Large scale flavivirus vaccine production and manufacture

Abstract

The present invention provides methods for large scale flavivirus vaccine production and manufacture. The methods provided herein are specifically contemplated for large scale production and manufacture of live attenuated flavivirus vaccines, such as live attenuated dengue virus vaccines. In addition, the methods provided herein relate to the formulation of live, attenuated, monovalent, divalent, trivalent, or tetravalent viral vaccine products.

Description

Technical Field

[0001] The present invention provides methods for large-scale flavivirus vaccine production and manufacturing. The methods provided herein are specifically contemplated for large-scale production and manufacturing of live, attenuated flavivirus vaccines, such as live, attenuated dengue virus vaccines. In addition, the methods provided herein relate to the formulation of live, attenuated, monovalent, bivalent, trivalent, or quadrivalent viral vaccine products. Background Art

[0002] Expanding vaccine manufacturing capacity is crucial to stopping the global spread of the disease and preventing the emergence of new variants of the virus. One of the biggest challenges in vaccine production and manufacturing is economically utilizing resources to meet the increasing challenge of delivery to billions of people worldwide.

[0003] Flaviviruses are a class of viruses that continue to pose an ever-growing threat to public health. These are vector-borne RNA viruses that can unexpectedly emerge in human populations and cause a range of potentially serious diseases, including hepatitis, vascular shock syndrome, encephalitis, acute flaccid paralysis, congenital anomalies, and fetal death. This epidemiological pattern has occurred numerous times over the past 70 years, including epidemics of dengue virus and West Nile virus, and more recently, the explosive Zika virus epidemic in the Americas. Flaviviruses are now found worldwide and infect up to 400 million people annually. Significantly, outbreaks of other, less well-characterized flaviviruses have been reported in humans and animals in various parts of the world. Little is known about the potential for these viruses to continue to spread among humans. Therefore, 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.

[0004] Early signs of supply pressure are being observed across all vaccine manufacturing steps, such as the working virus used to infect the first production culture, the bioreactors used to produce the vaccine components, the chromatography and filtration steps used to purify the virus, or the filters and vials that contain the vaccine components. These individual challenges can have a compounding effect: the lack of a single input can disrupt the entire manufacturing process. Therefore, there is an urgent need to reinvent large-scale vaccine production and manufacturing by providing economically tailored processes at each step that keep pace with the growing demand for vaccines. Summary of the Invention

[0005] The present invention provides 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 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. The infection medium may optionally contain a low MOI of the flavivirus.

[0006] The present invention provides 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 comprising the flavivirus (optionally 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 format.

[0007] The present invention provides 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 comprising the flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least one further harvesting step, and 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.

[0008] The present invention provides 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 infection medium comprising the flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least one further harvesting step, and 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. In this context, the infection method in step (ii) is static, and the cells are infected as a monolayer.

[0009] The present invention provides 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 comprising the flavivirus (optionally 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 wherein step (v) comprises the following sequential steps: (va) ion exchange chromatography to obtain a purified harvest, and (vb) ultrafiltration.

[0010] The present invention provides 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 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 in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein the difference between (a) the maximum pH occurring at any time point in steps (iii) to (v) and (b) the minimum pH occurring at any time point in steps (iii) to (v) is no greater than 0.4 or 0.3 units.

[0011] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0012] - Harvest step to obtain the harvest,

[0013] - Bulk stock obtained from harvest,

[0014] - Processing the bulk stock solution, including the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution.

[0015] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0016] - provide cells in growth medium,

[0017] - Infect cells with medium containing flavivirus,

[0018] - Harvest step to obtain the harvest,

[0019] - Bulk stock obtained from harvest,

[0020] - Processing the bulk stock solution, comprising the step of filtering the bulk stock solution, followed by the step of freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtration step. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 to less than 0.008 or 0.1 to 0.008.

[0021] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0022] - at least two harvesting steps to obtain at least two harvests,

[0023] - at least two bulk stocks from at least two harvests,

[0024] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, followed by freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stock solution.

[0025] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0026] - provide cells in growth medium,

[0027] - Infect cells with medium containing flavivirus,

[0028] - at least two harvesting steps to obtain at least two harvests,

[0029] - at least two bulk stocks from at least two harvests,

[0030] - Processing at least two bulk stock solutions, comprising the step of filtering each of the at least two bulk stock solutions, followed by the step of freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solutions does not involve a freezing step prior to the filtering step. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 to less than 0.008 or 0.1 to 0.008.

[0031] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps:

[0032] (i) providing cells in a growth medium,

[0033] (ii) infecting the cells of step (i) with medium comprising the flavivirus (optionally at a low MOI),

[0034] (iii) a first harvest to obtain a first harvest,

[0035] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0036] (v) purifying the processed harvest of step (iv), comprising at least one chromatography step to obtain a purified harvest,

[0037] (vi) an ultrafiltration step to obtain a first stock solution,

[0038] (vi) processing the stock solution obtained in step (v) comprising subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain the bulk stock solution,

[0039] wherein a second harvesting step is performed, and the above steps are repeated to obtain a second bulk stock solution,

[0040] (vii) processing of bulk stock solutions,

[0041] wherein processing the bulk stock solution includes the step of filtering the bulk stock solution before the step of freezing the bulk stock solution,

[0042] And wherein after the step of freezing the bulk stock solution, the bulk stock solution is thawed and pooled.

[0043] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps:

[0044] (i) providing cells in a growth medium,

[0045] (ii) infecting the cells of step (i) with medium comprising the flavivirus (optionally at a low MOI),

[0046] (iii) a first harvest to obtain a first harvest,

[0047] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0048] (v) purifying the processed harvest of step (iv), comprising at least one chromatography step to obtain a purified harvest,

[0049] (vi) an ultrafiltration step to obtain a first stock solution,

[0050] (vi) processing the stock solution obtained in step (v) comprising subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain the bulk stock solution,

[0051] wherein a second harvesting step is performed, and the above steps are repeated to obtain a second bulk stock solution,

[0052] (vii) processing of bulk stock solutions,

[0053] wherein steps (iii) to (vii) are performed on the same day,

[0054] Wherein processing the bulk stock solution includes the step of filtering the bulk stock solution before the step of freezing the bulk stock solution.

[0055] According to the present invention, preferably the total surface area of ​​the production culture can be on the order of 35,000 cm 2 or larger, such as, for example, 50,000 cm 2 to 1,000,000 cm 2 、100,000 cm 2 to 1,000,000 cm 2 , 50,000 cm 2 to 450,000 cm 2 、100,000 cm 2 to 450,000 cm 2 .

[0056] According to the present invention, preferably the flavivirus is a dengue virus, preferably the dengue virus is selected from the group consisting of live, attenuated dengue-2 virus serotype, dengue 2 / 1 chimera, dengue 2 / 3 chimera and dengue 2 / 4 chimera.

[0057] The present invention also provides a method for large-scale flavivirus vaccine production and manufacturing, comprising formulating a formulation. The formulation preferably comprises a tetravalent dengue virus vaccine comprising a live, attenuated dengue-2 virus serotype, a dengue 2 / 1 chimera, a dengue 2 / 3 chimera, and a dengue 2 / 4 chimera. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1- Example Overview of Flavivirus Vaccine Production and Manufacturing Process. In this figure, an example of a manufacturing process for a tetravalent dengue virus vaccine according to the present invention is shown. References to individual serotypes as Den-1, Den-2, Den-3, or Den-4 can be interpreted as non-chimeric dengue serotypes 1, 2, 3, and 4, respectively, or as one or more chimeric dengue serotypes, such as four chimeric dengue serotypes or three chimeric dengue serotypes, such as the dengue 2 / 1 chimera or TDV-1 represented by SEQ ID NO: 1 and / or SEQ ID NO: 2, the dengue 2 serotype or TDV-2 represented by SEQ ID NO: 3 and / or SEQ ID NO: 4, the dengue 2 / 3 chimera or TDV-3 represented by SEQ ID NO: 5 and / or SEQ ID NO: 6, and the dengue 2 / 4 chimera or TDV-4 represented by SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0059] Figure 2 - Example overview of the production and manufacturing process of flavivirus vaccines. In this figure, an example of a manufacturing process for a quadrivalent dengue virus vaccine according to the present invention is shown. In the step "Processing BDS," filtration can occur before and / or after freezing and storage of the BDS. The reference to a single serotype as Den-1, Den-2, Den-3, or Den-4 can be interpreted as non-chimeric dengue serotypes 1, 2, 3, and 4, respectively, or as one or more chimeric dengue serotypes, such as four chimeric dengue serotypes or three chimeric dengue serotypes, such as the dengue 2 / 1 chimera or TDV-1 represented by SEQ ID NO: 1 and / or SEQ ID NO: 2, the dengue 2 serotype or TDV-2 represented by SEQ ID NO: 3 and / or SEQ ID NO: 4, the dengue 2 / 3 chimera or TDV-3 represented by SEQ ID NO: 5 and / or SEQ ID NO: 6, and the dengue 2 / 4 chimera or TDV-4 represented by SEQ ID NO: 7 and / or SEQ ID NO: 8. 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 differ in the scale of manufacture and / or processing of bulk stock. 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.

[0060] Figure 3- Graph of viral titers obtained after infection with a low MOI of 0.005 (Group 1) and a high MOI of 0.05 (Group 2). Individual viral titers on days 5, 6, and 9 are shown (bars from left to right).

[0061] Figure 4 - Graph of viral titers obtained after infection with a low MOI of 0.001 (Group 1) and a high MOI of 0.05 (Group 2). Individual viral titers on days 5, 6, and 9 are shown (bars from left to right).

[0062] Figure 5 - Graph showing the dependence of virus yield (grams) on pH fluctuations - the lower the fluctuations, the higher the virus yield.

[0063] Figure 6 - A graph showing the average viral titers (PFU / mL) over 36 months according to processes A and B. Points at the upper end of the graph, indicated by large curly brackets on the right side of the graph, relate to process B, while points at the lower end of the graph, indicated by small curly brackets on the right side of the graph, relate to process A. DETAILED DESCRIPTION

[0064] Throughout the specification, references to the word "may" (such as the phrase "may be") should not be construed as limiting the invention to specific embodiments, but rather are illustrative and non-limiting examples.

[0065] Throughout the specification, reference to performing multiple steps "in sequence" means that the steps must be performed in that order. However, when the word "in sequence" is not mentioned, the steps may or may not be performed in that order.

[0066] The methods of the present 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 isolated viruses that can be obtained from a starting sample volume of approximately thousands of liters or thousands of square centimeters. From another perspective, large-scale refers to a starting production culture of at least 1000 liters or more, or at least 35,000 cm 2 or more. In the context of the present invention, large-scale production means a "larger" number of tissue culture dishes and a "larger" surface area for cell expansion and growth. For large-scale production and manufacturing of flavivirus vaccines according to the present invention, culture dishes such as Corning® CellSTACK® culture chambers or Nunc™ EasyFill™ Cell Factory™ systems can be used.

[0067] For example, a Corning® CellSTACK® culture chamber with 1 layer (CF1) and a cell growth area of ​​636 cm can be used. 2; 2 layers (CF2), cell growth area 1,272 cm 2 ; 5-layer (CF5), cell growth area 3,180 cm 2 10 layers (CF10), cell growth area 6,360 cm 2 ; 40 layers (CF40), cell growth area 25,440 cm 2 The recommended volume of culture medium required for each of these vessels is: 130 to 200 mL for CF1; 260 to 400 mL for CF2; 650 to 1,000 mL for CF5; 1,300 to 2,000 mL for CF10; and 5,200 to 8,000 mL for 40 layers.

[0068] Thus, for large-scale production and manufacture of flavivirus vaccines, a "larger" number of culture vessels can be any combination of the above-mentioned culture vessels or other commercially available culture vessels, with which the total volume of the production culture can be on the order of at least 1000 liters or with which the total surface area of ​​the production culture can be on the order of 35,000 cm. 2 or larger, such as, for example, 50,000 cm 2 to 1,000,000 cm 2 、100,000 cm 2 to 1,000,000 cm 2 , 50,000 cm 2 to 450,000 cm 2 、100,000 cm 2 to 450,000 cm 2 .

[0069] Thus, for example, for large-scale production and manufacture of flavivirus vaccines, 5 to 20 CF10 chambers, 10 to 20 CF10 chambers, 15 to 20 CF10 chambers may be used, or 1 to 20 CF40 chambers, 4 CF40 chambers, 5 to 20 CF40 chambers, 10 to 20 CF40 chambers, 15 to 20 CF40 chambers, or any combination thereof may be used. It will be understood that each individual possibility can be combined with another possibility to yield a combination of features, and that each possibility and each combination represents a separate embodiment of the present invention.

[0070] It will be appreciated by those skilled in the art that large-scale production is designed specifically to meet the demands of viral vaccine production and manufacturing while economically improving process resources. Therefore, the method of the present invention is specifically directed to large-scale production and manufacturing of viral vaccines, for which scientific and economic considerations are completely different from small-scale processes. Therefore, the present invention preferably excludes small-scale viral vaccine production processes.

[0071] The skilled artisan will also understand that the methods of the present invention involve the "production" of flavivirus vaccines using "seed" starting materials. The terms "production culture" and "seed culture" are well known in the art. A seed culture generally refers to a pure culture of the desired strain, i.e., the primary strain, and an inoculum refers to a seed culture that is inoculated into a culture medium for propagation and is then considered a production culture. Therefore, the skilled artisan will understand that methods used to develop seed starting materials or methods typically used for seed culture are not applicable or relevant and cannot be used for large-scale production and manufacture of viral vaccines.

[0072] The methods of the present invention also relate to the large-scale production and manufacture of "attenuated" viral vaccines. The large-scale production and manufacture of attenuated viral vaccines is challenging because the replication rate of attenuated viruses can be compromised. As used herein, the term "replication" refers to the process by which complementary strands of nucleic acid molecules are synthesized by polymerases. In the context of the present invention, the term "replication" with respect to viruses refers to the completion of the flavivirus life cycle, in which infectious viral particles or virions attach to the surface of a host cell (usually bound to specific cell surface molecules, which determine the specificity of infection). Once inside the cell, the virions shed their coats and the flavivirus genes begin to be expressed, leading to the synthesis of proteins required for genome replication and the synthesis of new proteins to prepare new capsids and nuclei, thereby leading to the assembly of progeny infectious viral particles, which themselves are capable of infecting and replicating in new host cells. Therefore, the viral life cycle is complete only when infection of one or more viral particles or virions within a single cell proceeds to produce fully infectious progeny viral particles. The term "replication rate" refers to the multiple times the flavivirus population grows. Therefore, as used herein, the term "attenuated" means that the viral replication rate has been compromised. Several methods are known to those skilled in the art to assess the attenuation of a particular virus. For example, dengue virus attenuation can be measured using methods including temperature sensitivity, plaque size, reduced replication in mosquito C6136 cell culture, reduced replication in intact mosquitoes, and reduced incidence of viremia in monkeys.

[0073] The method of the present invention can be used for large-scale production and manufacturing of flaviviruses. The genus Flavivirus includes enveloped positive-strand 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, Tyuleniy 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.

[0074] The genus Flavivirus includes highly pathogenic and potentially hemorrhagic fever viruses (such as yellow fever virus and dengue virus), Zika virus, encephalitis viruses (such as Japanese encephalitis virus, Murray Valley encephalitis virus, and West Nile virus), as well as several less pathogenic viruses. The flavivirus genome comprises, from 5' to 3' direction: a 5'-noncoding region (5'-NCR), a capsid protein (C) coding region, a pre-membrane protein (prM) coding region, an envelope protein (E) coding region, regions encoding nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), and a 3' noncoding region (3'-NCR). The flavivirus structural proteins are C, prM, and E, and the nonstructural proteins are NS1 to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and flavivirus proteases.

[0075] Preferred flavivirus vaccines manufactured according to the present invention are Zika and dengue vaccines. Therefore, the method of the present invention can be used for the large-scale production and manufacture of dengue virus, i.e., each dengue serotype. As used herein, dengue virus is a single-stranded positive-sense RNA virus of the Flaviviridae family. The Flaviviridae family includes three genera: flavivirus, hepacivirus, and pestivirus. As used herein, the term "dengue serotype" refers to a dengue virus species that is defined by its cell surface antigens and can therefore be distinguished from other dengue serotypes by serological methods known in the art. Currently, there are four known dengue virus serotypes, i.e., dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3), and dengue serotype 4 (DENV-4). Therefore, the method of the present invention can also be used for large-scale production and manufacture of four live, attenuated dengue virus strains alone.

[0076] As used herein, the term "live attenuated dengue virus" refers to a viable and infective dengue virus whose virulence has been reduced by mutation. The live attenuated dengue virus can be a dengue virus whose components are all derived from the same dengue serotype, or can be a chimeric dengue virus with parts from two or more dengue serotypes. "Viral strain" and especially "dengue virus strain" are genetic subtypes of viruses (especially dengue viruses) characterized by specific nucleic acid sequences. Dengue serotypes can include different strains with different nucleic acid sequences, which have the same cell surface antigens and are therefore recognized by the same antibodies. Dengue virus strains can be a dengue virus whose components are all derived from the same dengue serotype, or can be a chimeric dengue virus with parts from two or more dengue serotypes.

[0077] 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).

[0078] TDV-1, TDV-2, TDV-3 and TDV-4 together form a tetravalent dengue virus composition called "TDV" or "TAK-003", a dengue vaccine sold under the trade name "Qdenga".

[0079] In one embodiment, the tetravalent dengue virus composition "TAK-003" comprises:

[0080] Live attenuated dengue virus serotype 1,

[0081] Live attenuated dengue virus serotype 2,

[0082] Live attenuated dengue virus serotype 3, and

[0083] Live attenuated dengue virus serotype 4;

[0084] wherein 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;

[0085] The dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 and differs from the wild-type by at least three nucleotides as follows:

[0086] a) 5'-noncoding region (NCR)-57 (nt-57)

[0087] b) NS1-53 Gly-to-Asp (nt-2579)

[0088] c) NS3-250 Glu-to-Val (nt-5270);

[0089] Three of the chimeric dengue strains were derived from dengue serotype 2 strains by replacing the structural proteins prM and E of the serotype 2 strain with the corresponding structural proteins of other dengue serotypes, resulting in the following chimeric dengue strains:

[0090] DENV-2 / 1 chimera,

[0091] DENV-2 / 3 chimera, and

[0092] DENV-2 / 4 chimera.

[0093] "Chimeric viruses" or "chimeric strains" or "chimeric virus strains" typically comprise portions from at least two different viruses. For example, a chimeric virus may comprise the prM and E proteins of dengue virus and other proteins from another flavivirus. A chimeric virus may comprise the 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 may comprise the prM and E proteins of dengue virus and other proteins from yellow fever virus strain YF-17D. Such chimeric viruses are found in the commercial product Dengvaxia® and are described, for example, in WO 98 / 37911, WO 03 / 101397, WO 2007 / 021672, WO 2008 / 007021, WO 2008 / 047023, and WO 2008 / 065315. Thus, certain embodiments of the present invention include making a tetravalent composition of four chimeric dengue strains, and / or a tetravalent composition of four live, attenuated dengue strains, such as, for example, four live, attenuated chimeric dengue strains.

[0094] "Chimeric dengue viruses" or "chimeric dengue serotype strains" or "chimeric dengue strains" preferably comprise portions from at least two different dengue strains or two different dengue serotypes, i.e., dengue-dengue chimeras. Such chimeric dengue viruses are described, for example, in WO 01 / 060847 A2, WO 2014 / 150939 A2, and WO 2017 / 179017 A1.

[0095] 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, said DEN-2 PDK-53 variant having a triple mutation at NS1-53, 5'NCR-57 and NS3-250 such that amino acid position 250 of the NS3 proteinopathy contains a valine residue, and wherein the chimera has said DEN-2 PDK-53 genome as the viral backbone and one or more structural protein genes encoding capsid, premembrane / membrane or envelope, or a combination thereof, of said DEN-2 PDK-53 genome are replaced by one or more corresponding structural protein genes from DEN-1, DEN-3 or DEN-4.

[0096] In a preferred embodiment, the dengue 2 / 1 chimera or DENV-2 / 1 chimera or TDV-1 has two additional 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, wherein the live attenuated dengue-2 virus serotype or DENV-2 or TDV-2 has two additional 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, wherein the dengue-2 / 3 chimera or DENV-2 / 3 chimera or TDV-3 has the DEN-2 genome as the viral backbone and the DEN-2 PDK-53 is present. The prM-E gene (nt -457 to -2373) of the PDK-53 genome is replaced by the corresponding prM-E gene from wild-type DEN-3 16562, and wherein the dengue-2 / 3 chimera or DENV-2 / 3 chimera or TDV-3 has one additional mutation at 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, and wherein 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 wherein the dengue-2 / 4 chimera or DENV-2 / 4 chimera or TDV-4 is of mixed genotype with respect to containing either an arginine or lysine residue at amino acid position 99 of the NS2A protein.

[0097] Thus, the dengue-2 / 1 chimera or DENV-2 / 1 chimera or TDV-1 has the DEN-2PDK-53 genome as the viral backbone and the prM-E gene (nt-457 to 2379) of the DEN-2 PDK-53 genome is replaced by the corresponding prM-E gene from wild-type DEN-1 16007, wherein 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 prM-E gene (nt-457 to 2373) of the DEN-2 PDK-53 genome is replaced by the corresponding prM-E gene from wild-type DEN-3 16562, and wherein the dengue-2 / 4 chimera or DENV-2 / 4 chimera or TDV-4 has the DEN-2 PDK-53 genome as the viral backbone and the DEN-2 The prM-E gene (nt-457 to 2379) of the PDK-53 genome was replaced by the corresponding prM-E gene from wild-type DEN-4 1036.

[0098] Thus, the 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; the 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; the dengue 2 / 3 chimera or DENV-2 / 3 chimera or TDV-3 has nonstructural proteins from a modified, live, attenuated dengue-2 virus serotype and structural proteins 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 from a modified, live, attenuated dengue-2 virus serotype and structural proteins from a dengue-4 virus serotype, represented by the polynucleotide of SEQ ID NO: 7 or the polypeptide of SEQ ID NO: 8.

[0099] RNA viruses, such as those contained in TDV, can be characterized by their RNA sequences. Alternatively or additionally, it is common practice to characterize the genome of an RNA virus by its corresponding DNA sequence, and the corresponding DNA sequence is readily understood to reflect the RNA genome of the virus. Therefore, throughout this disclosure, references to "t" or "thymine" should be understood as references to "u" or "uracil," respectively, when referring to a genomic RNA viral sequence.

[0100] Provide cells in growth medium

[0101] According to the present invention, the method for large-scale flavivirus vaccine production and manufacturing preferably includes 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 the 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.

[0102] This step also involves expanding the cells to a sufficient number in growth medium. For the purpose of large-scale production and manufacture of flavivirus vaccines, a number of different cell lines can be used, including Madin-Darby canine kidney cells, monkey cell lines pMK, Vero, and human cell lines HEK 293, MRC 5, Per.C6, PMK, LLCMK2, BHK, and WI-38. All of these cell lines are well characterized and have a proven safety profile. In addition, these cell lines are suitable for supporting the replication of attenuated vaccine strains. Each of these cell lines represents a separate embodiment of the present invention.

