drug product formulation. Bags may be selected based on specific parameters such as size, manufacturer, material of construction, and storage temperature range, thawing conditions, ability to maintain sterile conditions, ease of transport and use in previous processes and clinical trials.

[0293] The temperature range supported for the bag can be, for example, within the range of -84°C to 45°C. The freezing options supported by the bag used in the method of the present invention can be ultra-low temperature freezer, blast freezer, plate freezing and thawing. As used herein, "ultra-low temperature freezer" or "ULT" refers to a freezer that operates within the range of -50°C to -80°C, with ULTs being used to store a variety of analytes and products from biological samples to enzymes and drugs. As used herein, "blast freezer" refers to a specialized freezer that allows products to be rapidly frozen over a short period of time. The temperature of a blast freezer can vary from -10°C to -120°C. As used herein, "plate freezing" is a method of rapid freezing when a product is in direct contact with two cold metal surfaces.

[0294] Thawing options supported by the bags used in certain embodiments of the method of the present invention may be water bath, plasma thaw bath, shaker incubator, plate freeze-thaw. The term "water bath" refers to a bath containing a heating medium with at least 50% water by volume. The maximum heating temperature of the water bath can be controlled by selecting the composition of the heating medium. As used herein, "plasma thaw" refers to a thawing method using a device designed to rapidly and uniformly thaw fresh frozen plasma (FFP) bags at 37°C. Typically, the device is made from a stainless steel chamber and equipped with a digital temperature controller and a water circulation pump. Considerations for handling and transport of the bags used in the present invention may be the bottle and hard casting to minimize damage, the EVOH gas barrier with an EVA film layer, the rigidity of the ULDPE film. The material compatibility and sterility properties of the bag include animal component-free, gamma irradiation, and USP standards. "EVOH" barrier refers to an environmentally friendly substance used as a barrier plastic. Unlike PVDC, EVOH does not contain chlorine, dioxins, metals, or other elements that may cause endocrine disorders. As used herein, "GULPED" refers to very low density polyethylene.

[0295] Thus, suitable bags are commercially available bags that can be used to freeze bulk drug substances including Nalgene PETG / PFA bottles (50 mL to 2 L), Sartorius-Stedim Celsius-Pak / FFT / FFTp, 2D bags (30 mL to 16 L) or Pall Allegro 2D Biocontainers (125 mL to 50 L).

[0296] Once a bag is selected, the freezing and thawing conditions must be optimized to minimize loss of viral potency. These conditions include the freeze setpoint and rate, the thaw setpoint and rate, and optionally the cooling setpoint.

[0297] Thus, large scale production and manufacturing of flavivirus vaccines includes processing the bulk drug substance, such as freezing and storing the bulk drug substance. If a bivalent, trivalent, or tetravalent final drug product is intended, the bulk drug substance at this step still includes each individual monovalent bulk drug substance. It should be understood that in the context of the present invention, each of the individual method steps and / or component elements described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0298] Bulk Drug Formulation After the bulk drug substance is frozen, the next step is (ix) formulation of the bulk drug product.

[0299] This involves, for example, two major sub-steps: (ix-a) Thawing the bulk drug substance (ix-b) mixing the thawed bulk drug substance of step (ix-a) with a formulation buffer to obtain the bulk drug product.

[0300] If a bivalent, trivalent or tetravalent final drug product is intended, at this stage two or more monovalent bulk drug substances are mixed to obtain a final bulk drug product comprising a bivalent, trivalent or tetravalent drug product. Thus, in one embodiment, after step (ix-a) and before step (ix-b), two, three or four thawed bulk drug substances are mixed. In a preferred embodiment, the flavivirus vaccine is a tetravalent dengue vaccine comprising all four live attenuated dengue serotypes, including dengue serotype 1, such as dengue 2 / 1 chimera or TDV-1 represented by SEQ ID NO:1 and / or SEQ ID NO:2, dengue serotype 2 or TDV-2 represented by SEQ ID NO:3 and / or SEQ ID NO:4, dengue serotype 3, such as dengue serotype 2 / 3 or TDV-3 represented by SEQ ID NO:5 and / or SEQ ID NO:6, dengue serotype 4, such as dengue serotype 2 / 4 or TDV-4 represented by SEQ ID NO:7 and / or SEQ ID NO:8.

[0301] The formulation buffer includes two buffers: a first excipient buffer (FEB) and a second excipient buffer (SEB). Qualitatively, the components of each of the FEB and SEB may be the same or different. However, quantitatively, the components of each of the FEB and SEB are different.

[0302] As used herein, the term "excipient" refers to a substance added to a liquid pharmaceutical composition in addition to a biologically active agent. This may include substances used to enhance the stability of the active agent, salts, carbohydrates (e.g., sugars), surfactants, proteins, bulking agents, fillers, or agents that can be combined with the active agent to provide therapeutic enhancement of the composition. In particular, excipients may refer to salts, carbohydrates, non-ionic surfactants, and albumin. Excipients may also refer to buffers, such as, for example, phosphate buffers.

[0303] The FEB and SEB buffers may, for example, include four salts, a sugar, a non-ionic surfactant, and a protein. The FEB and SEB buffers may also include water for injection (WFI) as needed. The salts used in the buffers may be anhydrous or hydrated. The salts may be either one, two, three, or four sodium salts, or one, two, three, or four potassium salts, or one, two, three, or four magnesium salts. Each possibility may be combined with the other possibilities. Thus, each possibility represents a separate embodiment of the present invention.

[0304] For example, the sodium salt can be sodium fluoride, or sodium chloride, or sodium bromide, or sodium iodide, or sodium sulfate, or disodium salt, such as disodium phosphate, or sodium dihydrogen phosphate, or disodium hydrogen phosphate, or disodium hydrogen phosphate dihydrate, or sodium bicarbonate, or sodium carbonate.For example, the potassium salt can be potassium fluoride, or potassium chloride, or potassium iodide, or potassium sulfate, or potassium dihydrogen phosphate, or potassium dihydrogen sulfate, or potassium bicarbonate, or potassium carbonate.For example, the magnesium salt can be magnesium fluoride, or magnesium chloride, or magnesium iodide, or magnesium sulfate, or magnesium dihydrogen phosphate, or magnesium bicarbonate, or magnesium carbonate.

[0305] Each of the above salts can be used, for example, in the range of 0.9 mM to 130 mM in SEB buffer.

[0306] For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the range of 0.9-6 mM. For example, the SEB buffer may contain potassium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the range of 0.9-6 mM. For example, the SEB and SEB buffer may contain magnesium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the range of 0.9-6 mM.

[0307] For example, the SEB buffer may contain sodium fluoride in the ranges of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the ranges of 0.9-6 mM. For example, the SEB buffer may contain potassium fluoride in the ranges of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the ranges of 0.9-6 mM.

[0308] For example, the SEB buffer may contain sodium carbonate in the ranges of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the ranges of 0.9-6 mM. For example, the SEB buffer may contain potassium carbonate in the ranges of 100-130 mM, 110-130 mM, 120-130 mM, and three other salts in the ranges of 0.9-6 mM.

[0309] For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium chloride in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM. For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, magnesium chloride in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM. For example, the SEB buffer may contain potassium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, magnesium chloride in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM.

[0310] For example, the SEB buffer may contain sodium fluoride in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium chloride in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM. For example, the SEB buffer may contain sodium fluoride in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium carbonate in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM. For example, the SEB buffer may contain sodium fluoride in the range of 100-130 mM, 110-130 mM, 120-130 mM, magnesium chloride in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM. For example, the SEB buffer may contain sodium iodide in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium carbonate in the range of 1 mM-2 mM, 1 mM-1.5 mM, and two other salts in the range of 0.9-6 mM.

[0311] For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium chloride in the range of 1 mM-2 mM, 1 mM-1.5 mM, potassium dihydrogen phosphate in the range of 0.9-1.2 mM, 0.9-1.0 mM, and one other salt in the range of 4-6 mM, 5-6 mM. For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium phosphate in the range of 1 mM-2 mM, 1 mM-1.5 mM, potassium dihydrogen sulfate in the range of 0.9-1.2 mM, 0.9-1.0 mM, and one other salt in the range of 4-6 mM, 5-6 mM. For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, 120-130 mM, potassium phosphate in the range of 1 mM-2 mM, 1 mM-1.5 mM, sodium dihydrogen sulfate in the range of 0.9-1.2 mM, 0.9-1.0 mM, and one other salt in the range of 4-6 mM, 5-6 mM.

[0312] For example, the SEB buffer may contain sodium chloride in the range of 100-130 mM, 110-130 mM, or 120-130 mM, potassium chloride in the range of 1 mM-2 mM, or 1 mM-1.5 mM, potassium dihydrogen phosphate in the range of 0.9-1.2 mM, or 0.9-1.0 mM, and disodium hydrogen phosphate dihydrate in the range of 4-6 mM, or 5-6 mM. For example, the SEB buffer may contain potassium chloride in the range of 100-130 mM, 110-130 mM, or 120-130 mM, magnesium chloride in the range of 1 mM-2 mM, or 1 mM-1.5 mM, sodium dihydrogen phosphate in the range of 0.9-1.2 mM, or 0.9-1.0 mM, and disodium hydrogen phosphate dihydrate in the range of 4-6 mM, or 5-6 mM. For example, the SEB buffer may contain potassium chloride in the range of 100-130 mM, 110-130 mM, or 120-130 mM, magnesium chloride in the range of 1 mM-2 mM, or 1 mM-1.5 mM, sodium dihydrogen phosphate in the range of 0.9-1.2 mM, or 0.9-1.0 mM, and magnesium carbonate in the range of 4-6 mM, or 5-6 mM.

[0313] Each of the above salts can be used in the range of 3 mM to 50 mM in FEB buffer.

[0314] For example, the FEB buffer may contain sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the range of 3-25 mM. For example, the FEB buffer may contain potassium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the range of 3-25 mM. For example, the FEB buffer may contain magnesium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the range of 3-25 mM.

[0315] For example, FEB buffers may contain sodium fluoride in the ranges of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the ranges of 3-25 mM. For example, FEB buffers may contain potassium fluoride in the ranges of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the ranges of 3-25 mM.

[0316] For example, FEB buffers may contain sodium carbonate in the ranges of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the ranges of 3-25 mM. For example, FEB buffers may contain potassium carbonate in the ranges of 20-50 mM, 30-50 mM, 40-50 mM, and three other salts in the ranges of 3-25 mM.

[0317] For example, the FEB buffer may contain sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium chloride in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM. For example, the FEB buffer may contain sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, magnesium chloride in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM. For example, the FEB buffer may contain potassium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, magnesium chloride in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM.

[0318] For example, the FEB buffer may contain sodium fluoride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium chloride in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM. For example, the FEB buffer may contain sodium fluoride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium carbonate in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM. For example, the FEB buffer may contain sodium fluoride in the range of 20-50 mM, 30-50 mM, 40-50 mM, magnesium chloride in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM. For example, FEB buffer may contain sodium iodide in the range of 20-50 mM, 30-50 mM, 40-50 mM, and potassium carbonate in the range of 4-6 mM, 5-6 mM, and two other salts in the range of 3-25 mM.

