Low-temperature filtration of oil-in-water emulsion adjuvants

Abstract

To provide a method for filtering emulsions at low temperatures. [Solution] Specifically, the cryogenic filtration of emulsion adjuvants for vaccine production is discussed. A method is provided for improving the filtration of an oil-in-water emulsion through a membrane filter, the method comprising the steps of filtering the oil-in-water emulsion through a membrane filter at a temperature below or equal to 10°C, wherein the filter throughput is increased compared to the filtration of an oil-in-water emulsion at a temperature above 10°C.

Description

[Technical Field]

[0001] cross reference This application claims priority under U.S. Provisional Patent Application No. 63,045,949, filed June 30, 2020 (the entirety of which is incorporated herein by reference).

[0002] field This invention relates to the field of producing oil-in-water emulsions for vaccines. This disclosure relates to a method for filtering oil-in-water emulsions at low temperatures. Furthermore, the filtration of oil-in-water emulsions at low temperatures for vaccine production is considered. [Background technology]

[0003] background Drugs or immunological agents that enhance the immune response to antigens are important for vaccine production (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). Oil-in-water emulsions, which can be used as adjuvants, are one such example of drugs that enhance the immune response (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). The use of these adjuvants in vaccine formulations is advantageous because the adjuvants in the vaccine formulation enhance, promote, and prolong vaccine efficacy (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4; Onraedt et al. (2010) BioPharm International Supplement, Issue 8). Adjuvants are also described as dose-saving because they induce a more rapid and broader response during a pandemic outbreak (Onraedt et al. (2010) BioPharm International Supplement, Issue 8). Oil-in-water emulsions and liposomal adjuvants are being purchased by vaccine manufacturers worldwide as a cost-effective mechanism to meet global vaccine demand, for example (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4).

[0004] Emulsions have previously been described as thermodynamically unstable (Raposo et al. (2013) Pharm Dev Technol 1-13). Potential stabilizers include polyfunctional excipients (e.g., surfactants, co-emulsifiers, polymers, biomolecules, and colloidal particles) (Raposo et al. (2013) Pharm Dev Technol 1-13; Tamilvanan et al. (2010) J. Excipients and Food Chem 1(1):11-29).

[0005] One such water-in-oil adjuvant is known as "MF59" (登録商標) (WO90 / 14837; Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203; Podda (2001) Vaccine 19:2673-2680). MF59 (登録商標)It is a submicron oil-in-water emulsion of squalene, polysorbate 80 (also known as Tween® 80), and sorbitan trioleate (also known as Span® 85). It may also contain citrate ions (for example, 10 mM sodium citrate buffer (Vaccine Design: The Subunit and Adjuvant Approach (ed. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X; Vaccine Adjuvants: Preparation Methods and Research Protocols (Vol. 42 of the Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Edited by O'Hagan; New Generation Vaccines (ed. Levine et al.). 3rd edition, 2004. ISBN 0-8247-4071-8)). The volumetric composition of the emulsion may be approximately 5% squalene, approximately 0.5% Tween® 80, and approximately 0.5% Span® 85 (Vaccine Design: The Subunit and Adjuvant Approach (ed. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X; Vaccine Adjuvants: Preparation Methods and Research Protocols (Vol. 42 of the Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Edited by O'Hagan; New Generation Vaccines (Edited by Lev (ine et al.). 3rd edition, 2004. ISBN 0-8247-4071-8).

[0006] MF59 (登録商標)It is manufactured on a commercial scale by dispersing Span® 85 in the squalene phase and Tween® 80 in the aqueous phase, followed by high-speed mixing to form a coarse emulsion (O’Hagan (2007) Expert Rev Vaccines 6(5):699-710). This coarse emulsion is then passed repeatedly through a microfluidizer to generate an emulsion with a homogeneous oil droplet size (O’Hagan (2007) Expert Rev Vaccines 6(5):699-710). The microfluidized emulsion is then filtered through a 0.22 μm membrane to remove large oil droplets, and the average droplet size of the resulting emulsion remains unchanged over at least three years at 4°C (New Generation Vaccines (ed. Levine et al.). 3rd Edition, 2004. ISBN 0-8247-4071-8). The squalene content of the final emulsion is then measured (EP-B-2029170).

[0007] In representative filtration applications, the throughput of water-in-oil emulsions passing through membranes can be affected by many factors, including membrane structure, viscosity of the adjuvant suspension, adjuvant particle size, adjuvant particle concentration, and resistance of the filter material (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). The overall throughput of the filter is determined by flux and durability (Rogers et al. (2010) BioPharm (International Supplement, Issue 1:1-4). Flux is determined by the driving force (e.g., inlet pressure), flow properties (viscosity), and membrane structure (e.g., pore size, asymmetry) (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). Reduced flux can significantly affect processing time (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). Endurance is determined by the membrane structure and the characteristics of the process flow (e.g., adjuvant particle loading) (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). Asymmetric membranes and increased pressure have long been associated with increased membrane endurance (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4).

[0008] Regarding viscosity, suspensions are generally less viscous at higher temperatures, but their viscosity is higher than that of water at all temperatures (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). The flux of more viscous solutions is higher than that of aqueous solutions (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). 1:1-4).

[0009] Membrane clogging is another factor worth considering during emulsion filtration. Flow typically decreases rapidly after the start of filtration due to membrane particle clogging and adjuvant particle characteristics (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). Therefore, obstruction of membrane pores is a significant factor in filter durability and is the main mechanism of flux attenuation (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4). Flow of smaller particles has previously been associated with increased membrane durability (Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4).

[0010] Retention of exogenous contaminants such as bacteria is another important consideration during membrane filtration of emulsions. Numerous factors have been associated with influencing bacterial retention, including adjuvant-bacteria-membrane interactions; membrane clogging; adjuvant surface tension; membrane properties; temperature; and operating pressure (Onraedt et al. (2010) BioPharm International Supplement, Issue 8). Bacterial coating in emulsions is associated with less strong retention due to interference with membrane pores and low adjuvant surface tension (Onraedt et al. (2010) BioPharm International Supplement, Issue 8). Increased temperature was associated with increased retention (Onraedt et al. (2010) BioPharm International Supplement, Issue 8). One of the mechanisms disclosed in the prior art, used to cleverly circumvent many of the problems associated with membrane filtration of emulsions, involves heating the emulsion before filtration (Tamilvanan et al. (2010) J. Excipients and Food Chem 1(1):11-29). While increasing the emulsion temperature has been associated with enhanced filtration, it can significantly impair the integrity of the emulsion and their subsequent performance in vaccines. Oil-in-water emulsion (e.g., MF59) (登録商標) The preparation of these products typically involves numerous levels of filtration, such as bioburden reduction filtration, sterilization filtration, and particle size filtration. In the context of manufacturing, these filtration processes utilize a large number of filter membranes. In light of this, improvements in filtration methods and systems are needed. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 90 / 14837 [Patent Document 2] European Patent No. 2029170 [Non-patent literature]

[0012] [Non-Patent Document 1] Rogers et al. (2010) BioPharm International Supplement, Issue 1:1-4 [Non-Patent Document 2] Onraedt et al. (2010) BioPharm International Supplement, Issue 8 [Non-Patent Document 3] Raposo et al. (2013) Pharm Dev Technol 1-13) [Non-Patent Document 4] Tamilvanan et al. (2010) J. Excipients and Food Chem 1(1):11-29 [Non-Patent Document 5] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203 [Non-Patent Document 6] Podda (2001) Vaccine 19:2673-2680 [Non-Patent Document 7] Vaccine Design: The Subunit and Adjuvant Approach (Eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X [Non-Patent Document 8] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of the Methods in Molecular Medicine series). ISBN: 1-59259-083-7 [Non-Patent Document 9] Edited by O'Hagan; *New Generation Vaccines* (edited by Levine et al.). 3rd edition, 2004. ISBN 0-8247-4071-8 [Non-Patent Document 10] O'Hagan (2007) Expert Rev Vaccines 6(5):699-710 [Non-Patent Document 11] New Generation Vaccines (ed., Levine et al.). 3rd edition, 2004. ISBN 0-8247-4071-8) [Overview of the project] [Means for solving the problem]

[0013] Abstract This disclosure provides emulsion adjuvants subjected to membrane filtration at low temperatures.