[0103] Therefore, for the purpose of the present invention, two cell banks, i.e., master cell bank (MCB) and working cell bank (WCB), can be developed using cell lines (such as selected from Madin-Darby canine kidney cells, monkey cell lines pMK, Vero and human cell lines HEK 293, MRC 5, Per.C6, PMK, LLCMK2, BHK and WI-38). As used herein, the term "master cell bank" refers to a cell culture (e.g., fully characterized cells) grown and stored under cryopreservation conditions from a single clone. Cells from MCB are used to develop working cell banks. As used herein, the term "working cell bank" refers to a cell culture (e.g., fully characterized cells) grown and stored under cryopreservation conditions from the MCB of a single bottle or from the master cell banks of two pooled bottles. Cells from WCB are later used to culture production cells.

[0104] The methods of the present invention also include expanding the cells using a growth medium and additive formulation that can be the same as or different from the medium used to develop the WCB and MCB. According to the present invention, the growth medium used for cell expansion comprises one or more of a growth medium, glutamine or GlutaMAX™, and serum. In some embodiments, the medium is serum-free.

[0105] The term "growth medium" refers to a culture medium for culturing cells containing nutrients that maintain cell viability and support proliferation. Growth medium can contain appropriate amounts and any of the following nutrients combined: one or more salts, one or more buffers, amino acids, glucose or other one or more sugars, antibiotics and other components (such as peptide growth factors, etc.). "Growth medium" is known in the art and can be classified as natural or artificial cell culture medium. The example of the cell culture medium that can be used for the method of the present invention includes minimum essential medium (MEM), Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, DMEM), DMEM / F-12 and Roswell Park Memorial Institute culture medium (Roswell Park Memorial Institute Medium, RPMI). Different culture mediums have different pH, glucose concentrations, growth factors and other supplements, which can be used for different cell types or different applications. In some embodiments, custom cell culture medium or commercially available cell culture medium can be used, such as Dulbecco's Modified Eagle's Medium, minimum essential medium, RPMI culture medium, HA or HAT culture medium, or other culture mediums that can be obtained from other commercial sources. The growth medium can include one or more antibiotics (e.g., actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, penicillin, streptomycin, polymyxin B, streptomycin, tetracycline or any other suitable antibiotic or any combination of two or more thereof). The growth medium can include one or more salts (e.g., balanced salts, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, etc.). The growth medium can also include one or more buffers (e.g., HEPES or other suitable buffers). The growth medium can also include differentiation factors. Growth or differentiation factors (e.g., WNT family proteins, BMP family proteins, IGF family proteins, etc.) can be added alone or in combination, for example, as a differentiation mixture comprising the different factors that cause differentiation to a specific lineage. It should be understood that each individual possibility can be combined with another possibility to derive the combination of features, and each possibility and each combination represent separate embodiments of the present invention.

[0106] The concentration of each individual component in the growth medium can be adjusted according to the needs of the cell line used and the flavivirus vaccine to be produced. For example, the glucose concentration 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 glucose concentration 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 present invention.

[0107] If glutamine or GlutaMAX™ is added, its concentration can be 3.5 mM to 4.5 mM glutamine or GlutaMAX™, 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 to 4.2 mM, 4.2 to 4.3 mM, 4.3 to 4.4 mM, or 4.4 to 4.5 mM. The concentration of glutamine or GlutaMAX™ 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.

[0108] As used herein, "serum" refers to mammalian serum and can be selected from human serum, equine serum, bovine serum, or ovine serum. Serum is widely used as a supplement for cell growth medium for adherent mammalian cell culture because it provides a variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water-insoluble components, and other compounds necessary for cell growth in vitro, such as hormones and attachment factors. The term "serum" also encompasses serum-based solutions containing buffers. It can include adding substances such as salts, buffers, sugars, chelating agents, preservatives, and protease inhibitors to the serum used in the present invention. As used herein, the term "equine serum" refers to serum obtained from horses. As used herein, the term "bovine serum" refers to serum obtained from bovines, and bovine serum commonly used in the art is referred to as "fetal bovine serum (FBS)." As used herein, the term "ovine 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, equine serum, porcine serum, or other serum). In some embodiments, the cell culture medium does not contain serum. In some embodiments, the cell culture medium comprises human platelet lysate (hPL).

[0109] In some embodiments, culture medium is serum-free. If serum is added, the serum concentration can be in the range of 1% to 10%, for example, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9% or 9% to 10%. The concentration of serum can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. Therefore, the concentration of FBS in culture medium can be in the range of 1% to 10%, for example, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9% or 9% to 10%. Therefore, the concentration of equine serum in culture medium can be in the range of 1% to 10%, for example, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9% or 9% to 10%. Thus, the concentration of sheep serum in the culture medium may be in the range of 1% to 10%, for example, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, or 9% to 10%. Thus, the concentration of porcine serum in the culture medium may be in the range of 1% to 10%, for example, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, or 9% to 10%. Each of these possibilities represents a separate embodiment of the present invention.

[0110] Other 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 culture medium can be indicated by a shift to a lower pH value, which will trigger a color change in the pH indicator. Instead of or in addition to a pH indicator, visual microscopic examination of microbial contamination can also be performed. Each possibility has been tested and represents a separate embodiment of the present invention.

[0111] The growth medium may or may not contain a nonionic surfactant, such as a poloxamer. Preferably, the growth medium does not contain a poloxamer. Thus, according to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing includes the step of providing cells in a growth medium, wherein the growth medium does not contain a nonionic surfactant, such as a poloxamer.

[0112] In the context of the present invention, the growth medium preparation strategy is also a factor in controlling microbial growth. For the purposes of the present invention, the growth medium can be prepared by "aseptic mixing" and homogenizing all components, or by mixing the components and then "filtering" the components, depending on the virus being produced. The term "sterile" should be understood to mean a state of being substantially free of the growth of harmful or pathogenic organisms and substantially free of the accumulation of debris or other media in which such organisms could reproduce.

[0113] The present invention encompasses preparing the growth medium in either of the two ways described above. Furthermore, the present invention encompasses preparing the growth medium for one flavivirus using one way and preparing the growth medium for another flavivirus using another way. Furthermore, if flaviviruses with different serotypes are produced, the present invention encompasses producing one serotype in a growth medium prepared in one way and producing the other serotype in a growth medium prepared in another way. For example, for the purposes of the present invention, if dengue serotypes are produced separately, serotype 4 can be produced using filtered growth medium, while serotype 1 can be produced using sterile mixing, or vice versa. Each possibility represents a separate embodiment of the present invention. When filtration is used, it is preferred to use a filter of less than 1 µm, and more preferably, a filter of less than 0.5 µm.

[0114] Another optional additive that can be included is a dissociation agent for adherent cell passage, to remove cells from the surface. As used herein, the term "dissociation agent" refers to a solution or fluid that contacts anchorage-dependent cells and causes cells to dissociate (loosen their cell attachment and possibly fall off) from the surface to which they adhere. Therefore, if adherent cells are used for the purposes of the present invention, growth medium can also include a dissociation agent, such as one or more chelating agents (ethylenediaminetetraacetic acid, "EDTA"; ethylene glycol-bis-β-aminoethyl ether N, N, N', N'-tetraacetic acid, "EGTA"; versen; etc.). Growth medium can alternatively include one or more proteolytic enzymes (such as, ficin, pepsin, trypsin, chymotrypsin, papain, etc., wherein trypsin is a preferred enzyme). The proteolytic enzyme used in the present invention can be recombinant, such as commercially available TrypLE Select, or can be mammalian in origin, such as the trypsin of porcine origin. Growth medium can also include a combination of chelating agents and proteolytic enzymes. Each possibility represents a separate embodiment of the present invention.

[0115] The target volume of dissociation reagent is determined by the surface area of ​​the culture dish. The volume to surface area ratio of the dissociation reagent can be 0.01 to 0.02 mL / cm 2 or 0.011 to 0.016 mL / cm 2 Thus, for example, a proteolytic enzyme may have a volume to surface area ratio of 0.01 to 0.02 mL / cm 2 , or 0.011 to 0.016 mL / cm2. 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 / cm2. 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 / cm2. Thus, for example, porcine-derived trypsin may have a volume to surface area ratio of 0.01 to 0.02 mL / cm 2 , or 0.011 to 0.016 mL / cm2.

[0116] The duration of treatment with a dissociating agent can 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 can 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 can 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 can 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-derived trypsin can 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.

[0117] If dissociation reagent is used and growth medium comprises serum, then preferably washing cell before using dissociation reagent.Preferably, before adding dissociation reagent, carry out one, twice or three washings.Can use any washing buffer solution to wash, such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS) or Tris buffered saline (TBS).As used herein, " PBS " is a kind of water-based salt solution, and it contains sodium hydrogen phosphate, sodium chloride, and contains potassium chloride and potassium dihydrogen phosphate in some formulas.As used herein, " Dulbecco's phosphate buffered saline (DPBS) " is a kind of balanced salt solution, is used for multiple cell culture application, such as washing cell, transport cell or tissue sample, diluting cell and counting and preparing reagent before dissociation.

[0118] After all cells have been removed from the culture dish surface, wash the cells with the growth medium used for cell expansion. The target volume of growth medium used per wash is determined by the surface area of ​​the culture dish. The volume to surface area ratio of growth medium used for washing can be 0.04 to 0.06 mL / cm 2 or 0.046 to 0.053 mL / cm2.

[0119] In the context of the present invention, it should be understood that each of the individual components of the culture medium described above may be considered alone or in combination with one another, depending on the cell line used and the virus to be produced. These components provide all the necessary nutrients, growth factors, and other proteins for the growth of adherent cells on the culture dish surface.

[0120] For the purposes of the present invention, it is necessary to use an operating range that supports robust cell growth for large-scale production and manufacturing of flavivirus vaccines.

[0121] One of the parameters is the cell thawing process. The term "thawing" refers to raising the temperature of a cryopreserved composition or biomaterial (in this case, cells) to 0°C or above, preferably to 4°C or above. The term "thawing" can also refer to raising the temperature of a cryopreserved composition or biomaterial to a temperature at which all or part of the cryopreserved composition or biomaterial is free of or substantially free of ice crystals. Therefore, the term "thawing" includes complete thawing and partial thawing. The duration of the cell thawing process can be in the range of 1 to 5 minutes, or 3 to 5 minutes. Thus, for example, when the cells used are Vero cells, the thawing duration can be in the range of 1 to 5 minutes or 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 at 20 to 22°C. The storage duration of the thawed cells can be extended to 5, 6, 7 or 8 minutes. Thus, for example, when the cells used are Vero cells, the storage duration of the thawed cells can be extended to 5, 6, 7 or 8 minutes.

[0122] Other parameters are the carbon dioxide (CO2) percentage and temperature during the incubation of the cells in the expansion process. For the purposes of the present invention, the CO2 percentage may be 2% to 8%, 4% to 6%, or less than 6%, such as, for example, 5% or 6%, and the temperature range may be a temperature of 36°C to 39°C, or less than 39°C, such as, for example, 37°C or 38°C.

[0123] Another parameter is to determine the time allowed for cells to remain "dry" during passaging (i.e., when using adherent cells, the cell monolayer is not covered with solution, such as after the growth medium or DPBS is removed). The time can be up to two hours, preferably less than two hours, such as, for example, 30 minutes, 45 minutes, 60 minutes. As used herein, the term "passaging" refers to the transfer of one or more cells from a first growth environment to a second growth environment, wherein the cell density of the second growth environment is less than the cell density of the first growth environment.

[0124] Another parameter is the maximum process duration of the passaging, i.e. the time period from the time the cells leave the incubator, are passaged and maintained with the growth medium. The maximum process duration of the passaging can be up to 20 hours, preferably less than 20 hours, or less than 15 hours, or less than 10 hours, such as, for example, 5 hours, 6 hours, 7 hours, 8 hours or 9 hours.

[0125] Another parameter is the maximum cell division ratio, which can be set as high as 1:8 regardless of the culture dish used. This parameter depends largely on the cell density before infection and the confluence of the cells during expansion. For the method of the present invention, the minimum confluence of the cells before infection is 80% to 90%, preferably greater than 80%. Therefore, for the method of the present invention, the cell seeding density is 1.5 x 10 4 Up to 3 x 10 4 cells / cm 2 range, such as 1.75 x 10 4 or 2 x 10 4 cells / cm 2 , and the cell density before infection was 9 x 10 4 to 2 x 10 5 cells / cm 2 within the range.

[0126] Therefore, for large-scale flavivirus vaccine production and manufacturing, the duration of the cell thawing process can be in the range of 3 to 10 minutes, the incubation CO2 percentage can be 2% to 8%, the cell drying period can be two hours, the maximum process duration for passaging can be up to 20 hours, and the maximum cell division ratio can be as high as 1:8. Therefore, for large-scale flavivirus vaccine production and manufacturing, the duration of the cell thawing process can be in the range of 3 to 8 minutes, the incubation CO2 percentage can be 4% to 6%, the cell drying period can be less than two hours, the maximum process duration for passaging can be up to 20 hours, and the maximum cell division ratio can be as high as 1:8. Therefore, for large-scale flavivirus vaccine production and manufacturing, the duration of the cell thawing process can be in the range of 3 to 5 minutes, the incubation CO2 percentage can be less than 6%, the cell drying period can be less than two hours, the maximum process duration for passaging can be up to 20 hours, and the maximum cell division ratio can be as high as 1:8.

[0127] Thus, in one embodiment of the method for large-scale production and manufacture of a flavivirus vaccine, the step of providing cells in a poloxamer-free growth medium includes expanding the cells to a sufficient number. In the context of the present invention, it should be understood that each individual method step and / or component described above can be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0128] Infected cells

[0129] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, and (ii) infecting the cells of step (i) with a medium containing the flavivirus (optionally at a low MOI). 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. 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 the 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.

[0130] After the cells have been expanded to a sufficient number in the production vessel, as described in the previous section, the cells are infected with a culture medium containing a working virus seed (WVS) containing the flavivirus. This culture medium is called an infection medium. The infection medium may be the same as the growth medium or different. The purpose of this step is to ensure that the virus infects the cells and replicates to sufficient potency for harvesting and purification. In addition, when an attenuated virus is used, another goal is that the virus retains its attenuated locus. WCB is used to make the main virus seed MVS and the working virus seed WVS, respectively. As used herein, the term "main virus seed (MVS)" within the meaning of the present disclosure is a virus intended for use in the production of a vaccine. First, MVS is used to inoculate the WVS, and then the WVS is used to inoculate the production culture.

[0131] As used herein, a "production vessel" refers to a "culture dish" inoculated with infection medium containing WVS for large-scale production and manufacture of flavivirus vaccines. The term "culture dish" can refer to any container in which cells can be cultured. Culture dishes include, but are not limited to, tissue culture flasks, 96-well plates, culture dishes, culture slides, and rotating wall containers. For the purposes of the present invention, tissue culture flasks that can be used are Corning® CellSTACK® culture chambers or Nunc™ EasyFill™ Cell Factory™ systems. For example, a Corning® CellSTACK® culture chamber that can be used alone or in combination is a 1-layer (CF1) with a cell growth area of ​​636 cm 2 ; 2 layers (CF2), cell growth area 1,272 cm 2; 5-layer (CF5), cell growth area 3,180 cm 2 10 layers (CF10), cell growth area 6,360 cm 2 ; 40 layers (CF40), cell growth area 25,440 cm 2 .

[0132] For the purposes of the present invention, it is particularly important to maintain cell viability while promoting viral replication in the cells and the stability of the produced virus. Therefore, all method steps of the present invention are performed specifically to achieve these advantages. The methods of the present invention also include infecting the cells with an infection medium comprising a WVS containing a flavivirus. Thus, the present invention includes infecting the cells with an infection medium comprising a flavivirus. In the context of the methods of the present invention, the infection medium may be the same as or different from the growth medium used for cell expansion. In some embodiments, the culture medium is serum-free.

[0133] The infection medium may comprise one or more growth media and additionally contain glutamine or GlutaMAX™ and a nonionic surfactant.

[0134] The term "nonionic surfactant" means a surfactant that does not contain either a positively charged functional group or a negatively charged functional group. Unlike anionic and cationic surfactants, nonionic surfactants do not ionize in solution. Nonionic surfactants can be selected from block copolymers, sorbitan esters, ethoxylated or propoxylated sorbitan esters, alkyl polyglycosides (APGs), alkoxylated mono- or dialkylamines, 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 (glucosamides (GAs) or fatty acid glucosamides (FAGAs)) and combinations thereof.

[0135] The nonionic surfactant can be a high molecular weight nonionic surfactant. "High molecular weight" means a molecular weight of 1500 or above. 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 polyethylene oxide (-CH2CHO-designated EO) and polypropylene oxide (-CH2CHCH3O-designated PO) blocks. In an EOx-POy-Eox arrangement, the PO block can be flanked by two EO blocks. Since the PO component is hydrophilic and the EO component is hydrophobic, the overall hydrophilicity, molecular weight, and surfactant properties of the copolymer can be adjusted by varying the x and y in the EOx-POy-Eox block structure. In aqueous solution, the EO-PO block copolymer will self-assemble into micelles having a PO core and a hydrophilic EO group corona.

[0136] The nonionic surfactant can be a poloxamer. Poloxamers are nonionic triblock copolymers consisting of a central poly(propylene oxide) hydrophobic chain flanked by two poly(ethylene oxide) hydrophilic chains. The length of the polymer blocks can be tailored to produce different poloxamers with slightly different properties.

[0137] As used herein, "EO-PO block copolymer" may refer to a copolymer composed of poly(ethylene oxide) and poly(propylene oxide) blocks. Additionally, as used herein, "Pluronic" may refer to an EO-PO block copolymer in the form of EOx-POy-EOx. This configuration of EO-PO block copolymers is also referred to as a "poloxamer."

[0138] Thus, the nonionic surfactant can 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 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 comprising a central propylene glycol block having about 56 repeating units and two flanking hydrophilic polyethylene glycol blocks each comprising about 101 repeating units. "Poloxamer 407" is also known by its trade names Pluronic F127 and Synperonic PE / F127. As used herein, "Poloxamer 188" (P188) is a nonionic linear copolymer with an average molecular weight of 8400 Daltons and is also known as Pluronic F68, FLOCOR, and RheothRx. Pluronic P123 is a symmetrical triblock copolymer comprising poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) in an alternating linear arrangement of PEO-PPO-PEO. Pluronic P85 is a primary hydroxyl-terminated difunctional block copolymer surfactant.

[0139] Therefore, according to one embodiment of the present invention, the preparation of infection medium can comprise adding glutamine or GlutaMAX and nonionic surfactant, thereby produce the glucose of specific concentration in infection medium, glutamine or GlutaMAX and nonionic surfactant.The glucose concentration in infection medium can be 3 g / L to 4 g / L of 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 glucose concentration 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 present invention.

[0140] The concentration of glutamine or GlutaMAX™ in the infection medium 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 to 4.2 mM, 4.2 to 4.3 mM, 4.3 to 4.4 mM, or 4.4 to 4.5 mM. The concentration of glutamine or GlutaMAX™ 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.

[0141] In order to implement some embodiment of the method of the present invention, compared with the infection culture medium that does not add nonionic surfactant, the nonionic surfactant that adds specific concentration strengthens the generation of virus.For example, the concentration of nonionic surfactant can be 0.05% to 2.0% (w / v) in the culture 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).Every kind of possibility all represents independent 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 culture 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).Every kind of possibility all represents independent embodiment of the present invention. In some embodiments, the concentration of poloxamer 403 and / or F68 can be 0.05% to 2.0% (w / v) in the culture 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). Every 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 culture 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). Every 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 culture 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.

[0142] Infection media can be used with or without pH indicators. Phenol red can be used as a pH indicator for infection media. If a pH indicator is not used, such as if the media does not contain phenol red, visual microbiological assessment can be performed.

[0143] In the context of the present invention, the infection medium preparation strategy is also a factor in controlling microbial growth. The method of preparing the infection medium can affect viral growth or potency. For the purposes of the present invention, the infection medium can be prepared by aseptic mixing and homogenizing all components, or by mixing the components and then filtering them, depending on the flavivirus being produced. The present invention encompasses preparing the infection medium in either of these two ways. Furthermore, the present invention encompasses preparing the infection medium for one flavivirus using one method and preparing the growth medium for another flavivirus using another method. Furthermore, if viruses with different serotypes are used, the present invention encompasses producing one serotype in an infection medium prepared in one method and another serotype in an infection medium prepared in another method. For example, for the purposes of the present invention, if Dengue serotypes are produced alone, serotype 4 can be produced using filtered infection medium, while serotype 1 can be produced using aseptic mixing, and vice versa. Each possibility represents a separate embodiment of the present invention. When filtration is used, it is preferred to use a filter with a diameter of less than 1 µm, and more preferably, a filter with a diameter of less than 0.5 µm.

[0144] Cells provided in growth medium are infected with a medium containing the flavivirus WVS. This means that each flavivirus and each serotype of each flavivirus has its own WVS, from which a separate production culture is initiated for large-scale production and manufacture of flavivirus vaccines. Infection is a complex biological process involving various operational parameters that will affect infection efficiency / harvest efficacy, i.e., virus attachment, entry, and infection of cells.

[0145] It has surprisingly been found that the infection parameters used in the method of the present invention are reproducible and allow for the large-scale production of flaviviruses in general as well as the large-scale production of attenuated flaviviruses.

[0146] In order to achieve high infection efficiency / high harvest efficacy / and / or high virus titer, the method of the present invention includes specific infection parameters.

[0147] One of the infection parameters is the multiplicity of infection ("MOI"). MOI refers to the ratio defined by the number of infectious viral particles deposited in a vessel and / or container divided by the number of target cells present in the vessel and / or container.

[0148] The probability of a given infectious virus particle infecting a target cell is given by Poisson's distribution,

[0149]

[0150] That is, the infections are independent of each other.

[0151] m is the multiplicity of infection or MOI,

[0152] n is the number of infectious virus particles, and

[0153] P(n) is the probability that a target cell is infected by n infectious virus particles.

[0154] If n = 0, the probability that the target cell will not be infected can be calculated. Therefore, the probability that the target cell will be infected by the virus particle is 1-P(0).

[0155]

[0156] Thus, the above formula gives the average percentage of cells that will be infected due to inoculation at a given MOI. For example, for an MOI of 1, 63.2% of cells will be infected. Thus, the skilled artisan understands that the MOI will also affect the efficiency of virus production calculated based on the viral titer.

[0157] 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 the 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 this assay is based on the classic viral plaque assay, in which serial dilutions of virus are plated onto a monolayer of adherent cells from a suitable host. After a period of time for the infectious virions to bind to and be taken up by the cells, an overlay medium containing a thickening agent is added to prevent the virions from spreading. As a result, progeny virions can only infect cells adjacent to the originally infected cell. This results in a roughly circular focus of infection for each viral infectious unit. The immunofocus assay differs from the classic plaque assay in that the infection focus is detected by immunostaining rather than visual observation of cytopathic effects. Specifically, the term "viral titer" refers to the number of infectious viral particles or "transduction units" that cause infection of the target cell. Viral titer in the context of the present invention can be measured using the IFA assay discussed above. Viral titer determined by IFA assay is expressed in plaque forming units (PFU). In one example, the immune focus assay can be performed in a manner described in detail in Section 2.5 of Brewoo et al. (Vaccine. 2012 Feb 14; 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 functional assays such as those 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.