[0319] For example, the FEB buffer may include sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium chloride in the range of 4-6 mM, 5-6 mM, potassium dihydrogen phosphate in the range of 3-4 mM, 3.2-4 mM, and one other salt in the range of 18-22 mM. For example, the FEB buffer may include sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium phosphate in the range of 4-6 mM, 5-6 mM, potassium dihydrogen sulfate in the range of 3-4 mM, 3.2-4 mM, and one other salt in the range of 18-22 mM. For example, the FEB buffer may contain sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium phosphate in the range of 4-6 mM, 5-6 mM, sodium dihydrogen sulfate in the range of 3-4 mM, 3.2-4 mM, and one other salt in the range of 18-22 mM.

[0320] For example, the FEB buffer may contain sodium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, potassium chloride in the range of 4-6 mM, 5-6 mM, potassium dihydrogen phosphate in the range of 3-4 mM, 3.2-4 mM, and disodium hydrogen phosphate dihydrate in the range of 18-22 mM, 19-22 mM, 20-22 mM. For example, the FEB buffer may contain potassium chloride in the range of 20-50 mM, 30-50 mM, 40-50 mM, magnesium chloride in the range of 4-6 mM, 5-6 mM, sodium dihydrogen phosphate in the range of 3-4 mM, 3.2-4 mM, and disodium hydrogen phosphate dihydrate in the range of 18-22 mM, 19-22 mM, 20-22 mM. For example, the FEB buffer may contain potassium chloride in the range of 20-50 mM, 30-50 mM, or 40-50 mM, magnesium chloride in the range of 4-6 mM, or 5-6 mM, sodium dihydrogen phosphate in the range of 3-4 mM, or 3.2-4 mM, and magnesium carbonate in the range of 18-22 mM, 19-22 mM, or 20-22 mM.

[0321] The sugars in the FEB and SEB buffers can be monosaccharides (e.g., glucose, galactose, ribose, mannose, rhamnose, talose, xylose, or allose arabinose), disaccharides (e.g., trehalose, sucrose, maltose, isomaltose, cellibiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, or fructose), trisaccharides (e.g., acarbose, raffinose, melizitose, panose, or cellotriose), and sugar polymers (e.g., dextran, xanthan, pullulan, cyclodextrin, amylose, amylopectin, starch, cello-oligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan, or chitin). The sugar in the FEB and SEB buffers can be a sugar alcohol, such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, and glycerol. The sugar in the FEB and SEB buffers can be a non-reducing sugar. The sugar in the FEB and SEB buffers can be sucrose, trehalose, or a hydrate thereof, such as trehalose dihydrate.

[0322] Each of the above sugars can be used in the range of 120-160 g / L in SEB buffer and 180-220 g / L in FEB buffer. For example, sucrose can be in the range of 120-160 g / L, 130-160 g / L, 140-150 g / L in SEB buffer, and 180-220 g / L, 190-210 g / L, 200-210 g / L in FEB buffer. For example, trehalose can be in the range of 120-160 g / L, 130-160 g / L, 140-150 g / L in SEB buffer, and 180-220 g / L, 190-210 g / L, 200-210 g / L in FEB buffer. For example, trehalose dihydrate can be in the range of 120-160 g / L, 130-160 g / L, 140-150 g / L in SEB buffer, and 180-220 g / L, 190-210 g / L, 200-210 g / L in FEB buffer.

[0323] The non-ionic surfactants in the FEB and SEB buffers may be selected from block copolymers, sorbitan esters, ethoxylated or propoxylated sorbitan esters, alkyl-polyglycosides (APGs), alkoxylated mono- or di-alkylamines, fatty acid monoethanolamides (FAMAs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GAs, or fatty acid glucamides, FAGAs), and combinations thereof.

[0324] The nonionic surfactant in the FEB and SEB buffers can be a high molecular weight nonionic surfactant. The nonionic surfactant can be a nonionic triblock copolymer. The surfactant can be a nonionic, hydrophilic, polyoxyethylene-polyoxypropylene block copolymer (or EO-PO block copolymer). The EO-PO block copolymer can include blocks of polyethylene oxide (-CH2CHO-designated EO) and polypropylene oxide (-CH2CHCHO-designated PO). The PO block can be adjacent to two EO blocks in an EOx-POy-Eox configuration. Because the PO component is hydrophilic and the EO component is hydrophobic, the overall hydrophilicity, molecular weight, and surfactant properties of the copolymer can be tuned by varying x and y in the EOx-POy-Eox block structure. In aqueous solution, the EO-PO block copolymers self-assemble into micelles with a PO core and a corona of hydrophilic EO groups.

[0325] The non-ionic surfactant in the FEB and SEB buffers can be a poloxamer. Poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of poly(propylene oxide) flanked by two hydrophilic chains of poly(ethylene oxide). The length of the polymer blocks can be customized, resulting in different poloxamers with slightly different properties. The non-ionic surfactant in the FEB and SEB buffers can be Pluronic F127 (poloxamer 407), Pluronic F68 (poloxamer 403), Pluronic P123, Pluronic P85, other polyethylene oxide-polypropylene oxide (EO-PO) block copolymers over 3,000-4,000 MW, or combinations thereof.

[0326] The concentrations of the nonionic surfactant in the FEB and SEB buffers may be the same. The concentrations of the nonionic surfactant in the FEB and SEB buffers may be in the range of 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively. The concentrations of the high molecular weight nonionic surfactant in the FEB and SEB buffers may be in the range of 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively. The concentrations of the poloxamer in the FEB and SEB buffers may be in the range of 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively. The concentration of Pluronic F127 (poloxamer 407) in the FEB and SEB buffers may range from 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively. The concentration of Pluronic F68 (poloxamer 403) in the FEB and SEB buffers may range from 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively. The concentration of Pluronic P123 in the FEB and SEB buffers may range from 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively. The concentration of Pluronic P85 in the FEB and SEB buffers can range from 2.0-12 g / L, 4-12 g / L, 6-12 g / L, 8-12 g / L, or 10-12 g / L, respectively.

[0327] The protein in the FEB and SEB buffers can be any protein that is essentially inert and does not react with the virus. In particular, the protein does not affect the structure or infectivity of the virus. Thus, the protein can be a structural protein or a serum protein. The protein can be selected from albumin, human serum albumin, collagen, hydrolyzed collagen, gelatin, and hydrolyzed gelatin.

[0328] The protein concentrations in the FEB and SEB buffers may be the same. The protein concentrations in the FEB and SEB buffers may be in the ranges of 0.5-1.5 g / L, 0.7-1.2 g / L, and 0.8-1.0 g / L, respectively. The protein concentrations in the FEB and SEB buffers may be in the ranges of 0.5-1.5 g / L, 0.7-1.2 g / L, and 0.8-1.0 g / L, respectively. The albumin concentrations in the FEB and SEB buffers may be in the ranges of 0.5-1.5 g / L, 0.7-1.2 g / L, and 0.8-1.0 g / L, respectively. The human serum albumin concentrations in the FEB and SEB buffers may be in the ranges of 0.5-1.5 g / L, 0.7-1.2 g / L, and 0.8-1.0 g / L, respectively. The collagen concentrations in the FEB and SEB buffers may be in the ranges of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, and 0.8 to 1.0 g / L, respectively. The gelatin concentrations in the FEB and SEB buffers may be in the ranges of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, and 0.8 to 1.0 g / L, respectively.

[0329] In the context of the present invention, it is to be understood that each of the individual components of the buffer solutions described hereinabove is contemplated alone or in combination with each other. Once the bulk drug substance is thawed, it is mixed with the FEB and SEB to formulate the bulk drug product.

[0330] Thus, one embodiment of large-scale production and manufacturing of flavivirus vaccines includes the formulation of bulk drug products. If a bivalent, trivalent, or tetravalent final drug product is intended, the drug product obtained at this stage includes a bivalent, trivalent, or tetravalent bulk drug product. It should be understood that in the context of the present invention, each of the individual method steps and / or component elements described hereinabove are contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0331] sterile filtration The formulated and mixed bulk drug substance may be filtered through a sterile filtration assembly. The filter pore size may be up to 0.2 μm. The specific types and characteristics of filters that may be used have been previously discussed under harvest clarification and may also be used in this step.

[0332] For purposes of the present invention, sterile filtration of the bulk drug product may be performed via a peristaltic pump characterized by a maximum 260 rpm and a maximum inlet pressure of 1.0-1.2 bar. The filtered drug product may be collected in a separate container that is kept at a temperature in the range of 2°C to 8°C. For purposes of the present invention, the sterile filtered bulk drug product may be mixed at about 100-150 rpm for at least 10 minutes and then further sampled for sterilization.

[0333] Thus, large scale production and manufacturing of flavivirus vaccines may include sterile filtration of bulk pharmaceutical product. It should be understood that in the context of the present invention, each of the individual process steps and / or components described hereinabove is contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0334] Filling and Loading The filling process can be carried out using any commercially available scale isolator filling line known in the art. Filling machine isolators are designed to enclose the filling, stoppering, and capping operations of a powder or liquid filling machine while providing a sterile environment for the process. This improves product safety by reducing the risk of contamination with no direct contact between the operator and the product. Isolators also provide an excellent environmentally friendly decontamination option.

[0335] According to the method of the present invention, the temperature of the isolator filling line is maintained between 10°C and 25°C, more preferably between 15°C and 25°C during filling.

[0336] The method of the present invention utilizes a vial that can be used to fill the filtered pharmaceutical product. The vial can be either a plastic or glass vial. In a preferred embodiment, the vial is a glass vial. When using a glass vial, either a USP Type I, II or III glass vial can be used. In a preferred embodiment, a USP Type I glass vial is used.

[0337] As used herein, "glass" refers to any suitable glass. For example, neutral glass is a borosilicate glass that contains significant amounts of borate, aluminum oxide, alkali and / or alkaline earth oxides. It has high hydrolysis resistance and high thermal shock resistance. Soda-lime-silica glass is a silica glass that contains alkali metal oxides, primarily sodium oxide, and alkaline earth oxides, primarily calcium oxide. It has only moderate hydrolysis resistance.

[0338] According to their hydrolytic resistance, glass containers are classified as follows: Type I glass containers, a neutral glass with high hydrolytic resistance, suitable for most preparations, regardless of whether they are for parenteral use or not; Type II glass containers, usually soda-lime-silica glass with high hydrolytic resistance resulting from suitable treatment of the surface. These are suitable for most acidic and neutral aqueous preparations, with or without parenteral use, Type III glass containers, which are usually soda-lime-silica glasses that have only moderate hydrolytic resistance. These are generally suitable for non-aqueous preparations for parenteral use, powders for parenteral use (excluding lyophilized preparations), and preparations not for parenteral use.