[0014] This disclosure also provides a method for filtering emulsion adjuvants at low temperatures. (Item 1) A method for improving the filtration of an oil-in-water emulsion through a membrane filter, the method comprising the steps of filtering the oil-in-water emulsion through a membrane filter at a temperature below 10°C or equal to 10°C, wherein the filter throughput is increased compared to the filtration of an oil-in-water emulsion at a temperature above 10°C. (Item 2) A method for preparing an oil-in-water emulsion, the method comprising the steps of filtering the oil-in-water emulsion through a membrane filter at a temperature below 10°C or equal to 10°C, wherein the filter throughput is increased compared to a temperature higher than 10°C. (Item 3) The step of filtering the oil-in-water emulsion is the method according to any one of items 1 to 2, which reduces the amount of bioburden in the oil-in-water emulsion. (Item 4) The step of filtering the oil-in-water emulsion is the method according to any one of items 1 to 2, wherein the step of filtering the oil-in-water emulsion includes sterilization by filtration of the oil-in-water emulsion. (Item 5) The step of filtering the oil-in-water emulsion is the method according to any one of items 1 to 2, wherein the step of filtering the oil-in-water emulsion includes particle size reduction filtration of the oil-in-water emulsion. (Item 6) The method according to any one of items 1 to 5, comprising the step of filtering the oil-in-water emulsion through a membrane filter at a temperature of 2 to 8°C. (Item 7) An oil-in-water emulsion prepared according to the method described in any one of items 1 to 6. (Item 8) The oil-in-water emulsion is an adjuvant, as described in item 7. (Item 9) The oil-in-water emulsion is an oil-in-water emulsion according to any one of items 7 to 8, comprising squalene. (Item 10) The oil-in-water emulsion is an oil-in-water emulsion according to any one of items 7 to 9, including (a) a submicron oil-in-water emulsion comprising squalene, polysorbate 80 and sorbitan trioleate, or (b) squalene, tocopherol and polysorbate 80. (Item 11) The aforementioned oil-in-water emulsion is MF59 (登録商標) An oil-in-water emulsion as described in any one of items 7 to 10. (Item 12) A vaccine composition comprising an oil-in-water emulsion prepared according to the method described in any one of items 1 to 6. (Item 13) The vaccine composition is the vaccine composition according to item 12, which specifically targets the influenza virus. (Item 14) The aforementioned oil-in-water emulsion is MF59 (登録商標) A vaccine composition as described in any one of items 12 to 13. (Item 15) A method for preparing an oil-in-water emulsion adjuvant, the method comprising the step of filtering the oil-in-water emulsion adjuvant through a membrane filter at an adjuvant throughput that is greater at a temperature of 5°C than at a temperature of 10°C. (Item 16) An oil-in-water emulsion adjuvant prepared according to the method described in item 15. (Item 17) The aforementioned oil-in-water emulsion adjuvant is MF59 (登録商標) The oil-in-water emulsion adjuvant described in item 16. (Item 18) A vaccine composition comprising an oil-in-water emulsion adjuvant prepared according to the method described in item 15. (Item 19) The vaccine composition is the vaccine composition according to item 18, which specifically targets the influenza virus. (Item 20) The aforementioned oil-in-water emulsion adjuvant is MF59 (登録商標) A vaccine composition as described in any one of items 18 to 19. (Item 21) A method for preparing an oil-in-water emulsion, wherein the method is: A step of mixing oil, aqueous components, and a surfactant to form the oil-in-water emulsion; A step of microfluidizing the mixture to reduce the average droplet size of the oil-in-water emulsion; and A method comprising the step of filtering the aforementioned microfluidized oil-in-water emulsion through a membrane filter at a temperature below 10°C or equal to 10°C, wherein the filter throughput is increased compared to the filtration of the oil-in-water emulsion at a temperature above 10°C. (Item 22) The oil-in-water emulsion is the method according to item 21, comprising squalene. (Item 23) The method according to any one of items 21 to 22, wherein the step of mixing the oil, aqueous components, and surfactant includes a step of homogenizing the components. (Item 24) An oil-in-water emulsion prepared according to the method described in any one of items 21 to 23. (Item 25) The aforementioned oil-in-water emulsion is MF59 (登録商標) The oil-in-water emulsion described in item 24. (Item 26) A vaccine composition comprising an oil-in-water emulsion prepared according to the method described in any one of items 21 to 23. (Item 27) The vaccine composition described above is the vaccine composition described in item 26, which specifically targets the influenza virus. (Item 28) The aforementioned oil-in-water emulsion is MF59 (登録商標) A vaccine composition as described in any one of items 26 to 27. (Item 29) The method according to any one of items 1 to 23, further comprising the step of mixing the oil-in-water emulsion with an antigen compound. (Item 30) The method according to item 29, wherein the antigen compound is an influenza virus antigen. (Item 31) The method according to any one of items 29 to 30, further comprising the step of packaging the oil-in-water emulsion together with an antigen compound into a kit.

Brief Description of the Drawings

[0015] Description of the Drawings [Figure 1] Figure 1 shows the throughput of the SHF film at temperatures of 5°C, 30°C, and 40°C.

[0016] [Figure 2] Figure 2 shows the throughput of the SHC film at 5°C, 30°C, and 40°C.

[0017] [Figure 3] Figure 3 shows the throughput of the ECV membrane at 5°C and 40°C. [Modes for carrying out the invention]

[0018] Detailed explanation Many modifications and other embodiments of the disclosure shown herein will remind those skilled in the art that such disclosures have the benefit of teaching as shown in the preceding description and accompanying drawings. It should therefore be understood that this disclosure should not be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be within the scope of the appended claims. Certain terms are used herein, but they are used only in a general and descriptive sense, and not for limiting purposes.

[0019] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context otherwise clearly indicates. Furthermore, the terms “including,” “includes,” “having,” “has,” “with,” or their variations are intended to be as comprehensive as the term “comprising,” to the extent that they are used in any of the detailed description and / or claims.

[0020] The terms “comprise,” “have,” and “include” are unrestricted linking verbs. Any one or more forms or tenses of these verbs (e.g., “comprises,” “comprising,” “has,” “having,” “includes,” and “including”) are also unrestricted. For example, any method “comprises,” “has,” or “includes” one or more steps is not limited to having only those one or more steps, but also covers other unlisted steps. Similarly, any composition “comprises,” “has,” or “includes” one or more features is not limited to having only those one or more features, but also covers other unlisted features. Any and all examples or illustrative language provided herein with respect to any particular embodiment herein is intended solely to better illustrate the disclosure and does not impose any limitation on the scope of the claimed disclosure.

[0021] Oil-in-water emulsion adjuvant The method of the present invention is used for the production of oil-in-water emulsions. These emulsions contain three core components: oil; aqueous components; and a surfactant.

[0022] Oil-in-water emulsions have been found to be suitable for use as adjuvants in influenza virus vaccines. Various such emulsions are known, and they typically contain at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion generally have a diameter of less than 5 μm, and may even have a submicron diameter; these small sizes are achieved in microfluidizers to provide a stable emulsion. Droplets with a size of less than 220 nm are preferred because they can be subjected to filter sterilization.

[0023] Oils can be derived from animal (e.g., fish) or plant sources. Since emulsions are intended for pharmaceutical use, oils are typically biodegradable (metabolizable) and biocompatible. Sources of plant oils include nuts, seeds, and grains. Peanut oil, soybean oil, coconut oil, and olive oil (the most commonly available) are examples of nut oils. Jojoba oil can be used (e.g., obtained from jojoba seeds). Seed oils include safflower oil, cottonseed oil, sunflower oil, and sesame oil. In the grain group, corn oil is the most readily available, but oils from other grains (e.g., wheat, oats, rye, rice, teff, rye, etc.) may also be used. Fatty acid esters of glycerol and 1,2-propanediol with 6 to 10 carbon atoms are not naturally present in seed oils, but may be prepared from nut and seed oils by hydrolysis, separation, and esterification of suitable substances. Fats and oils derived from mammalian milk are metabolizable and therefore can be used in the implementation of the present invention. Procedures for separation, purification, saponification, and other means necessary to obtain pure oils from animal sources are well known in the art. Many branched-chain oils are biochemically synthesized from 5-carbon isoprene units and are generally called terpenoids. Shark liver oil contains branched unsaturated terpenoids known as squalene and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Squalane (a saturated analog of squalene) is another example of an oil. The oil of the present invention may include, for example, a mixture (or combination) of oils comprising squalene and at least one further oil. Fish oil (containing squalene and squalane) is readily available from commercial sources or can be obtained by methods known in the art.

[0024] Other useful oils include tocopherol, particularly tocopherol in combination with squalene. When the oil phase of the emulsion contains tocopherol, any of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, ε-tocopherol, or ζ-tocopherol may be used, but α-tocopherol is preferred. Both D-α-tocopherol and DL-α-tocopherol can be used. The preferred α-tocopherol is DL-α-tocopherol. Tocopherol can take several forms, e.g., different salts and / or isomers. Examples of salts include organic salts (e.g., succinates, acetates, nicotinates, etc.). When a salt of this tocopherol is to be used, the preferred salt is succinate. Combinations of oils containing squalene and α-tocopherol (e.g., DL-α-tocopherol) may be used.

[0025] The aqueous component may be fresh water (e.g., water for injection) or may contain further components (e.g., solutes). For example, it may contain salts that form the buffer (e.g., citrates or phosphates (e.g., sodium salts)). Typical buffers include phosphate buffers, Tris buffers, borate buffers, succinate buffers, histidine buffers, or citrate buffers. Buffers are typically in the 5–20 mM range.