[0158] As used herein, "high viral titer" refers to a viral titer greater than 7.0 log 10 PFU / mL, preferably greater than 7.5log 10 PFU / mL.

[0159] One of the biggest challenges for large-scale vaccine production and manufacturing is economically preserving MVS and WVS stocks while achieving high virus titers to meet population needs in a timely manner. This challenge is further exacerbated in the large-scale production and manufacturing of attenuated virus vaccines, as the replication rate of the attenuated virus may be compromised.

[0160] It has surprisingly been discovered that, for certain embodiments of the methods of the present invention, infection media containing a low MOI of flavivirus can be used for large-scale production and manufacture of flavivirus vaccines. Even more surprisingly, it has been discovered that a low MOI can be used for large-scale production and manufacture of attenuated flavivirus vaccines. In this context, the flavivirus can be dengue virus. In one embodiment, using the methods of the present invention, a low MOI can be used for large-scale production and manufacture of flavivirus vaccines, providing a higher average viral titer than an identical method comprising the same process steps but using a higher MOI. Thus, using the methods of the present invention, a low MOI can be used for large-scale production and manufacture of attenuated flavivirus vaccines, providing a higher average viral titer than an identical method comprising the same process steps but using a higher MOI. In this context, a high MOI refers to an MOI greater than 0.008. In this context, the flavivirus can be dengue virus. As used herein, "average viral titer" refers to the average viral titer of at least two harvest steps.

[0161] As used herein, "low MOI" refers to an MOI of 0.008 and 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, such as 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0110, 0.011112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0. 0.015, 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. Thus, for the purposes of the present invention, a low MOI in the range of 0.008 or less can be used. Thus, 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, infection with each serotype can be performed within the low MOI range. It is also possible that one serotype is infected with a medium containing a flavivirus at a low MOI in the range of 0.008 or less and 0.005 or less, and another serotype is infected with a medium containing a flavivirus at another low MOI in the range of 0.008 or less and 0.005 and less. Each possibility represents a separate embodiment of the present invention.

[0162] Thus, by using the methods of the present invention, a low MOI can be used for large-scale flavivirus vaccine production and manufacturing, thereby providing high viral titers. Thus, by using the methods of the present invention, a low MOI can be used for large-scale attenuated flavivirus vaccine production and manufacturing with high viral titers. In this context, the flavivirus can be dengue virus. In one embodiment, by using the methods of the present invention, a low MOI can be used for large-scale flavivirus vaccine production and manufacturing, providing a higher average viral titer than the same method comprising the same process steps but using a higher MOI. Thus, by using the methods of the present invention, a low MOI can be used for large-scale attenuated flavivirus vaccine production and manufacturing, providing a higher average viral titer than the same method comprising the same process steps but using a higher MOI. In this context, a high MOI refers to an MOI greater than 0.008. In this context, the flavivirus can be dengue virus. As used herein, "average viral titer" refers to the average viral titer of at least two harvest steps.

[0163] In one embodiment, Vero cells in more than ten CF-10 flasks are infected at a low MOI with dengue serotype 1, such as the dengue 2 / 1 chimera represented by SEQ ID NO: 1 and / or SEQ ID NO: 2, or TDV-1.

[0164] In one embodiment, more than ten CF-10 flasks of Vero cells 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.

[0165] In one embodiment, Vero cells in more than ten CF-10 flasks or CF-40 flasks are infected at a low MOI with dengue serotype 3, such as the dengue 3 / 1 chimera or TDV-3 represented by SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0166] In one embodiment, Vero cells in more than ten CF-40 flasks are infected at a low MOI with dengue serotype 4, such as a dengue 2 / 4 chimera or TDV-4 represented by SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0167] According to certain embodiments of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, and (ii) infecting the cells of step (i) with a medium comprising the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0168] In one embodiment, large-scale production and manufacturing of flavivirus vaccines at high viral titers involves the use of cell lines that are rich in flavivirus receptors. In another embodiment, large-scale production and manufacturing of attenuated flavivirus vaccines at high viral titers involves the use of cell lines that are rich in flavivirus receptors. In this context, the cells can be Vero cells. In this context, the flavivirus can be dengue virus and the flavivirus receptor can be a dengue receptor.

[0169] As used herein, "flavivirus receptor" refers to a cell surface receptor. A variety of cell surface receptors are involved in the entry of flaviviruses into different cell types, such as the αvβ3 integrin C-type lectin receptor (CLR), the 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 receptors refers to a cell line that has a certain degree of overlap between the flavivirus receptors and the flavivirus receptors in mosquito cell lines (such as C636), for example, at least one or two common receptors.

[0170] As used herein, "dengue virus vaccine" refers to 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).

[0171] As used herein, "dengue virus receptor" refers to a cell surface receptor. These include carbohydrate molecules, lectins, and tight junction protein-1 cell receptors. Carbohydrate molecules, such as glycosaminoglycans (GAGs), sulfated polysaccharides, and glycosphingolipids (GSLs) are widely expressed cell surface co-receptors for DENV entry and are believed to enhance viral entry efficiency. As used herein, a cell line "rich in" dengue virus receptors refers to a dengue virus receptor that overlaps to a certain extent with the dengue virus receptors in a mosquito cell line (such as C636), for example, a cell line with at least one or two common receptors.

[0172] According to one aspect of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells enriched with flavivirus receptors in a growth medium, and (ii) infecting the cells of step (i) with a medium containing the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0173] Another infection parameter is the volume of infection medium per production vessel surface area. For the methods of the present invention, when using a low MOI range, the volume of infection medium per production vessel surface area is such that it covers the cell monolayer in the tissue culture dish. However, whether the infection medium covers the cell monolayer can depend on the physical method of infection, i.e., static or agitated.

[0174] Generally, for the purpose of large-scale vaccine production, it is understood that a shaking / oscillation infection method should be employed to ensure infection efficiency and viral potency. "Shaking / oscillation, as used herein, refers to manually shaking the tissue culture dish or using a shaking platform. The term "static" infection means that the tissue culture dish is not moved during the infection process, so that the infection medium is not mechanically dispersed throughout the cell monolayer. The term "shaking" infection, on the other hand, means that the tissue culture dish is kept on a platform that is gently moved along a horizontal axis, or the tissue culture dish is manually moved, so that the infection medium is mechanically dispersed throughout the monolayer.

[0175] It has been surprisingly discovered that static infection methods can be used for large-scale flavivirus vaccine production and manufacturing. Even more surprisingly, it has been discovered that static infection methods can be used for large-scale attenuated flavivirus vaccine production and manufacturing. In this context, the flavivirus can be dengue virus.

[0176] Therefore, for large-scale flavivirus vaccine production and manufacturing, static infection provides the same infection efficiency and / or viral titer, i.e., provides higher viral titers, compared to shaking infection methods. Therefore, for large-scale flavivirus vaccine production and manufacturing, static infection provides the same infection efficiency and / or viral titer, i.e., provides higher viral titers, compared to shaking infection methods, 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 nearly completely immersed in the infection medium. This will largely depend on the volume of infection medium used and the tissue culture dish used. Therefore, whenever "static infection" is mentioned, it should be understood that the volume of infection medium is such that it covers the cell monolayer.

[0177] Furthermore, it has been surprisingly found that static infection combined with a low MOI range provides high infection efficiency and / or high virus titers for large-scale flavivirus vaccine production, and even more surprisingly, provides high infection efficiency and / or high virus titers for large-scale attenuated flavivirus vaccine production. In this context, the flavivirus can be dengue virus.

[0178] According to one aspect of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, and (ii) statically infecting the cells of step (i) with a medium containing the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0179] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells enriched with flavivirus receptors in a growth medium, and (ii) statically infecting the cells of step (i) with a medium containing the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0180] Another infection parameter within the meaning of the infection method is to infect suspended cells or monolayer cells. The term "suspension" refers to cells dispersed in a culture medium. In this context, cells can be adherent cells that have been dissociated from a culture dish using a dissociation agent. Typically, for the purpose of large-scale vaccine production, it will be understood that cells should be infected immediately after using a dissociation agent (when cells are still in suspension) to ensure infection efficiency.

[0181] Surprisingly, it has been discovered that infected cell monolayers can be used for large-scale flavivirus vaccine production and manufacturing. Even more surprisingly, it has been discovered that infected cell monolayers can be used for large-scale attenuated flavivirus vaccine production and manufacturing. In this context, the cells can be Vero cells. In this context, the flavivirus can be dengue virus.

[0182] Furthermore, it has been surprisingly found that infection of cell monolayers in combination with a low MOI provides high infection efficiency for large-scale flavivirus vaccine production, and even more surprisingly, for large-scale attenuated flavivirus vaccine production. In this context, the cells can be Vero cells. In this context, the flavivirus can be dengue virus.

[0183] Furthermore, it has been surprisingly discovered that infecting monolayer cells, combined with low MOI and static infection, provides high infection efficiency for large-scale flavivirus vaccine production, and even more surprisingly, for large-scale attenuated flavivirus vaccine production. In this context, the cells can be Vero cells. In this context, the flavivirus can be dengue virus.

[0184] As used herein, the term "monolayer" refers to a layer of cells in which no or substantially no cells are growing on top of another cell, but rather all cells are growing side by side and often in contact with each other on the same growth surface.

[0185] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, and (ii) statically infecting the cells from step (i) in monolayer form with a medium containing the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0186] Thus, infected cell monolayers can be used for large-scale flavivirus vaccine production and manufacturing in cell lines rich in flavivirus receptors. Furthermore, infected cell monolayers combined with a low MOI can be used for large-scale flavivirus vaccine production and manufacturing with high infection efficiency and / or high viral titers in cell lines rich in flavivirus receptors. Furthermore, infected cell monolayers combined with a low MOI and static infection can be used for large-scale flavivirus vaccine production and manufacturing with high infection efficiency and / or high viral titers in cell lines rich in flavivirus receptors.

[0187] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells enriched with flavivirus receptors in a growth medium, and (ii) statically infecting the cells of step (i) in monolayer form with a medium containing the flavivirus at a low MOI. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0188] Another infection parameter is the infection duration and infection temperature. The infection duration can be in the following ranges: 50 to 250 minutes, 60 to 200 minutes, 70 to 150 minutes, 70, 80, 90, 100, 110, 120, 130, 140 or 150 minutes. The cells can be incubated at a temperature between 36 and 39° C., preferably below 39° C. during infection.

[0189] After infection, the cells may be washed or not. If washed, the cells may be washed once, twice, three times, or four times. Any washing buffer may be used to wash, such as phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS), or Tris buffered saline (TBS).

[0190] Thus, one aspect of a method for large-scale production and manufacture of a flavivirus vaccine comprises the step of infecting cells with a medium containing the flavivirus at a low MOI. Within the context of the present invention, it should be understood that each individual method step and / or ingredient component described above can be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0191] Harvest to obtain harvest

[0192] After infection of the cells, a harvesting step can be performed to obtain a harvest. Thus, according to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells from 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 nonionic surfactant. 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. 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 the 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.

[0193] As used herein, the term "harvest" refers to a composition obtained by harvesting (eg, collecting the supernatant). The harvest refers to any intermediate composition from the time the supernatant is collected until the first processing step.

[0194] According to the present invention, "harvesting" refers to the act of collecting at least a portion of the culture medium from a production culture that produces and releases virus. As used herein, a "portion" of the culture medium refers to the supernatant. Thus, "harvesting" refers to collecting the supernatant from infected cells. In the context of the present invention, "collecting the supernatant" or "harvesting step" is immediately followed by the addition of fresh culture medium, such as the growth medium described above. In a preferred embodiment, the fresh culture medium refers to the growth medium described above.

[0195] In the context of the present invention, harvesting may comprise a first harvesting step and at least one further harvesting step.

[0196] According to some embodiments of the present invention, before " first results step ", carry out culture medium replacement, comprise collecting supernatant, discard the supernatant collected and add fresh culture medium 12-30 hours, 24 hours, 48 ​​hours, 96 hours or 120 hours.Preferably, culture medium replacement is carried out 12 to 30 hours before the first results step.Therefore, according to one embodiment of the present invention, results comprise the first results step and at least one further results step.In the context of the present invention, the interval between each results step is preferably at least 20-30 hours, 21-27 hours, 24 hours, 48 ​​hours or 96 hours.

[0197] Thus, according to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium comprising the virus at a low MOI, and (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least one further harvesting step, and wherein the medium is changed at least 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0198] According to one embodiment of the inventive method, medium exchange is performed 12-30 hours, 24 hours, 48 ​​hours, 96 hours or 120 hours before the first harvesting step, i.e., medium exchange is performed on the 1st, 2nd, 3rd or 4th day after infection and the first harvesting step is performed on the 1st, 2nd, 3rd or 4th day after medium exchange or on the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th or 14th day 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 harvesting step is at least 20-30 hours, 21-27 hours, 24 hours, 48 ​​hours or 96 hours.

[0199] Therefore according to one embodiment of the method of the present invention, the first harvesting step is carried out on the 2nd day after infection, and a further harvesting step is carried out on the 3rd day after infection subsequently, or the first harvesting step is carried out on the 3rd day after infection, and a further harvesting step is carried out on the 4th day after infection subsequently, or the first harvesting step is carried out on the 4th day after infection, and a further harvesting step is carried out on the 5th day after infection subsequently, or the first harvesting step is carried out on the 5th day after infection, and a further harvesting step is carried out on the 6th day after infection subsequently, or the first harvesting step is carried out on the 6th day after infection, and a further harvesting step is carried out on the 7th day after infection subsequently. In this context, culture medium replacement is carried out 12-30 hours before the first harvesting step. Fresh culture medium is added to the cells immediately after the first harvesting step.

[0200] Therefore according to an embodiment of the inventive method, the first harvesting step is carried out on the 2nd day after infection, and two further harvesting steps are carried out on the 3rd and 4th day after infection subsequently, or the first harvesting step is carried out on the 3rd day after infection, and two further harvesting steps are carried out on the 4th and 5th day after infection subsequently, or the first harvesting step is carried out on the 4th day after infection, and two further harvesting steps are carried out on the 5th day after infection subsequently, or the first harvesting step is carried out on the 5th day after infection, and two further harvesting steps are carried out on the 6th and 7th day after infection subsequently, or the first harvesting step is carried out on the 6th day after infection, and two further harvesting steps are carried out on the 7th and 8th day after infection subsequently.In this context, culture medium is replaced 12-30 hours before the first harvesting step.All immediately add fresh culture medium to the cells after each harvesting step.

[0201] Therefore according to an embodiment of the inventive method, the first harvesting step is carried out on the 2nd day after infection, and at the 3rd day, the 4th day and the 5th day after infection, three further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 3rd day after infection, and at the 4th day, the 5th day and the 6th day after infection, three further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 4th day after infection, and at the 5th day, the 6th day and the 7th day after infection, three further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 5th day after infection, and at the 6th day, the 7th day and the 8th day after infection, three further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 6th day after infection, and at the 7th day, the 8th day and the 9th day after infection, three further harvesting steps are carried out subsequently.In this context, culture medium is replaced 12-30 hours before the first harvesting step.All immediately in the cells, add fresh culture medium after each harvesting step.

[0202] Therefore according to an embodiment of the inventive method, the first harvesting step is carried out on the 2nd day after infection, and at the 3rd day, the 4th day, the 5th day and the 6th day after infection, four further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 3rd day after infection, and at the 4th day, the 5th day, the 6th day and the 7th day after infection, four further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 4th day after infection, and at the 5th day, the 6th day, the 7th day and the 8th day after infection, four further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 5th day after infection, and at the 6th day, the 7th day, the 8th day and the 9th day after infection, four further harvesting steps are carried out subsequently, or the first harvesting step is carried out on the 6th day after infection, and at the 7th day, the 8th day, the 9th day and the 10th day after infection, four further harvesting steps are carried out subsequently.In this context, culture medium is replaced within 12-30 hours before the first harvesting step.All immediately add fresh culture medium to the cell after each harvesting step.

[0203] Thus, according to one embodiment of the method of the present invention, the first harvesting step is performed on day 2 after infection, followed by five further harvesting steps on day 3, day 4, day 5, day 6, and day 7 after infection, or the first harvesting step is performed on day 3 after infection, followed by five further harvesting steps on day 4, day 5, day 6, day 7, and day 8 after infection, or the first harvesting step is performed on day 4 after infection, followed by five further harvesting steps on day 5, day 6, day 7, day 8, day 9, and day 10 after infection. In a preferred embodiment, the first harvesting step is performed on day 5 after infection, followed by five further harvesting steps on day 6, day 7, day 8, day 9, day 10, and day 10 after infection. In one embodiment, the first harvesting step is performed on day 6 after infection, followed by five further harvesting steps on day 7, day 8, day 9, day 10, and day 11 after infection. In this context, the medium is changed 12-30 hours before the first harvesting step. Fresh culture medium was added to the cells immediately after each harvesting step.

[0204] Therefore, according to one embodiment of the method of the present invention, the first harvesting step is carried out on the 2nd day after infection, followed by six further harvesting steps on the 3rd day, 4th day, 5th day, 6th day, 7th day and 8th day after infection, or the first harvesting step is carried out on the 3rd day after infection, followed by six further harvesting steps on the 4th day, 5th day, 6th day, 7th day, 8th day and 9th day after infection, or the first harvesting step is carried out on the 4th day after infection, followed by six further harvesting steps on the 5th day, 6th day, 7th day, 8th day, 9th day and 10th day after infection, or the first harvesting step is carried out on the 5th day after infection, followed by six further harvesting steps on the 6th day, 7th day, 8th day, 9th day, 10th day and 11th day after infection, or the first harvesting step is carried out on the 6th day after infection, followed by six further harvesting steps on the 7th day, 8th day, 9th day, 10th day, 11th day and 12th day after infection. In this context, the medium was changed 12-30 hours before the first harvest step. Fresh medium was added to the cells immediately after each harvest step.

[0205] Thus, according to one embodiment of the method of the invention, the first harvesting step is performed on day 2 after infection, followed by seven further harvesting steps on days 3, 4, 5, 6, 7, 8 and 9 after infection, or the first harvesting step is performed on day 3 after infection, followed by seven further harvesting steps on days 4, 5, 6, 7, 8, 9 and 10 after infection, or the first harvesting step is performed on day 4 after infection, followed by seven further harvesting steps on days 5, 6, 7, 8, 9 and 10 after infection. The 6th day, 7th day, 8th day, 9th day, 10th day and 11th day carried out seven further results-gathering steps, or the first results-gathering step was carried out on the 5th day after infection, and subsequently on the 6th day, 7th day, 8th day, 9th day, 10th day, 11th day and 12th day after infection, carried out seven further results-gathering steps, or the first results-gathering step was carried out on the 6th day after infection, and subsequently on the 7th day, 8th day, 9th day, 10th day, 11th day, 12th day and 13th day after infection, carried out seven further results-gathering steps. In this context, medium replacement was carried out in the first results-gathering step 12-30 hours before. Fresh culture medium was all added to the cells immediately after each results-gathering step.

[0206] Thus, according to one embodiment of the method of the invention, the first harvesting step is performed on day 2 after infection, followed by eight further harvesting steps on days 3, 4, 5, 6, 7, 8, 9 and 10 after infection, or the first harvesting step is performed on day 3 after infection, followed by eight further harvesting steps on days 4, 5, 6, 7, 8, 9, 10 and 11 after infection, or the first harvesting step is performed on day 4 after infection, followed by eight further harvesting steps on days 5, 6, 7, 8, 9, 10 and 11 after infection. In some embodiments, the first step of gathering the crops is carried out on the 5th day after infection, and on the 6th day, 7th day, 8th day, 9th day, 10th day, 11th day and 12th day after infection, eight further steps of gathering the crops are carried out; or the first step of gathering the crops is carried out on the 6th day after infection, and on the 7th day, 8th day, 9th day, 10th day, 11th day, 12th day and 13th day after infection, eight further steps of gathering the crops are carried out; or the first step of gathering the crops is carried out on the 6th day after infection, and on the 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day and 14th day after infection, eight further steps of gathering the crops are carried out. In this context, medium replacement was carried out in the first step of gathering the crops before 12-30 hours. Fresh culture medium is all added immediately to the cells after each step of gathering the crops.

[0207] Thus, according to one embodiment of the method of the invention, the first harvesting step is performed on day 2 after infection, followed by nine further harvesting steps on days 3, 4, 5, 6, 7, 8, 9, 10 and 11 after infection, or the first harvesting step is performed on day 3 after infection, followed by nine further harvesting steps on days 4, 5, 6, 7, 8, 9, 10, 11 and 12 after infection, or the first harvesting step is performed on day 4 after infection, followed by nine further harvesting steps on days 5, 6, 7, 8, 9, 10, 11 and 12 after infection. In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.In some embodiments, the culture medium of the present invention is used as the culture medium of the cell.

[0208] According to one embodiment of the present invention, large-scale production and manufacturing of flavivirus vaccines may involve a medium exchange step 12-30 hours before the first daily harvest. Another challenge for large-scale vaccine production and manufacturing is maintaining a balance between high viral titers and the removal of process-related impurities. This is because the process of removing process-related impurities may also adversely affect viral titers. Surprisingly, it was discovered that exchanging the medium 12-30 hours (i.e., less than 48 hours) before the first daily harvest achieved an optimal balance between wasted viral titers and reduced impurity concentrations.

[0209] 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 are produced by or during the production, separation and / or purification process. Other process-related impurities, particularly in large-scale vaccine production, include defective interfering particles or DIPs. DIPs are natural byproducts of replicating viruses. DIPs interfere with the reproduction and spread of infectious standard viruses (STVs), reduce virus yields by competing for viral and cellular resources, and induce antiviral responses. Surprisingly, the method of the present invention removes process-related impurities, which significantly improves virus yield, as can be seen from the significant increase in viral titers.

[0210] Surprisingly, it was found that in a flavivirus vaccine production and manufacturing method, a medium change 12-30 hours (i.e., less than 48 hours) before the first harvesting step does not significantly affect the virus titer compared to not changing the medium. Furthermore, it was surprisingly found that in a flavivirus vaccine production and manufacturing method, a medium change 12-30 hours before the first harvesting step combined with at least two, at least three, at least four, or most preferably at least five further harvesting steps (such as seven or more further harvesting steps) does not significantly affect the virus titer compared to not changing the medium. In this context, the flavivirus can be a dengue virus. In this context, the vaccine can be a live attenuated virus vaccine.

[0211] Thus, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, combined with infection of the cells at a low MOI, provides high viral titers. Furthermore, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, followed by at least three further harvest steps, such as five further harvest steps, such as seven further harvest steps or more, combined with infection of the cells at a low MOI range, provides high viral titers. 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.

[0212] Thus, according to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (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, wherein the harvesting comprises a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven further harvesting steps or more, and wherein the medium is changed 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0213] Thus, for large-scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvest step, combined with static infection of cells at a low MOI range, provides high viral titers. Furthermore, for large-scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvest step, followed by at least three further harvest steps, such as five further harvest steps, such as seven further harvest steps or more, combined with static infection of cells at a low MOI, provides high viral titers. 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.