[0339] In a preferred embodiment, the vial used in the present invention is made of borosilicate glass. In a preferred embodiment, the vial used in the present invention has high hydrolytic resistance and high thermal shock resistance. Thus, in a preferred embodiment, the vial used is a USP Type I glass vial.

[0340] USP Type I glass vials that can be used in the present invention can be either 2R or 4R tubular glass vials with volumes of 2 mL or 4 mL, respectively.

[0341] Thus, according to an embodiment of the present invention, USP Type I 2R vials or USP Type 1 2R vials can be used to fill the filtered bulk drug product. Several steps can be performed prior to filling, including, but not limited to, one or more of vial washing, vial sterilization, or vial depyrogenation. Alternatively, the method can use pre-sterilized glass that is supplied directly to the filling station as an alternative to save costs and space for operating a washing and sterilization line. The pre-sterilized glass vials are washed and depyrogenated by the supplier, double bagged, and then gamma irradiated to sterilize before being shipped to the end user for use.

[0342] As the vials are processed, they are read for filling. At this time, the sterilized filtered bulk is aseptically connected to a single-use filling manifold. Prior to filling, the bulk can be mixed at about 100-150 rpm, 110-120 rpm, 130-140 rpm, or 150 rpm for at least 20-30 minutes, 15-20 minutes, or 15 minutes. In a preferred embodiment, mixing is performed at 140-150 rpm for about 15 minutes.

[0343] In a preferred embodiment of the invention, mixing and filling continue in parallel with each other. Preferably, mixing continues for 15 minutes until about 60-80% of the vials are filled, for example 70-80%, 75-80% of the vials are filled. In another embodiment, mixing is switched off after the first 15 minutes or after the first 20 minutes of mixing and filling. In this embodiment, mixing is not continued in parallel with further filling. Thus, in one embodiment, at least 60-80% of the vials, for example 70-80%, 75-80% of the vials are filled in parallel with the mixing of the bulk pharmaceutical product.

[0344] The fill range is one of the critical parameters of this step in certain embodiments of the method of the present invention. The critical vial fill volume can be set depending on the situation and the required use. For example, the critical vial fill volume can be set to 0.5-0.8 mL, 0.6-0.7 mL, 0.65-0.66 mL, 0.651, 0.652, 0.653, 0.654, or 0.655 mL. Those skilled in the art know how to set the critical vial fill volume by manually setting the pump seed of the fill needle in the isolator fill line at the beginning of the fill run and then maintaining the volume by automatic feedback control. In embodiments of the method of the present invention, additional checks are incorporated to ensure that the contents remain within the critical fill volume. These checks can be, for example, weight checks.

[0345] Thus, the method of the present invention may or may not include a weight check to ensure that the volume is within the critical fill volume. If a weight check is used, a standard deviation of ±5-7% can be considered to meet the critical fill volume requirement.

[0346] Once filling is complete, an embodiment of the method of the present invention includes stoppering the vials. In one embodiment, the filling machine may partially stopper the vials. In this embodiment, partial stoppering is accomplished before transferring the vials to the lyophilization unit. The stoppers may or may not undergo a sterilization step before being transferred to the filler. In one preferred embodiment, the transfer is performed aseptically. After the partial stoppering step, a "good stopper" inspection step may or may not be performed. At the end of filling, the minimum product yield is confirmed to be at least 90%, 95%, or 97% good vials out of the total number of vials filled.

[0347] The total time in solution after formulation (post-TIS) is defined as the time starting from the end of the transfer of the last lot per serotype and ending with the start of the lyophilization cycle. Post-TIS can be in the range of 10-30 hours, more preferably 12-29 hours. The post-TIS of one serotype can be different compared to the post-TIS of another serotype. In one embodiment, the post-TIS of TDV-4 is the longest, the post-TIS of TDV2 is the shortest or the same as TDV-1 and TDV-3, and the post-TIS of TDV-1 and TDV-3 is the same. In this embodiment, the post-TIS of TDV-4 is in the range of 15-30 hours, 16-30 hours, or 16-29 hours, the post-TIS of TDV2 is in the range of 10-24 hours, or 12-24 hours, and the post-TIS of TDV-1 and TDV-3 is 11-25 hours, or 12-25 hours.

[0348] Thus, large scale production and manufacturing of flavivirus vaccines may include filling and loading bulk pharmaceutical products into vials. It should be understood that in the context of the present invention, each of the individual method steps and / or components described hereinabove is contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0349] Freeze-drying and sealing Pharmaceuticals can be freeze-dried, such as in a commercial scale freeze dryer. Freeze-drying involves manipulating the temperature and pressure of a solution so that the phase of the solution can move directly from a frozen state to a gaseous state without moving through a liquid phase / state. This is accomplished by cooling the solution and reducing the pressure below the triple point of water (the temperature and pressure at which water can exist in equilibrium in liquid, solid, and gaseous states, i.e., 0.01°C). This allows the solvent to be removed from the product without exposing it to intense heat.

[0350] The freeze-drying process cycle of a freeze-dried liquid, for example, consists of five major stages: equilibration, freezing, primary drying, secondary drying, and stopping, each controlled by three fundamental process parameters: temperature, pressure, and time.

[0351] Thus, one embodiment of the method of the present invention includes a freeze-drying method for preparing a dried composition, the method comprising the steps of: (i) providing an aqueous composition; (ii) freezing the aqueous composition to form a frozen composition; (iii) subjecting the frozen composition to a primary drying step in an apparatus with a shelf temperature in the range of from above the Tg of the composition to not more than 15°C above the Tg and a pressure of less than 0.3 mbar to form a primary dried product; and (iv) subjecting the primary dried product to a secondary drying step at a temperature above the temperature of the primary drying step to form a secondary dried product.

[0352] According to one embodiment of the method of the present invention, the primary drying step (iii) is carried out in an apparatus having a shelf temperature of -33°C to -10°C, or -33°C to -20°C, or -31°C to -23°C, or 29°C to -25°C, such as about -27°C. In this context, the primary drying step (c) can be carried out in an apparatus having a shelf temperature above the collapse temperature (Tc) of the composition. Furthermore, the primary drying step (iii) is carried out for a period of 100 hours to 10 hours, or 60 hours to 30 hours, or 50 hours to 40 hours, such as about 48 hours. Furthermore, the primary drying step (iii) can be carried out at a pressure of less than 0.1 mbar, or less than 0.05 mbar, or less than 0.025 mbar, and optionally more than about 0.01 mbar. Furthermore, the secondary drying step (iv) can be carried out at a temperature of at least 20°C, or at least 25°C.

[0353] Thus, large scale production and manufacturing of flavivirus vaccines may include lyophilization of the pharmaceutical. It is to be understood that in the context of the present invention, each of the individual method steps and / or components described hereinabove is contemplated alone or in combination with each other. Thus, each possibility described hereinabove, alone and in combination with each other, represents a separate embodiment of the present invention.

[0354] Unloading and capping Once secondary drying is complete, the vials can be capped. For example, the chamber pressure can be backfilled to -0.125 to -0.075 bar (partial vacuum) using dry sterile nitrogen gas (N2). The vials can be capped at 6.5 N / cm 2 Allow the stopper to stand at above pressure for 60 s, then allow the shelf temperature to be reduced to 3 °C-7 °C until unloaded into the Grade A environment.

[0355] The vials may be sealed, for example with aluminum / plastic, in a sterile isolator.

[0356] Visual inspection All vials are visually inspected through a manual or semi-automated process for product, glass, crimp, stopper or other potential defects, including but not limited to cake appearance, cap defects, glass defects, and visible particles.

[0357] Freezing of final medicines The pharmaceutical products are preferably frozen for storage. Each pharmaceutical product batch can be transferred to an additional storage location, for example at 2°C to 8°C, before freezing, or the product batch can be transferred directly to a dedicated blast freezer for blast freezing, for example at a temperature of -15°C to -35°C, which can last for at least 36 hours in the blast freezer. The batch can, for example, remain in the blast freezer for at least 36 hours.

[0358] After blast freezing, the drug product batch can be transferred to long term storage, for example at -15°C to -25°C. EXAMPLES

[0359] Example 1 - Low MOI Range This example shows that high virus titers can be obtained in large-scale flavivirus vaccine production and manufacturing when monolayer cells are infected with infection medium containing flavivirus at a low MOI (less than 0.008 or less than 0.005), regardless of the volume of infection medium used.

[0360] A vial of Vero WCB is thawed in a 37° C. water bath. Thawing should not take longer than 5 minutes. The thawed cells are then transferred to an appropriate tissue culture vessel. Vero cells were grown in tissue culture vessels containing growth medium.

[0361] Dulbecco's Growth Medium (DGM), the main component of Vero cell growth medium, consists of Dulbecco's Modified Eagle's Medium (DMEM), glutamine, and fetal bovine serum (FBS). The growth medium contained DMEM + 4.0 mM glutamine + 3.52 g / L glucose + 10% fetal bovine serum (FBS). Vero cells were cultured at 37°C and 5% CO2 with operating ranges of 36°C to 39°C and 4% to 6%, respectively.

[0362] Vero cells were grown to at least 90% confluency on the surface of the culture vessel prior to TDV infection. Once the cells were confluent, they were removed from the surface by TrypLE Select within 10–30 min. Once the desired cell density was reached, infection and virus production was performed using infection medium. Infection medium contains DMEM + 4.0 mM glutamine + 3.88 g / L glucose + 0.1% (w / v) F127. The volume of infection medium was 0.014–0.114 mL / cm. 2 It was.

[0363] The infection medium described above containing monovalent TDV-1, 2, 3, or 4 working virus seeds (WVS) at low MOI (0.005 and 0.001 in this example) and high MOI (0.01 and 0.05 in this example) was used to infect Vero cells in monolayers in individual tissue culture vessels.

[0364] The volume of infection medium used was 0.014–0.114 mL / cm 2 and the infection method was rocking / shaking of the tissue culture vessel. Thus, the following groups result, as shown in Table 1 below: [Table 1]

[0365] Representative mean titers obtained for TDV-1 serotypes are shown in Table 2 below. [Table 2]

[0366] Table 2 shows that high flavivirus titers can be obtained in the low MOI range in large-scale flavivirus vaccine production and manufacturing, regardless of the volume of infection medium used.

[0367] Therefore, it can be concluded that infection of vero cell monolayers with a low MOI (less than 0.008, eg, 0.005 or less) still provides high virus titers for large-scale production and manufacturing of flavivirus vaccines.

[0368] Example 2 - Low MOI range + static infection This example shows that high virus titers can be obtained in large-scale flavivirus vaccine production and manufacturing when monolayer cells are infected with an infection medium containing a flavivirus at a low MOI of less than 0.008, for example 0.005 or less, and when the infection method is static.

