CIRCULATION OF COMPONENTS DURING HOMOGENEIZATION OF EMULSIONS

专利摘要:
circulation of the components during the homogenization of emulsions an improved method for the manufacture of an oil-in-water emulsion involving the circulation of the components of the emulsion between a first container and a second container through a homogenizer and / or through a microfluidization. usefully, all components of the emulsion from the first container are emptied, before being returned.
公开号:BR112012013427B1
申请号:R112012013427-2
申请日:2010-12-03
公开日:2021-03-30
发明作者:Harald Rueckl；Barbara Santry；Hanno Scheffczik
申请人:Novartis Ag；
IPC主号:

专利说明:

This application claims the benefit of United States Provisional Patent Application 61 / 283,518 filed on December 3, 2009, the complete contents of which are hereby incorporated by reference for all purposes. TECHNICAL FIELD
This invention is in the field of manufacturing oil-in-water emulsion adjuvants for microfluidization vaccines. BACKGROUND TECHNIQUE
The vaccine adjuvant known as 'MF59' [1-3] is an oil-in-water emulsion of squalene submicron, polysorbate 80 (also known as Tween 80), and sorbitan trioleate (also known as Span 85). It can also include citrate ions, for example, 10 mM sodium citrate buffer. The composition of the volume emulsion can be about 5% squalene, about 0.5% Tween 80 and about 0.5% Span 85. The adjuvant and its production are described in more detail in Chapter 10 of reference 4, chapter 12 of reference 5 and chapter 19 of reference 6.
As described in reference 7, MF5 9 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 thicker emulsion. This thicker emulsion is then passed several times through a microfluidizer to produce an emulsion having a uniform oil droplet size. As described in reference 6, the microfluidized emulsion is then filtered through a 0.22 µm membrane to remove any large oil droplets, and the average droplet size of the resulting emulsion remains unchanged for at least 3 years at 4 ° Ç. The squalene content of the final emulsion can be measured as described in reference 8. - - ___ „. „„
Oil-in-water emulsions contain oil droplets. The largest oil droplets contained in these emulsions can act as nucleation sites for aggregation, leading to degradation of the emulsion during storage.
It is an object of the invention to provide additional and improved methods for the production of microfluidized oil-in-water emulsions (such as MF59), in particular methods that are suitable for use on a commercial scale, and that provide improved homogenization and microfluidization to provide emulsions with less large particles. DESCRIPTION OF THE INVENTION
The invention provides a method for making an oil-in-water emulsion comprising squalene, the method comprising the step of (i) forming a first emulsion having a first medium oil droplet size using a homogenizer, wherein the first emulsion it is formed by circulating the first components of the emulsion through a homogenizer, a plurality of times.
The invention also provides a method for making an oil-in-water emulsion comprising squalene, the method comprising the step of: (b) microfluidizing a first emulsion having a first medium oil droplet size to form a second emulsion having a second medium oil droplet size which is smaller than the first medium oil droplet size, where the second emulsion is formed by circulating the second emulsion components by transferring the second emulsion components from a first container of the emulsion, through a first microfluidization device to a second emulsion container, and then through a second microfluidization device, the first and second microfluidization devices being the same.
Optionally, the method of the present invention comprises a previous step of (a) forming a first emulsion having a first average oil droplet size.
Optionally, the method of the present invention comprises the step of (c) filtering the second emulsion.
As described in more detail below, the first emulsion can have an average oil droplet size of 5000nm or less, for example, an average size between 300nm and 800nm. The number of oil droplets in the first emulsion with a size> 1.2 μm can be 5 x 101: L / ml or less, as described below. Oil droplets with a size> 1.2 μm are disadvantageous since they can cause instability of the emulsion due to the agglomeration and coalescence of the droplets of [14].
After formation, the first emulsion can then be subjected to at least one microfluidization pass to form the second emulsion having a reduced average oil droplet size. As described below, the average oil droplet size of the second emulsion is 500 nm or less. The number of oil droplets in the second emulsion having a size> 1.2 μm can be 5 x 10 / ml or less, as described below. To achieve these characteristics it may be necessary to pass the components of the emulsion through the microfluidization device a plurality of times, for example, 2, 3, 4, 5, 6, 7 times.— -— The second emulsion_. ~ PQde. ^ Ejitãa__ S-X filtered, for example, through a hydrophilic polyethersulfone membrane, to produce an oil-in-water emulsion that may be suitable for use as a vaccine adjuvant. The average oil droplet size of the oil-in-water emulsion produced after filtration can be 220 nm or less, for example, between 135-175 nm, between 145-165 nm, or about 155 nm. The number of oil droplets having a size> 1.2 μm present in the oil-in-water emulsion produced after filtration may be 5 x 108 / ml or less, some components may still occur and such low temperatures are preferred. Emulsion Components
The average oil droplet size (that is, the average numerical diameter of oil emulsion droplets) - can be measured using a dynamic light dispersion technique, as described in reference 13. An example of a dispersion measuring machine dynamic light is the Nicomp 380 Sub-micron Particle Size Analyzer (from Particle Sizing Systems).
The number of particles having a size> 1.2 μm can be measured using a particle counter, such as the Accusizer ™ 770 (from Particle Sizing Systems).
The methods of the invention are used for the manufacture of oil-in-water emulsions. These emulsions include three essential ingredients: an oil; an aqueous component, and a surfactant.
Since the emulsions are intended for pharmaceutical use, then the oil will typically be biodegradable (metabolizable) and biocompatible.