[0214] Thus, according to one embodiment of the present invention, large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) with a medium comprising the flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven further harvesting steps or more, and wherein a medium change is performed 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0215] Thus, for large-scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvest step, combined with static infection of the cell monolayer at a low MOI, provides high virus titers. Furthermore, for large-scale flavivirus vaccine production and manufacturing, a medium change 12-30 hours before the first harvest step, followed by at least three further harvest steps, such as five further harvest steps, such as seven further harvest steps or more, combined with static infection of the cell monolayer at a low MOI, provides high flavivirus titers. 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.

[0216] Thus, according to one embodiment of the present invention, large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) statically infecting the cells of step (i) in monolayer form with a medium containing the flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least three further harvesting steps in this context, such as five further harvesting steps in this context, such as seven further harvesting steps or more, and wherein a medium change is performed 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0217] Thus, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, combined with infection of cells rich in flavivirus receptors at a low MOI range, provides high viral titers. Furthermore, according to the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, followed by at least three further harvest steps, such as five further harvest steps, such as seven further harvest steps or more, combined with infection of cells rich in flavivirus receptors at a low MOI range, provides high viral titers. 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.

[0218] Thus, according to one embodiment of the present invention, large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells containing an enriched flavivirus receptor in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven further harvesting steps or more, and wherein a medium change is performed 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0219] Thus, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, combined with static infection of cells containing abundant flavivirus receptors at a low MOI range, provides high flavivirus titers. Furthermore, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, followed by at least three further harvest steps, such as five further harvest steps, such as seven further harvest steps or more, combined with static infection of cells containing abundant flavivirus receptors at a low MOI range, provides high flavivirus titers. 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.

[0220] Thus, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, combined with static infection of a cell monolayer containing abundant flavivirus receptors at a low MOI range, provides high flavivirus titers. Furthermore, according to one embodiment of the methods of the present invention, for large-scale flavivirus vaccine production and manufacturing, a medium exchange 12-30 hours prior to the first harvest step, followed by at least three further harvest steps, such as five further harvest steps, such as seven further harvest steps or more, combined with static infection of a cell monolayer containing abundant flavivirus receptors at a low MOI range, provides high flavivirus titers. 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.

[0221] Thus, according to one embodiment of the present invention, large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells enriched with flavivirus receptors in a growth medium, (ii) statically infecting the cells of step (i) in monolayer form with medium containing the flavivirus at a low MOI, and (iii) harvesting to obtain a harvest, wherein the harvesting comprises a first harvesting step and at least three further harvesting steps, such as five further harvesting steps, such as seven further harvesting steps or more, and wherein a medium change is performed 12-30 hours before the first harvesting step. Preferably, the growth medium of step (i) does not contain a nonionic surfactant. 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.

[0222] In certain embodiments, the pH throughout the above steps is maintained in the range of 7.6 to 8.1.

[0223] Thus, one embodiment of a method for large-scale production and manufacture of a flavivirus vaccine comprises a harvesting step comprising a first harvesting step and at least one further harvesting step, wherein a medium exchange is performed at least 12-30 hours prior to the first harvesting step. Within the context of the present invention, it should be understood that each individual method step and / or ingredient component described above can be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0224] Processing the harvest

[0225] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus at a low MOI, (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii). 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. 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 the 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.

[0226] As discussed above, the term "harvest" as used herein refers to the composition obtained by the act of harvesting (e.g., collecting the supernatant). "Harvest" or "crude harvest" are used interchangeably and refer to any intermediate composition from the time the supernatant is collected until the first processing step.

[0227] The term "processing the harvest" refers to one or more manipulation steps performed on the harvest obtained. Thus, processing the harvest can include one or all of the following steps: clarification, stabilization, freezing, thawing, and pooling. In a preferred embodiment, processing the harvest refers to clarification and stabilization, in that order. In a preferred embodiment, processed harvest refers to harvest that has been clarified and stabilized, in that order.

[0228] Processing the harvest: Clarification of the harvest

[0229] In the context of the present invention, processing harvest can comprise the clarification of harvest.Can clarify harvest to remove cells and cell debris that may accumulate in the infection process.The clarification of harvest can comprise centrifugal, and / or one or more non-membrane filtration steps, such as depth filtration.These steps are carried out to harvest.

[0230] Depth filtration uses porous filter media to separate particles and solids from liquids. Filter media used for depth filtration include cellulose acetate, polypropylene, cellulose acetate protected by glass wool, and 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.

[0231] The filter used to clarify the harvest can be a heterogeneous double-layer polyethersulfone filter with a pore size of 0.2 μm and 0.45 μm. Thus, for the purpose of harvest clarification, several heterogeneous double-layer filters can be used, such as Sartoclean (cellulose acetate) 0.8+0.65 μm, Sartopure (polypropylene) 0.65 μm, Sartoclean (glass wool) 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 present invention.

[0232] At the end of the clarification step, the composition obtained is called "clarified harvest", which refers to the harvest that has been subjected to the clarification process step.

[0233] Thus, according to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii), including clarifying the harvest. 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.

[0234] Processing the harvest: Stabilization of the harvest

[0235] In the context of the present invention, processing harvests can include stabilization.For method of the present invention, one or more buffered excipient compositions comprising surfactant, sugar and protein can be used.Buffered excipient compositions can include TRIS buffer, phosphate buffer, sodium citrate buffer, 2-(N-morpholino) ethanesulfonic acid (MES) buffer, 3-morpholination propane-1-sulfonic acid (MOPS) buffer and salt (such as, sodium chloride, magnesium chloride or calcium chloride). In some embodiments, buffer can be protein-free. In one embodiment, the buffered excipient compositions are included in nonionic surfactants, sugar and protein in phosphate buffered saline (PBS). The buffered excipient compositions are also referred to as stabilizing buffer. Therefore, the term buffered excipient compositions and stabilizing buffer are used interchangeably.

[0236] In some embodiments, the present invention provides a method for the treatment of a stabilizing agent comprising a phosphate buffered saline (PBS) or a mixture of phosphate buffered saline (PBS) and ...

[0237] As used herein, the term "sugar" includes monosaccharides (such as glucose, galactose, ribose, mannose, rhamnose, talose, xylose or allose arabinose), disaccharides (such as trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribiose, xylobiose, mannobiose, lactose or fructose), trisaccharides (such as acarbose, raffinose, melezitose, panose or cellotriose) and sugar polymers (such as dextran, xanthan gum, pullulan, cyclodextrin, amylose, amylopectin, starch, cellooligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan or chitin). In addition, as used herein, the term "sugar" also includes sugar alcohols such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, polyols, ethylene glycol, polyols, polyethylene glycol, polypropylene glycol and glycerol.

[0238] The sugar used to stabilize the buffer can be a non-reducing sugar. Non-reducing sugars do not contain aldehyde or ketone groups that can be oxidized. Examples of non-reducing sugars include sucrose, trehalose, or a hydrate thereof, such as trehalose dihydrate.

[0239] The sugar concentration in the stabilization buffer may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, 35% to 50% (w / v). The non-reducing sugar concentration in the stabilization buffer may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, 35% to 50% (w / v). The sucrose concentration in the stabilization buffer may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, 35% to 50% (w / v). The trehalose concentration in the stabilization buffer may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, 35% to 50% (w / v). The concentration of trehalose dihydrate in the stabilizing buffer may be 20% to 60% (w / v), 25% to 55% (w / v), or 35% to 50%, or 45% (w / v). Preferably, 35% to 50% (w / v).

[0240] In order to implement the embodiment of the inventive method, the surfactant in the stabilizing buffer can be a nonionic surfactant. The concentration of the nonionic surfactant in the stabilizing buffer can 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 poloxamer 407 and / or F127 can 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 poloxamer 403 and / or F123 can 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 P123 can 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 can 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.

[0241] 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. Specifically, 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.

[0242] Albumins are a family of globular, non-glycosylated proteins found particularly in the blood of vertebrates. They are soluble in water, moderately soluble in concentrated saline solutions, and undergo thermal denaturation. Suitable albumins for use in the methods 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. The human serum albumin may be recombinant human serum albumin or it may be human serum albumin purified from human serum. Preferably, human serum albumin purified from human serum is used.

[0243] As used herein, the term "collagen" is used to refer to a family of extracellular fibrous proteins characterized by their rigid triple-helical structure. Three collagen polypeptide chains ("α chains") are intertwined to form this helical molecule. The term is also intended to encompass the various types of collagen, but the preferred form is type I collagen.

[0244] 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 hydrolysis. Gelatin is obtained by boiling skin, tendon, bone, and ligaments in water. Gelatin is a colorless or slightly yellowish, transparent sheet, flake, or powder that absorbs approximately 5 to 10 times its weight in water to form a gel in solution.

[0245] The protein concentration in the stabilization 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 albumin concentration in the stabilization 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 human serum albumin or human serum albumin purified from human serum in the stabilization 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 collagen in the stabilization 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 gelatin in the stabilization 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).

[0246] The excipient concentration of the stabilization buffer in the stabilized harvest can be 1 / 7 to 1 / 15 times, preferably 1 / 12 to 1 / 15 times, of the excipient concentration in the stabilization buffer. For example, if the excipient concentration of the stabilization buffer in the stabilized harvest is 1 / 14 times the excipient concentration in the stabilization buffer, the following exemplary concentrations will be obtained in the stabilized harvest.

[0247] The sugar concentration 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 non-reducing sugar concentration 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 sucrose concentration 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 trehalose concentration 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 dihydrate in the stable 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).

[0248] In order to implement the embodiment of the inventive method, the nonionic 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.

[0249] The concentration of albumin in the stabilized harvest can be 0.007% to 0.04% (w / v), 0.01% to 0.03% (w / v), 0.01% to 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 can be 0.007% to 0.04% (w / v), 0.01% to 0.03% (w / v), 0.01% to 0.075% (w / v), or 0.02% (w / v). The concentration of collagen in the stabilized harvest can be 0.007% to 0.04% (w / v), 0.01% to 0.03% (w / v), 0.01% to 0.075% (w / v), or 0.02% (w / v). The gelatin concentration in the stabilized harvest can be 0.007% to 0.04% (w / v), 0.01% to 0.03% (w / v), 0.01% to 0.075% (w / v), or 0.02% (w / v).

[0250] For other ratios, such as 1 / 7 to 1 / 15 or 1 / 12 to 1 / 15, the skilled person can easily calculate the resulting concentration of excipient in the stabilized harvest.

[0251] A composition referred to as a "stabilized harvest" refers to a crude harvest that has been subjected to a stabilization process step or a crude harvest that has been subjected to a clarification and stabilization process step, in either order. Thus, the terms clarified and stabilized harvest refer to a crude harvest that has been clarified and stabilized, in that order. Thus, the terms stabilized and clarified harvest refer to a crude harvest that has been stabilized and clarified, in that order.

[0252] Thus, according to one embodiment of the present invention, a method for large-scale viral vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus (optionally 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 dengue virus. In this context, the vaccine may be a live, attenuated viral vaccine. Thus, according to one embodiment of the present invention, a method for large-scale viral vaccine production and manufacturing comprises the following sequential steps: (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 clarification and stabilization 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 viral vaccine. Thus, according to one embodiment of the present invention, a method for large-scale viral vaccine production and manufacturing comprises the following sequential steps: (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 can be Vero cells. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated viral vaccine.

[0253] Processing the harvest: freezing, thawing and pooling

[0254] Freezing is another processing mode and can be used to store and freeze the composition at -30°C or below at any time during processing.

[0255] Thawing is another type of processing and refers to allowing the frozen composition to acclimate to room temperature.

[0256] Pooling is another mode of processing and involves pooling one or more compositions at any time during processing in order to subject the pooled compositions to further processing steps or to subject the pooled processed harvest to purification steps.

[0257] According to one embodiment of the present invention, each "harvest" or "crude harvest" can be frozen immediately after the harvesting step and subsequently thawed, and the processed harvest can then be subjected to one or more processing steps or purification steps. In addition, according to one embodiment of the method according to the present invention, the first harvest obtained from the first harvesting step can be frozen immediately after the harvesting step, subsequently thawed and pooled with at least one further harvest obtained from at least one further harvesting step, and then subjected to one or more further processing steps or purification steps.

[0258] Furthermore, according to one embodiment of the method according to the invention, the first harvest obtained from the first harvesting step may be clarified immediately, then immediately frozen, subsequently thawed and pooled with at least one further clarified harvest obtained from at least one further harvesting step (which may or may not have been frozen and thawed) and then subjected to one or more further processing steps or purification steps.

[0259] Furthermore, according to one embodiment of the method of the present invention, the first harvest obtained from the first harvesting step may be immediately clarified and stabilized, then immediately frozen, subsequently thawed and pooled with at least one further clarified and stabilized harvest obtained from at least one further harvesting step (which may or may not have been frozen and thawed) and then subjected to one or more further processing steps or purification steps.

[0260] In a preferred embodiment, the first harvest obtained from the first harvesting step is immediately clarified and stabilized on the same day.

[0261] As used throughout this disclosure, "immediately on the same day" or "on the same day" refers to a time interval of 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, 5 hours or less, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less.

[0262] Purification of processed harvest: at least one chromatography step

[0263] In a preferred embodiment, the first harvest obtained from the first harvesting step is immediately clarified, stabilized and purified using at least one chromatography step on the same day.

[0264] Purification of the processed harvest may include a step to remove host cell DNA. The primary purpose of this step is to remove host cell DNA (HC DNA) while ensuring maximum viral recovery. "Host cell DNA" originates from cells that produce infectious viral particles. Host cell DNA can be distinguished from viral nucleic acid by differences in its nucleic acid sequence from that of the viral nucleic acid. Several methods are known in the art for removing HC DNA. These methods are either enzymatic or a combination of enzymatic and chromatographic methods.

[0265] In the context of the present invention, enzymatic methods can be used to remove 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 nucleotide / nucleic acid fragments and endonucleases can ultimately be easily removed from the process during subsequent downstream processing.

[0266] In the context of the present invention, purification of the processed harvest may necessarily include a chromatography step. Regardless of whether enzymatic methods are used to remove HC DNA, a chromatography step is preferably included and may be ion exchange chromatography (ie, cation exchange chromatography or anion exchange chromatography).

[0267] For one embodiment of the method of the present invention, the chromatography step for removing host cell DNA 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 groups, while impurities do not bind. By changing one or more chromatographic conditions, such as the salt conditions in the buffer or the pH of the buffer, the substance to be purified can be eluted from the anion exchange groups. In the flow-through mode, impurities bind to the anion exchange groups, while the substance to be purified does not bind, but can be collected directly from the flow-through.

[0268] Ion exchange chromatography (i.e., each of cation and anion exchange chromatography) relies on charge-charge interactions between components in the sample and the charge 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, while in cation exchange chromatography, the bound ions present in the sample are positive and the immobilized functional groups are negative.

[0269] Commonly used anion exchange functional groups are Q-resins, quaternary amines, and DEAE resins (diethylaminoethane). Commonly used cation exchange groups are CM (quaternary amine), S (methyl methanesulfonate), and SP (sulfonyl). However, in general, the anion or cation exchange chromatography step can be performed with all common commercially available anion or cation exchange resins or membranes.

[0270] Typical strong anion exchange groups that can be used for the purposes of the present invention include the following functional groups: quaternary aminoethyl (QAE) moiety, primary amine (PA), quaternary ammonium (Q) moiety and trimethylammoniumethyl (TMAE) group. Resins with quaternary aminoethyl (QAE) moieties include, for example, Toyopearl QAE (available from Tosoh Bioscience, Germany), Selectacel QAE (a quaternary aminoethyl derivative of cellulose, available from Polysciences Inc., Pennsylvania USA), etc. 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), MacroPrep 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 trimethylammoniumethyl (TMAE) groups include, for example, Fractogel EMD TMAE (available from Merck, Germany). Resins with primary amine groups include Sartobind STIC primary amine resins (available from Sartorius, Germany).

[0271] For example, the anion exchange chromatography step can be performed using an anion exchange chromatography membrane having quaternary ammonium groups. The membrane substrate can be selected from stabilized reinforced cellulose and polyethersulfone. The membrane substrate can be stabilized reinforced cellulose, and the functional groups are quaternary ammonium groups. The anion exchange chromatography step may or may not involve the use of a monolithic support. The nominal pore size of the membrane can be from 0.5 μm to 5 μm or greater than 3 μm. The membrane area can be from 20 to 50 cm 2 , 25 to 45 cm 2 , 30 to 40 cm 2 , such as for example 32 cm 2 、34 cm 2 、36 cm 2 or 38 cm 2 Depending on the column used, the flow rate can be 1 to 50 ml / min, or 10 to 40 ml / min.

[0272] For example, the cation exchange chromatography step can be performed using a cation exchange chromatography membrane having quaternary amine groups. The membrane substrate 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 from 0.5 μm to 5 μm or greater than 3 μm. The membrane area can be from 20 to 50 cm 2 , 25 to 45cm 2 , 30 to 40 cm 2 The flow rate may be 1 to 50 ml / min, or 10 to 40 ml / min.

[0273] In certain embodiments of the methods of the present invention, host cell DNA is removed from a sample comprising viral particles and host cell DNA. The term "removing host cell DNA" means that the content of host cell DNA after carrying out the method of the present invention is lower than the content of host cell DNA before carrying out the method of the present invention. In one embodiment, the method of the present invention causes the host cell DNA content to decrease by at least ten times. Preferably, the method of the present invention causes the host cell DNA content to decrease by at least 12 times or 15 times, more preferably at least 18 times or 20 times, and most preferably at least 22 times. The reduction fold can be calculated by dividing the host cell DNA content in the sample before carrying out the method by the host cell DNA content in the sample after carrying out the method. Preferably, the content of host cell DNA after carrying out the method of the present invention is lower than the detection limit of the assay method for measuring the host cell DNA content. In one embodiment, the content of host cell DNA after carrying out the method of the present invention is 50 ng / ml or lower.

[0274] Methods for determining the host cell DNA content in a biological sample obtained from a host cell are known in the art and include quantitative PCR using primers that specifically bind to host cell DNA but not to viral nucleic acid. Kits for determining host cell DNA content are commercially available from, for example, ThermoFisher. Preferably, Picogreen® dye is used to determine host cell DNA content.

[0275] Generally, for large-scale vaccine production, first concentrate harvest or clarified harvest or clarified and stable harvest or stabilized and clarified harvest with ultrafiltration step, then load it onto chromatographic column such as hydrophobic interaction chromatographic column or ion exchange chromatography column (that is, anion exchange chromatography column or cation exchange chromatography column).This is mainly because before column chromatography, concentrated harvest can make loading faster and increase column capacity, thereby improve productivity.And, it should be generally understood that, before chromatographic step, ultrafiltration is carried out and the efficiency of chromatography is enhanced.In addition, it should be generally understood that, before chromatographic step, harvest or clarified harvest or clarified and stable harvest or stabilized and clarified harvest is carried out ultrafiltration and needs less dilution to regulate pH and the conductivity of chromatographic step, this finally increases column productivity and reduces the use of consumables, thereby helps to improve process economy.As used herein, " concentrated " means that the cumulative volume of harvest reduces, thereby increases the concentration of component in harvest.

[0276] Ultrafiltration (UF) as used herein refers to a technique that relies on the use of polymer membranes with highly defined pore sizes to separate molecules according to size. In simple terms, the UF procedure relies on the use of fluid pressure to drive smaller molecules to migrate through the UF membrane while retaining larger molecules. Ultrafiltration can be performed in one of two modes of operation: direct flow filtration (DFF) or tangential flow filtration (TFF).

[0277] It has surprisingly been found that the harvest, or the clarified harvest, or the clarified and stabilized harvest, or the stabilized and clarified harvest can be subjected to a chromatography step directly, i.e. without any previous chromatography step and / or without a previous ultrafiltration step, without resulting in a significant loss of viral titer compared to when the harvest is first concentrated in a membrane filtration step or by a previous chromatography step.

[0278] As used herein, the term "feed composition" refers to the solution introduced into the chromatography column or ultrafiltration membrane. The feed composition is directly related to the way the harvest is processed prior to the chromatography step (e.g., by clarification, stabilization, or clarification and stabilization). Thus, it has been surprisingly found that performing a chromatography step directly after harvest clarification, stabilization, or clarification and stabilization does not result in a significant loss of viral titer compared to when the same feed composition is first concentrated in a membrane filtration step (such as a TFF step).

[0279] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to a chromatography step followed by ultrafiltration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using a chromatography step, and subsequent ultrafiltration are all performed on the same day.

[0280] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: performing a chromatography step directly on the clarified and stabilized harvest, followed by tangential flow filtration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using a chromatography step, and subsequent tangential flow filtration are all performed on the same day.

[0281] Thus, according to one aspect of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to an ion exchange chromatography step, followed by ultrafiltration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using ion exchange chromatography, and subsequent ultrafiltration are all performed on the same day.

[0282] Thus, according to one aspect of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to an ion exchange chromatography step, followed by tangential flow filtration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using ion exchange chromatography, and subsequent tangential flow filtration are all performed on the same day.

[0283] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to an anion exchange chromatography step, followed by ultrafiltration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using anion exchange chromatography, and subsequent ultrafiltration are all performed on the same day.

[0284] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to an anion exchange chromatography step, followed by tangential flow filtration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using anion exchange chromatography, and subsequent tangential flow filtration are all performed on the same day.

[0285] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to a cation exchange chromatography step, followed by ultrafiltration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated virus vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using cation exchange chromatography, and subsequent ultrafiltration are all performed on the same day.

[0286] Thus, according to one aspect of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially includes the following steps: directly subjecting the clarified and stabilized harvest to a cation exchange chromatography step, followed by tangential flow filtration. In this context, the flavivirus can be dengue virus. In this context, the vaccine can be a live, attenuated viral vaccine. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest using cation exchange chromatography, and subsequent tangential flow filtration are all performed on the same day.

[0287] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially comprises the following steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii), including clarifying and stabilizing the harvest, and (v) purifying the processed harvest of step (iv) by the following sequential steps: (va) directly subjecting the processed harvest to ion exchange chromatography, and (vb) subjecting the purified harvest obtained in step (va) to tangential flow filtration. 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.

[0288] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially comprises the following steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii), including clarifying and stabilizing the harvest, and (v) purifying the processed harvest of step (iv) by the following sequential steps: (va) directly subjecting the processed harvest to anion exchange chromatography, and (vb) subjecting the purified harvest obtained in step (va) to tangential flow filtration. 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.

[0289] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially comprises the following steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii), including clarifying and stabilizing the harvest, and (v) purifying the processed harvest of step (iv) by the following sequential steps: (va) directly subjecting the processed harvest to cation exchange chromatography, and (vb) subjecting the purified harvest obtained in step (va) to tangential flow filtration. 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.

[0290] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially comprises the following steps: (i) providing cells in growth medium, (ii) statically infecting the cells of step (i) in monolayer form with virus-containing medium (optionally at a low MOI), (iii) harvesting to obtain a harvest, (iv) processing the harvest of step (iii), including clarifying and stabilizing the harvest, and (v) purifying the processed harvest of step (iv) by the following sequential steps: (va) directly subjecting the processed harvest to ion exchange chromatography, and (vb) subjecting the purified harvest obtained in step (va) to tangential flow filtration. 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. In this context, harvesting comprises a first harvesting step and at least three further harvesting steps. In this context, a medium exchange is performed 12-30 hours before the first harvesting step. Preferably, steps (iii), (iv), and (v) are all performed on the same day.