[0369] The same parameters were used as in Example 1, except that the infection method was static instead of rocking / shaking. Table 3 below shows representative mean titers for TDV-1 serotypes. [Table 3]

[0370] Table 3 shows that high flavivirus titers can still be obtained in large-scale flavivirus vaccine production and manufacturing when the infection medium contains flavivirus at a low MOI and the infection method is static rather than rocking / shaking. To further confirm the above findings, TDV-3 was used as a representative serotype, and the infection volume was set at 0.057 mL / cm. 2 ~0.063mL / cm 2 The study was carried out while maintaining a low MOI of 0.004. The daily harvest titers of each TDV-3 from days 5 to 9 were still within 8.0 log 10 Potency values ​​of greater than PFU / mL were achieved.

[0371] Example 2A - Low MOI infection and improved titers In this experiment, three groups were set up according to the table below. JPEG2025504521000007.jpg37165

[0372] All other parameters were kept constant. The results are shown in Figure 3. As can be seen in Figure 3, all titers measured at low MOI (group 1) were greater than 1.0 x 10 8 The titer in group 2 was greater than 1.0 × 10 8 PFU / mL. However, comparing Group 1 and Group 2, the mean titers in Group 1, i.e., the average of days 5, 6 and 9 post-infection, were higher than in Group 2. This means that the low MOI provided consistently higher titers up to day 9 post-infection, i.e., higher titers on each harvest day, and the low MOI (Group 1) provided higher titers on average compared to the high MOI (Group 2).

[0373] Another experiment was set up as shown in the table below. JPEG2025504521000008.jpg31165

[0374] All other parameters were kept constant. The results are shown in Figure 4. Figure 4 also shows that low MOI consistently provided higher titers up to day 9 post-infection, with low MOI (group 1) providing higher titers on average compared to high MOI (group 2).

[0375] Example 3 - Medium changes and daily harvests This example shows that high virus titers can be obtained in large-scale flavivirus vaccine production and manufacturing when monolayer cells are infected with infection medium containing flavivirus at a low MOI (less than 0.008, e.g., 0.005 or less), and when the infection method is static, and when a medium change is performed 20 hours before the first harvesting step in combination with at least two or at least three further harvesting steps.

[0376] Table 4 below shows representative mean titers for TDV-1 serotypes, especially when infection medium containing flavivirus at a low MOI is used in combination with the static infection method, and when a medium change is performed 20 hours before the first harvesting step and at least two further harvesting steps are performed. [Table 4]

[0377] Table 5 below shows representative mean titers for TDV-1 serotypes, especially when infection medium containing flavivirus at a low MOI is used in combination with the static infection method, and when medium exchange is performed 20 hours before the first harvesting step and at least three further harvesting steps are performed. [Table 5]

[0378] Example 4 - No substantial virus loss when anion exchange chromatography is performed prior to the tangential flow filtration step A proof-of-concept study was designed to evaluate whether a chromatography step could be performed directly on the clarified, stabilized harvest prior to the ultrafiltration step without substantial loss of viral titer.

[0379] Table 6 below shows the loss in viral titer when the clarified and stabilized harvest was subjected to anion exchange chromatography compared to the loss in viral titer when the TFF retentate was subjected to anion exchange chromatography. The experiment was replicated on two different chromatography columns.

[0380] It has surprisingly been found that subjecting the clarified, stabilized harvest directly to anion exchange chromatography prior to the ultrafiltration step does not result in substantial loss of titer.

[0381] Even more surprisingly, it was found that subjecting the clarified, stabilized harvest directly to anion exchange chromatography resulted in higher virus titers compared to performing an ultrafiltration step immediately prior to the anion exchange chromatography step. [Table 6]

[0382] Example 5 - Completion of the process Each of the four viral serotypes is manufactured separately and constitutes one of the active ingredients of the drug product. The manufacturing process flows for all four serotypes are similar.

[0383] The main unit operations are as follows: 1. Vero Cell Expansion 2. Infection of Vero Cells 3. Clarification of Daily Collections 4. Stabilization of daily collections through FTA 5. AEX for daily collections 6. TFF of daily collections 7. Processing of API to obtain bulk API 8. Bulk drug substance processing, including filtration of bulk drug substances 9. Bulk drug substance handling, including freezing and storage of bulk drug substances 10. Pharmaceutical formulation 11.Sterile filtration 12. Filling and Loading 13. Freeze-drying and sealing 14.Unloading and capping 15. Visual Inspection 16. Freezing of final pharmaceutical products 17. Labelling and Packaging

[0384] The order of the steps above is exemplary and can be adjusted. For example, another variation of these steps could be: 1. Vero Cell Expansion 2. Infection of Vero Cells 3. Clarification of Daily Collections 4. Stabilization of daily collections through FTA 5. AEX for daily collections 6. TFF of daily collections 7. Processing of API to obtain bulk API 8. Bulk drug substance handling, including freezing and storage of bulk drug substances 9. Processing of bulk drug substances, such as filtration of the bulk drug substance followed by final freezing and storage 10. Pharmaceutical formulation 11.Sterile filtration 12. Filling and Loading 13. Freeze-drying and sealing 14.Unloading and capping 15. Visual Inspection 16. Freezing of final pharmaceutical products 17. Labelling and Packaging

[0385] Example 5.1 - Vero Cell Expansion The use of Vero cell lines for the manufacture of viral vaccines has been widely accepted by regulatory authorities. Continuous mammalian cell lines are well characterized with a demonstrated safety profile as they are used in several licensed vaccines. Vero cell lines were shown to support the replication of attenuated dengue vaccine strains and therefore were used for the manufacture of master virus seed (MVS) and working virus seed (WVS), as well as the production of monovalent drug substances.

[0386] In this example, a vial of Vero WCB (Working Cell Bank) is thawed in a water bath at 37°C. The thawing time is controlled within 5 minutes. The thawed cells are then transferred to a vessel already containing DMEM with 3.52 g / L glucose + 10% FBS + 4 mM glutamine and cultured at 37°C and 5% CO2, respectively. The medium was mixed aseptically and homogenized before use. In this example, the vessel used for cell expansion contained a 16×CF10 chamber. It has a volume of 101,760 cm 2When the cells become confluent, they are removed from the surface by TrypLE Select within 40 minutes. During the cell passaging process, the allowable cell "drying" duration and maximum duration for cell passaging are defined up to 1 hour and 8.0 hours, respectively.

[0387] The recombinant trypsin dissociation enzyme TrypLE Select was used to dissociate cells from the surface of the culture vessel. Once all cells were removed from the surface of the culture vessel, DGM was added to neutralize the activity of TrypLE Select.

[0388] Example 5.2 - Infection and harvesting of Vero cells After the Vero cells are expanded to sufficient numbers in the TDV production vessel, the cells are subsequently infected with monovalent dengue WVS. The infection medium used was DMEM without phenol red. The final concentrations of glucose, glutamine and F127 in the infection medium were 3.88 g / L, 4.0 mM and 0.1% (w / v), respectively.

[0389] DMP was prepared by aseptically mixing sterile liquid components including DMEM, F127 and glutamine, and the mixed liquid components were further filtered using a Sartopore 2 0.45+0.2 μm filter.

[0390] Vero cells are grown to at least 80% confluency on the surface of the culture vessel prior to TDV infection. Vero cells are grown at approximately 1.0×10 5 cells / cm 2 A minimum of 75% confluence was required to achieve a cell density of 1000. Once the desired cell density was reached, infection and virus production was performed using DMP.

[0391] Monovalent TDV working virus seeds (WVS) were used to infect Vero cells. Vero cells with at least 85% confluency in a vessel were infected in monolayer with WVS at an MOI of 0.001 and plated with 1 cm 2The infection volume per unit volume is 0.057 mL / cm 2 The infection method was static, i.e., no rocking mechanism was used on the vessel during the infection process. Infection and virus production parameters, temperature and CO2 set points were maintained at 38°C and 4%, respectively. The duration of infection was set to 70 min.

[0392] Three DPBS washing steps were performed post-infection, and fresh DMP was added to the vessel to facilitate virus production after the post-infection DPBS washes.

[0393] In this example, six daily harvest steps were performed between days 5 and 10 post-infection. Each harvest step was performed by recovering the supernatant from the chamber. 28 hours before the first harvest step, a medium change was performed. After each harvest step, fresh DMP was added to the cells. The daily harvest volume was up to about 12.8 L per day, with a total harvest volume of up to 76.8 L. All daily harvests were 7.0 log 10 achieved greater than PFU / mL.

[0394] Example 5.3 - Clarification of daily harvests Sartopore 2 (0.45+0.2um) was used for clarification of the daily harvest. For this filter, 20.454mL / cm 2 A throughput capacity of 800 L / m was determined for the harvest clarification step using this filter. 2 A conservative filtration flow rate of 100 / h was chosen to provide a high flow rate, short treatment time, and a reasonable safety margin.

[0395] Prior to clarification, the turbidity of the harvest ranged from 1 to 9 specific turbidity units (NTU). Upon being subjected to the clarification step, a reduction in turbidity (below 1 NTU) is observed. This indicates that cellular debris from the harvest is effectively removed in this clarification step. The average filter backpressure ranged from 0.1 to 0.3 bar. The robustness of the clarification step to reduce turbidity to low levels, at relatively low operating backpressures, ensured that the clarification process operated consistently and away from the process boundary.

[0396] Example 5.4 - Harvest stabilization The stabilization buffer was a stock solution of 3xFTA that was added to the harvest to a final concentration of 0.2xFTA (0.2% F127, 3% trehalose dihydrate, 0.02% HSA in PBS). A small amount of 3xFTA (calculated based on 1 / 14th the total volume of the clarified harvest) was used to obtain a concentration of 0.2xFTA.

[0397] This is the only time that the buffered excipient composition is added to the harvest during the entire purification process until the drug substance (the retentate of TFF) is obtained. This means that the 0.2x FTA clarified and stabilized harvest is the feed for both the anion exchange chromatography step and the tangential flow filtration step. At the end of the tangential flow filtration step, the retentate is obtained, which is the drug substance.

[0398] Example 5.5 - Anion Exchange Chromatography The main objective of this step is to remove host cell DNA (HC DNA) while ensuring maximal virus recovery.

[0399] In this step, an AEX column operated in flow-through mode is used. It was found that the stabilized harvest described above could be used directly as the feed material for the AEX column. No buffer exchange or buffer concentration steps were required. In this step, a Sartobind Q mini column was used, which efficiently removed HC DNA with minimal virus loss.

[0400] In this example, two 7 mL AEX capsules were used at a scale of 16×CF10. The flow rate was 230 mL / min.

[0401] The DBC for 7 mL membranes of Sartobind Q ranged from 2.15 to 3.69 mg of DNA per mL of membrane bed volume (MV).

[0402] The AEX step was sufficient to reduce the amount of host cell DNA to a specification value of 50 ng / mL or less.

[0403] Example 5.6 - Tangential Flow Filtration In this example, a TFF membrane with a molecular weight cut-off (MWCO) of 300 kDa polyethersulfone (PES) was used. The pore size is smaller than the size of the dengue virus particles, which is about 50 nm, and therefore capable of retaining the virus. A feed flow rate of 300 LMH was used.