[0026] The surfactants are preferably biodegradable (metabolizable) and biocompatible. Surfactants can be classified by their "HLB" (hydrophilic / lipophilic balance), in which case an HLB in the range of 1 to 10 generally means that the surfactant is more soluble in oil than in water, and an HLB in the range of 10 to 20 means that it is more soluble in water than in oil. The emulsion preferably contains at least one surfactant having an HLB of at least 10 (e.g., at least 15), or preferably at least 16.

[0027] The present invention can be used with surfactants, including but not limited to the following: polyoxyethylene sorbitan ester surfactants (commonly known as Tweens), particularly polysorbate 20 and polysorbate 80; DOWFAX TM Copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trade name of (e.g., linear EO / PO block copolymer); octoxynol (where the number of repeating ethoxy(oxy-1,2-ethanediyl) groups can vary, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being particularly important); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids (e.g., phosphatylcholine (lecithin)); polyoxyethylene fatty ethers obtained from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants) (e.g., triethylenegrochol monolauryl ether (Brij 30)); polyoxyethylene-9-lauryl ether; and sorbitan esters (generally known as SPAN®) (e.g., sorbitan trioleate (Span®) 85) and sorbitan monolaurate. Preferred surfactants to include in the emulsion are polysorbate 80 (Tween® 80; polyoxyethylene sorbitan monooleate), Span® 85 (sorbitan trioleate), lecithin, and Triton X-100.

[0028] A mixture of surfactants may be included in the emulsion (e.g., Tween® 80 / Span® 85 mixture, or Tween® 80 / Triton-X 100 mixture). Combinations of polyoxyethylene sorbitan esters (e.g., polyoxyethylene sorbitan monooleate (Tween® 80)) and octoxynol (e.g., t-octylphenoxy-polyethoxyethanol (Triton X-100)) are also suitable. Another useful combination involves laureth-9 and polyoxyethylene sorbitan ester and / or octoxynol. Useful mixtures may include surfactants with HLB values ​​in the range of 10-20 (e.g., Tween® 80, with HLB 15.0) and surfactants with HLB values ​​in the range of 1-10 (e.g., Span® 85, with HLB 1.8).

[0029] The appropriate amounts (by weight) of surfactants are as follows: polyoxyethylene sorbitan ester (e.g., Tween® 80) 0.01-1%, especially about 0.1%; octyl- or nonylphenoxy polyoxyethanol (e.g., Triton X-100, or other detergents in the Triton series) 0.001-0.1%, especially 0.005-0.02%; polyoxyethylene ether (e.g., Laureth 9) 0.1-20%, preferably 0.1-10% and especially 0.1-1% or about 0.5%.

[0030] Regardless of the choice of oil and surfactant, the surfactant is included in excess of the amount required for emulsification, and as a result, free surfactant remains in the aqueous phase. The free surfactant in the final emulsion can be detected by various assays. For example, sucrose gradient centrifugation can be used to separate emulsion droplets from the aqueous phase, and the aqueous phase can then be analyzed. Centrifugation can be used to separate the two phases. The oil droplets coalesce and float to the surface, and the surfactant content of the aqueous phase can then be determined, for example, using HPLC or any other suitable analytical technique.

[0031] Specific oil-in-water emulsion adjuvants pursuant to this disclosure include, but are not limited to, the following: • Submicron emulsion of squalene, Tween® 80, and Span® 85. The volumetric composition of the above emulsion may be approximately 5% squalene, approximately 0.5% polysorbate 80, and approximately 0.5% Span® 85. In terms of weight, these ratios are 4.3% squalene, 0.5% polysorbate 80, and 0.48% Span® 85. This adjuvant is "MF59". (登録商標) It is publicly known as MF59. (登録商標) The emulsion advantageously contains citrate ions, for example, 10 mM sodium citrate buffer. In some embodiments, the oil-in-water emulsion adjuvant is a squalene-in-water emulsion adjuvant having 9.75 mg of squalene. • Emulsion of squalene, tocopherol, and Tween® 80. The above emulsion may contain phosphate-buffered saline. It may also contain Span® 85 (e.g., in 1%) and / or lecithin. These emulsions may have 2-10% squalene, 2-10% tocopherol, and 0.3-3% Tween® 80, with a squalene:tocopherol weight ratio preferably ≤1, because this provides a more stable emulsion. Squalene and Tween® 80 may exhibit a volume ratio of about 5:2. One such emulsion may be prepared by dissolving Tween® 80 in PBS to give a 2% solution, then mixing 90 mL of this solution with a mixture of (5 g DL-α-tocopherol and 5 mL squalene), and then microfluidizing the mixture. The resulting emulsion may have submicron oil droplets having an average diameter between 100 nm and 250 nm, preferably about 180 nm. • An emulsion of squalene, tocopherol, and Triton detergent (e.g., Triton X-100). The emulsion may also contain 3d-MPL. The emulsion may also contain phosphate buffer. • An emulsion comprising polysorbate (e.g., polysorbate 80), Triton detergent (e.g., Triton X-100), and tocopherol (e.g., α-tocopherol succinate). The emulsion may contain these three components in a mass ratio of approximately 75:11:10 (e.g., 750 μg / mL polysorbate 80, 110 μg / mL Triton X-100, and 100 μg / mL α-tocopherol succinate), and their concentrations should include any contribution of these components derived from the antigen. The emulsion may also contain squalene. The emulsion may also contain 3d-MPL. The aqueous phase may contain phosphate buffer. • Squalane, polysorbate 80 and poloxamer 401 ("Pluronic") TM An emulsion of "L121". The above emulsion can be formulated in phosphate-buffered saline (pH 7.4). This emulsion is a useful delivery vehicle for muramyl dipeptide and is used with threonyl MDP in the "SAF-1" adjuvant (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without Thr-MDP, as in the "AF" adjuvant (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). An emulsion containing 0.5-50% oil, 0.1-10% phospholipids, and 0.05-5% nonionic surfactant. Preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, and cardiolipin. Submicron droplet sizes are advantageous. • Non-metabolizable oils (e.g., light mineral oil) and at least one surfactant (e.g., lecithin, Tween® 80, or Span®) 80) Submicron oil-in-water emulsions. Additives may be included (e.g., Quil A saponin, cholesterol, saponin-lipophilic conjugates produced by adding an aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid (e.g., GPI-0100)), dimethyldioctadecylammonium bromide and / or N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine). • Emulsions in which saponins (e.g., QuilA or QS21) and sterols (e.g., cholesterol) are associated as helical micelles.

[0032] Emulsion formation Emulsion components can be mixed to form an emulsion.

[0033] Oil droplets in an emulsion may have an average size of 5000 nm or less, for example, 4000 nm or less, 3000 nm or less, 2000 nm or less, 1200 nm or less, or 1000 nm or less, for example, an average size between 800 and 1200 nm or between 300 nm and 800 nm.

[0034] The number of oil droplets in an emulsion with a size > 1.2 μm is 5 × 10⁻⁶. 11 / ml or less, for example, 5 × 10 10 / ml or less, or 5 × 10 9 It may be / ml or less.

[0035] The average droplet size of the emulsion can be achieved by mixing the components of the first emulsion in a homogenizer. The homogenizer can operate in a vertical and / or horizontal configuration. For convenience in commercial situations, an in-line homogenizer is preferred.

[0036] For commercial-scale production, the homogenizer described above should ideally have a flow rate of at least 300 L / hour, for example, ≥400 L / hour, ≥500 L / hour, ≥600 L / hour, ≥700 L / hour, ≥800 L / hour, ≥900 L / hour, ≥1000 L / hour, ≥2000 L / hour, ≥5000 L / hour, or even ≥10000 L / hour. Suitable high-durability homogenizers are commercially available.

[0037] A preferred homogenizer is 3 × 10 5 ~1 × 10 6 s -1 For example, 3 x 10 5 ~7×10 5 s -1 During, 4 x 10 5 ~6×10 5 s -1 For example, during the period of approximately 5 x 10 5 s -1 Provides the shear rate.

[0038] In some embodiments, emulsion components can be homogenized multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 times, or more). To avoid the need for long connections between containers and homogenizers, emulsion components can be circulated. In particular, emulsions can be formed by circulating the first emulsion components through a homogenizer multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 times, etc.). However, too many cycles may be undesirable as they can lead to recombination (Jafari et al. (2008) Food Hydrocolloids 22:1191-1202). Therefore, the size of the oil droplets can be monitored when homogenizer circulation is used to check that the desired droplet size has been reached and / or that recombination is not occurring.

[0039] Circulation through a homogenizer is advantageous because it can reduce the average size of oil droplets in the emulsion. Circulation is also advantageous because it can reduce the number of oil droplets with a size >1.2 μm in the first emulsion. These reductions in average droplet size and the number of droplets >1.2 μm in the first emulsion can provide advantages in downstream processes. In particular, circulation of emulsion components through a homogenizer can result in an improved microfluidization process, which in itself can provide improved filtration performance. Improved filtration performance means less loss of contents during filtration, for example, oil-in-water emulsions can MF59 (登録商標) In that case, the loss of squalene, Tween® 80, and Span® 85 can be minimized.