[0291] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially comprises the following steps: (i) providing cells in growth medium, (ii) statically infecting the cells from step (i) in monolayer form with virus-containing medium at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest from step (iii), including clarifying and stabilizing the harvest, and (v) purifying the processed harvest from step (iv) by the following sequential steps: (va) directly subjecting the processed harvest to anion exchange chromatography, and (vb) subjecting the purified harvest obtained in step (va) to tangential flow filtration. 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. In this context, harvesting comprises a first harvesting step and at least three further harvesting steps. In this context, a medium exchange is performed 12-30 hours prior to the first harvesting step. Preferably, steps (iii), (iv), and (v) are all performed on the same day.

[0292] Thus, according to one embodiment of the present invention, high viral titers can be achieved when large-scale flavivirus vaccine production and manufacturing sequentially comprises the following steps: (i) providing cells in growth medium, (ii) statically infecting the cells from step (i) in monolayer form with virus-containing medium at a low MOI, (iii) harvesting to obtain a harvest, (iv) processing the harvest from step (iii), including clarifying and stabilizing the harvest, and (v) purifying the processed harvest from step (iv) by the following sequential steps: (va) directly subjecting the processed harvest to cation exchange chromatography, and (vb) subjecting the purified harvest obtained in step (va) to tangential flow filtration. 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. In this context, harvesting comprises a first harvesting step and at least three further harvesting steps. In this context, a medium exchange is performed 12-30 hours prior to the first harvesting step. Preferably, steps (iii), (iv), and (v) are all performed on the same day.

[0293] At the end of one or more chromatography steps, the composition obtained is referred to as a "purified harvest" which refers to a processed harvest that has been subjected to at least one chromatography step.

[0294] The term "clarified and purified harvest" refers to a crude harvest that has been subjected to a clarification step and one or more chromatography steps, in that order. The term "clarified, stabilized and purified harvest" refers to a crude harvest that has been subjected to a clarification step, a stabilization step, and one or more chromatography steps, in that order. The term "stabilized, clarified and purified harvest" refers to a crude harvest that has been subjected to a clarification step, one or more chromatography steps, and a stabilization step, in that order.

[0295] In one embodiment, by using the methods of the present invention as described above, a low MOI can be used for large-scale flavivirus vaccine production and manufacturing, providing a higher average virus titer than the same method comprising the same process steps but using a higher MOI. Thus, by using the methods of the present invention, a low MOI can be used for large-scale attenuated flavivirus vaccine production and manufacturing, providing a higher average virus titer than the same method comprising the same process steps but using a higher MOI. In this context, a high MOI refers to an MOI greater 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 harvest steps.

[0296] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0297] (i) providing cells in a growth medium,

[0298] (ii) infecting the cells of step (i) with infection medium containing the flavivirus,

[0299] (iii) harvesting to obtain a harvest,

[0300] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0301] (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step,

[0302] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0303] (a) The maximum pH at any time point in steps (iii) to (v)

[0304] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0305] The difference between them is no more than 0.4 units.

[0306] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2 higher. Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0307] (i) providing cells in a growth medium,

[0308] (ii) infecting the cells of step (i) with infection medium containing the flavivirus,

[0309] (iii) harvesting to obtain a harvest,

[0310] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0311] (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step,

[0312] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0313] (a) The maximum pH at any time point in steps (iii) to (v)

[0314] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0315] The difference between them is no more than 0.3 units.

[0316] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0317] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0318] (i) providing cells in a growth medium,

[0319] (ii) infecting the cells of step (i) with infection medium containing the flavivirus at a low MOI,

[0320] (iii) harvesting to obtain a harvest,

[0321] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0322] (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step,

[0323] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0324] (a) The maximum pH at any time point in steps (iii) to (v)

[0325] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0326] The difference between them is no more than 0.4 units.

[0327] In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method produces a higher virus yield compared to the same method with the same process steps but allowing the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0328] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0329] (i) providing cells in a growth medium,

[0330] (ii) infecting the cells of step (i) with infection medium containing the flavivirus at a low MOI,

[0331] (iii) harvesting to obtain a harvest,

[0332] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0333] (v) purifying the processed harvest of step (iv), wherein step (v) comprises at least one chromatography step,

[0334] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0335] (a) The maximum pH at any time point in steps (iii) to (v)

[0336] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0337] The difference between them is no more than 0.3 units.

[0338] In certain embodiments, "low MOI" refers to less than 0.008.

[0339] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0340] When using one type of flask, the calculation of virus yield (in mg / cm2) can be performed as follows:

[0341] (i) Total number of flasks x area of ​​each flask (in cm2) = total area

[0342] (ii) Total virus yield (in mg) / total area obtained in step (i) above (in cm2) = virus yield / cm2

[0343] When more than one type of flask is used, calculation of viral yield (in mg / cm2) can be performed as follows:

[0344] (i) (Number of Type 1 flasks) x (area of ​​each Type 1 flask in cm2) + (Number of Type 2 flasks) x (area of ​​each Type 2 flask in cm2) etc. = Total area

[0345] (ii) Total virus yield (in mg) / total area obtained in step (i) above (in cm2) = virus yield / cm2

[0346] Purification of processed products: Ultrafiltration

[0347] In the context of the present invention, the step of purifying the harvest may comprise an ultrafiltration step to obtain a stock solution.

[0348] Thus, the feed composition, ie, the input to the ultrafiltration step, is the clarified and purified harvest, the clarified, stabilized and purified harvest, the stabilized, clarified and purified harvest, the stabilized and purified harvest, or simply the purified harvest.

[0349] The purpose of implementing the ultrafiltration step in the method of the present invention is to minimize the harvest volume without affecting the virus potency / recovery. According to one embodiment of the present invention, the ultrafiltration step can be a direct flow filtration (DFF) or a tangential flow filtration (TFF) step.

[0350] According to one embodiment of the present invention, a method for large-scale viral vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium comprising a flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), including at least one chromatography step and an ultrafiltration step. Preferably, steps (iii), (iv), and (v) are all performed on the same day.

[0351] According to one embodiment of the present invention, a method for large-scale viral vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium comprising a flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), including at least one chromatography step and a direct flow filtration step. Preferably, steps (iii), (iv) and (v) are all performed on the same day.

[0352] According to one embodiment of the present invention, a method for large-scale viral vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing a flavivirus (optionally at a low MOI), (iii) harvesting to obtain a harvest, and (iv) processing the harvest of step (iii) to obtain a processed harvest, and (v) purifying the processed harvest of step (iv), including at least one chromatography step and a tangential flow filtration step. 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 viral vaccine. Preferably, steps (iii), (iv), and (v) are all performed on the same day.

[0353] Direct-flow filtration (DFF) is used when all the fluids to be filtered are driven in a direction perpendicular to the filter surface due to the supply pressure. Contaminants are trapped in the filter medium or accumulate on the surface, resulting in contaminant blockage and an increase in the pressure differential across the filter during the duration of the filtration process. The filtrate leaves the filter from the downstream side. Once a certain pressure differential is reached, the fluid flow rate decreases and / or the filter reaches its terminal pressure differential, filtration is stopped, and the filter is discarded or sometimes 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 liquid flow to pass through the membrane (filtrate), while the remaining portion (retentate) is recirculated back to the feed tank. Therefore, in TFF, most of the feed travels tangentially across the filter surface rather than entering the filter.

[0354] After at least one processing step, the composition obtained may be subjected to one or more tangential flow filtration (TFF) steps, one TFF step and one DFF step, or one TFF step. This step is used to concentrate the sample to ensure sufficient potency and remove small molecular weight impurities.

[0355] Suitable filter materials for TFF can be cross-linked cellulose and polyethersulfone. Preferably, a cross-linked cellulose-based polymer available from Sartorius under the name Hydrosart is used. The cutoff size of the filter material used for tangential flow filtration determines whether a certain compound is present in the filtrate or the retentate. The cutoff size may be less than 1 / 3 to 1 / 5 of the size of the virus to be purified. For one embodiment of the method of the present invention, the cutoff size can be 50 to 300 kDa. Within the range of 50 to 300 kDa, the cutoff size can be 100 kDa. Therefore, for the purposes of the present invention, a cross-linked cellulose-based polymer filter with a cutoff size of 100 kDa can be used in the TFF step. The typical TFF membrane used in this step includes a polyethersulfone (PES) with a molecular weight cutoff (MWCO) size of 100 kDa (Hydrosart) and 300 kDa.

[0356] The TFF process can be characterized by flow rate and transmembrane pressure. Flow rate is directly proportional to transmembrane pressure and inversely proportional to the resistance of the membrane and filter cake. Flow rate is expressed as feed flow rate per unit membrane area, i.e., liters / square meter / hour (LMH). In the TFF step of the present invention, the flow rate can be 100 to 500 LMH. Within the range of 100 to 500 LMH, the flow rate can be 120 to 400 LMH, 150 to 300 LMH, or 300 LMH. Transmembrane pressure (TMP) is the pressure difference between the two sides of the membrane. TMP can be 0.2 to 0.5 bar, 0.25 to 0.45 bar, 0.30 to 0.45 bar, 0.35 to 0.45 bar, or 0.35 to 0.40 bar, or 0.30 to 0.50 bar.

[0357] TFF typically uses two basic filter configurations: cartridge filters and cassette filters. In a cartridge filter (often called a hollow fiber filter), the membrane forms a set of parallel hollow fibers. The feed stream passes through the fiber lumen and the permeate is collected from the outside of the fibers. In a cassette filter, several flat membrane sheets are separated from each other and from a cassette housing by a supporting screen. The feed stream enters the space between the two sheets and the permeate is collected from the other side of the sheets. In embodiments of the TFF step in the method of the present invention, both cartridge filters 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.

[0358] During the TFF step, the virus-containing solution used in flavivirus vaccine production is in the retentate. The retentate obtained is also the stock solution. After the TFF step, the feed volume can be concentrated, for example, 2 to 10 times, 3 to 8 times, 4 to 6 times, or 5 times. At the end of the TFF step, a stock solution 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.

[0359] In certain embodiments, the pH throughout the above steps is maintained in the range of 7.6 to 8.1.

[0360] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0361] (i) providing cells in a growth medium,

[0362] (ii) infecting the cells of step (i) with infection medium containing the flavivirus,

[0363] (iii) harvesting to obtain a harvest,

[0364] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0365] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0366] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0367] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0368] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0369] (a) The maximum pH at any time point in steps (iii) to (v)

[0370] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0371] The difference between them is no more than 0.4 units.

[0372] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0373] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0374] (i) providing cells in a growth medium,

[0375] (ii) infecting the cells of step (i) with infection medium containing the flavivirus,

[0376] (iii) harvesting to obtain a harvest,

[0377] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0378] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0379] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0380] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0381] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0382] (a) The maximum pH at any time point in steps (iii) to (v)

[0383] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0384] The difference between them is no more than 0.3 units.

[0385] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0386] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0387] (i) providing cells in a growth medium,

[0388] (ii) infecting the cells of step (i) with infection medium containing the flavivirus at a low MOI,

[0389] (iii) harvesting to obtain a harvest,

[0390] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0391] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0392] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0393] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0394] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0395] (a) The maximum pH at any time point in steps (iii) to (v)

[0396] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0397] The difference between them is no more than 0.4 units.

[0398] In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method produces a higher virus yield compared to the same method with the same process steps but allowing the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0399] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0400] (i) providing cells in a growth medium,

[0401] (ii) infecting the cells of step (i) with infection medium containing the flavivirus at a low MOI,

[0402] (iii) harvesting to obtain a harvest,

[0403] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0404] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0405] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0406] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0407] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0408] (a) The maximum pH at any time point in steps (iii) to (v)

[0409] (b) the minimum pH occurring at any time during steps (iii) to (v),

[0410] The difference between them is no more than 0.3 units.

[0411] In certain embodiments, "low MOI" refers to less than 0.008.

[0412] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0413] Thus, one embodiment of a large-scale flavivirus vaccine production and manufacturing method for a flavivirus vaccine comprises a step of processing the harvest and a step of purifying the processed harvest, the purification step comprising at least one chromatography step followed by an ultrafiltration step. The purification step comprises an ultrafiltration step after the chromatography step. At the end of the ultrafiltration step, a stock solution is obtained. If a bivalent, trivalent, or quadrivalent final formulation is desired, the stock solution of this step still comprises each individual monovalent stock solution. In the context of the present invention, it should be understood that each individual method step and / or ingredient component described above can be considered individually or in combination with one another. Therefore, each possibility described above, both individually and in combination, represents a separate embodiment of the present invention.

[0414] Processing of stock solution to obtain bulk stock solution

[0415] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus (optionally 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), including at least one chromatography step to obtain a purified harvest (v), an ultrafiltration step to obtain a stock solution, and (vi) processing the stock solution obtained in step (v), including at least one rinsing step of the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and the stock solution, wherein the composition is a bulk stock solution. Preferably, the ultrafiltration is tangential flow filtration. Preferably, the chromatography step is 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 the 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.

[0416] For the purposes of the present invention, the TFF step may or may not be followed by diafiltration. Diafiltration is a fractionation process that removes smaller molecules by membrane washing while leaving larger molecules in the retentate, ultimately without changing the concentration. It can be used to remove salts or exchange buffers. In a preferred embodiment, the method of the present invention includes the step of rinsing the filter membrane with a buffer once, twice, three times, four times, or five times to obtain a composition comprising a buffered rinse and a stock solution, the composition being a bulk stock solution. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest by a chromatographic step, and tangential flow filtration, followed by a rinse step, are all performed on the same day.

[0417] For purposes of the present invention, the TFF step may or may not be followed by diafiltration. Diafiltration is a fractionation process that removes smaller molecules by membrane washing while leaving larger molecules in the retentate, ultimately without changing concentration. It can be used to remove salts or exchange buffers. In an exemplary embodiment, the method of the present invention comprises the step of rinsing the filter membrane with a buffer solution once, twice, three times, four times, or five times to obtain a buffered rinse. Preferably, the harvesting step, clarification of the harvest, stabilization of the harvest, purification of the clarified and stabilized harvest with a chromatographic step, and tangential flow filtration, followed by a rinse step, are all performed on the same day.

[0418] After the TFF step, the composition comprising the stock solution and the buffered rinses is the bulk stock solution. Thus, according to one embodiment of the present invention, the bulk stock solution is a composition comprising the stock solution (TFF retentate) and at least two buffered rinses of the TFF membrane. Thus, according to one embodiment of the present invention, the bulk stock solution is a composition comprising the stock solution (TFF retentate) and at least three buffered rinses of the TFF membrane. Thus, according to one embodiment of the present invention, the bulk stock solution is a composition comprising the stock solution (TFF retentate) and at least four buffered rinses of the TFF membrane.

[0419] After the TFF step, the stock solution and the buffered rinse can be mixed to obtain a bulk stock solution. Thus, according to an exemplary embodiment of the present invention, the bulk stock solution is a composition comprising the stock solution (TFF retentate) and at least two buffered rinses of the TFF membrane. Thus, according to an embodiment of the present invention, the bulk stock solution is a composition comprising the stock solution (TFF retentate) and at least three buffered rinses of the TFF membrane. Thus, according to an embodiment of the present invention, the bulk stock solution is a composition comprising the stock solution (TFF retentate) and at least four buffered rinses of the TFF membrane.

[0420] The wash buffer comprises the same buffer excipient composition / stabilization buffer that is added during the processing step of the harvest, more specifically during the stabilization step of the harvest, except that the excipient concentration in the wash buffer is lower compared to the buffer excipient composition / stabilization buffer.

[0421] The flushing buffer comprises one or more excipients, such as nonionic surfactants, non-reducing sugars, and albumin, all of which are described in the section on stabilization of the harvest. The flushing buffer used for flushing can comprise F 127, trehalose dihydrate, and human serum albumin. The buffer used for flushing preferably comprises 0.5% to 5% nonionic surfactant, 20% to 40% non-reducing sugar, and 0.05% to 0.3% albumin. The buffer used for flushing preferably comprises 0.5% to 5% F127, 20% to 40% trehalose dihydrate, and 0.05% to 0.3% human serum albumin.

[0422] The wash buffer may comprise the same excipients as the stabilizing buffer added in step (ii) of the method, including sugars, nonionic surfactants and proteins. Thus, in a preferred embodiment, the wash buffer comprises the same excipients as the stabilizing buffer added in step (ii) of the method, including sugars, nonionic surfactants and proteins, except that the excipients in the wash buffer are at a lower concentration than those in the stabilizing buffer.

[0423] The flushing buffer may comprise the same excipients as the harvest that is stable or purified, including sugars, nonionic surfactants, and proteins. Therefore, in a preferred embodiment, the flushing buffer comprises the same excipients as the harvest that is stable or purified, including sugars, nonionic surfactants, and proteins, except that the excipients in the flushing buffer are at a higher concentration than the excipients in the harvest that is stable or purified.

[0424] The wash buffer may comprise the same excipients as the stock solution, including sugars, nonionic surfactants, and proteins. In a preferred embodiment, the wash buffer comprises the same excipients as the stock solution, including sugars, nonionic surfactants, and proteins, except that the sugar in the wash buffer is at a higher concentration than that in the stock solution.

[0425] Thus, one embodiment of the method for large-scale production and manufacturing of flavivirus vaccines comprises a further step (vi): processing the bulk solution, including at least one rinsing step of the ultrafiltration membrane with a wash buffer, to obtain a composition comprising a buffered wash solution and the bulk solution, wherein the wash buffer comprises a sugar, a surfactant, and a protein. Preferably, steps (iii), (iv), (v), and (vi) are all performed on the same day.

[0426] In an exemplary embodiment, the method for large-scale production and manufacturing of a flavivirus vaccine comprises a further step (vi): processing the stock solution, including at least one washing step of the ultrafiltration membrane with a wash buffer to obtain a buffered wash solution, wherein the wash buffer comprises a sugar, a surfactant, and a protein. Preferably, steps (iii), (iv), (v), and (vi) are all performed on the same day.

[0427] According to one embodiment of the inventive method, the stock solution and the bulk stock solution contain the same excipients as the stabilizing buffer added in step (ii) of the method, including sugars, nonionic surfactants and proteins. In a preferred embodiment, the concentrations of the excipients (including sugars, nonionic surfactants and proteins) in the stock solution and the bulk stock solution are the same.

[0428] In one embodiment, the concentration of the excipients in the bulk stock solution from the buffered excipient composition / stabilization buffer added in step (ii) is two to five times the concentration of these excipients in the stabilized harvest or purified harvest. In a preferred embodiment, the concentration of these excipients in the bulk stock solution is five times the concentration of these excipients in the stabilized harvest or purified harvest.

[0429] In one embodiment, the concentration of excipients (including sugars, proteins and / or non-ionic excipients) in the buffer excipient composition / stabilization buffer added in step (ii) in the bulk stock solution is two to five times the concentration of these excipients in the stabilized harvest or purified harvest. In a preferred embodiment, the concentration of these excipients in the bulk stock solution is five times the concentration of these excipients in the stabilized harvest or purified harvest.

[0430] In certain embodiments, the pH throughout the above steps is maintained in the range of 7.6 to 8.1.

[0431] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0432] (i) providing cells in a growth medium,

[0433] (ii) infecting the cells of step (i) with infection medium containing the flavivirus,

[0434] (iii) harvesting to obtain a harvest,

[0435] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0436] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0437] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0438] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0439] (vi) processing the stock solution obtained from step (vb) to obtain a bulk stock solution,

[0440] wherein the pH in steps (iii) to (vi) is maintained in the range of 7.6 to 8.1, and wherein

[0441] (a) The maximum pH at any time point in steps (iii) to (vi)

[0442] (b) the maximum pH occurring at any time point in steps (iii) to (vi),

[0443] The difference between them is no more than 0.4 units.

[0444] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0445] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0446] (i) providing cells in a growth medium,

[0447] (ii) infecting the cells of step (i) with infection medium containing the flavivirus,

[0448] (iii) harvesting to obtain a harvest,

[0449] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0450] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0451] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0452] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0453] (vi) processing the stock solution obtained from step (vb) to obtain a bulk stock solution,

[0454] wherein the pH in steps (iii) to (vi) is maintained in the range of 7.6 to 8.1, and wherein

[0455] (a) The maximum pH at any time point in steps (iii) to (vi)

[0456] (b) the maximum pH occurring at any time point in steps (iii) to (vi),

[0457] The difference between them is no more than 0.3 units.

[0458] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0459] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0460] (i) providing cells in a growth medium,

[0461] (ii) infecting the cells of step (i) with infection medium containing the flavivirus at a low MOI,

[0462] (iii) harvesting to obtain a harvest,

[0463] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0464] (v) purifying the processed harvest of step (iv), wherein step (vi) comprises the following steps in sequence:

[0465] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0466] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0467] (vi) processing the stock solution obtained from step (vb) to obtain a bulk stock solution,

[0468] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0469] (a) The maximum pH at any time point in steps (iii) to (vi)

[0470] (b) the maximum pH occurring at any time point in steps (iii) to (vi),

[0471] The difference between them is no more than 0.4 units.

[0472] In certain embodiments, "low MOI" refers to less than 0.008. In certain embodiments, the method produces a higher virus yield compared to the same method with the same process steps but allowing the difference between (a) and (b) to be greater than 0.4 units. In certain embodiments, higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0473] Thus, in certain embodiments, a method for large-scale flavivirus vaccine production and manufacturing is provided, comprising the following sequential steps:

[0474] (i) providing cells in a growth medium,

[0475] (ii) infecting the cells of step (i) with infection medium containing the flavivirus at a low MOI,

[0476] (iii) harvesting to obtain a harvest,

[0477] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0478] (v) purifying the processed harvest of step (iv), wherein step (v) comprises the following steps in sequence:

[0479] (va) ion exchange chromatography to obtain a purified harvest, preferably anion exchange chromatography,

[0480] (vb) ultrafiltration to obtain a stock solution, preferably tangential flow filtration,

[0481] (vi) processing the stock solution obtained from step (vb) to obtain a bulk stock solution,

[0482] wherein the pH in steps (iii) to (v) is maintained in the range of 7.6 to 8.1, and wherein

[0483] (a) The maximum pH at any time point in steps (iii) to (vi)

[0484] (b) the maximum pH occurring at any time point in steps (iii) to (vi),

[0485] The difference between them is no more than 0.3 units.

[0486] In certain embodiments, "low MOI" refers to less than 0.008.

[0487] In certain embodiments, the method produces a higher virus yield compared to the same method having the same process steps but allowing the difference between (a) and (b) to be greater than 0.3 units. In certain embodiments, a higher virus yield refers to a virus yield that is at least 1 mg / cm2 higher, such as 1.5 mg / cm2.