[0404] The flow-through from the AEX can be carried out directly as the feed composition for the TFF step.

[0405] The resulting TFF residue is the drug substance.

[0406] Example 5.7 - Processing of Drug Substance to Obtain Bulk Drug Substance Three flushing steps of the TFF membrane were performed to obtain the bulk drug substance.

[0407] The flush buffer was 1×FTA, however, to compensate for trehalose dihydrate passing through the TFF membrane, the buffer flush composition was composed of a trehalose dihydrate concentration of 27%, resulting in a trehalose dihydrate concentration of 15% in the monovalent bulk drug substance.

[0408] Analytical data for FTA content of bulk drug substance for all serotypes produced during the production scale runs were close to the theoretical value of 1× FTA concentration.

[0409] Example 5.8 - Bulk Drug Substance Processing, Including Filtration of Bulk Drug Substance In this step, for bulk drug substances for TDV-3 and TDV-4, filtration of the bulk drug substance was performed immediately.

[0410] Filtration of the bulk drug substance also employed Sartopore 2 filters, which are hydrophilic polyethersulfone membranes with pore sizes of 0.45 + 0.2 μm.

[0411] For situations where concentrated individual harvests are pooled prior to filtration, 525.0 cm 2 For situations where a filter size of 87.6 cm is used and each daily collection is filtered separately, 2 The closest membrane area sizes available from the filter manufacturers were 0.1 m 2 and 150cm 2 It is.

[0412] Analytical data for FTA content of filtered bulk drug substance for all serotypes produced during the production scale runs were close to the theoretical value of 1× FTA concentration.

[0413] Example 5.9 - Freezing and Storage of Bulk Drug Substance After the filtration step for TDV-3 and TDV-4, the bulk drug substance is frozen and stored. For TDV-1 and TDV-2, the bulk drug substance for each collection day was frozen and thawed on the day the last bulk drug substance was obtained from the last collection step, then all bulk drug substances were pooled and then subjected to a filtration step, followed by final freezing and storage. The purpose of the freezing step is to ensure that the potency and other quality attributes of the drug substance are maintained for the material to be used in drug product formulation.

[0414] The 6L FFtp bag was used for this purpose as it offers the possibility to utilize different freeze / thaw options while maintaining the same product contact surface, ensuring that transportation and product compatibility at different fill volumes is sufficiently easy and robust.

[0415] The bags of bulk drug substance are stored frozen until thawed for use in drug product formulation.

[0416] Example 5.10 - Pharmaceutical Formulation After freezing the bulk drug substance, the next step was formulation of the bulk drug substance product, which included thawing the bulk drug substance and mixing the thawed bulk drug substance (i.e., for each of TDV-1, TDV-2, TDV-3, and TDV-4) with formulation buffer to obtain the bulk drug product.

[0417] The formulation buffer contained two buffers: a first excipient buffer (FEB) and a second excipient buffer (SEB). Qualitatively, the components of each of FEB and SEB are the same. However, quantitatively, the components of each of FEB and SEB are different.

[0418] After sterile filtration of the drug product (step 5.11), it was filled and loaded into vials (step 5.12) and moved directly to the next step.

[0419] Example 5.13 - Freeze-drying and sealing An exemplary lyophilization cycle is shown in Table 8 below. [Table 7]

[0420] After unloading and capping (step 5.14) and visual inspection (step 5.15), the final drug product is frozen (step 5.16) and finally labeled and packaged in step 5.17.

[0421] Example 6 - Completion of the process Each of the four viral serotypes is manufactured separately and constitutes one of the active ingredients of the drug product. The manufacturing process flows for all four serotypes (drug substances) are similar, with differences between the individual serotypes being driven by potency and volume needs.

[0422] The main unit operations are as follows: 1. Vero Cell Expansion 2. Infection of Vero Cells 3. Clarification of Daily Collections 4. Stabilization of daily collections through FTA 5. AEX for daily collections 6. TFF of daily collections 7. Processing of API to obtain bulk API 8. Bulk drug substance processing, such as bulk drug substance filtration 9. Bulk drug substance handling, including freezing and storage of bulk drug substances 10. Pharmaceutical formulation 11.Sterile filtration 12. Filling and Loading 13. Freeze-drying and sealing 14.Unloading and capping 15. Visual Inspection 16. Freezing of final pharmaceutical products 17. Labelling and Packaging

[0423] The order of the steps above is exemplary and can be adjusted. For example, another variation of these steps could be: 1. Vero Cell Expansion 2. Infection of Vero Cells 3. Clarification of Daily Collections 4. Stabilization of daily collections through FTA 5. AEX for daily collections 6. TFF of daily collections 7. Processing of API to obtain bulk API 8. Bulk drug substance handling, including freezing and storage of bulk drug substances 9. Processing of bulk drug substances, such as filtration of the bulk drug substance followed by final freezing and storage 10. Pharmaceutical formulation 11.Sterile filtration 12. Filling and Loading 13. Freeze-drying and sealing 14.Unloading and capping 15. Visual Inspection 16. Freezing of final pharmaceutical products 17. Labelling and Packaging

[0424] Example 6.1 - Vero Cell Expansion A vial of Vero WCB is thawed in a water bath at 37°C. In this example, the thawing duration is controlled within 8 minutes. The thawed cells are then transferred to a vessel already containing DMEM with 4.0 g / L glucose + 5% FBS + 4.5 mM glutamine, and cultured at 38°C and 4% CO2, respectively. Phenol red was omitted from the medium and a visual microscope check was used. The used medium was mixed together and filtered to maintain microbial control before use. In this example, the vessel used for cell expansion contains 4 x CF40 chambers. It has a 101,760 cm 2 When the cells become confluent, they are removed from the surface by TrypLE Select within 20 minutes. During the cell passaging process, the allowable cell "drying" duration and maximum duration for cell passaging are defined up to 30 minutes and 5.0 hours, respectively.

[0425] The recombinant dissociation trypsin enzyme TrypLE Select was used to dissociate cells from the surface of the culture vessel, as in Example 5.1. However, in this example, before adding TrypLE Select, the cells are washed with DPBS to remove residual FBS. Once all cells have been removed from the surface of the culture vessel, DGM is added to neutralize the activity of TrypLE Select.

[0426] Example 6.2 - Infection of Vero cells After the Vero cells have expanded to sufficient numbers in the production vessel, the cells are subsequently infected with monovalent dengue WVS (working virus seed). The infection medium (DMP) was DMEM without phenol red. The final concentrations of glucose, glutamine, and F68 in the infection medium were 3 g / L, 4.5 mM, and 0.2% (w / v), respectively. In this example, the DMP was filtered using a Sartopore 2 0.45+0.2 μm filter with which the liquid components were aseptically mixed.

[0427] In this example, Vero cells are grown to at least 90% confluency on the surface of a culture vessel prior to infection, where the Vero cells are approximately 2×10 cells / cm 2 A minimum of 90% confluence was required to achieve a cell density of 1000. Once the desired cell density was reached, infection and virus production was performed using DMP.

[0428] Monovalent TDV working virus seeds (WVS) were used to infect Vero cells. Vero cells with at least 90% confluency were infected in monolayer with WVS at an MOI of 0.0014 and plated with 1 cm 2 The infection volume per unit volume is 0.014 mL / cm 2 Manual shaking of CF10 during the infection process was performed. In this example, a DPBS wash step was performed prior to infection as part of the serial dilution steps. Infection and virus production parameters, temperature and CO2 set points were maintained at 37°C and 5%, respectively. Duration of infection was set to 90 min.

[0429] Three DPBS washing steps were also performed post-infection. Fresh DMP was added to facilitate virus production after the post-infection DPBS washes.

[0430] In this example, three daily harvest steps were performed between days 5 and 7 post-infection. The harvest steps included recovering the supernatant from the chamber. A medium change was performed 12 hours before the first harvest step. Fresh DMP was added to the cells after each harvest step. The daily harvest volume was approximately 12.8 L maximum per day, with a total harvest volume of 38.4 L maximum. All daily harvests were 7.0 log 10 achieved greater than PFU / mL.

[0431] Example 6.3 - Clarification of daily harvests Sartopore 2 (0.45+0.2um) was used for clarification of the daily harvest. For this filter, 20.454mL / cm 2 The throughput capacity (Pmax) of the Sartopore 2 150 cm 2 Filter: 300mL / min (1200L / m 2 / h [LMH]). The harvest clarification step using this filter involved a flow rate of 800 L / m 2 A conservative filtration flow rate of 100 / h was chosen to provide a high flow rate, short treatment time, and a reasonable safety margin.

[0432] Prior to clarification, the harvest ranged from 1 to 9 turbidity units (NTU). Upon being subjected to the clarification step, a reduction in turbidity (below 1 NTU) is observed, indicating that cellular debris from the harvest is effectively removed in this clarification step. The average filter backpressure ranged from 0.2 to 0.4 bar. The robustness of the clarification step to reduce turbidity to low levels at relatively low operating backpressures ensured that the clarification process operated consistently and away from the process boundary.

[0433] Example 6.4 - Harvest stabilization The stabilization buffer was a stock solution of 3xFTA that was added to the harvest to a final concentration of 0.2xFTA (0.2% F123, 3% trehalose dihydrate, 0.02% HSA in PBS). A smaller amount of 3xFTA (calculated based on 1 / 14th the total volume of the clarified harvest) was used to obtain a concentration of 0.2xFTA.

[0434] This is the only time that the buffered excipient composition is added to the harvest during the entire purification process until the drug substance is obtained. This means that the 0.2xFTA harvest is the feed for both the anion exchange chromatography step and the tangential flow filtration step. At the end of the tangential flow filtration step, a retentate is obtained which is the drug substance.

[0435] Example 6.5 - Anion Exchange Chromatography The main objective of this step is to remove host cell DNA (HC DNA) while ensuring maximal virus recovery.

[0436] In this step, an AEX column operated in flow-through mode is used. It was found that the stabilized harvest described above could be used directly as the feed material for the AEX column. No buffer exchange or buffer concentration steps were required. In this step, a Mustang Q column was used, which efficiently removed HC DNA with minimal virus loss.

[0437] In this example, two 7 mL AEX capsules were used. The flow rate was 230 mL / min. The AEX step was sufficient to reduce the amount of host cell DNA to the specified value of 50 ng / mL or less.

[0438] The DBC of Mustang Q membranes ranged from 2.15 to 3.69 mg of DNA per mL of membrane bed volume (MV).

[0439] Example 6.6 - Tangential Flow Filtration In this example, a TFF membrane (Hydrosart) with a molecular weight cut-off (MWCO) of 100 kDa was used. The pore size is smaller than the size of the dengue virus particles, which is about 50 nm, and therefore should be able to retain the virus. A feed flow rate of 180 LMH was used.

[0440] The flow-through from the AEX can be carried out directly as the feed composition for the TFF step.