[0040] The methods of the present invention can be used on a large scale. Accordingly, one method may involve preparing a first emulsion with a volume greater than 1 liter, for example, ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.

[0041] microfluidization After its formation, the emulsion may be microfluidized to reduce its average droplet size and / or to reduce the number of droplets having a size >1.2 μm.

[0042] Microfluidizers reduce the average droplet size by driving the flow of input components at high pressure and high speed through geometrically fixed channels. The pressure upon entering the interaction chamber (also called the "first pressure") may be substantially constant (i.e., ±15%; e.g., ±10%, ±5%, ±2%) for at least 85% of the time while the components are being supplied to the microfluidizer, for example, at least 87%, at least 90%, at least 95%, at least 99%, or 100% of the time while the emulsion is being supplied to the microfluidizer.

[0043] A microfluidization apparatus typically includes at least one intensifier pump (preferably two pumps, which may be synchronized) and an interaction chamber. The intensifier pump (ideally electrohydraulic driven) provides high pressure (i.e., a first pressure) to push the emulsion through it into the interaction chamber. The synchronous nature of the intensifier pump can be used to provide a substantially constant pressure for the emulsion considered above. This means that all emulsion droplets are exposed to substantially the same level of shear force during microfluidization.

[0044] Reducing the average oil droplet size and the number of oil droplets with a size >1.2 μm in the emulsion can provide improved filtration performance. Improved filtration performance can also mean, for example, reduced loss of contents during filtration, e.g., if the emulsion is MF59 (登録商標) In that case, the loss of squalene, Tween® 80, and Span® 85 can be minimized.

[0045] Preferred microfluidization devices operate at pressures between 170 bar and 2750 bar (approximately 2500 psi and 40000 psi), for example, at around 345 bar, 690 bar, 1380 bar, and 2070 bar.

[0046] A preferred microfluidization device is 1 × 10 6 s -1 For example, ≥2.5 × 10 6 s -1 , ≥ 5 × 10 6 s -1 , ≥10 7 s -1 It has an interaction chamber that provides shear rates such as those mentioned above.

[0047] A microfluidization device may include multiple interaction chambers used in parallel (e.g., two, three, four, five, or more), but including a single interaction chamber is more useful.

[0048] The result of microfluidization may be an oil-in-water emulsion with an average droplet size of 500 nm or less. This average size is particularly useful because it facilitates filtration sterilization of the emulsion. Emulsions in which at least 80% of the number of droplets have an average size of 500 nm or less, for example, 400 nm or less, 300 nm or less, 200 nm or less, or 165 nm or less, are particularly useful. Furthermore, the number of droplets in emulsions with a size > 1.2 μm is 5 × 10⁻⁶. 10 / ml or less, for example, 5 × 10 9 / ml or less, 5 x 10 8 / ml or less, or 2 × 10 8 / ml or less.

[0049] In a microfluidication apparatus, the emulsion container can be maintained under an inert gas, such as nitrogen up to 0.5 bar. This prevents oxidation of the emulsion components. This is particularly advantageous when one of the emulsion components is squalene. This results in increased emulsion stability.

[0050] The methods of the present invention can be used on a large scale. Thus, one method may involve microfluidizing volumes greater than 1 liter, for example, ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.

[0051] filtration Following microfluidization, the emulsion is filtered. Filtration removes any large oil droplets remaining from the homogenization and microfluidization procedures. Although small in number overall, these droplets can be large in volume and can act as nucleation sites for aggregation, leading to emulsion degradation during storage. Furthermore, filtration can achieve filter sterilization.

[0052] Filtration of oil-in-water emulsions in a manufacturing process may involve one or more levels and / or types of filtration steps. Some of these may include bioburden reduction filtration, sterilization filtration, particle size filtration, and the like. Accordingly, in various embodiments, this disclosure describes methods for improving the filtration of emulsions, preferably oil-in-water emulsions. In one or more preferred aspects, the types of filtration embodied by this disclosure include, but are not limited to, bioburden reduction filtration, sterilization filtration, and particle size filtration.

[0053] The appropriate filtration membrane depends on the fluid properties of the emulsion and the required degree of filtration. The characteristics of the filter can affect its suitability for filtering microfluidized emulsions. For example, the pore size and surface properties of the filter are important, and may be particularly important when filtering squalene-based emulsions.

[0054] The pore size of the membrane used in conjunction with this invention should allow the passage of desired droplets while retaining unwanted droplets. For example, it should retain droplets having a size of ≥1 μm while allowing the passage of droplets <200 nm. A 0.2 μm or 0.22 μm filter is ideal, as it can also achieve filtration.

[0055] The emulsion described above can be pre-filtered, for example, through a 0.45 μm filter. Pre-filtration and filtration can be achieved in one step by using a known double-layer filter comprising a first membrane having larger pores and a second membrane having smaller pores. Double-layer filters are particularly useful in the present invention. The first layer ideally has a pore size of >0.3 μm (e.g., between 0.3 and 2 μm or between 0.3 and 1 μm, or between 0.4 and 0.8 μm, or between 0.5 and 0.7 μm). A pore size of ≤0.75 μm in the first layer is preferred. Therefore, the first layer may have a pore size of, for example, 0.6 μm or 0.45 μm. The second layer ideally has a pore size less than 75% (and ideally less than half) of the pore size of the first layer (e.g., between 25-70% or 25-49% of the pore size of the first layer, e.g., between 30-45%, e.g., 1 / 3 or 4 / 9 of the pore size of the first layer). Thus, the second layer may have a pore size <0.3 μm (e.g., between 0.15-0.28 μm or 0.18-0.24 μm, e.g., a second layer with a pore size of 0.2 μm or 0.22 μm). In one example, the first film layer with the larger pores provides a 0.45 μm filter, while the second film layer with the smaller pores provides a 0.22 μm filter.

[0056] The above-mentioned filtration membrane and / or pre-filtration membrane may be asymmetrical. An asymmetrical membrane is one in which the pore size varies from one side to the other (for example, the pore size is larger on the inlet side than on the outlet side). One side of the asymmetrical membrane may be called a "coarse pored surface," while the other side may be called a "fine pored surface." In a double-layer filter, one or (ideally) both layers may be asymmetrical.

[0057] The above-mentioned filtration membrane can be porous or homogeneous. A homogeneous membrane is typically a dense film ranging from 10 to 200 μm in thickness. A porous membrane has a porous structure. In one embodiment, the above-mentioned filtration membrane is porous. In a double-layer filter, both layers may be porous, both layers may be homogeneous, or one layer may be porous and the other homogeneous. A preferred double-layer filter is one in which both layers are porous.

[0058] In one embodiment, the emulsion is pre-filtered through an asymmetric hydrophilic porous membrane and then filtered through another asymmetric hydrophilic porous membrane having smaller pores than the pre-filtered membrane. A double-layer filter may be used.

[0059] The above filter membrane may be autoclaved before use to ensure sterility.

[0060] Filtration membranes are typically made from polymer-supported materials such as PTFE (polytetrafluoroethylene), PES (polyethersulfone), PVP (polyvinylpyrrolidone), PVDF (polyvinylidene fluoride), nylon (polyamide), PP (polypropylene), cellulose (including cellulose esters), PEEK (polyetheretherketone), and nitrocellulose. These materials have various properties; some supports are inherently hydrophobic (e.g., PTFE), while others are inherently hydrophilic (e.g., cellulose acetate). However, these inherent properties can be modified by treating the membrane surface. For example, it is known that hydrophilic or hydrophobic membranes can be prepared by coating the membrane surface with other materials (e.g., other polymers, graphite, silicone, etc.) (WO90 / 04609). In double-layer filters, the two membranes can be made from different materials or (ideally) the same material.

[0061] An ideal filter for use with this invention has a hydrophilic surface rather than a hydrophobic (polysulfone) surface (Baudner et al. (2009) Pharm Res.). 26(6):1477-85; Dupuis et al. (1999) Vaccine 18:434-9; Dupuis et al. (2001) Eur J Immunol 31:2910-8; Burke et al. (1994) J Infect Dis 170:1110-9). Filters having a hydrophilic surface can be formed from a hydrophilic material or by making a hydrophobic material hydrophilic, and a preferred filter for use with the present invention is a hydrophilic polyethersulfone film. Several different methods are known for converting a hydrophobic PES film to a hydrophilic PES film. This has often been achieved by coating the film with a hydrophilic polymer. To provide permanent adhesion of the hydrophilic polymer to the PES, the hydrophilic coating layer is typically subjected to either a crosslinking reaction or grafting. A process for modifying the surface properties of a hydrophobic polymer having functionalizable chain ends comprises the steps of contacting the polymer with a solution of a linker moiety to form a covalent bond, and then contacting the reacted hydrophobic polymer with a solution of a modifying agent (WO90 / 04609). A method for making a PES film hydrophilic by direct film coating is further used, comprising the steps of pre-wetting with alcohol and then immersing in an aqueous solution containing a hydrophilic monomer, a polyfunctional monomer (crosslinking agent), and a polymerization initiator (U.S. Patent No. 4,618,533). The monomer and crosslinking agent are then polymerized using heat-initiated or UV-initiated polymerization to form a crosslinked hydrophilic polymer coating on the film surface (U.S. Patent No. 4,618,533). Similar methods include coating a PES film by immersing it in an aqueous solution of a hydrophilic polymer (polyalkylene oxide) and at least one polyfunctional monomer (crosslinking agent), and then polymerizing the monomer to provide a non-extractable hydrophilic coating (U.S. Patent No. 6,193,077; U.S. Patent No. 6,495,050).Subsequently, the PES film can be made hydrophilic by a grafting reaction in which the PES film is subjected to low-temperature helium plasma treatment, followed by the grafting of the hydrophilic monomer, N-vinyl-2-pyrrolidine (NVP), onto the film surface (Chen et al. (1999) Journal of Applied Polymer Science). 72:1699-1711).