[0488] Thus, one embodiment of a method for large-scale production and manufacture of a flavivirus vaccine includes a step of processing a stock solution, including mixing the stock solution with a buffered rinse to obtain a bulk stock solution. The rinse step produces a composition comprising the buffered rinse and the stock solution, which is the bulk stock solution. If a bivalent, trivalent, or quadrivalent final formulation is desired, the bulk stock solution of this step still includes each individual monovalent bulk stock solution. In the context of the present invention, it should be understood that each individual method step and / or component or ingredient described above can be considered individually or in combination with one another. Therefore, each possibility described above, both individually and in combination, represents a separate embodiment of the present invention.

[0489] Processing bulk liquid

[0490] According to one embodiment of the present invention, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with a medium containing the flavivirus (optionally 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), including at least one chromatography step to obtain a purified harvest (v), an ultrafiltration step to obtain a stock solution, (vi) processing the stock solution obtained in step (v), preferably including at least one washing step of the ultrafiltration membrane with a washing buffer, to obtain a bulk stock solution, and (vii) processing the bulk stock solution. Preferably, the ultrafiltration is tangential flow filtration. Preferably, the chromatography step is 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 the 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.

[0491] Processing of the bulk stock may include a depth filtration step. This filtration step may be performed in the same manner as the clarification step of the harvest.

[0492] Depth filtration uses porous filter media to separate particles and solids from liquids. Filter media used for depth filtration include cellulose acetate, polypropylene, cellulose acetate protected by glass wool, and 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.

[0493] The filter used to clarify the harvest can be a heterogeneous double-layer polyethersulfone filter with a pore size of 0.2 μm and 0.45 μm. Thus, for the purpose of harvest clarification, several heterogeneous double-layer filters can be used, such as Sartoclean (cellulose acetate) 0.8+0.65 μm, Sartopure (polypropylene) 0.65 μm, Sartoclean (glass wool) 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 present invention.

[0494] According to the method of the present invention, preferably the bulk stock solution after this step comprises the same excipients as were present in the bulk stock solution before the processing step.

[0495] Freezing is another way to process bulk stock solutions and can be used to store and freeze bulk stock solutions, preferably below -30°C.

[0496] Thawing is another way of processing bulk stock and refers to allowing the frozen bulk stock to acclimate to room temperature.

[0497] Pooling is another way of processing bulk stocks and involves combining two or more bulk stocks.

[0498] In one embodiment of the method of the present invention, the first harvest obtained from the first harvesting step is clarified and stabilized on the same day. The clarified and stabilized harvest is also purified by at least one chromatography step, preferably anion exchange chromatography, on the same day. The purified harvest is also passed through an ultrafiltration step, preferably tangential flow filtration, on the same day, to obtain the first stock solution. The first stock solution is also mixed with flushing buffer on the same day to obtain a composition comprising a buffered flushing solution and the first stock solution, which is the first bulk stock solution. In one embodiment, a second harvesting step is carried out 20-30 hours after the first harvesting step, and the above steps are repeated to obtain the second bulk stock solution. In one embodiment, up to ten harvesting steps are carried out, and the above steps are repeated to obtain up to ten bulk stock solutions, wherein the interval between each harvesting step is 20-30 hours.

[0499] In a preferred embodiment, six harvesting steps are performed and repeated on six different days to obtain six bulk stocks, wherein the interval between each harvesting step is 20-30 hours.

[0500] 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 the 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.

[0501] Thus, in a preferred embodiment, for dengue serotype 1, six bulk stock solutions are obtained from six harvesting steps, each obtained at the end of each day's harvest, with an interval of 20-30 hours between each harvesting step. Thus, in a preferred embodiment, for dengue serotype 2, six bulk stock solutions are obtained from six harvesting steps, each obtained at the end of each day's harvest, with an interval of 20-30 hours between each harvesting step. Thus, in a preferred embodiment, for dengue serotype 3, six bulk stock solutions are obtained from six harvesting steps, each obtained at the end of each day's harvest, with an interval of 20-30 hours between each harvesting step. Thus, in a preferred embodiment, for dengue serotype 4, six bulk stock solutions are obtained from six harvesting steps, each obtained at the end of each day's harvest, with an interval of 20-30 hours between each harvesting step.

[0502] In one embodiment, each bulk stock solution can be frozen on the same day, or undergo a depth filtration step on the same day and then frozen on the same day.

[0503] In a preferred embodiment, each dengue serotype 1 bulk stock solution is immediately frozen on the same day. In a preferred embodiment, each frozen dengue serotype 1 bulk stock solution is thawed, pooled, and then subjected to a depth filtration step. In a preferred embodiment, six dengue serotype 1 bulk stock solutions are thawed, pooled, and then subjected to a depth filtration step.

[0504] In a preferred embodiment, each dengue serotype 2 bulk stock solution is immediately frozen on the same day. In a preferred embodiment, each frozen dengue serotype 2 bulk stock solution is thawed, pooled, and then subjected to a depth filtration step. In a preferred embodiment, six dengue serotype 2 bulk stock solutions are thawed, pooled, and then subjected to a depth filtration step.

[0505] In a preferred embodiment, each dengue serotype 3 bulk stock solution is immediately subjected to a depth filtration step and then frozen on the same day. In a preferred embodiment, six dengue serotype 3 bulk stock solutions are individually subjected to a depth filtration step and then individually frozen on the same day.

[0506] In a preferred embodiment, each dengue serotype 4 bulk stock solution is immediately subjected to a depth filtration step and then frozen on the same day. In a preferred embodiment, six dengue serotype 4 bulk stock solutions are individually subjected to a depth filtration step and then individually frozen on the same day.

[0507] In a preferred embodiment, for dengue serotype 1, such as represented by SEQ ID NO: 1 and / or SEQ ID NO:2 represents a dengue 2 / 1 chimera or TDV-1, the method of the present invention comprises the following steps: (i) providing cells in a growth medium as explained in the previous section, (ii) infecting the cells with a medium comprising dengue serotype 1 (optionally 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, comprising clarifying and stabilizing the harvest on the same day, (v) purifying the clarified and stabilized harvest on the same day, comprising (va) at least one chromatography step, preferably anion exchange chromatography, and (vb) also subjecting the purified harvest to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain a first stock solution, (vi) also processing the first stock solution on the same day, preferably mixing it with a wash buffer on the same day to obtain a composition comprising a buffered wash solution and the first stock solution, said composition being a first bulk stock solution, (vii) processing the first bulk stock solution, comprising (vii-a) freezing the first bulk stock solution to obtain a frozen first bulk stock solution. In a preferred embodiment, six harvesting steps are performed and repeated on six different days to obtain six bulk stock solutions, wherein the interval between each harvesting step is 20-30 hours. In one embodiment, the method comprises the steps of (vii-b) thawing the six bulk stock solutions, and (vii-c) pooling the six bulk stock solutions, and (vii-d) performing a depth filtration step on the pooled six bulk stock solutions, and (vii-e) freezing the filtered bulk stock solution.

[0508] In a preferred embodiment, for dengue serotype 2 or TDV-2 represented by SEQ ID NO: 3 and / or SEQ ID NO: 4, the method of the present invention comprises the following steps: (i) providing cells in a growth medium as explained in the previous section, (ii) infecting the cells with a medium comprising dengue serotype 2 (optionally 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, comprising clarifying and stabilizing the harvest on the same day, (v) purifying the clarified and stabilized harvest on the same day, comprising (va) at least one chromatography step, preferably anion exchange chromatography, and (vb) also subjecting the purified harvest on the same day to an ultrafiltration step, preferably tangential flow filtration, to obtain a first stock solution, (vi) also processing the first stock solution on the same day, preferably mixing with a wash buffer on the same day to obtain a composition comprising a buffered wash solution and the first stock solution, said composition being a first bulk stock solution, (vii) processing the first bulk stock solution, comprising (vii-a) freezing the first bulk stock solution to obtain a frozen first bulk stock solution. In a preferred embodiment, six harvesting steps are performed and repeated on six different days to obtain six bulk stock solutions, wherein the interval between each harvesting step is 20-30 hours. In one embodiment, the method comprises the steps of (vii-b) thawing the six bulk stock solutions, and (vii-c) pooling the six bulk stock solutions, and (vii-d) performing a depth filtration step on the pooled six bulk stock solutions, and (vii-e) freezing the filtered bulk stock solution.

[0509] In a preferred embodiment, for the sequence of SEQ ID NO: 5 and / or SEQ ID NO:6 represents dengue serotype 3 or TDV-3, the method of the present invention comprises the following steps: (i) providing cells in a growth medium as explained in the previous section, (ii) infecting the cells with a medium comprising 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, comprising clarifying and stabilizing the harvest on the same day, (v) purifying the clarified and stabilized harvest on the same day, comprising (va) at least one chromatography step, preferably anion exchange chromatography, and (vb) also subjecting the purified harvest to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain a first stock solution, (vi) also processing the first stock solution on the same day, preferably mixing it with a wash buffer on the same day to obtain a composition comprising a buffered wash solution and the first stock solution, the composition being a first bulk stock solution, (vii) processing the first bulk stock solution, comprising (vii-a) subjecting the first bulk stock solution to a depth filtration step, and (vii-b) freezing the filtered first bulk stock solution. In a preferred embodiment, six harvesting steps are performed and repeated on six different days to obtain six bulk stocks, wherein the interval between each harvesting step is 20-30 hours.

[0510] In a preferred embodiment, for the sequence of SEQ ID NO: 7 and / or SEQ ID NO:8 represents dengue serotype 4 or TDV-4, the method of the present invention comprises the following steps: (i) providing cells in a growth medium as explained in the previous section, (ii) infecting the cells with a medium comprising 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, comprising clarifying and stabilizing the harvest on the same day, (v) purifying the clarified and stabilized harvest on the same day, comprising (va) at least one chromatography step, preferably anion exchange chromatography and (vb) also subjecting the purified harvest to an ultrafiltration step, preferably tangential flow filtration, on the same day to obtain a first stock solution, (vi) also processing the first stock solution on the same day, preferably mixing it with a wash buffer on the same day to obtain a composition comprising a buffered wash solution and the first stock solution, the composition being a first bulk stock solution, (vii) processing the first bulk stock solution, comprising (vii-a) subjecting the first bulk stock solution to a depth filtration step, and (vii-b) freezing the filtered first bulk stock solution. In a preferred embodiment, six harvesting steps are performed and repeated on six different days to obtain six bulk stocks, wherein the interval between each harvesting step is 20-30 hours.

[0511] Thus, one embodiment of a method for large-scale production and manufacture of a flavivirus vaccine includes a step of processing a bulk stock solution, including a depth filtration step. If a bivalent, trivalent, or quadrivalent final formulation is desired, the bulk stock solution of this step still includes each individual monovalent bulk stock solution. In the context of the present invention, it should be understood that each individual method step and / or component or ingredient described above can be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0512] Preferably processing bulk stocks to obtain high virus titers

[0513] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0514] - Harvest step to obtain the harvest,

[0515] - Bulk stock obtained from harvest,

[0516] - processing the bulk stock solution, comprising the step of filtering the bulk stock solution, followed by the step of freezing the bulk stock solution to obtain a frozen bulk stock solution.

[0517] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0518] - Harvest step to obtain the harvest,

[0519] - Bulk stock obtained from harvest,

[0520] - Processing the bulk stock, comprising the step of filtering the bulk stock and then freezing the bulk stock to obtain a frozen bulk stock. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock.

[0521] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0522] - Harvest step to obtain the harvest,

[0523] - Bulk stock obtained from harvest,

[0524] - Processing the bulk stock solution, comprising the step of filtering the bulk stock solution, followed by the step of freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step.

[0525] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0526] - Harvest step to obtain the harvest,

[0527] - Bulk stock obtained from harvest,

[0528] - Processing the bulk stock, including the steps of filtering the bulk stock and then freezing the bulk stock to obtain a frozen bulk stock. In this context, processing the bulk stock does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock.

[0529] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0530] - Harvest step to obtain the harvest,

[0531] - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0532] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0533] - processing the bulk stock solution, comprising the step of filtering the bulk stock solution, followed by the step of freezing the bulk stock solution to obtain a frozen bulk stock solution.

[0534] In this context, processing the bulk stock does not involve a freezing step prior to the filtration step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock.

[0535] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0536] - Harvest step to obtain the harvest,

[0537] - processing the harvest to obtain processed harvest,

[0538] - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0539] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0540] - Processing the bulk stock solution, including the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution.

[0541] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0542] - Harvest step to obtain the harvest,

[0543] - processing the harvest to obtain processed harvest,

[0544] - purification, including at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0545] - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0546] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0547] - Processing the bulk stock solution, including the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution.

[0548] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0549] - Harvest step to obtain the harvest,

[0550] - processing the harvest, including clarifying and stabilizing the harvest, to obtain a clarified and stabilized harvest,

[0551] - purifying the clarified and stabilized harvest, comprising at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0552] - subjecting the purified harvest to ultrafiltration, such as tangential flow filtration, to obtain a stock solution, and

[0553] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0554] - Processing the bulk stock solution, including the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution.

[0555] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0556] - provide cells in growth medium,

[0557] - Infect cells with medium containing flavivirus.

[0558] - Harvest step to obtain the harvest,

[0559] - Bulk stock obtained from harvest,

[0560] - Processing the bulk stock, comprising the step of filtering the bulk stock, followed by the step of freezing the bulk stock to obtain a frozen bulk stock. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0561] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0562] - provide cells in growth medium,

[0563] - Infect cells with medium containing flavivirus,

[0564] - Harvest step to obtain the harvest,

[0565] - Bulk stock obtained from harvest,

[0566] - Processing the bulk stock solution, comprising the step of filtering the bulk stock solution, followed by the step of freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtration step. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0567] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0568] - provide cells in growth medium,

[0569] - Infect cells with medium containing flavivirus.

[0570] - Harvest step to obtain the harvest,

[0571] - Bulk stock obtained from harvest,

[0572] - Processing the bulk stock solution, comprising the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0573] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0574] - provide cells in growth medium,

[0575] - Infect cells with medium containing flavivirus.

[0576] - Harvest step to obtain the harvest,

[0577] - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0578] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0579] - Processing the bulk stock solution, comprising the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0580] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0581] - provide cells in growth medium,

[0582] - Infect cells with medium containing flavivirus.

[0583] - Harvest step to obtain the harvest,

[0584] - processing the harvest to obtain processed harvest,

[0585] - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0586] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0587] - Processing the bulk stock solution, comprising the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0588] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0589] - provide cells in growth medium,

[0590] - Infect cells with medium containing flavivirus.

[0591] - Harvest step to obtain the harvest,

[0592] - processing the harvest to obtain processed harvest,

[0593] - purification, including at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0594] - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0595] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0596] - Processing the bulk stock solution, comprising the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0597] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0598] - provide cells in growth medium,

[0599] - Infect cells with medium containing flavivirus.

[0600] - Harvest step to obtain the harvest,

[0601] - processing the harvest, including clarifying and stabilizing the harvest, to obtain a clarified and stabilized harvest,

[0602] - purifying the clarified and stabilized harvest, comprising at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0603] - subjecting the purified harvest to ultrafiltration, such as tangential flow filtration, to obtain a stock solution, and

[0604] - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0605] Processing the bulk stock solution, including the steps of filtering the bulk stock solution and then freezing the bulk stock solution to obtain a frozen bulk stock solution. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, the harvest does not undergo a freezing step until the step of processing the bulk stock solution. In this context, infecting the cells with the medium comprising the flavivirus optionally includes infecting the cells at an MOI of 0.1 or less. In some embodiments, the cells are Vero cells.

[0606] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing as described above in this section provides high viral titers.

[0607] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing as described above in this section provides higher virus titers compared to a method that differs only in that processing the bulk stock includes freezing the bulk stock prior to filtering the bulk stock.

[0608] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing as described above in this section provides higher virus titers compared to a method in which processing the bulk stock includes freezing the bulk stock prior to filtering the bulk stock.

[0609] In some embodiments, the filtration step comprises agitating the bulk stock solution. Agitation herein means, for example, shaking the bulk stock solution during filtration.

[0610] In some embodiments, the filtering step comprises agitating the bulk stock solution for at least 1 minute, such as 5 minutes, for example 5 to 15 minutes. Agitation herein means, for example, shaking the bulk stock solution during filtration.

[0611] In some embodiments, the freezing step comprises freezing the bulk stock solution at a temperature of less than -65°C.

[0612] In some embodiments, the bulk stock solution weighs at least 100 g, such as 100 to 1000 g, such as 2000 g or 3000 g.

[0613] In some embodiments, the growth medium is serum-free. In some embodiments, the infection medium is serum-free.

[0614] In some embodiments, the viral vaccine is bivalent, trivalent, or quadrivalent, preferably wherein the viral vaccine is the quadrivalent dengue vaccine TAK-003.

[0615] In some embodiments, large scale includes a surface area of ​​35,000 cm 2 or larger, 50,000 cm 2or larger, 100,000 cm 2 or larger production cultures.

[0616] In some embodiments, 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.

[0617] In some embodiments, 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.

[0618] In some embodiments, the MOI is 0.1 or less, 0.1 to less than 0.008, 0.1 to 0.0001, 0.1 to greater than 0.008, 0.1 to 0.008, 0.0001 to 0.008, 0.0001 to 0.005, 0.001 to 0.005.

[0619] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0620] - at least two harvesting steps to obtain at least two harvests,

[0621] - at least two bulk stocks from at least two harvests,

[0622] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, and then freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions.

[0623] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0624] - at least two harvesting steps to obtain at least two harvests,

[0625] - at least two bulk stocks from at least two harvests,

[0626] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, and then freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions.

[0627] In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stock.

[0628] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0629] - at least two harvesting steps to obtain at least two harvests,

[0630] - at least two bulk stocks from at least two harvests,

[0631] - Processing at least two bulk stock solutions, comprising the step of filtering each of the at least two bulk stock solutions, followed by the step of freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solutions does not involve a freezing step prior to the filtering step.

[0632] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0633] - at least two harvesting steps to obtain at least two harvests,

[0634] - at least two bulk stocks from at least two harvests,

[0635] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, followed by freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stock solution.

[0636] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0637] - at least two harvesting steps to obtain at least two harvests, and

[0638] Perform the following steps for each of at least two harvests:

[0639] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0640] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0641] to obtain at least two stock solutions and at least two bulk stock solutions,

[0642] - Processing at least two bulk stocks, comprising a step of filtering each of the at least two bulk stocks, followed by a step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks, wherein processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks.

[0643] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0644] - at least two harvesting steps to obtain at least two harvests, and

[0645] Perform the following steps for each of at least two harvests:

[0646] o processing the harvest to obtain a processed harvest,

[0647] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0648] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0649] to obtain at least two processed harvests, at least two stock solutions, and at least two bulk stock solutions,

[0650] - Processing at least two bulk stocks, comprising a step of filtering each of the at least two bulk stocks, followed by a step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks, wherein processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks.

[0651] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0652] - at least two harvesting steps to obtain at least two harvests, and

[0653] Perform the following steps for each of at least two harvests:

[0654] o processing the harvest to obtain a processed harvest,

[0655] o purification, including at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0656] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0657] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0658] to obtain at least two processed harvests, at least two purified harvests, at least two stock solutions, and at least two bulk stock solutions,

[0659] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, followed by freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stock solution.

[0660] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0661] - at least two harvesting steps to obtain at least two harvests, and

[0662] Perform the following steps for each of at least two harvests:

[0663] o processing the harvest, including clarifying and stabilizing the harvest, to obtain a clarified and stabilized harvest,

[0664] o purifying the clarified and stabilized harvest, comprising at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0665] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0666] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0667] to obtain at least two clarified and stabilized harvests, at least two purified harvests, at least two stock solutions, and at least two bulk stock solutions,

[0668] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, followed by freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solution does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stock solution.

[0669] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0670] - provide cells in growth medium,

[0671] - Infect cells with medium containing flavivirus.

[0672] - at least two harvesting steps to obtain at least two harvests,

[0673] - at least two bulk stocks from at least two harvests,

[0674] - Processing the at least two bulk stocks, comprising the step of filtering each of the at least two bulk stocks, followed by the step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks. In this context, each of the at least two harvests is not subjected to the freezing step until the step of processing the bulk stocks. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0675] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0676] - provide cells in growth medium,

[0677] - Infect cells with medium containing flavivirus,

[0678] - at least two harvesting steps to obtain at least two harvests,

[0679] - at least two bulk stocks from at least two harvests,

[0680] - Processing at least two bulk stock solutions, comprising the steps of filtering each of the at least two bulk stock solutions, and then freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions.

[0681] In this context, each of the at least two harvests is not subjected to a freezing step until the step of processing the bulk stock.In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0682] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0683] - provide cells in growth medium,

[0684] - Infect cells with medium containing flavivirus,

[0685] - at least two harvesting steps to obtain at least two harvests,

[0686] - at least two bulk stocks from at least two harvests,

[0687] - Processing the at least two bulk stock solutions, comprising the step of filtering each of the at least two bulk stock solutions, followed by the step of freezing each of the at least two bulk stock solutions to obtain at least two frozen bulk stock solutions. In this context, processing the bulk stock solutions does not involve a freezing step prior to the filtering step. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0688] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0689] - provide cells in growth medium,

[0690] - Infect cells with medium containing flavivirus,

[0691] - at least two harvesting steps to obtain at least two harvests,

[0692] - at least two bulk stocks from at least two harvests,

[0693] - Processing at least two bulk stocks, comprising the step of filtering each of the at least two bulk stocks, followed by the step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks. In this context, processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0694] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0695] - provide cells in growth medium,

[0696] - Infect cells with medium containing flavivirus,

[0697] - at least two harvesting steps to obtain at least two harvests, and

[0698] Perform the following steps for each of at least two harvests:

[0699] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0700] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0701] to obtain at least two stock solutions and at least two bulk stock solutions,

[0702] - Processing at least two bulk stocks, comprising a step of filtering each of the at least two bulk stocks, followed by a step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks, wherein processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0703] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0704] - provide cells in growth medium,

[0705] - Infect cells with medium containing flavivirus,

[0706] - at least two harvesting steps to obtain at least two harvests, and

[0707] Perform the following steps for each of at least two harvests:

[0708] o processing the harvest to obtain a processed harvest,

[0709] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0710] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0711] to obtain at least two processed harvests, at least two stock solutions, and at least two bulk stock solutions,

[0712] - Processing at least two bulk stocks, comprising a step of filtering each of the at least two bulk stocks, followed by a step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks, wherein processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0713] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0714] - provide cells in growth medium,

[0715] - Infect cells with medium containing flavivirus,

[0716] - at least two harvesting steps to obtain at least two harvests, and

[0717] Perform the following steps for each of at least two harvests:

[0718] o processing the harvest to obtain a processed harvest,

[0719] o purification, including at least one chromatography step, such as anion exchange chromatography, to obtain purified,

[0720] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0721] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0722] to obtain at least two processed harvests, at least two purified harvests, at least two stock solutions, and at least two bulk stock solutions,

[0723] - Processing at least two bulk stocks, comprising the step of filtering each of the at least two bulk stocks, followed by the step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks. In this context, processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0724] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises,

[0725] - provide cells in growth medium,

[0726] - Infect cells with medium containing flavivirus,

[0727] - at least two harvesting steps to obtain at least two harvests, and

[0728] Perform the following steps for each of at least two harvests:

[0729] o processing the harvest, including clarifying and stabilizing the harvest, to obtain a clarified and stabilized harvest,

[0730] o purifying the clarified and stabilized harvest, comprising at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest,

[0731] ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and

[0732] o performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a bulk stock solution,

[0733] to obtain at least two clarified and stabilized harvests, at least two purified harvests, at least two stock solutions, and at least two bulk stock solutions,

[0734] - Processing at least two bulk stocks, comprising the step of filtering each of the at least two bulk stocks, followed by the step of freezing each of the at least two bulk stocks to obtain at least two frozen bulk stocks. In this context, processing the bulk stocks does not involve a freezing step prior to the filtering step. In this context, each of the at least two harvests does not undergo a freezing step until the step of processing the bulk stocks. In this context, infecting the cells with the medium comprising the flavivirus optionally comprises infecting the cells at an MOI of 0.1 or less.