[0441] The resulting TFF residue is the drug substance.

[0442] Example 6.7 - Processing of Drug Substance Three flushing steps of the TFF membrane were performed to obtain the bulk drug substance.

[0443] The flush buffer was 1×FTA, however, to compensate for trehalose dihydrate passing through the TFF membrane, the buffer flush composition was composed of a trehalose dihydrate concentration of 27%, resulting in a trehalose dihydrate concentration of 15% in the monovalent bulk drug substance.

[0444] Analytical data for FTA content of bulk drug substance for all serotypes produced during the production scale runs were close to the theoretical value of 1× FTA concentration.

[0445] Example 6.8 - Bulk Drug Substance Processing For bulk drug substances for TDV-3 and TDV-4, filtration of the bulk drug substance was performed immediately.

[0446] Filtration of the bulk drug substance also employed Sartopore 2 filters, which are hydrophilic polyethersulfone membranes with pore sizes of 0.45 + 0.2 μm.

[0447] If the concentrated individual harvests are pooled prior to filtration, the 2 If a filter size of 87.6 cm is used and each daily collection is filtered separately, 2The closest membrane area sizes available from the filter manufacturers were 0.1 m 2 and 150cm 2 It is.

[0448] Example 6.9 - Freezing and Storage of Bulk Drug Substance After the filtration step for TDV-3 and TDV-4, the bulk drug substance is frozen and stored. For TDV-1 and TDV-2, the bulk drug substance for each collection day was frozen and thawed on the day the last bulk drug substance was obtained from the last collection step, then all bulk drug substances were pooled and then subjected to a filtration step, followed by final freezing and storage. The purpose of the freezing step is to ensure that the potency and other quality attributes of the drug substance are maintained for the material to be used in drug product formulation.

[0449] Bulk drug substances are stored frozen using a plate freezer until thawed for use in drug product formulation.

[0450] Example 6.10 - Pharmaceutical Formulation After freezing the bulk drug substance, the next step was formulation of the bulk drug substance product, which included thawing the bulk drug substance and mixing the thawed bulk drug substance (i.e., for each of TDV-1, TDV-2, TDV-3, and TDV-4) with formulation buffer to obtain the bulk drug product.

[0451] The formulation buffer contained two buffers: a first excipient buffer (FEB) and a second excipient buffer (SEB). Qualitatively, the components of each of FEB and SEB are the same. However, quantitatively, the components of each of FEB and SEB are different.

[0452] After sterile filtration of the drug product (step 6.11), it was filled and loaded into vials (step 6.12) and moved directly to the next step.

[0453] Example 6.13 - Freeze-drying and sealing An exemplary lyophilization cycle is shown in Table 11 below. [Table 8]

[0454] After unloading and capping (step 6.14) and visual inspection (step 6.15), the final drug product is frozen in step 6.16 and finally labeled and packaged in step 6.17.

[0455] Example 7: pH Variation and Virus Yield The optimal pH for the entire purification process ranged from 7.6 to 8.1. To test whether variation in pH affected the virus yield, we set up an experiment in two groups as follows.

[0456] Group A: pH was maintained within 7.6-8.1 but varied by 0.5 units.

[0457] Group B: pH was maintained within 7.6-8.1 but fluctuated by less than 0.5 units (0.3 units in this group).

[0458] All other conditions that would not allow a fair comparison between the two groups were kept constant.

[0459] The results are shown in Table 9 below. [Table 9]

[0460] The allowable variation in pH units according to Table 9 above, and the yields obtained for each group are shown in Table 10 below. [Table 10]

[0461] It has surprisingly been found that virus yield can be significantly improved not only by maintaining the pH throughout the process steps at a given pH range (here 7.6-8.1), but also by ensuring that the pH does not fluctuate by more than 0.3 units.

[0462] Virus yield (mg / cm 2 ) is calculated as follows: Group A (i) Total number of CF10 flasks (93) × area of ​​each CF10 flask (6360 cm 2 ) = Total Area (ii) Total virus yield in milligrams (23,951,000 g) / total area obtained from step (i) above (591,480 cm 2 )=40.5mg / cm 2 Group B (i) Total number of CF10 flasks (93) × area of ​​each CF10 flask (6360 cm 2 ) = Total Area (ii) Total virus yield in milligrams (25,052,000 g) / total area obtained from step (i) above (591,480 cm 2 )=42.4mg / cm 2 .

[0463] In addition, this application references the following:

[0464] Item 1: A method for large scale production and manufacturing of a flavivirus vaccine comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing a flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), The method, wherein step (v) comprises at least one chromatography step. Item 1A: A method for large scale production and manufacturing of a flavivirus vaccine comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing a flavivirus; (iii) collecting to obtain a collection; (iv) processing the harvest of step (iii) to obtain a processed harvest; (v) purifying the treated harvest of step (iv), wherein step (v) comprises at least one chromatography step; Throughout steps (iii) to (v), the pH is maintained in the range of 7.6 to 8.1; (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is The method according to claim 1, wherein the amount is 0.4 units or less.

[0465] Item 1B: The method according to Item 1A, wherein the difference between Item 1A(a) and Item 1A(b) is 0.3 units or less.

[0466] Item 1C: The method according to item 1A, wherein the method results in a higher virus yield compared to the same method having the same process steps, but the difference between item 1A(a) and item 1A(b) is more than 0.4 units.

[0467] Item 1D: The method according to item 1B, wherein the method results in a higher virus yield compared to the same method having the same process steps, but allows the difference between item 1A(a) and item 1A(b) to be more than 0.3 units.

[0468] Item 1E: The increase in virus yield is at least about 1 mg / cm2 , e.g., 1.5 mg / cm 2 The method according to item 1C or 1D,

[0469] Item 2: The method according to items 1, 1A, 1B, 1C, 1D, or 1E, wherein the infection method in step (ii) is static and / or the cells are infected in a monolayer.

[0470] Item 3: The method of items 1, 1A, 1B, 1C, 1D, 1E, or 2, wherein the low MOI is less than 0.008.

[0471] Item 4: The method according to item 3, wherein the low MOI is 0.005 or less.

[0472] Item 5: The method of any one of items 1, 1A, 1B, 1C, 1D, 1E, or 2-4, wherein collecting includes a first collecting step and, optionally, at least one further collecting step.

[0473] Item 6: The method of any one of items 1, 1A, 1B, 1C, 1D, 1E, or 2 to 5, wherein the collecting in step (iii) comprises recovering supernatant from the cells obtained in step (ii).

[0474] Item 7: The method according to Item 6, wherein fresh medium is added immediately after the supernatant is collected.

[0475] Item 8: The method according to any one of Items 5 to 7, wherein medium exchange is performed at least 12 to 30 hours prior to the first collecting step.

[0476] Item 9: The method of item 8, wherein the medium exchange comprises discarding the supernatant and adding fresh medium.

[0477] Item 10: The method according to any one of items 5 to 9, wherein the first collecting step is performed on the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth day after infection.

[0478] Item 11: The method according to any one of Items 5 to 10, wherein the at least one further collecting step includes up to 9, preferably 5, further collecting steps.

[0479] Item 12: The method according to Item 11, wherein the interval between each collecting step is at least 20 to 30 hours.

[0480] Item 13: The method of any one of the preceding items, wherein step (i) comprises expanding the cells in a growth medium.

[0481] Item 14: The method of item 13, wherein the growth medium does not contain a non-ionic surfactant.

[0482] Item 15: The method of any one of the preceding items, wherein the cells are adherent cells.

[0483] Item 16: The method of any one of the preceding items, wherein the cells contain abundant flavivirus receptors.

[0484] Item 17: The method of any one of the preceding items, wherein the cells contain abundant dengue virus receptors.

[0485] Item 18: The method of item 15, wherein the cells are selected from the group consisting of Madin-Darby canine kidney cells, monkey cell line pMK, Vero cells, and human cell lines HEK293, MRC5, Per.C6, PMK, and WI-38.

[0486] Item 19: The method of item 18, wherein the cell is a Vero cell.

[0487] Item 20: The method according to any one of 2 to 19, wherein the infection medium of step (ii) covers a cell monolayer.

[0488] Item 21: The method of any one of the preceding items, wherein step (ii) comprises incubating the cells for 50 to 250 minutes, 60 to 200 minutes, or 70 to 150 minutes.

[0489] Item 22: The method according to Item 21, wherein the cells are incubated at a temperature range of 36 to 39°C.

[0490] Item 23: The method of any one of the preceding items, wherein step (ii) is immediately followed by 1, 2, 3, or 4 washing step(s).

[0491] Item 24: The method of item 23, wherein the cells are washed with PBS, DPBS, or TBS.

[0492] Item 25: The method of any one of the preceding items, wherein step (iv) comprises clarification of the harvest.

[0493] Item 26: The method of item 25, wherein the clarification of the harvest comprises one or more depth filtration steps.

[0494] Item 27: The method of item 26, wherein the one or more depth filtration steps comprise filters containing pore sizes between 0.1 μm and 1 μm.

[0495] Item 28: The method of items 26 or 27, wherein the one or more depth filtration steps comprise a heterogeneous bilayer filter.

[0496] Item 29: The method according to any one of items 25 to 28, wherein immediately after clarification, the harvest is frozen.

[0497] Item 30: The method of item 6, wherein the harvest is frozen immediately after recovery of the supernatant.

[0498] Item 31: The method of item 30, wherein the clarification is performed on a pooled harvest resulting from a first collecting step and at least one further collecting step.

[0499] Item 32: The method of any one of the preceding items, wherein step (iv) comprises clarification of the harvest.

[0500] Item 33: The method of item 32, wherein stabilization comprises adding a stabilization buffer to the harvest or the clarified harvest.

[0501] Item 34: The method according to item 33, wherein the stabilization buffer comprises a sugar, a surfactant, and a protein.

[0502] Item 35: Step (v) comprises the following successive steps: (va) ion exchange chromatography to obtain a purified harvest; (vb) The method of any one of the preceding items, comprising ultrafiltration.

[0503] Item 36: The method according to Item 35, wherein the ion exchange chromatography is anion exchange chromatography.

[0504] Item 37: The method of items 35 or 36, wherein step (va) is performed on a stabilized harvest.

[0505] Item 38: The method according to any one of Items 35 to 7, wherein the ultrafiltration in step (vb) is tangential flow filtration.

[0506] Item 39: The method according to any one of items 35 to 38, wherein the purified collection obtained after step (va) contains 50 ng / mL or less of host cell DNA.

[0507] Item 40: The method according to any one of Items 35 to 39, wherein the drug substance is obtained after the ultrafiltration of step (vb).

[0508] Item 41: The method according to any one of Items 35 to 40, further comprising a step (vi) of treating the drug substance, preferably comprising a buffer flush and at least one flushing step of the ultrafiltration membrane with a flushing buffer to obtain a composition comprising the drug substance.

[0509] Item 42: The method of item 41, wherein the composition comprising the drug substance and the buffer flush is a bulk drug substance.