[0062] In a method that does not rely on coating, PES may be dissolved in a solvent and blended with soluble hydrophilic additives, and the blended solution is then used to cast a hydrophilic film, for example, by precipitation or by initiating copolymerization (US Patent Nos. 4,943,374; 6,071,406; 4,705,753; 5,178,765; 6,495,043; 6,039,872; 5,277,812). For example, a method can be used to prepare a hydrophilic charge-modified membrane having low membrane extractability, enabling rapid recovery of ultrapure water resistance, and having a cross-linked interpenetrating polymer network structure, by preparing a polymer solution of a blend of PES, PVP, polyethyleneimine, and aliphatic diglycidyl ether, forming a thin film of the solution, and precipitating the film as a membrane (U.S. Patent No. 5,277,812; U.S. Patent No. 5,531,893).

[0063] A hybrid approach may be used. In this approach, a hydrophilic additive is present during film formation and added later as a coating (U.S. Patent No. 4,964,990).

[0064] Making PES films hydrophilic can also be achieved by low-temperature plasma treatment, including modifying the hydrophilicity of PES films by treatment with low-temperature CO2 plasma (Wavhal & Fisher (2002) Journal of Polymer Science Part B: Polymer Physics 40:2473-88).

[0065] Making a PES film hydrophilic can also be achieved by oxidation (WO2006 / 044463). This method involves pre-wetting a hydrophobic PES film in a liquid with low surface tension, exposing the wetted PES film to an aqueous solution of an oxidizing agent, and then heating it (WO2006 / 044463).

[0066] Opposition inversion can also be used (Espinoza-Gomez et al. (2003) Revista de la Sociedad Quimica de Mexico 47:53-57).

[0067] An ideal hydrophilic PES film can be obtained by treating PES (hydrophobic) with PVP (hydrophilic). It has been found that treating with PEG (hydrophilic) instead of PVP yields a hydrophilic PES film. This PES film clogs easily (especially when using squalene-containing emulsions) and, undesirably, releases formaldehyde during autoclaving.

[0068] A preferred double-layer filter has a first hydrophilic PES film and a second hydrophilic PES film.

[0069] Known hydrophilic films include Bioassure (manufactured by Cuno) and EverLUX. TM Polyethersulfone; STyLUX TM Polyethersulfone (both from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200, and Durapore CVGL01TP3 films (from Millipore); Fluorodyne TM EX EDF membrane, SuporTM EAV; Supor TM EBV, Supor TM ECV, Supor TM EKV (all manufactured by Pall); Sartopore TM Examples include hydrophilic PES films from Sartorius, Sterlitech, and WFPES PES films from Wolftechnik.

[0070] During filtration, the emulsion may be maintained at a temperature of 40°C or lower, for example, 30°C or lower, for example, 20°C or lower, for example, 10°C or lower, for example, 2-8°C or lower, for example, 5°C or lower, to promote successful filtration sterilization. Depending on the emulsion, it may not need to pass through the sterilization filter if it is present at a temperature higher than 40°C.

[0071] It is advantageous to perform the filtration process within 24 hours of the formation of the second emulsion, for example, within 18 hours, 12 hours, 6 hours, 2 hours, or 30 minutes. This is because after this time, there is a possibility that the second emulsion may not be able to pass through the sterile filter without clogging the filter (Lidgate et al. (1992) Pharmaceutical Research 9(7):860-863).

[0072] The methods of the present invention can be used on a large scale. Thus, one method may involve filtering volumes greater than 1 liter, for example, ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.

[0073] In one or more aspects, as described herein, membranes suitable for reduced bioburden may be used in the manner described herein. These membranes include, but are not limited to, Millipore Milliguard, Pall Supor EAV, Pall Fluorodyne II DBL, and Sartorius Sartoguard.

[0074] In further context, membranes suitable for filtration sterilization, as described herein, may be used in the manner described herein. These membranes may include, but are not limited to, Millipore Durapore, Millipore Express SHC, Millipore Express SHF, Pall Supor EBV, Pall Supor ECV, Pall Supor EKV, Pall Emflon II, Pall Fluorodyne II, Pall Fluorodyne EDF, Sartorius Sartopore 2, Sartorius Sartopore 2 XLG, and Sartorius Sartopore Platinum.

[0075] In further context, membranes suitable for particle size filtration, as described herein, may be used in the manner described herein. These membranes include Millipore Milliguard, Pall Supor EAV, and Pall Fluorodyne II. Examples include, but are not limited to, DBL, Pall HDC, Pall Posidyne, Pall PreFlow, Sartorius Sartoguard, and Sartorius Sartoclear.

[0076] Final emulsion The results of microfluidization and filtration show that the average size of the oil droplets is less than 220 nm, for example, 155 ± 20 nm, 155 ± 10 nm, or 155 ± 5 nm, and the number of oil droplets with a size > 1.2 μm is 5 × 10⁻¹⁰. 8 / ml or less, for example, 5 × 10 7 / ml or less, 5 x 10 6 / ml or less, 2 × 10 6 / ml or less, or 5 × 10 5 It is an oil-in-water emulsion that may be less than or equal to / ml.

[0077] The average oil droplet size of the emulsions described herein is generally 50 nm or larger.

[0078] The method of the present invention can be used on a large scale. Accordingly, one method may involve preparing a final emulsion in a volume greater than 1 liter, for example, ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.

[0079] Once the oil-in-water emulsion described above has formed, it can be transferred to a sterile glass bottle. The glass bottle may be 5L, 8L, or 10L in size. Alternatively, the oil-in-water emulsion can be transferred to a sterile flexible bag (flex bag). The flex bag may be 50L, 100L, or 250L in size, etc. Furthermore, the flex bag may be fitted with one or more sterile connectors to connect it to a system. Using a flex bag and sterile connectors is advantageous compared to a glass bottle because the flex bag is larger than a glass bottle, and it may not be necessary to change the flex bag to store all the emulsion produced in one batch. This can provide a sterile closed system for emulsion production, which can reduce the chance of impurities being present in the final emulsion. This is important when the final emulsion is used for pharmaceutical purposes, for example, if the final emulsion is MF59 (登録商標) This can be especially important when it comes to adjuvants.

[0080] The preferred amount (volume %) of oil in the final emulsion is between 2 and 20%, for example, about 10%. Squalene content of about 5% or about 10% is particularly useful. Squalene content (w / v) between 30 and 50 mg / ml is useful (for example, between 35-45 mg / ml, 36-42 mg / ml, 38-40 mg / ml, etc.).

[0081] The preferred amounts (wt%) of surfactants in the final emulsion are as follows: polyoxyethylene sorbitan ester (e.g., Tween® 80) 0.02-2%, particularly about 0.5% or about 1%; sorbitan ester (e.g., Span® 85) 0.02-2%, particularly about 0.5% or about 1%; octyl- or nonylphenoxy polyoxyethanol (e.g., Triton X-100) 0.001-0.1%, particularly 0.005% or 0.02%; polyoxyethylene ether (e.g., Laureth 9) 0.1-20%, preferably 0.1-10% and particularly 0.1-1% or about 0.5%. A polysorbate 80 content (w / v) between 4-6 mg / ml is useful (e.g., between 4.1-5.3 mg / ml). A sorbitan trioleate content (w / v) of 4-6 mg / ml is useful (for example, between 4.1 and 5.3 mg / ml).