[0735] In some embodiments, up to ten harvesting steps are performed.

[0736] In some embodiments, the interval between the two harvesting steps is 20-30 hours.

[0737] In some embodiments, the cells are Vero cells.

[0738] In some embodiments, the harvesting step is followed by a medium change, as explained above in the Harvest section.

[0739] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing as described above in this section provides high viral titers.

[0740] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing as described above in this section provides higher virus titers compared to a method that differs only in that processing the bulk stock includes freezing the bulk stock prior to filtering the bulk stock.

[0741] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing as described above in this section provides higher virus titers compared to a method in which processing the bulk stock includes freezing the bulk stock prior to filtering the bulk stock.

[0742] In some embodiments, the filtration step comprises agitating the bulk stock solution. Agitation herein means, for example, shaking the bulk stock solution during filtration.

[0743] In some embodiments, the filtering step comprises agitating the bulk stock solution for at least 1 minute, such as 5 minutes, for example 5 to 15 minutes. Agitation herein means, for example, shaking the bulk stock solution during filtration.

[0744] In some embodiments, the freezing step comprises freezing the bulk stock solution at a temperature of less than -65°C.

[0745] In some embodiments, the bulk stock solution weighs at least 100 g, such as 100 to 1000 g, such as 2000 g or 3000 g.

[0746] In some embodiments, the growth medium is serum-free. In some embodiments, the infection medium is serum-free. In some embodiments, the viral vaccine is bivalent, trivalent, or quadrivalent, preferably wherein the viral vaccine is the quadrivalent dengue vaccine TAK-003.

[0747] In some embodiments, large scale includes a surface area of ​​35,000 cm 2 or larger, 50,000cm 2 or larger, 100,000cm 2 or larger production cultures.

[0748] In some embodiments, 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.

[0749] In some embodiments, 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.

[0750] In some embodiments, the MOI is 0.1 or less, 0.1 to less than 0.008, 0.1 to 0.0001, 0.1 to greater than 0.008, 0.1 to 0.008, 0.0001 to 0.008, 0.0001 to 0.005, 0.001 to 0.005.

[0751] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps:

[0752] (i) providing cells in a growth medium,

[0753] (ii) infecting the cells of step (i) with medium containing the flavivirus at a low MOI,

[0754] (iii) a first harvest to obtain a first harvest,

[0755] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0756] (v) purifying the processed harvest of step (iv), comprising at least one chromatography step to obtain a purified harvest,

[0757] (vi) an ultrafiltration step to obtain a first stock solution,

[0758] (vi) processing the stock solution obtained in step (v) comprising subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain the bulk stock solution,

[0759] wherein a second harvesting step is performed, and the above steps are repeated to obtain a second bulk stock solution,

[0760] (vii) processing of bulk stock solutions,

[0761] wherein processing the bulk stock solution includes the step of filtering the bulk stock solution before the step of freezing the bulk stock solution,

[0762] And wherein after the step of freezing the bulk stock solution, the bulk stock solution is thawed and pooled.

[0763] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps:

[0764] (i) providing cells in a growth medium,

[0765] (ii) infecting the cells of step (i) with medium comprising the flavivirus (optionally at a low MOI),

[0766] (iii) a first harvest to obtain a first harvest,

[0767] (iv) processing the harvest of step (iii) to obtain a processed harvest,

[0768] (v) purifying the processed harvest of step (iv), comprising at least one chromatography step to obtain a purified harvest,

[0769] (vi) an ultrafiltration step to obtain a first stock solution,

[0770] (vi) processing the stock solution obtained in step (v) comprising subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain the bulk stock solution,

[0771] wherein a second harvesting step is performed, and the above steps are repeated to obtain a second bulk stock solution,

[0772] (vii) processing of bulk stock solutions,

[0773] wherein steps (iii) to (vii) are performed on the same day,

[0774] Wherein processing the bulk stock solution includes the step of filtering the bulk stock solution before the step of freezing the bulk stock solution.

[0775] In some embodiments, up to ten harvesting steps are performed.

[0776] In some embodiments, the interval between the two harvesting steps is 20-30 hours.

[0777] In some embodiments, a method for large-scale flavivirus vaccine production and manufacturing comprises the following sequential steps:

[0778] (i) providing cells in a growth medium,

[0779] (ii) infecting the cells of step (i) with infection medium comprising the flavivirus (optionally at a low MOI),

[0780] (iii) a first harvesting step to obtain a first harvest,

[0781] (iv) processing the first harvest obtained in step (iii), including clarifying and stabilizing the harvest, to obtain a first clarified and stabilized harvest,

[0782] (v) purifying the clarified and stabilized harvest obtained in step (iv), comprising

[0783] (va) at least one chromatography step, such as anion exchange chromatography, to obtain a first purified harvest, and

[0784] (vb) subjecting the purified harvest of step (va) to ultrafiltration, such as tangential flow filtration, to obtain a first stock solution, and

[0785] (vi) performing at least one washing step on the ultrafiltration membrane with a wash buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being a first bulk stock solution,

[0786] Preferably, up to ten harvesting steps are performed and the above steps are repeated to obtain up to ten bulk stocks, wherein the interval between each harvesting step is 20-30 hours,

[0787] (vii) the first bulk solution obtained in process (vi), comprising:

[0788] (vii-a) filtering the bulk stock solution to obtain a filtered first bulk stock solution, and then

[0789] (vii-b) freezing the filtered bulk stock solution obtained from step (vii-a) to obtain a frozen bulk stock solution,

[0790] wherein all steps (iii) to (vi) are performed on the same day, preferably wherein steps (vii-a) and (vii-b) are performed on the same day as step (vi).

[0791] In some embodiments, processing the bulk stock does not involve a freezing step prior to the filtration step.

[0792] In some embodiments, frozen bulk stocks are thawed and pooled.

[0793] In some embodiments, the low MOI is less than 0.005 or preferably 0.008 or less.

[0794] In some embodiments, the harvesting step is followed by a medium change, as explained above in the Harvest section.

[0795] In some embodiments, the method provides high viral titers.

[0796] In some embodiments, the viral vaccine is bivalent, trivalent, or quadrivalent, preferably wherein the viral vaccine is the quadrivalent dengue vaccine TAK-003.

[0797] In some embodiments, large scale includes a surface area of ​​35,000 cm 2 or larger, 50,000cm 2 or larger, 100,000cm 2 or larger production cultures.

[0798] In some embodiments, 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.

[0799] In some embodiments, 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.

[0800] In some embodiments, the MOI is 0.0001 to 0.008, 0.0001 to 0.005, 0.001 to 0.005.

[0801] Processing the bulk stock solution includes a filtration step according to the above embodiments. In one embodiment, the filtration step is a depth filtration step. In some embodiments, the filtration step can be performed in the same manner as the filtration step in the harvest clarification step.

[0802] Depth filtration uses porous filter media to separate particles and solids from liquids. In some embodiments, filter media useful for depth filtration can be cellulose acetate, polypropylene, cellulose acetate protected by glass wool, and polyethersulfone. In some embodiments, filters used for depth filtration can have a pore size of 0.1 μm to 1 μm, 0.2 μm to 0.8 μm, or 0.2 μm to 0.45 μm.

[0803] In some embodiments, the filtering step comprises using a heterogeneous double layer filter. In some embodiments, the filtering step comprises using a heterogeneous double layer filter of 0.1 μm to 1 μm (such as 0.45 μm) and 0.1 μm to 1 μm (such as 0.2 μm). In some embodiments, the filtering step comprises using a heterogeneous double layer filter of 0.45 μm to 0.2 μm. In some embodiments, the filtering step comprises using a heterogeneous double layer filter of polyethersulfone. In some embodiments, the filtering step comprises using a heterogeneous double layer filter of polyethersulfone with a pore size of 0.2 μm to 0.45 μm. Several heterogeneous double-layer filters can be used, such as Sartoclean (cellulose acetate) 0.8+0.65 μm, Sartopure (polypropylene) 0.65 μm, Sartoclean (glass wool) 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 present invention.

[0804] According to the method of the present invention, preferably the bulk stock solution after this step comprises the same excipients as were present in the bulk stock solution before the processing step.

[0805] Freezing and storage of bulk stock solutions

[0806] The method of the present invention also provides an apparatus and method for freezing and storing bulk stock solution.

[0807] The stock solution can be frozen and stored at any time. One such time point is after BDS filtration. After the filtration step, the bulk stock solution can be frozen and stored. The purpose of the freezing step is to ensure viral potency so that the material can be used for formulation. In this step, any commercially available bag can be used to freeze and store the bulk stock solution.

[0808] In one embodiment, the bulk stock is frozen and stored after BDS filtration as discussed in the previous section, e.g., for TDV-3 and TDV-4. In one embodiment, the bulk stock can be frozen and stored immediately after obtaining the bulk stock from each harvest step, after which the bulk stock from each harvest step is thawed, pooled, and filtered as discussed above, and then frozen and stored, e.g., for TDV-1 and TDV-2.

[0809] The bags are used to freeze bulk stock solutions and store them until they are thawed for formulation. The bags can be selected based on certain parameters such as size, manufacturer, construction material and storage temperature range, thawing conditions, ability to maintain sterile conditions, ease of transportation, and use in previous processes and clinical studies.

[0810] The temperature range supported by the bag can be, for example, in 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 an ultra-low temperature freezer, a flash freezer, flat plate freezing and thawing. As used herein, "ultra-low temperature freezer" or "ULT" refers to a refrigerator that operates in the range of -50°C to -80°C, and ULT is used to store a variety of analytes and products, from biological specimen samples to enzymes and drugs. As used herein, "flash freezer" refers to a specialized refrigerator that allows for rapid freezing of products in a short period of time. The temperature of a flash freezer can vary from -10°C to -120°C. "Plate freezing" as used herein is a rapid freezing method once the product is in direct contact with two cold metal surfaces.

[0811] Thawing options supported by the bags used in certain embodiments of the present methods can include a water bath, plasma thawing bath, shaking incubator, or plate freeze-thaw. The term "water bath" refers to a bath containing a heating medium that is 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 thawing" refers to a method of thawing using equipment designed for rapid and uniform thawing of fresh frozen plasma (FFP) bags at 37°C. Typically, the apparatus is constructed from a stainless steel chamber and equipped with a digital temperature controller and a water circulation pump. Considerations for handling and shipping the bags used in the present invention may include the rigidity of the bottles and the hard casting to minimize damage, the EVOH gas barrier with an EVA film layer, and the ULDPE film. Material compatibility and sterility characteristics of the bags include being free of animal components, gamma-irradiated, and meeting USP standards. The "EVOH" barrier refers to an environmentally friendly material used as a barrier plastic. Unlike PVDC, EVOH does not contain chlorine, dioxins, metals, or other elements that may cause endocrine disruption. As used herein, "GULPED" refers to ultra-low-density polyethylene.

[0812] Thus, suitable bags are commercially available bags that can be used for freezing bulk stock solutions, including Nalgene PETG / PFA bottles (50 mL-2 L), Sartorius-Stedim Celsius-Pak / FFT / FFTp 2D bags (30 mL-16 L), or PallAllegro 2D Biocontainer (125 mL-50 L).

[0813] Once the bag is selected, the freezing and thawing conditions must be optimized to minimize the loss of viral potency. These conditions include the freezing set point and rate, the thawing set point and rate, and optionally the cooling set point.

[0814] Thus, large-scale production and manufacture of flavivirus vaccines involves processing bulk stock solutions, such as freezing and storing them. If a bivalent, trivalent, or quadrivalent final formulation is desired, the bulk stock solution for this step still includes each individual monovalent bulk stock solution. Within the context of the present invention, it should be understood that each individual process step and / or ingredient component described above can be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0815] Preparation of bulk dosage forms

[0816] After the bulk stock solution is frozen, the next step is (ix) preparation of the bulk formulation.

[0817] This may for example comprise two main sub-steps:

[0818] (ix-a) Thawing of bulk stock solution

[0819] (ix-b) The stock solution thawed in step (ix-a) was mixed with formulation buffer to obtain a bulk formulation.

[0820] If a bivalent, trivalent or quadrivalent final formulation is desired, two or more monovalent bulk stock solutions are mixed at this stage to obtain a final bulk formulation comprising a bivalent, trivalent or quadrivalent formulation. Thus, in one embodiment, two, three or four thawed bulk stock solutions are mixed after step (ix-a) and before step (ix-b). 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 the 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.

[0821] The formulation buffer comprises two buffers, a first excipient buffer (FEB) and a second excipient buffer (SEB). Qualitatively, the components of FEB and SEB may be the same or different. However, quantitatively, the components of FEB and SEB are different.

[0822] As used herein, the term "excipient" refers to a substance added to a liquid pharmaceutical composition in addition to a bioactive agent. This can include substances for the purpose of stabilizing an agent, such as an enhancing agent, salts, carbohydrates (such as sugars), surfactants, proteins, leavening agents, fillers, or agents that, in combination with an active agent, can impart therapeutic enhancement to the composition. Specifically, an excipient can refer to salts, carbohydrates, nonionic surfactants, and albumin. An excipient can also refer to a buffer, such as a phosphate buffer.

[0823] FEB and SEB buffers can, for example, comprise four salts, sugar, nonionic surfactants, and proteins. As required, FEB and SEB buffers can also comprise water for injection (WFI). The salt used in the buffer can be anhydrous or a hydrate. The salt can be one, two, three, or four sodium salts, or one, two, three, or four potassium salts, or one, two, three, or four magnesium salts. Every possibility can be combined with other possibilities. Therefore, every possibility represents a separate embodiment of the present invention.

[0824] For example, the sodium salt can be sodium fluoride, or sodium chloride, or sodium bromide, or sodium iodide, or sodium sulfate, or a disodium salt such as disodium hydrogen 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.

[0825] Each of the above salts can be used in SEB buffer, for example, in the range of 0.9 mM to 130 mM.

[0826] For example, SEB buffer can include sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and three other salts in the range of 0.9 to 6 mM. For example, SEB buffer can include potassium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and three other salts in the range of 0.9 to 6 mM. For example, SEB and SEB buffer can include magnesium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and three other salts in the range of 0.9 to 6 mM.

[0827] For example, SEB buffer can contain sodium fluoride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM and three other salts in the range of 0.9 to 6 mM. For example, SEB buffer can contain potassium fluoride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM and three other salts in the range of 0.9 to 6 mM.

[0828] For example, SEB buffer can contain sodium carbonate in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM and the other three salts in the range of 0.9 to 6 mM. For example, SEB buffer can contain potassium carbonate in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM and the other three salts in the range of 0.9 to 6 mM.

[0829] For example, SEB buffer can include sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, and two other salts in the range of 0.9 to 6 mM. For example, SEB buffer can include sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and magnesium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, and two other salts in the range of 0.9 to 6 mM. For example, SEB buffer can include potassium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and magnesium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, and two other salts in the range of 0.9 to 6 mM.

[0830] For example, SEB buffer can include sodium fluoride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, and two other salts in the range of 0.9 to 6 mM. For example, SEB buffer can include sodium fluoride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium carbonate in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, and two other salts in the range of 0.9 to 6 mM. For example, SEB buffer can include sodium fluoride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and magnesium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, and two other salts in the range of 0.9 to 6 mM. For example, SEB buffer can contain sodium iodide in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium carbonate in the range of 1 mM to 2 mM, 1 mM to 1.5 mM and two other salts in the range of 0.9 to 6 mM.

[0831] For example, SEB buffer can include sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, potassium dihydrogen phosphate in the range of 0.9 to 1.2 mM, 0.9 to 1.0 mM, and one other salt in the range of 4 to 6 mM, 5 to 6 mM. For example, SEB buffer can include sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium phosphate in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, potassium dihydrogen sulfate in the range of 0.9 to 1.2 mM, 0.9 to 1.0 mM, and one other salt in the range of 4 to 6 mM, 5 to 6 mM. For example, SEB buffer can contain sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium phosphate in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, sodium dihydrogen sulfate in the range of 0.9 to 1.2 mM, 0.9 to 1.0 mM and one other salt in the range of 4 to 6 mM, 5 to 6 mM.

[0832] For example, the SEB buffer may include sodium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and potassium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, potassium dihydrogen phosphate in the range of 0.9 to 1.2 mM, 0.9 to 1.0 mM, and disodium hydrogen phosphate dihydrate in the range of 4 to 6 mM, 5 to 6 mM. For example, the SEB buffer may include potassium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and magnesium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, sodium dihydrogen phosphate in the range of 0.9 to 1.2 mM, 0.9 to 1.0 mM, and disodium hydrogen phosphate dihydrate in the range of 4 to 6 mM, 5 to 6 mM. For example, SEB buffer can include potassium chloride in the range of 100 to 130 mM, 110 to 130 mM, 120 to 130 mM, and magnesium chloride in the range of 1 mM to 2 mM, 1 mM to 1.5 mM, sodium dihydrogen phosphate in the range of 0.9 to 1.2 mM, 0.9 to 1.0 mM, and magnesium carbonate in the range of 4 to 6 mM, 5 to 6 mM.

[0833] Each of the above salts can be used in the FEB buffer in the range of 3 mM to 50 mM.

[0834] For example, FEB buffer may contain sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and three other salts in the range of 3 to 25 mM. For example, FEB buffer may contain potassium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and three other salts in the range of 3 to 25 mM. For example, FEB buffer may contain magnesium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and three other salts in the range of 3 to 25 mM.

[0835] For example, FEB buffer can contain sodium fluoride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and three other salts in the range of 3 to 25 mM. For example, FEB buffer can contain potassium fluoride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and three other salts in the range of 3 to 25 mM.

[0836] For example, FEB buffer can contain sodium carbonate in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and the other three salts in the range of 3 to 25 mM. For example, FEB buffer can contain potassium carbonate in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM and the other three salts in the range of 3 to 25 mM.

[0837] For example, FEB buffer may contain sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium chloride in the range of 4 to 6 mM, 5 to 6 mM, and two other salts in the range of 3 to 25 mM. For example, FEB buffer may contain sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range of 4 to 6 mM, 5 to 6 mM, and two other salts in the range of 3 to 25 mM. For example, FEB buffer may contain potassium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range of 4 to 6 mM, 5 to 6 mM, and two other salts in the range of 3 to 25 mM.

[0838] For example, FEB buffer may include sodium fluoride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium chloride in the range of 4 to 6 mM, 5 to 6 mM, and two other salts in the range of 3 to 25 mM. For example, FEB buffer may include sodium fluoride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium carbonate in the range of 4 to 6 mM, 5 to 6 mM, and two other salts in the range of 3 to 25 mM. For example, FEB buffer may include sodium fluoride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range of 4 to 6 mM, 5 to 6 mM, and two other salts in the range of 3 to 25 mM. For example, FEB buffer may contain sodium iodide in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium carbonate in the range of 4 to 6 mM, 5 to 6 mM and two other salts in the range of 3 to 25 mM.

[0839] For example, the FEB buffer may comprise sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium chloride in the range of 4 to 6 mM, 5 to 6 mM, potassium dihydrogen phosphate in the range of 3 to 4 mM, 3.2 to 4 mM, and one other salt in the range of 18 to 22 mM. For example, the FEB buffer may comprise sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium phosphate in the range of 4 to 6 mM, 5 to 6 mM, potassium dihydrogen sulfate in the range of 3 to 4 mM, 3.2 to 4 mM, and one other salt in the range of 18 to 22 mM. For example, FEB buffer may contain sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium phosphate in the range of 4 to 6 mM, 5 to 6 mM, sodium dihydrogen sulfate in the range of 3 to 4 mM, 3.2 to 4 mM and one other salt in the range of 18 to 22 mM.

[0840] For example, the FEB buffer may comprise sodium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and potassium chloride in the range of 4 to 6 mM, 5 to 6 mM, potassium dihydrogen phosphate in the range of 3 to 4 mM, 3.2 to 4 mM, and disodium hydrogen phosphate dihydrate in the range of 18 to 22 mM, 19 to 22 mM, 20 to 22 mM. For example, the FEB buffer may comprise potassium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range of 4 to 6 mM, 5 to 6 mM, sodium dihydrogen phosphate in the range of 3 to 4 mM, 3.2 to 4 mM, and disodium hydrogen phosphate dihydrate in the range of 18 to 22 mM, 19 to 22 mM, 20 to 22 mM. For example, the FEB buffer may comprise potassium chloride in the range of 20 to 50 mM, 30 to 50 mM, 40 to 50 mM, and magnesium chloride in the range of 4 to 6 mM, 5 to 6 mM, sodium dihydrogen phosphate in the range of 3 to 4 mM, 3.2 to 4 mM, and magnesium carbonate in the range of 18 to 22 mM, 19 to 22 mM, 20 to 22 mM.

[0841] The sugar in FEB and SEB buffer can be a monosaccharide (such as glucose, galactose, ribose, mannose, rhamnose, talose, xylose or allose arabinose), a disaccharide (such as trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribiose, xylobiose, mannobiose, lactose or fructose), a trisaccharide (such as acarbose, raffinose, melezitose, panose or cellotriose) and a sugar polymer (such as dextran, xanthan gum, pullulan, cyclodextrin, amylose, amylopectin, starch, cellooligosaccharides, cellulose, maltooligosaccharides, glycogen, chitosan or chitin). The sugar in FEB and SEB buffer can be a sugar alcohol such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, polyol, ethylene glycol, polyol, polyethylene glycol, polypropylene glycol and glycerol. The sugar in FEB and SEB buffer may be a non-reducing sugar. The sugar in FEB and SEB buffer may be sucrose, trehalose or a hydrate thereof, such as trehalose dihydrate.

[0842] Each of the above sugars can be used in a range of 120 to 160 g / L in SEB buffer and in a range of 180 to 220 g / L in FEB buffer. For example, sucrose can be used in a range of 120 to 160 g / L, 130 to 160 g / L, or 140 to 150 g / L in SEB buffer, and in a range of 180 to 220 g / L, 190 to 210 g / L, or 200 to 210 g / L in FEB buffer. For example, trehalose can be used in a range of 120 to 160 g / L, 130 to 160 g / L, or 140 to 150 g / L in SEB buffer, and in a range of 180 to 220 g / L, 190 to 210 g / L, or 200 to 210 g / L in FEB buffer. For example, trehalose dihydrate may be in the range of 120 to 160 g / L, 130 to 160 g / L, 140 to 150 g / L in SEB buffer, and may be in the range of 180 to 220 g / L, 190 to 210 g / L, 200 to 210 g / L in FEB buffer.