[0510] Item 43: The method according to Item 41 or 42, wherein the flushing buffer comprises a sugar, a surfactant, and a protein.

[0511] Item 44: The method according to any one of Items 35 to 45, wherein the volume of the drug substance in step (vb) is 2 to 10 times lower than the volume obtained after step (va).

[0512] Item 45: step (iii) comprises a first collecting step to obtain a first collection; step (iv) comprises processing the first harvest obtained in step (iii) to obtain a first clarified and stabilized harvest, the step comprising clarification and stabilization of the harvest; Step (v) is (va) at least one chromatography step, such as anion exchange chromatography, to obtain a first purified harvest; (vb) purification of the clarified and stabilized harvest obtained in step (iv) comprising ultrafiltration, such as tangential flow filtration, on the purified harvest of step (va) to obtain a first drug substance; step (vi) buffer flushing and at least one flushing step of said ultrafiltration membrane with a flushing buffer to obtain a composition comprising a drug substance, said drug substance being a first bulk drug substance; 45. The method according to any one of items 42 to 44, wherein all of steps (iii) to (vi) are carried out on the same day to obtain the first bulk drug substance.

[0513] Item 46: The method of item 45, wherein a second collecting step is carried out 20 to 30 hours after the first collecting step of (iii), and steps (iv) to (vi) are repeated on the same day to obtain a second bulk drug substance.

[0514] Item 47: The method according to Item 46, wherein a total of up to 10 collecting steps are performed to obtain 10 bulk drug substances, preferably a total of up to 6 collecting steps are performed to obtain 6 bulk drug substances, and the interval between each collecting step is 20 to 30 hours.

[0515] Item 48: The method of any one of items 1, 1A, 1B, 1C, 1D, or 1E, or 2 to 47, wherein the flavivirus is a live, attenuated dengue virus selected from the group consisting of dengue serotype 1 (DENV-1), such as dengue 2 / 1 chimera, dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), such as dengue 2 / 3 chimera, and dengue serotype 4 (DENV-4), such as dengue 2 / 4 chimera.

[0516] Item 49: The method according to item 1, or any one of items 1A, 1B, 1C, 1D, or 1E, or 2 to 48, wherein the flavivirus is a live attenuated dengue virus selected from the group consisting of TDV-1 represented by SEQ ID NO: 1 and / or 2, TDV-2 represented by SEQ ID NO: 3 and / or 4, TDV-3 represented by SEQ ID NO: 5 and / or 6, and TDV-4 represented by SEQ ID NO: 7 and / or 8.

[0517] Item 50: (vii-a) filtering the bulk drug substance to obtain a filtered bulk drug substance; (vii-b) freezing the filtered bulk drug substance obtained from step (vii-a) to obtain a frozen bulk drug substance; The method according to any one of items 42 to 47, wherein steps (vii-a) and (vii-b) are preferably carried out on the same day as step (vi).

[0518] Item 51: The method of item 50, comprising a step (viii) of storing the frozen bulk drug substance obtained from step (vii-b), preferably wherein step (viii) is performed on the same day as steps (vii-a) and (vii-b).

[0519] Item 52: The method of item 50 or 51, wherein the flavivirus is a live attenuated dengue virus selected from the group consisting of dengue serotype 3 (DENV-3), such as dengue 2 / 3 chimera, and dengue serotype 4 (DENV-4), such as dengue 2 / 4 chimera.

[0520] Item 53: The method according to any one of items 50 to 52, wherein the flavivirus is a live attenuated dengue virus selected from the group consisting of TDV-3 represented by SEQ ID NO: 5 and / or 6, and TDV-4 represented by SEQ ID NO: 7 and / or 8.

[0521] Item 54: (vii-a) freezing the bulk drug substance obtained from the first collecting step and at least one further collecting step to obtain a frozen bulk drug substance, preferably freezing the bulk drug substance obtained from a total of up to ten collecting steps to obtain ten frozen bulk drug substance, more preferably freezing the bulk drug substance obtained from a total of six collecting steps to obtain six frozen bulk drug substance; (vii-b) thawing the bulk drug substance of step (vii-a) to obtain a thawed bulk drug substance; (vii-c) pooling the thawed bulk drug substance of step (vii-b) to obtain a pooled bulk drug substance. (vii-d) subjecting the pooled bulk drug substance obtained from step (vii-c) to a filtration step to obtain a filtered drug substance; (vii-e) processing the bulk drug substance from step (vii-d) comprising freezing the filtered drug substance to obtain a frozen bulk drug substance; The method according to any one of items 42 to 47, wherein steps (vii-a) to (vii-e) are preferably carried out on the same day as step (vi).

[0522] Item 55: The method of Item 54, comprising a step (viii) of storing the frozen bulk drug substance obtained from step (vii-e), preferably wherein step (viii) is performed on the same day as steps (vii-a) to (vii-e).

[0523] Item 56: The method of item 54 or 55, wherein the flavivirus is a live attenuated dengue virus selected from the group consisting of dengue serotype 1 (DENV-1), such as dengue 2 / 1 chimera, and dengue serotype 2 (DENV-2).

[0524] Item 57: The method according to any one of Items 54 to 56, wherein the flavivirus is a live attenuated dengue virus selected from the group consisting of TDV-1 represented by SEQ ID NO: 1 and / or 2, TDV-2 represented by SEQ ID NO: 3 and / or 4.

[0525] Item 58: The method according to any one of Items 51 to 53 or 55 to 57, further comprising the step (ix) of formulating a pharmaceutical agent.

[0526] Item 59: The formulation of a medicinal product comprises the following successive steps: (ix-a) thawing the bulk drug substance; (ix-b) mixing the thawed bulk drug substance of step (ix-a) with a formulation buffer.

[0527] Item 60: The method of item 59, wherein the formulation buffer comprises a first excipient buffer (FEB) and a second excipient buffer (SEB).

[0528] Item 61: The method according to Item 60, wherein the FEB and SEB each contain four salts, a sugar, a nonionic surfactant, and a protein.

[0529] Item 62: The method of any one of items 59 to 61, wherein step (ix-a) comprises thawing two, three, or four bulk drug substances each comprising a different dengue serotype selected from the group consisting of live attenuated dengue viruses selected from the group consisting of dengue serotype 1 (DENV-1), such as a dengue 2 / 1 chimera, dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), such as a dengue 2 / 3 chimera, and dengue serotype 4 (DENV-4), such as a dengue 2 / 4 chimera.

[0530] Item 63: The method according to any one of items 59 to 62, wherein step (ix-a) comprises thawing two, three, or four bulk drug substances, each comprising a different dengue serotype selected from the group consisting of TDV-1 represented by SEQ ID NO:1 and / or 2, TDV-2 represented by SEQ ID NO:3 and / or 4, TDV-3 represented by SEQ ID NO:5 and / or 6, and TDV-4 represented by SEQ ID NO:7 and / or 8.

[0531] Item 64: The method of items 62 or 63, wherein immediately after step (ix-a) and before step (ix-b), two, three, or four thawed bulk drug substances are mixed.

[0532] Item 65: The method according to any one of items 62 to 64, wherein the viral vaccine is bivalent, trivalent, or quadrivalent, preferably, the viral vaccine is the quadrivalent dengue vaccine TAK-003.

[0533] Item 66: The method of any one of items 48 to 65, wherein the live attenuated dengue-2 virus serotype is in the form of a DEN-2 PDK-53 variant from DENV-2 16681 having a triple mutation in NS1-53, 5'NCR-57, and NS3-250 such that amino acid position 250 of the NS3 protein contains a valine residue, and the chimera has the DEN-2 PDK-53 genome as a viral backbone and one or more structural protein genes encoding the capsid, pre-membrane / membrane, or envelope of the DEN-2 PDK-53 genome replaced with one or more corresponding structural protein genes from DEN-1, DEN-3, or DEN-4.

[0534] Item 67: The dengue-2 / 1 chimera has two further mutations such that amino acid position 116 of the NS2A protein contains a leucine residue and amino acid position 92 of the NS2B protein contains an aspartic acid residue; the live attenuated dengue-2 virus serotype has two further mutations such that amino acid position 52 of the prM protein contains a glutamic acid residue and amino acid position 412 of the NS5 protein contains a valine residue; the dengue-2 / 3 chimera has the DEN-2 PDK-53 genome as a viral backbone and has the prM-E gene (nt -457 to -2373) of the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild-type DEN-3 16562; and the dengue-2 / 3 chimera has the wild-type DEN-3 16562 prM-E gene (nt -457 to -2373) such that amino acid position 223 of the E protein contains a serine residue. 67. The method according to any one of items 48 to 66, wherein the dengue-2 / 4 chimera has one further mutation in the corresponding prM-E gene from 16562, such that amino acid position 66 of the NS2A protein contains a glycine residue and amino acid position 21 of the NS4A protein contains a valine residue, and wherein the dengue-2 / 4 chimera is of mixed genotype for amino acid position 99 of the NS2A protein containing an arginine or lysine residue.

[0535] Item 68: The dengue-2 / 1 chimera has the DEN-2 PDK-53 genome as a viral backbone and the prM-E gene (nt -457 to -2379) of the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild type DEN-1 16007; the dengue-2 / 3 chimera has the DEN-2 PDK-53 genome as a viral backbone and the prM-E gene (nt -457 to -2373) of the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild type DEN-3 16562; and the dengue-2 / 4 chimera has the DEN-2 PDK-53 genome as a viral backbone and the DEN-2 PDK-53 genome replaced with the corresponding prM-E gene from wild type DEN-4 1036. 68. The method according to any one of items 48 to 67, comprising the prM-E gene (nt-457 to -2379) of PDK-53.

[0536] Item 69: The method according to any one of items 48 to 68, wherein the live, attenuated dengue-2 virus serotype is represented by a polynucleotide of SEQ ID NO: 3 or a polypeptide of SEQ ID NO: 4, the dengue 2 / 1 chimera has nonstructural proteins from a modified, live, attenuated dengue-2 virus serotype and structural proteins from a dengue-1 virus serotype represented by a polynucleotide of SEQ ID NO: 1 or a polypeptide of SEQ ID NO: 2, the dengue 2 / 3 chimera has nonstructural proteins from a modified, live, attenuated dengue-2 virus serotype and structural proteins from a dengue-3 virus serotype represented by a polynucleotide of SEQ ID NO: 5 or a polypeptide of SEQ ID NO: 6, and the dengue 2 / 4 chimera has nonstructural proteins from a modified, live, attenuated dengue-2 virus serotype and structural proteins from a dengue-4 virus serotype represented by a polynucleotide of SEQ ID NO: 7 or a polypeptide of SEQ ID NO: 8.

[0537] Item 70: The method of any one of the preceding items, wherein the method provides a high viral titer.

[0538] Item 71: The method according to any one of the preceding items, wherein the method provides a higher average virus titer compared to a method comprising the same process steps, but wherein in step (ii) the cells are infected with an infection medium comprising a flavivirus at a high MOI, preferably a high MOI refers to an MOI of more than 0.008.