[0082] The above process is particularly useful for preparing any of the following oil-in-water emulsions: • An emulsion containing squalene, polysorbate 80 (Tween® 80), and sorbitan trioleate (Span® 85). The volumetric composition of the above emulsion may be approximately 5% squalene, approximately 0.5% polysorbate 80, and approximately 0.5% sorbitan trioleate. In terms of weight, these amounts to 4.3% squalene, 0.5% polysorbate 80, and 0.48% sorbitan trioleate. This adjuvant is "MF59". (登録商標) It is publicly known as MF59. (登録商標)The emulsion is advantageous in that it contains citrate ions, for example, 10 mM sodium citrate buffer. • Emulsions containing squalene, α-tocopherol (ideally DL-α-tocopherol), and polysorbate 80. These emulsions may contain (by weight) 2-10% squalene, 2-10% α-tocopherol, and 0.3-3% polysorbate 80 (e.g., 4.3% squalene, 4.7% α-tocopherol, 1.9% polysorbate 80). The weight ratio of squalene to tocopherol is preferably ≤1 (e.g., 0.90) because this provides a more stable emulsion. Squalene and polysorbate 80 may be present in a volume ratio of about 5:2, or by weight of about 11:5. One such emulsion can be prepared by dissolving polysorbate 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g DL-α-tocopherol and 5 ml squalene), and then microfluidizing the mixture. The resulting emulsion may have submicron oil droplets, for example, having a size between 100 and 250 nm, preferably about 180 nm. • An emulsion of squalene, tocopherol, and Triton detergent (e.g., Triton X-100). The emulsion may also contain 3-O-deacetylated monophosphoryl lipid A ("3d-MPL"). The emulsion may contain phosphate buffer. An emulsion comprising squalene, polysorbate (e.g., polysorbate 80), Triton detergent (e.g., Triton X-100), and tocopherol (e.g., α-tocopherol succinate). The emulsion may contain these three components in a mass ratio of approximately 75:11:10 (e.g., 750 μg / ml polysorbate 80, 110 μg / ml Triton X-100, and 100 μg / ml α-tocopherol succinate), and their concentrations should include any contribution of these components derived from the antigen. The emulsion may also contain 3d-MPL. The emulsion may also contain saponin (e.g., QS21). The aqueous phase may contain phosphate buffer. An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g., polyoxyethylene cetostearyl ether), and a hydrophobic nonionic surfactant (e.g., sorbitan ester or mannide ester (e.g., sorbitan monooleate or "Span® 80")). The emulsion is preferably thermoreversible and / or has at least 90% oil droplets (by volume) having a size of less than 200 nm (U.S. Patent Publication 2007 / 0014805). The emulsion may also comprise one or more of the following: algitol; cryoprotectant (e.g., a sugar such as dodecyl maltoside and / or sucrose); and / or alkyl polyglycoside. It may also comprise a TLR4 agonist (e.g., one whose chemical structure does not contain a sugar ring) (WO2007 / 080308). Such an emulsion may be freeze-dried.

[0083] The composition of these emulsions (expressed as percentages above) can be altered by dilution or concentration (for example, by integers such as 2 or 3, or by fractions such as 2 / 3 or 3 / 4), while their ratios remain the same. For example, 2x concentrated MF59 (登録商標)However, it contains approximately 10% squalene, approximately 1% polysorbate 80, and approximately 1% sorbitan trioleate. The concentrated form can be diluted (e.g., with an antigen solution) to give the desired final concentration of the emulsion.

[0084] The emulsions of the present invention are ideally stored between 2°C and 8°C. They should not be frozen. Ideally, they should be stored away from direct light. In particular, the squalene-containing emulsions and vaccines of the present invention should be protected to avoid photochemical degradation of squalene. When the emulsions of the present invention are stored, this is preferably in an inert atmosphere (e.g., N2 or argon).

[0085] vaccine While it is possible to administer an oil-in-water emulsion adjuvant alone to a patient (for example, to provide an adjuvant effect with respect to an antigen administered separately to the patient), it is more common to mix the adjuvant and antigen before administration to form an immunogenic composition (e.g., a vaccine). Mixing of the emulsion and antigen can occur without preparation, at the time of use, or during vaccine production, before filling. The method of the present invention can be applied in both situations.

[0086] Accordingly, the method of the present invention may include a further process step of mixing the emulsion with the antigen component. Alternatively, the method may include a further step of packaging the adjuvant into a kit together with the antigen component as a kit component.

[0087] Accordingly, the present invention can be used in preparing a mixed vaccine or in preparing a kit containing an antigen and adjuvant ready for mixing. When mixing is performed during production, the volume of bulk antigen and emulsion to be mixed is typically greater than 1 liter (e.g., ≥5 liters, ≥10 liters, ≥20 liters, ≥50 liters, ≥100 liters, ≥250 liters, etc.). When mixing is performed at use, the volume to be mixed is typically less than 1 milliliter (e.g., ≤0.6 ml, ≤0.5 ml, ≤0.4 ml, ≤0.3 ml, ≤0.2 ml, etc.). In both cases, substantially equal solutions of emulsion and antigen solution are typically mixed (i.e., substantially 1:1 (e.g., between 1.1:1 and 1:1.1, preferably between 1.05:1 and 1:1.05, and more preferably between 1.025:1 and 1:1.025)). However, in some embodiments, excess emulsion and excess antigen may be used (WO2007 / 052155). When one component of the excess volume is used, the excess is generally at least 1.5:1, e.g., ≥2:1, ≥2.5:1, ≥3:1, ≥4:1, ≥5:1, etc.

[0088] When the antigen and adjuvant are presented as separate components within the kit, they are physically separated from each other within the kit, and this separation can be achieved in various ways. For example, the components may be in separate containers (e.g., vials). The contents of the two vials can then be mixed, if necessary, for example, by removing the contents of one vial and adding it to the other vial, or by separately removing the contents of both vials and mixing them in a third container.

[0089] In another configuration, one component of the kit is in a syringe and the other is in a container (e.g., a vial). The syringe may be used (e.g., with a needle) to insert its contents into a vial for mixing, and the mixture may then be drawn into the syringe. The mixed contents of the syringe may then be administered to the patient, typically via a new sterile needle. Packaging one component in a syringe eliminates the need to use a separate syringe for administration to the patient.

[0090] In another preferred combination, the two kit components are held together but separately within the same syringe (e.g., a dual-chamber syringe (WO2005 / 089837; U.S. Patent No. 6,692,468; WO00 / 07647; WO99 / 17820; U.S. Patent No. 5,971,953; U.S. Patent No. 4,060,082; EP-A-0520618; WO98 / 01174)). When the syringe is operated (e.g., during administration to a patient), the contents of its two chambers are mixed. This combination avoids the need for a separate mixing step during use.

[0091] The contents of various kit components are generally all in liquid form. In some combinations, components (typically antigen components rather than emulsion components) are in a dry form (e.g., lyophilized form), and other components are in liquid form. The two components can be mixed to reactivate the dry component and give a liquid composition for administration to a patient. Lyophilized components are typically placed in vials rather than syringes. Dry components may contain stabilizers (e.g., lactose, sucrose, or mannitol) and mixtures thereof (e.g., lactose / sucrose mixture, sucrose / mannitol mixture, etc.). One possible combination is to use liquid emulsion components in a pre-filled syringe and lyophilized antigen components in a vial.

[0092] If the vaccine includes components in addition to the emulsion and antigen, these additional components may be contained within one or two kit components, or they may be part of a third kit component.

[0093] Suitable containers for the mixed vaccine of the present invention or for individual kit components include vials and disposable syringes. These containers should be sterile.

[0094] If the composition / component is placed in a vial, the vial is preferably made of glass or plastic material. The vial is preferably sterilized before the composition is added thereto. To avoid problems associated with latex-sensitive patients, the vial is preferably sealed with a latex-free stopper, and it is preferable that no latex is present in any of the packaging materials. In one embodiment, the vial has a butyl rubber stopper. The vial may contain a single dose of the vaccine / component, or more than one dose ("multiple dose" vial), for example, 10 doses. In one embodiment, the vial contains an emulsion of 10 × 0.25 ml doses. A preferred vial is made of colorless glass.

[0095] The vial may have a cap (e.g., Luer lock) into which a pre-filled syringe can be inserted, into which the contents of the syringe can be dispensed into the vial (e.g., to reconstitute lyophilized material therein), and into which the contents of the vial can be returned to the syringe. After the syringe is removed from the vial, a needle can then be attached and the composition can be administered to a patient. The cap is preferably located inside a seal or cover so that the seal or cover must be removed before the cap can be accessed.

[0096] When the composition / component is packaged in a syringe, the syringe typically does not have a needle attached, but a separate needle may be supplied with the syringe for assembly and use. A safety needle is preferred. Typical needles are 1-inch 23 gauge, 1-inch 25 gauge, and 5 / 8-inch 25 gauge. The syringe may be supplied with a peel-off label on which a lot number, influenza season, and expiration date of the contents may be printed to facilitate record keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from accidentally coming off during aspiration. The syringe may have a latex rubber cap and / or plunger. Disposable syringes contain a single dose of vaccine. The syringe generally has a tip cap that seals the tip before the needle is attached, and the tip cap is preferably made of butyl rubber. When the syringe and needle are packaged separately, the needle is preferably fitted with a butyl rubber cover.

[0097] The above emulsion may be diluted with a buffer before packaging into vials or syringes. Typical buffers include phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer; or citrate buffer. Dilution can reduce the concentration of adjuvant components, for example, to provide an adjuvant with "half the strength" while maintaining the relative proportion of the adjuvant.

[0098] The container may be marked to indicate half-dose volumes, for example, to facilitate delivery to children. For example, a syringe containing a 0.5 ml dose may have a mark indicating a 0.25 ml volume.