[0843] The nonionic surfactant in FEB and SEB buffers can be selected from block copolymers, sorbitan esters, ethoxylated or propoxylated sorbitan esters, alkyl polyglycosides (APGs), alkoxylated mono- or dialkylamines, 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 (glucosamide (GA) or fatty acid glucosamide (FAGA)), and combinations thereof.

[0844] 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). EO-PO block copolymers can include polyethylene oxide (-CH2CH2O-, designated as EO) and polypropylene oxide (-CH2CHCH3O-, designated as PO) blocks. In an EOx-POy-Eox arrangement, the PO block can be flanked by two EO blocks. 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 adjusted by varying the x and y in the EOx-POy-Eox block structure. In aqueous solution, the EO-PO block copolymer will self-assemble into micelles with a PO core and a hydrophilic EO group corona.

[0845] The nonionic surfactant in FEB and SEB buffers can be a poloxamer. Poloxamers are nonionic triblock copolymers consisting of a central poly(propylene oxide) hydrophobic chain flanked by two poly(ethylene oxide) hydrophilic chains. The length of the polymer blocks can be customized to produce different poloxamers with slightly different properties. The nonionic surfactant in 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 greater than 3,000-4,000 MW, or combinations thereof.

[0846] The nonionic surfactant concentration in FEB and SEB buffer can be identical. The nonionic surfactant concentration in FEB and SEB buffer can be in the scope of 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L or 10 to 12 g / L respectively. The concentration of the high molecular weight nonionic surfactant in FEB and SEB buffer can be in the scope of 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L or 10 to 12 g / L respectively. The poloxamer concentration in FEB and SEB buffer can be in the scope of 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L or 10 to 12 g / L respectively. The concentration of Pluronic F127 (Poloxamer 407) in FEB and SEB buffers can each range from 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L, or 10 to 12 g / L. The concentration of Pluronic F68 (Poloxamer 403) in FEB and SEB buffers can each range from 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L, or 10 to 12 g / L. The concentration of Pluronic P123 in FEB and SEB buffers can each range from 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L, or 10 to 12 g / L. The concentration of Pluronic P85 in the FEB and SEB buffers can each range from 2.0 to 12 g / L, 4 to 12 g / L, 6 to 12 g / L, 8 to 12 g / L, or 10 to 12 g / L.

[0847] The proteins in the FEB and SEB buffers can be any protein that is inherently inert and non-reactive with the virus. Specifically, 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.

[0848] The protein concentrations in FEB and SEB buffers may be the same. The protein concentrations in FEB and SEB buffers may each be in the range of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, or 0.8 to 1.0 g / L. The protein concentrations in FEB and SEB buffers may each be in the range of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, or 0.8 to 1.0 g / L. The albumin concentrations in FEB and SEB buffers may each be in the range of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, or 0.8 to 1.0 g / L. The human serum albumin concentrations in FEB and SEB buffers may each be in the range of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, or 0.8 to 1.0 g / L. The collagen concentration in FEB and SEB buffers can each be in the range of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, 0.8 to 1.0 g / L. The gelatin concentration in FEB and SEB buffers can each be in the range of 0.5 to 1.5 g / L, 0.7 to 1.2 g / L, 0.8 to 1.0 g / L.

[0849] In the context of the present invention it is to be understood that each of the individual components of the buffer described above is to be considered alone or in combination with one another.Once the bulk stock solution is thawed it is mixed with FEB and SEB to prepare the bulk formulation.

[0850] Thus, one embodiment of large-scale production and manufacturing of flavivirus vaccines involves the preparation of bulk formulations. If a bivalent, trivalent, or quadrivalent final formulation is desired, the formulation obtained at this stage comprises a bivalent, trivalent, or quadrivalent bulk formulation. In the context of the present invention, it should be understood that each individual process step and / or ingredient component described above can be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0851] Sterile filtration

[0852] The formulated and blended bulk formulation material can be filtered through a sterile filtration assembly. Filters with pore sizes up to 0.2 µm are available. The specific types and characteristics of filters that can be used have been previously discussed for harvest clarification and can also be used in this step.

[0853] For the purposes of the present invention, sterile filtration of bulk formulations can be performed via a peristaltic pump characterized by a speed of up to 260 rpm and a maximum inlet pressure of 1.0 to 1.2 bar. The filtered formulation can be collected in a separate vessel, which is maintained at a temperature in the range of 2° C. to 8° C. For the purposes of the present invention, the sterile filtered bulk formulation can be mixed at approximately 100 to 150 rpm for at least 10 minutes before further sampling for sterility.

[0854] Thus, large-scale production and manufacturing of flavivirus vaccines may include sterile filtration of bulk formulations. In the context of the present invention, it should be understood that each individual process step and / or component described above may be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0855] Filling and loading

[0856] The filling process can be performed using any commercial-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 eliminating direct contact between operators and product, reducing the risk of contamination. Isolators also offer an environmentally friendly decontamination option.

[0857] According to the method of the present invention, during filling, the temperature of the isolator filling line is maintained at 10°C to 25°C, more preferably at 15°C to 25°C.

[0858] The method of the present invention utilizes a vial that can be used to fill the filtered formulation. The vial can be a plastic vial or a glass vial. In a preferred embodiment, the vial is a glass vial. When using a glass vial, any USP Type I, II, or III glass vial can be used. In a preferred embodiment, a USP Type I glass vial is used.

[0859] As used herein, "glass" refers to any suitable glass. For example, neutral glass is a borosilicate glass containing significant amounts of boron oxide, aluminum oxide, and alkali and / or alkaline earth metal oxides. It exhibits high resistance to hydrolysis and thermal shock. Soda-lime-silica glass is a silica glass containing alkali metal oxides (primarily sodium oxide) and alkaline earth metal oxides (primarily calcium oxide). It exhibits only moderate resistance to hydrolysis.

[0860] Based on their resistance to hydrolysis, glass containers can be classified as:

[0861] Type I glass containers, made of neutral glass, have high hydrolysis resistance and are suitable for most dosage forms, whether for parenteral use or not.

[0862] Type II glass containers, usually made of soda-lime-silica glass, have high resistance to hydrolysis due to appropriate surface treatment. They are suitable for most acidic and neutral aqueous preparations, whether for parenteral use or not.

[0863] Type III glass containers, typically made of soda-lime-silica glass, have only moderate resistance to hydrolysis. They are generally suitable for non-aqueous preparations for parenteral use, powders for parenteral use (except freeze-dried preparations), and preparations not for parenteral use.

[0864] In a preferred embodiment, the vials used in the present invention are made of borosilicate glass. In a preferred embodiment, the vials used in the present invention have high hydrolysis resistance and high thermal shock resistance. Therefore, in a preferred embodiment, the vials used are USP Type I glass vials.

[0865] USP Type I glass vials useful in the present invention may be 2R or 4R tubular glass vials, with a capacity of 2 mL or 4 mL, respectively.

[0866] Thus, according to one embodiment of the present invention, USP Type 1 2R vials or USP Type 1 2R vials can be used to fill the filtered bulk formulation. Prior to filling, multiple steps can be performed, including but not limited to one or more of the following: washing the vials, sterilizing the vials, or removing pyrogens from the vials. Alternatively, the method can use pre-sterilized glass supplied directly to the filling station as a cost- and space-saving alternative to operating a washing and sterilization line. The pre-sterilized glass vials are washed and depyrogens by the supplier, double-bagged, and then sterilized by gamma irradiation before being shipped to the end user for use.

[0867] Once the vials have been processed, they are read and filled. At this point, the sterile filtered bulk is aseptically connected to a disposable filling manifold. Prior to filling, the bulk can be mixed at about 100 to 150 rpm, 110 to 120 rpm, 130 to 140 rpm to 150 rpm for at least 20 to 30 minutes, 15 to 20 minutes, or 15 minutes. In a preferred embodiment, mixing is carried out at 140 to 150 rpm for about 15 minutes.

[0868] In a preferred embodiment of the present invention, mixing and filling continue in parallel with each other. Preferably, mixing continues for 15 minutes until about 60% to 80% of the vials are filled, for example, 70% to 80% or 75% to 80% of the vials are filled. In another embodiment, mixing is switched off after the first 15 minutes or 20 minutes of mixing and filling. In this embodiment, mixing does not continue in parallel with further filling. Thus, in one embodiment, at least 60% to 80% of the vials, for example, 70% to 80% or 75% to 80% of the vials are filled in parallel with mixing of the bulk formulation.

[0869] In certain embodiments of the inventive method, the filling range is one of the important parameters of this step. The vial critical filling volume can be set according to the specific circumstances and the desired use. For example, the vial critical filling volume can be set to 0.5 to 0.8 mL, 0.6 to 0.7 mL, 0.65 to 0.66 mL, 0.651, 0.652, 0.653, 0.654 or 0.655 mL. The technician knows how to set the vial critical filling volume by manually setting the pump type of the filling needle in the isolator filling line at the beginning of the filling run, and then maintain the volume by automatic feedback control. In one embodiment of the inventive method, additional checks are incorporated to ensure that the contents remain within the critical filling volume. These checks can, for example, be weight checks.

[0870] Therefore, the method of the present invention may or may not include a weight check to confirm that the volume is within the critical fill volume. If a weight check is used, a standard deviation of ±5% to 7% can be considered to meet the critical fill volume requirement.

[0871] Once filling is complete, one embodiment of the method of the present invention involves stoppering the vials. In one embodiment, the filler can partially stopper the vials. In this embodiment, partial stoppering is achieved before the vials are transferred to the freeze drying unit. The stoppers may or may not undergo a sterilization step before being transferred to the filler. In a preferred embodiment, the transfer is performed in an aseptic manner. After the partial stoppering step, a "correct stoppering" check step may or may not be performed. At the end of the filling, a minimum product yield is confirmed to be at least 90%, 95% or 97% of the total number of filled bottles as high-quality vials.

[0872] The total solution time after preparation (post-TIS) is defined as the time from the end of the last batch of transfers of each serotype to the end of the freeze-drying cycle. Post-TIS can be in the range of 10-30 hours, more preferably 12-29 hours. The post-TIS of a serotype may be different from 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 TDV 2 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.

[0873] Thus, large-scale production and manufacturing of flavivirus vaccines may include filling and loading bulk formulations into vials. Within the context of the present invention, it should be understood that each individual method step and / or component described above may be considered individually or in combination with one another. Thus, each of the possibilities described above, both individually and in combination, represents a separate embodiment of the present invention.

[0874] Freeze-dried and sealed

[0875] The formulation can be lyophilized, such as in a commercial-scale freeze dryer. Lyophilization involves manipulating the temperature and pressure of a solution so that the phase of the solution can be directly changed from a frozen state to a gaseous state without passing through a liquid / liquid phase. This is achieved by cooling the solution and reducing the pressure to 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 subjecting the product to severe heat.

[0876] The lyophilization process cycle for freeze-drying a liquid, for example, consists of five main stages: equilibration, freezing, primary drying, secondary drying, and sealing, each controlled by three basic process parameters: temperature, pressure, and time.

[0877] Thus, one embodiment of the method of the present invention comprises 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 having a shelf temperature ranging from above the Tg of the composition to no 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 greater than the primary drying step temperature to form a secondary dried product.

[0878] 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 higher than the collapse temperature (Tc) of the composition. In addition, 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. In addition, 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 higher than about 0.01 mbar. In addition, the secondary drying step (iv) can be carried out at a temperature of at least 20°C or at least 25°C.

[0879] Thus, large-scale production and manufacture of flavivirus vaccines may include freeze-drying of the formulation. In the context of the present invention, it should be understood that each individual method step and / or component described above can be considered individually or in combination with one another. Thus, each possibility described above, both individually and in combination, represents a separate embodiment of the present invention.

[0880] Unloading and capping

[0881] After 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 (N2) for stoppering. The vials can be stoppered with a pressure of ≥6.5 N / cm2 for 60 seconds, and then the shelf temperature can be reduced to 3°C to 7°C until unloading into a Grade A environment.

[0882] For example, while in a sterile isolator, the vial can be sealed, such as with aluminum / plastic.

[0883] Visual inspection

[0884] Each vial is visually inspected through a manual or semi-automated process for potential defects in the product, glass, crimp, closure, or other areas, including but not limited to caked appearance, cap defects, glass defects, and visible particles.

[0885] Freeze the final preparation

[0886] The formulation is preferably stored frozen. For example, each formulation batch can be transferred to an additional storage location at 2°C to 8°C and then frozen, or the product batch can be transferred directly to a dedicated quick freezer, for example at a temperature of -15°C to -35°C, and quick frozen, for example, for at least 36 hours. The batch can be kept in the quick freezer for at least 36 hours.

[0887] After quick freezing, the formulation batch can be transferred to long-term storage, for example at -15°C to -25°C.

[0888] Example

[0889] Example 1 - Low MOI Range

[0890] This example shows that when cell monolayers are infected with infection medium containing flavivirus at a low MOI (less than 0.008 or 0.005 or less), high viral titers can be obtained in large-scale flavivirus vaccine production and manufacturing, regardless of the volume of infection medium used.

[0891] Thaw a vial of ...

Claims

1. A method for large-scale production and manufacture of flavivirus vaccines, comprising: - Harvest step to obtain the harvest, - obtaining a bulk stock from said harvest, - processing said bulk stock solution, comprising the step of filtering said bulk stock solution, followed by the step of freezing said bulk stock solution to obtain a frozen bulk stock solution.

2. The method of claim 1, wherein the harvest is not subjected to a freezing step until the step of processing the bulk stock.

3. The method of any one of claims 1 or 2, wherein processing the bulk stock does not involve a freezing step prior to the filtering step.

4. The method of any one of claims 1 to 3, wherein the harvest is not subjected to a freezing step until the step of processing the bulk stock, and wherein processing the bulk stock does not involve a freezing step prior to the filtering step.

5. The method of any one of claims 1 to 4, wherein the filtering step comprises agitating the bulk stock solution.

6. The method of any one of claims 1 to 5, wherein the filtering step comprises agitating the bulk stock solution for at least 1 minute, such as 5 minutes.

7. The method of any one of claims 1 to 6, wherein the freezing step comprises freezing the bulk stock solution at a temperature of less than -65°C.

8. The method of any one of claims 1 to 7, wherein the weight of the frozen bulk stock is at least 100 g.

9. The method according to any one of claims 1 to 8, comprising - Harvest step to obtain the harvest, - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and - subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution.

10. The method according to any one of claims 1 to 9, comprising - Harvest step to obtain the harvest, - processing the harvest to obtain a processed harvest, - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and - subjecting said ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution.

11. The method according to any one of claims 1 to 10, comprising - Harvest step to obtain the harvest, - processing the harvest to obtain a processed harvest, - purification, including at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest, - Ultrafiltration such as tangential flow filtration to obtain a stock solution, and - subjecting said ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution.

12. The method according to any one of claims 1 to 11, comprising - Harvest step to obtain the harvest, - processing the harvest, including clarifying and stabilizing the harvest, to obtain a clarified and stabilized harvest, - purifying said clarified and stabilized harvest, comprising at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest, - subjecting the purified harvest to ultrafiltration, such as tangential flow filtration, to obtain a stock solution, and - subjecting said ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution.

13. The method of any one of claims 1 to 12, comprising the following sequential steps prior to the harvesting step: - provide cells in growth medium, - Infect cells with medium containing flavivirus.

14. The method of claim 13, wherein infecting the cells with a medium comprising a flavivirus comprises infecting the cells at an MOI of 0.1 to less than 0.

008.

15. The method of any one of the preceding claims, wherein the method provides high viral titers.

16. The method of any one of the preceding claims, wherein the method provides a higher viral titer than a method that differs only in that processing the bulk stock includes freezing the bulk stock prior to filtering the bulk stock.

17. A method for large-scale production and manufacture of flavivirus vaccines, comprising - at least two harvesting steps to obtain at least two harvests, - obtaining at least two bulk stocks from said at least two harvests, - processing said at least two bulk stock solutions, comprising the step of filtering each of said at least two bulk stock solutions, followed by the step of freezing each of said at least two bulk stock solutions to obtain at least two frozen bulk stock solutions.

18. The method of claim 17, wherein the at least two frozen bulk stocks are thawed and optionally pooled.

19. The method of claim 17 or 18, wherein each of the at least two harvests is not subjected to a freezing step until the step of processing the at least two bulk stocks.

20. The method of any one of claims 17 to 19, wherein processing the at least two bulk stock solutions does not involve a freezing step prior to the filtering step.

21. The method of any one of claims 17 to 20, wherein each of the at least two harvests is not subjected to a freezing step until the step of processing the at least two bulk stocks, and wherein processing the at least two bulk stocks does not involve a freezing step prior to the filtering step.

22. The method of any one of claims 17 to 21, wherein the filtering step comprises agitating the bulk stock solution.

23. A method as claimed in any one of claims 17 to 22, wherein the filtering step comprises agitating the bulk stock solution for at least 1 minute, such as 5 minutes.

24. The method of any one of claims 17 to 23, wherein the freezing step comprises freezing at a temperature of less than -65°C.

25. The method of any one of claims 17 to 24, wherein the at least two frozen bulk stocks each weigh at least 100 g.

26. The method of any one of claims 17 to 25, comprising - at least two harvesting steps to obtain at least two harvests, and For each of the at least two harvests, the following steps are performed: ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and o performing at least one washing step on the ultrafiltration membrane with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution, To obtain at least two stock solutions and at least two bulk stock solutions.

27. The method of any one of claims 17 to 26, comprising - at least two harvesting steps to obtain at least two harvests, and For each of the at least two harvests, the following steps are performed: o processing the harvest to obtain a processed harvest, ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and o performing at least one washing step on the ultrafiltration membrane with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution, To obtain at least two processed harvests, at least two stock solutions and at least two bulk stock solutions.

28. The method of any one of claims 17 to 27, comprising - at least two harvesting steps to obtain at least two harvests, and For each of the at least two harvests, the following steps are performed: o processing the harvest to obtain a processed harvest, o purification, including at least one chromatography step, such as anion exchange chromatography, to obtain purified, ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and o performing at least one washing step on the ultrafiltration membrane with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution, To obtain at least two processed harvests, at least two purified harvests, at least two stock solutions and at least two bulk stock solutions.

29. The method of any one of claims 17 to 28, comprising - at least two harvesting steps to obtain at least two harvests, and For each of the at least two harvests, the following steps are performed: o processing the harvest, including clarifying and stabilizing the harvest, to obtain a clarified and stabilized harvest, o purifying the clarified and stabilized harvest, comprising at least one chromatography step, such as anion exchange chromatography, to obtain a purified harvest, ○ Ultrafiltration such as tangential flow filtration to obtain a stock solution, and o performing at least one washing step on the ultrafiltration membrane with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being said bulk stock solution, To obtain at least two clarified and stabilized harvests, at least two purified harvests, at least two stock solutions, and at least two bulk stock solutions.

30. The method of any one of claims 17 to 29, comprising the following steps prior to the harvesting step: - provide cells in growth medium, - Infect cells with medium containing flavivirus.

31. The method of claim 30, wherein infecting the cells with a medium comprising a flavivirus comprises infecting the cells at an MOI of 0.1 to less than 0.

008.

32. The method of any one of the preceding claims, wherein the method provides high viral titers.

33. The method of any one of the preceding claims, wherein the method provides a higher viral titer than a method that differs only in that processing the bulk stock comprises freezing the bulk stock and then filtering the bulk stock.

34. A method for large-scale production and manufacture of flavivirus vaccines, comprising the following steps in sequence: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with medium containing the flavivirus at a low MOI, (iii) a first harvest to obtain a first 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, (vi) an ultrafiltration step to obtain a first stock solution, (vi) processing the stock solution obtained in step (v) comprising subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a bulk stock solution, wherein a second harvesting step is performed, and the above steps are repeated to obtain a second bulk stock solution, (vii) processing the bulk stock solution, wherein processing the bulk stock solution includes the step of filtering the bulk stock solution before the step of freezing the bulk stock solution, And wherein after the step of freezing the bulk stock solution, the bulk stock solution is thawed and optionally pooled.

35. A method for large-scale production and manufacture of flavivirus vaccines, comprising the following steps in sequence: (i) providing cells in a growth medium, (ii) infecting the cells of step (i) with medium containing the flavivirus at a low MOI, (iii) a first harvest to obtain a first 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, (vi) an ultrafiltration step to obtain a first stock solution, (vi) processing the stock solution obtained in step (v) comprising subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a bulk stock solution, wherein a second harvesting step is performed, and the above steps are repeated to obtain a second bulk stock solution, (vii) processing the bulk stock solution, wherein steps (iii) to (vii) are performed on the same day, And wherein processing the bulk stock solution includes the step of filtering the bulk stock solution before the step of freezing the bulk stock solution.

36. The method of claims 17 to 35, wherein at most ten harvesting steps are performed.

37. The method of any one of claims 17 to 36, wherein the interval between two harvesting steps is 20-30 hours.

38. The method of any one of claims 34 to 37, comprising the following steps in sequence: (vii) providing the cells in a growth medium, (viii) infecting the cells of step (i) with infection medium containing flavivirus at a low MOI, (ix) a first harvesting step to obtain a first harvest, (x) processing said first harvest obtained in step (iii), comprising clarifying and stabilizing said harvest to obtain a first clarified and stabilized harvest, (xi) purifying the clarified and stabilized harvest obtained in step (iv), comprising, (va) at least one chromatography step, such as anion exchange chromatography, to obtain a first purified harvest, and (vb) subjecting the purified harvest of step (va) to ultrafiltration, such as tangential flow filtration, to obtain a first stock solution, and (xii) subjecting the ultrafiltration membrane to at least one washing step with a washing buffer to obtain a composition comprising a buffered wash solution and a stock solution, said composition being the first bulk stock solution, Preferably, up to ten harvesting steps are performed and the above steps are repeated to obtain up to ten bulk stocks, wherein the interval between each harvesting step is 20-30 hours, (vii) the first bulk solution obtained in process (vi), comprising: (vii-a) filtering the bulk stock solution to obtain a filtered first bulk stock solution, and then (vii-b) freezing the filtered bulk stock solution obtained from step (vii-a) to obtain a frozen bulk stock solution, wherein all steps (iii) to (vi) are performed on the same day, preferably wherein steps (vii-a) and (vii-b) are performed on the same day as step (vi).

39. The method of any one of claims 34 to 38, wherein processing the bulk stock does not involve a freezing step prior to the filtering step.

40. The method of any one of claims 34 to 39, wherein the frozen bulk stock is thawed and optionally pooled.

41. The method of any one of claims 34 to 40, wherein the low MOI is less than 0.005, preferably less than 0.008 or less.

42. The method of any one of the preceding claims, wherein the method provides high viral titers.

43. The method of any one of claims 1-42, further comprising the step of formulating the formulation.

44. The method of any one of claims 1 to 43, wherein the viral vaccine is bivalent, trivalent or quadrivalent, preferably wherein the viral vaccine is the quadrivalent dengue vaccine TAK-003.

45. The method of any preceding claim, wherein large scale comprises a surface area of ​​35,000 cm 2 or larger, 50,000 cm 2 or larger, 100,000 cm 2 or larger production cultures.

46. ​​The method of any of the preceding claims, 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.

47. The method of any one of the preceding claims, 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.

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