[0539] Item 72: The method according to any one of the preceding items, wherein the method provides a higher average virus titer compared to a method comprising the same process steps, but wherein in step (ii) the cells are infected with an infection medium comprising a flavivirus at a high MOI and a medium change is performed 12 to 30 hours before the first harvesting step, preferably a high MOI refers to an MOI of more than 0.008.

[0540] Item 73: The high viral titer is 7.0 log 10 More than 7.5 log PFU / mL, preferably 10 71. The method of any one of items 70, wherein the titer is greater than PFU / mL.

[0541] Item 74: Large scale, surface area 35,000 cm 2 7. The method of any one of the preceding items, comprising said production culture.

[0542] Item 75: Large scale, surface area 50,000 cm 2 75. The method according to item 74, comprising the production culture described above.

[0543] Item 76: Large scale, surface area 100,000 cm 2 76. The method according to item 74 or 75, comprising the production culture.

[0544] Item 77: The method of any one of the preceding items, wherein the viral vaccine is a live attenuated tetravalent dengue vaccine, preferably TAK-003.

[0545] Item 78: The pH is maintained in the range of 7.6 to 8.1 throughout steps (iii) to (v); (a) the maximum pH occurring at any time through steps (iii) through (v); (b) The difference between the minimum pH occurring at any time throughout steps (iii) to (v) is 3. The method of any one of the preceding claims, wherein the amount of the compound is 0.4 units or less.

[0546] Item 79: The pH is maintained in the range of 7.6 to 8.1 throughout steps (iii) to (vi); (a) the maximum pH occurring at any time through steps (iii) to (vi); and (b) The difference between the maximum pH at any time point through steps (iii) to (vi) is 78. The method according to any one of items 45 to 77, wherein the amount of the ion exchange reaction is 0.4 units or less.

[0547] Item 80: The pH is maintained in the range of 7.6 to...

Claims

1. 1. A method for large scale flavivirus vaccine production and manufacturing comprising the following sequential steps: (i) providing cells in a growth medium; (ii) infecting the cells of step (i) with an infection medium containing a flavivirus at a low MOI; (iii) collecting to obtain a collection; (iv) processing the collection of step (iii) to obtain a processed collection; (v) purifying the treated harvest of step (iv); step (v) comprises at least one chromatography step; The method, wherein the flavivirus is a dengue virus.

2. 2. The method of claim 1, wherein the infection method in step (ii) is static and / or the cells are infected in a monolayer.

3. The method of claim 1 , wherein the low MOI is less than 0.

008.

4. The method of claim 1 , wherein the low MOI is 0.005 or less.

5. The method of claim 1 , wherein collecting comprises a first collecting step and, optionally, at least one further collecting step.

6. 2. The method of claim 1, wherein collecting in step (iii) comprises recovering supernatant from the cells obtained in step (ii).

7. 7. The method of claim 6, wherein fresh medium is added immediately after the supernatant is collected.

8. 6. The method of claim 5, wherein a medium change is performed at least 12 to 30 hours before the first harvesting step.

9. 9. The method of claim 8, wherein the medium change comprises discarding the supernatant and adding fresh medium.

10. 6. The method of claim 5, wherein the first harvesting step is performed on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, or day 10 post-infection.

11. The method of claim 5 , wherein the at least one additional acquisition step comprises five additional acquisition steps.

12. 12. The method of claim 11, wherein the interval between each collecting step is at least 20-30 hours.

13. 10. The method of claim 1, wherein step (i) comprises expanding the cells in a growth medium.

14. 14. The method of claim 13, wherein the growth medium is free of non-ionic surfactants.

15. The method of claim 1 , wherein the cells are adherent cells.

16. 16. The method of claim 15, wherein the cells are Vero cells.

17. 10. The method of claim 1, wherein step (iv) comprises clarification of the harvest.

18. 10. The method of claim 1, wherein step (iv) comprises stabilizing the harvest.

19. 20. The method of claim 18, wherein stabilization comprises adding a stabilization buffer to the harvest or the clarified harvest.

20. 20. The method of claim 19, wherein the stabilization buffer comprises a sugar, a surfactant, and a protein.

21. Step (v) comprises the following sequential steps: (va) ion exchange chromatography to obtain a purified harvest; The method of claim 1, comprising (vb) ultrafiltration.

22. 22. The method of claim 21, wherein the ion exchange chromatography is anion exchange chromatography.

23. 22. The method of claim 21, wherein step (va) is performed on the clarified, stabilized harvest.

24. 22. The method of claim 21, wherein the ultrafiltration in step (vb) is tangential flow filtration.

25. 22. The method of claim 21, wherein the purified harvest obtained after step (va) contains 50 ng / mL or less of host cell DNA.

26. 22. The method of claim 21, wherein the drug substance is obtained after the ultrafiltration of step (vb).

27. 22. The method of claim 21, further comprising step (vi) of processing the drug substance, comprising at least one step of flushing the ultrafiltration membrane with a buffer flush buffer to obtain a composition comprising the drug substance.

28. 28. The method of claim 27, wherein the composition comprising the drug substance and the buffer flush is a bulk drug substance.

29. 28. The method of claim 27, wherein the flushing buffer comprises a sugar, a surfactant, and a protein.

30. step (iii) comprises a first collecting step to obtain a first collection; step (iv) comprising processing the first harvest obtained in step (iii) to obtain a first clarified and stabilized harvest, wherein step (iv) comprises clarification and stabilization of the harvest; Step (v) (va) at least one chromatography step, such as anion exchange chromatography, to obtain a first purified collection; (v-b) purification of the clarified and stabilized harvest obtained in step (iv) comprising ultrafiltration, such as tangential flow filtration, on the purified harvest of step (va) to obtain a first drug substance; step (vi) comprising a buffer flush and at least one flushing step of said ultrafiltration membrane with a flushing buffer to obtain a composition comprising a drug substance, said drug substance being a first bulk drug substance; 30. The method of claim 28, wherein steps (iii) through (vi) are all performed on the same day to obtain the first bulk drug substance.

31. 31. The method of claim 30, wherein a second collecting step is performed 20 to 30 hours after the first collecting step of (iii), and steps (iv) to (vi) are repeated on the same day to obtain a second bulk drug substance.

32. 2. The method of claim 1, wherein the flavivirus is a live, attenuated dengue virus selected from the group consisting of dengue serotype 1 (DENV-1), such as a dengue 2 / 1 chimera, dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), such as a dengue 2 / 3 chimera, and dengue serotype 4 (DENV-4), such as a dengue 2 / 4 chimera.

33. 2. The method of claim 1, wherein the flavivirus is a live attenuated dengue virus selected from the group consisting of TDV-1 represented by SEQ ID NO: 1 and / or 2, TDV-2 represented by SEQ ID NO: 3 and / or 4, TDV-3 represented by SEQ ID NO: 5 and / or 6, and TDV-4 represented by SEQ ID NO: 7 and / or 8.

34. (vii-a) filtering the bulk drug substance to obtain a filtered bulk drug substance; 29. The method of claim 28, comprising a step (vii) of processing the bulk drug substance comprising: (vii-b) freezing the filtered bulk drug substance obtained from step (vii-a) to obtain a frozen bulk drug substance.

35. 35. The method of claim 34, comprising the step (viii) of storing the frozen bulk drug substance obtained from step (vii-b).

36. 36. The method of claim 35, further comprising the step (ix) of formulating the pharmaceutical product.

37. The formulation of a pharmaceutical product comprises the following successive steps: (ix-a) thawing the bulk drug substance; 37. The method of claim 36, comprising: (ix-b) combining the thawed bulk drug substance of step (ix-a) with a formulation buffer.

38. 38. The method of claim 37, wherein the formulation buffer comprises a first excipient buffer (FEB) and a second excipient buffer (SEB).

39. 39. The method of claim 38, wherein the FEB and SEB each comprise four salts, a sugar, a non-ionic surfactant, and a protein.

40. 40. The method of claim 39, wherein step (ix-a) comprises thawing two, three, or four bulk drug substances each comprising a different dengue virus selected from the group consisting of live attenuated dengue viruses selected from the group consisting of dengue serotype 1 (DENV-1), such as dengue 2 / 1 chimera, dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), such as dengue 2 / 3 chimera, and dengue serotype 4 (DENV-4), such as dengue 2 / 4 chimera.

41. 40. The method of claim 39, wherein step (ix-a) comprises thawing two, three, or four bulk drug substances, each comprising a different dengue serotype selected from the group consisting of TDV-1 represented by SEQ ID NO: 1 and / or 2, TDV-2 represented by SEQ ID NO: 3 and / or 4, TDV-3 represented by SEQ ID NO: 5 and / or 6, and TDV-4 represented by SEQ ID NO: 7 and / or 8.

42. 41. The method of claim 40, wherein immediately after step (ix-a) and before step (ix-b), two, three, or four thawed bulk drug substances are mixed.

43. 41. The method of claim 40, wherein the viral vaccine is bivalent, trivalent, or tetravalent.

44. 33. The method of claim 32, wherein the live attenuated dengue-2 virus serotype is in the form of a DEN-2 PDK-53 variant derived from DENV-2 16681 having a triple mutation in NS1-53, 5'NCR-57, and NS3-250 such that amino acid position 250 of the NS3 protein contains a valine residue, and the chimera has the DEN-2 PDK-53 genome as a viral backbone and one or more structural protein genes encoding the capsid, pre-membrane / membrane, or envelope of the DEN-2 PDK-53 genome replaced with one or more corresponding structural protein genes from DEN-1, DEN-3, or DEN-4.

45. 10. The method of claim 1, wherein the method provides a high viral titer.

46. 2. The method of claim 1, wherein the method provides a higher average virus titer compared to a method comprising the same process steps, but wherein in step (ii) the cells are infected with an infection medium comprising a flavivirus at a high MOI.

47. 2. The method of claim 1, wherein the method provides a higher average virus titer compared to a method comprising the same process steps, but wherein in step (ii) the cells are infected with an infection medium comprising a flavivirus at a high MOI, and a medium change is performed 12 to 30 hours before the first harvesting step.

48. The high viral titer is 7.0 log １０ 46. ​​The method of claim 45, wherein the titer is greater than PFU / mL.

49. Large scale, surface area 35,000 cm ２ The method of claim 1, comprising the above production culture.

50. Large scale, surface area 50,000 cm ２ 50. The method of claim 49, comprising the production culture described above.

51. Large scale, surface area 100,000 cm ２ 50. The method of claim 49, comprising the production culture described above.

52. 10. The method of claim 1, wherein the viral vaccine is a live attenuated tetravalent dengue vaccine.

53. The method described in claim 1, wherein the viral vaccine is TAK-003.

54. A virus composition obtained by the method of claim 1.

55. 44. A viral vaccine obtained by the method of claim 43.

56. A pharmaceutical package comprising a viral vaccine obtained by the method of claim 1.

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