[0099] When glass containers (e.g., syringes or vials) are used, it is preferable to use containers made from borosilicate glass rather than soda-lime glass.

[0100] Various antigens can be used with oil-in-water emulsions (including, but not limited to, viral antigens (e.g., viral surface proteins); bacterial antigens (e.g., protein and / or saccharide antigens); fungal antigens; parasitic antigens; and tumor antigens). The present invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, RSV, cytomegalovirus, Staphylococcus aureus, Chlamydia, SARS coronavirus, varicella-zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein-Barr virus, human papillomavirus, and others. For example: · Influenza virus antigenThese can take the form of live viruses or inactivated viruses. When inactivated viruses are used, the vaccine may contain whole virions, split virions, or purified surface antigens (containing hemagglutinin, and usually neuraminidase as well). Influenza antigens may also be presented in the form of virosomals. The above antigens may have any hemagglutinin subtype selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and / or H16. The vaccine may contain antigens derived from one or more (e.g., one, two, three, four or more) influenza virus strains, including influenza A virus and / or influenza B virus (e.g., monovalent A / H5N1 or A / H1N1 vaccine, or trivalent A / H1N1 + A / H3N2 + B vaccine). The influenza viruses described above may be reassortant strains and may be obtained by reverse genetics techniques (Hoffmann et al. (2002) Vaccine 20:3165-3170; Subbarao et al. (2003) Virology 305:192-200; Liu et al. (2003) Virology 314:580-590; Ozaki et al. (2004) J. Virol. 78:1851-1857; Webby et al. (2004) Lancet 363:1099-1103). Therefore, the viruses described above may contain one or more RNA segments derived from the A / PR / 8 / 34 virus (typically six segments from A / PR / 8 / 34, with the HA and N segments derived from the vaccine strain (i.e., 6:2 reassortment)). The viruses used as the source of the above antigens can be grown in either eggs (e.g., embryogenerating chicken eggs) or cell cultures. When cell cultures are used, the cell substrate is typically a mammalian cell line (e.g., MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; etc.).Preferred mammalian cell lines for culturing influenza viruses include: MDCK cells derived from Madin Darby canine kidney (WO97 / 37000; Brands et al. (1999) Dev Biol Stand 98:93-100; Halperin et al. (2002) Vaccine 20:1240-7; Tree et al. (2001) Vaccine 19:3444-50); Vero cells derived from African green monkey kidney (Istner et al. (1998) Vaccine 16:960-8; Kistner et al. (1999) Dev Biol Stand 98:101-110; Bruhl et al.). (2000) Vaccine 19:1149-58); or PER.C6 cells derived from human embryonic retinoblasts (Pau et al. (2001) Vaccine 19:2716-21). When the virus is grown in mammalian cell lines, the composition is advantageously free from egg proteins (e.g., ovalbumin and ovomucoid) and chicken DNA, thereby reducing allergenicity. The unit dose of the vaccine is typically standardized by reference to hemagglutinin (HA) content and typically measured by SRID. Existing vaccines typically contain about 15 μg of HA / strain, but lower doses may be used, especially when adjuvants are used. Fractional doses (fractional doses) (1 dose) (e.g., 'A' (i.e., 7.5 μg HA / strain), 'A' and Vs are used (WO01 / 22992; Hebe et al. (2004) Virus Res. 103(1-2):163-71). Higher doses are also used (e.g., 3× or 9× doses (Treanor et al. (1996) J Infect Dis 173:1467-70; Keitel et al. (1996) Clin Diagn Lab Immunol) 3:507-10). Therefore, the vaccine may contain HA between 0.1 and 150 μg per influenza strain, preferably between 0.1 and 50 μg, for example, 0.1 to 20 μg, 0.1 to 15 μg, 0.1 to 10 μg, 0.1 to 7.5 μg, 0.5 to 5 μg, etc. Specific doses include, for example, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5 per strain. · Human immunodeficiency virus (Including HIV-1 and HIV-2). These antigens are typically envelope antigens. · Hepatitis B virus surface antigen This antigen is preferably obtained by recombinant DNA method, for example, after expression in Saccharomyces cerevisiae yeast. Unlike the natural virus HBsAg, the recombinant yeast-expressed antigen is not glycosylated. It can be in the form of substantially spherical particles (average diameter about 20 nm) containing a lipid matrix with phospholipids. Unlike the natural HBsAg particles, the yeast-expressed particles may contain phosphatidylinositol. HBsAg may be derived from any of the subtypes aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq-, and adrq+. · hookworm, particularly as observed in dogs (Ancylostoma caninum). This antigen may be recombinant Ac-MTP-1 (astacin-like metalloproteinase) and / or aspartate hemoglobinase (Ac-APR-1), which can be expressed as a secreted protein in baculovirus / insect cell lines (Williamson et al. (2006) Infection and Immunity 74:961-7; Loukas et al. (2005) PLoS Med 2(10): e295). · Herpes simplex virus antigen (HSV). A preferred HSV antigen for use with the present invention is the membrane glycoprotein gD. It is preferable to use gD derived from the HSV-2 strain ("gD2" antigen). The composition is g, in which the C-terminal membrane anchor region is deleted. Form D, for example, the shortened form gD) containing amino acids 1-306 of the native protein with the addition of asparagine and glutamine at the C-terminus, may be used (EP-A-0139417). This form of the protein contains a signal peptide that is cleaved to produce a mature 283-amino acid protein. The deletion of the anchor allows the above protein to be prepared in a soluble form. · Human papillomavirus antigen(HPV). A preferred HPV antigen for use with the present invention is the LI capsid protein, which can be assembled to form a known structure as a virus-like particle (VLP). The VLP can be produced by recombinant expression of LI in yeast cells (e.g., in S. cerevisiae) or insect cells (e.g., in Spodoptera cells (e.g., S. frugiperda) or Drosophila cells). With respect to yeast cells, a plasmid vector can carry the LI gene; with respect to insect cells, a baculovirus vector can carry the LI gene. More preferably, the composition contains LI VLPs derived from both HPV-16 and HPV-18 strains. This bivalent combination has been shown to be very effective (Harper et al. (2004) Lancet 364(9447):1757-65). In addition to HPV-16 and HPV-18 strains, it is also possible to include LI VLPs derived from HPV-6 and HPV-11 strains. The use of oncogenic HPV strains is also possible. The vaccine may contain LI ranging from 20 to 60 μg / ml per HPV strain (for example, about 40 μg / ml). · anthrax antigen Anthrax is caused by Bacillus anthracis. Suitable B. anthracis antigens include the A components (lethal factor (LF) and edema factor (EF)), which together may share a common B component known as a protective antigen (PA) (J Toxicol Clin Toxicol (2001) 39:85-100; Demicheli et al. (1998) Vaccine 16:880-884; Stepanov et al. (1996) J Biotechnol 4AL55A60). These antigens can be detoxified as needed (J Toxicol Clin Toxicol (2001) 39:85-100; Demicheli et al. (1998) Vaccine 16:880-884; Stepanov et al. (1996) J Biotechnol 4ΑΛ55Α60). · S. aureus antigenVarious S. aureus antigens are known. Suitable antigens include capsular saccharides (e.g., derived from type 5 and / or type 8 strains) and proteins (e.g., IsdB, Hla, etc.). Capsular saccharide antigens are ideally conjugated to carrier proteins. · S.pneumoniae antigen Various S. pneumoniae antigens are known. Suitable antigens include capsular saccharides (e.g., derived from one or more of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and / or 23F) and proteins (e.g., pneumolysin, detoxified pneumolysin, polyhistidine triad protein D (PhtD), etc.). Capsular saccharide antigens are ideally conjugated to carrier proteins. · Cancer antigens Various tumor-specific antigens are known. The present invention can be used in conjunction with antigens that induce immunotherapeutic responses to lung cancer, melanoma, breast cancer, prostate cancer, and the like.

[0101] The antigen solution is typically mixed with an emulsion (for example, in a 1:1 volume ratio). This mixing may be done by the vaccine manufacturer before filling or by a healthcare professional at the time of use.

[0102] Pharmaceutical composition The antigen solution is typically mixed with an emulsion (for example, in a 1:1 volume ratio). This mixing may be done by the vaccine manufacturer before filling or by a healthcare professional at the time of use.

[0103] Compositions prepared using the method of the present invention are pharmaceutically acceptable. They may contain components in addition to emulsions and alternative antigens.

[0104] The above composition may contain a preservative (e.g., thimerosal or 2-phenoxyethanol). However, it is preferable that the vaccine be substantially mercury-free (i.e., less than 5 μg / ml) (e.g., thimerosal-free (Banzhoff)). (2000) Immunology Letters 71:91-96; WO02 / 097072). Mercury-free vaccines and components are more preferable.

[0105] The pH of the composition is generally between 5.0 and 8.1, more typically between 6.0 and 8.0, for example, between 6.5 and 7.5. The process of the present invention may therefore include a step of adjusting the pH of the vaccine before packaging.

[0106] The above composition is preferably sterile. The above composition is preferably free of pyrogens (e.g., containing <1 EU (endotoxin unit (standard scale)) per dose, and preferably <0.1 EU per dose). The above composition is preferably gluten-free.

[0107] The above composition may contain a substance for a single immunization or a substance for multiple immunizations (i.e., a "multi-dose" kit). The inclusion of a preservative is preferable in multi-dose combinations.

[0108] The vaccine is typically administered in a dose volume of approximately 0.5 ml, but half the dose (i.e., approximately 0.25 ml) may be administered to children.

[0109] Treatment methods and vaccine administration The vaccine is typically administered in a dose volume of approximately 0.5 ml, but half the dose (i.e., approximately 0.25 ml) may be administered to children.

[0110] The present invention provides kits and compositions prepared using the methods of the present invention. Compositions prepared according to the methods of the present invention are suitable for administration to human patients, and the present invention provides a method for inducing an immune response in a patient, the method comprising the step of administering such a composition to the patient.

[0111] The present invention also provides these kits and compositions for use as pharmaceuticals.

[0112] The present invention also provides (i) aqueous preparations of antigens; and (ii) the use of oil-in-water emulsions prepared according to the present invention in the manufacture of pharmaceuticals for inducing an immune response in patients.

[0113] The immune response induced by these methods and uses generally includes an antibody response, preferably a protective antibody response.

[0114] The above composition can be administered by various methods. The most preferred immune route is by intramuscular injection (e.g., into the arm or leg), but other available routes include subcutaneous injection and intranasal injection (Greenbaum et al. (2004) Vaccine 22:2566-77; Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304; Piascik (2003) J Am Pharm Assoc (Wash DC). 43:728-30), oral (Mann et al. (2004) Examples include Vaccine 22:2425-9), intradermal (Halperin et al. (1979) Am J Public Health 69:1247-50; Herbert et al. (1979) J Infect Dis 140:234-8), transcutaneous, and transdermal (Chen et al. (2003) Vaccine 21:2830-6).

[0115] Vaccines prepared according to the present invention may be used to treat both children and adults. Patients may be under 1 year of age, 1 to 5 years of age, 5 to 15 years of age, 15 to 55 years of age, or at least 55 years of age. The above patients may be the elderly (e.g., ≥50 years of age, preferably ≥65 years of age), young people (e.g., <5 years of age), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, patients with chronic illnesses, immunocompromised individuals, and people traveling abroad. The above vaccines are not suitable only for these groups, but more generally, they may be used in populations.

[0116] The vaccine of the present invention can be administered to a patient substantially simultaneously with other vaccines (for example, during the same medical consultation or visit to a healthcare professional).

[0117] intermediate process The present invention also provides a method for producing an oil-in-water emulsion, the method comprising the steps of: microfluidizing a first emulsion to form a second emulsion; and then filtering the second emulsion. The first emulsion has the above-described properties.

[0118] The present invention also provides a method for producing an oil-in-water emulsion, the method comprising filtering a second emulsion, i.e., a microfluidized emulsion, the microfluidized emulsion having the above-described properties.

[0119] The present invention also provides a method for producing a vaccine, the method comprising the step of combining an emulsion and an antigen, wherein the emulsion comprises the step of having the above-described properties.

[0120] Specific Embodiments Certain preferred embodiments of this disclosure are summarized in the following paragraphs. This list is illustrative and does not exhaustive of all embodiments provided by this disclosure.

[0121] Embodiment 1. A method for improving the filtration of an oil-in-water emulsion through a membrane filter, the method comprising the steps of filtering the oil-in-water emulsion through a membrane filter at a temperature below 10°C or equal to 10°C, wherein the filter throughput is increased compared to the filtration of an oil-in-water emulsion at a temperature above 10°C. Embodiment 2. A method for preparing an oil-in-water emulsion, the method comprising the steps of filtering the oil-in-water emulsion through a membrane filter at a temperature below 10°C or equal to 10°C, wherein the filter throughput is increased compared to a temperature higher than 10°C.

[0122] Embodiment 3. A method for preparing an oil-in-water emulsion, the method comprising the step of filtering the oil-in-water emulsion through a membrane filter with a greater filter throughput at temperatures below 10°C than at temperatures above 10°C.

[0123] Embodiment 4. A method for preparing an oil-in-water emulsion adjuvant, the method comprising the step of filtering the oil-in-water emulsion adjuvant through a membrane filter with a greater adjuvant throughput at a temperature of 5°C than at a temperature of 10°C.

[0124] Embodiment 5. A method for preparing an oil-in-water emulsion, the method comprising the steps of: mixing oil, aqueous components, and a surfactant to form an oil-in-water emulsion; microfluidizing the mixture to reduce the average droplet size of the oil-in-water emulsion; and filtering the microfluidized oil-in-water emulsion through a membrane filter at a temperature below 10°C or equal to 10°C, wherein the filter throughput is increased compared to filtering an oil-in-water emulsion at a temperature above 10°C.

[0125] Outline The present invention also provides a method for producing a vaccine, the method comprising the step of combining an emulsion and an antigen, wherein the emulsion comprises the step of having the above-described properties.

[0126] The terms "to include" and "to constitute" encompass both "including" and "consisting." For example, a composition "comprising" X may consist solely of X or it may contain something else (e.g., X + Y).

[0127] The phrase "substantially" does not exclude "completely." For example, "substantially does not include Y." The "free)" composition does not have to contain Y at all. Where necessary, the word "substantially" may be omitted from the definition of the present invention.

[0128] The term "about" in relation to a numerical value x is optional; for example, it can mean x + 10%.

[0129] Unless otherwise specifically stated, a process involving the mixing of two or more components does not require any specific order in which the components are mixed. Therefore, the components can be mixed in any order. For example, if there are three components, two components may be mixed with each other, and then that combination may be mixed with a third component.

[0130] When animal (and especially bovine) materials are used in cell culture, they should be obtained from sources free from infectious spongiform encephalopathy (TSE), and especially bovine spongiform encephalopathy (BSE). Overall, it is preferable to culture cells in the complete absence of animal-derived materials.

[0131] All of the claims in the claims are incorporated herein by reference in whole as further embodiments. [Examples]

[0132] Examples Example 1: MF59 (登録商標) Filter selection for filtration Several filters, including Express SHC, Express SHF, Durapore 0.22μm, and Durapore 0.45 / 0.22μm, are used with MF59. (登録商標) During filtration, testing was conducted to determine the optimal filter for enhancing filter durability during both the sizing and sterilization of the oil-in-water emulsion adjuvant described above. A description of the tested filters is shown in Table 1 below. [Table 1]

[0133] MF59 (登録商標) Filtration Vmax (L / m 2 The following measurements were taken for the above filters at various temperatures: 5°C, 30°C, and 40°C. All filters were isolated and run under a constant pressure of 43 psi. The low pressure was 22 psi and the high pressure was 50 psi.

[0134] In short, the protocol for Vmax filtration first involved installing the filter device in a pressure vessel having a stop-lock upstream of the filter. Next, 1000 L / m 2 The feed was added to the pressure vessel so that it could be filtered. The device was vented to properly remove air and pressurize the vessel.

[0135] Once filtration began, time and volume were recorded at regular intervals. The trial was terminated when all material had been used up or when >75% flux decay was observed.

[0136] For the tests conducted at 5°C, the test material was kept in a low-temperature room, then removed and immediately filtered at ambient temperature. For the tests conducted at 30°C and 40°C, the test material was warmed in a water bath, then removed and immediately filtered at ambient temperature.

[0137] The results of the Vmax test are shown in Table 2 below. [Table 2-1] [Table 2-2]

[0138] The Vmax test results showed that the sterile-grade filter achieved an MF of 59 with a pressure drop of 43 psi. (登録商標) Approximately 30 L / m³ 2 This indicates that filtration was possible up to a certain point. SHF, SHC, and Durapore filters showed improvement at 5°C compared to 30°C and 40°C. SHF had the best filtration capacity, followed by SHC. SHF performed slightly better at lower temperatures. Lot-to-lot variability between SHF and SHC was quite low. The above data suggests that increasing the pressure increases the filtration capacity. For example, SHC showed an improvement of approximately 1.5 × in durability when the pressure was increased from 42.9 psi to 49.9 psi.

[0139] conclusion Generally speaking, MF59 (登録商標) The filtration exhibits high clogging and shear viscosity reduction characteristics of sterile-grade filters. The Express SHF showed the most favorable filter hydrodynamics for all sterile-grade filters tested. The Express SHC High Area is a high-area device that provides twice the area per cartridge with similar performance compared to the MF59. (登録商標) We provided the minimum equipment necessary to process a 330L batch.

[0140] Separate filtration tests also showed that throughput significantly increased when performed at lower temperatures with various membranes, as shown in Tables 3 and 4 and Figures 1-3 below. [Table 3] [Table 4]

[0141] Table 5, shown below, also demonstrated that increasing the pressure further enhanced throughput at lower temperatures (e.g., 10°C). [Table 5]

[0142] References All references cited herein are incorporated herein by reference in their entirety.

Claims

[Claim 1] The invention described herein.

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