The oil used may comprise squalene, a shark liver oil, which is an unsaturated branched terpenoid (C3oH50; [(CH3) 2C [= CHCH2CH2C (CH3)] 2 = CHCH2-] 2; 2,6,10,15, 19,23-hexamethyl-2,6,10,14,18,22-tetracosaexane; CAS RN 7683-64-9). Squalene is particularly preferred for use in the present invention.
The oil of the present invention can comprise a mixture of oils (or combination), for example, comprising squalene and at least one additional oil.
Instead of (or in addition to) using squalene, an emulsion may comprise oil (s) including those from, for example, an animal (such as fish) or vegetable source. Sources of vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil and olive oil, the most commonly available, exemplary nut oils. Jojoba oil can be used, for example, obtained from jojoba grain. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but oil from other cereal grains, such as wheat, oats, rye, rice, teff, triticale and the like can also be used. Fatty acid esters of 6-10 carbons of glycerol and 1,2 propanediol, while not naturally occurring in seed oils, can be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the oils of nuts and seeds. Mammalian milk fats and oils are metabolizable and can thus be used. The procedures for separation, purification, saponification and other means necessary to obtain pure oils from animal sources are well known in the art.
Most fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils, and whale oil, such as spermaceti, exemplify several of the fish oils that can be used here. Various branched-chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Squalane, the saturated analog for squalene, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known in the art.
Other useful oils are tocopherols, particularly in combination with squalene. When the oil phase of an emulsion includes a tocopherol, any of the tocopherols a, p, Y ε or K can be used, however, a-tocopherols are preferred. D-α-tocopherol and DL-a-tocopherol both can be used. A preferred a-tocopherol is DL-a-tocopherol. Tocopherol can take many forms, for example, different salts and / or isomers. Salts include organic salts, such as succinate, acetate, nicotate, etc. If a salt of this tocopherol is to be used, the preferred salt is succinate. A combination of oil comprising squalene and tocopherol (for example, DL-a-tocopherol) can be used.
The aqueous component can be simple water (for example, w.f.i) or it can include additional components, for example, solutes. For example, it can include salts to form a buffer, for example, citrate or phosphate salts, such as sodium salts. Typical buffers include: a phosphate buffer; a buffer Tris; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20mM range.
The surfactant is preferably biodegradable (metabolizable) and biocompatible. Surfactants can be classified by their 'HLB' (hydrophilic / lipophilic balance), where an HLB in the range of 1-10- generally means that the surfactant is more soluble in oil than in water, and an HLB in the range of 10-20 it is more soluble in water than in oil. Emulsions preferably comprise at least one surfactant that has an HLB ———— of at least 10. for example, at least 15, or preferably at least 16.
The invention can be used with surfactants, including, but not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the trade name DOWFAX ™, such as linear EO / PO block copolymers; octoxynols, which may vary in the number of repetition of ethoxy groups (oxy-1,2-ethanedylyl), with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether, and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for inclusion in the emulsion are polysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
Mixtures of surfactants can be included in the emulsion, for example, mixtures of Tween 80 / Span 85, or mixtures of Tween 80 / Triton-X100. A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol as such. t-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises lauret 9 in addition to a polyoxyethylene sorbitan ester and / or an octoxynol. Useful mixtures may comprise a surfactant with an HLB value in the range of 10-20 (eg, Tween 80, with an HLB of 15.0) and a surfactant with an HLB value in the range of 1-10 (eg, Span 85, with an HLB of 1.8). Formation of the first emulsion
Before the microfluidization step, the components of the emulsion can be mixed to form a first emulsion.
Oil droplets in the first emulsion can have an average size of 5000 nm or less, for example, 4000 nm or less, 3000nm or less, 2000nm or less, 1200nm or less, 10000m or less, for example, an average size between 800 and 1200 nm, or between 300nm and 800nm.
In the first emulsion the number of oil droplets with a size> 1.2 μm can be 5 x 101: L / ml or less, for example, 5 x 1010 / ml or less, or 5 x 109 / ml or less.
The first emulsion can then be microfluidized to form a second emulsion having a smaller average oil droplet size than the first emulsion and / or less oil droplets> 1.2 µm in size.
The average oil droplet size of the first emulsion can be achieved by mixing the first components of the emulsion in a homogenizer. For example, as shown in Figure 1, they can be combined in a mixing vessel (12) and then the combined components can be introduced (13) into a mechanical homogenizer, such as a rotor-stator homogenizer (1).
Homogenizers can work in a vertical and / or horizontal way. For convenience in a commercial setting, in-line homogenizers are preferred.
The components are inserted in a rotor-stator homogenizer and find a fast rotating rotor containing grooves or holes. The components are discarded by centrifugation in a similar manner to the pump and pass through the grooves / holes. In some embodiments, the homogenizer includes multiple combinations of rotor and starter, —by and example, —a — concentric — arrangement — of — comb teeth rings, as shown by features (3) and (4), ( 5) and (6) and (7) and (8) in Figure 1 and Figure 2. Rotors in useful large-scale homogenizers may have comb teeth rings at the end of a horizontally oriented multi-laminated impeller (for example , characteristic (9) of Figure 1) aligned in close tolerance so that it satisfies the teeth in a static lining. The first emulsion is formed through a combination of cavitation, turbulence, and mechanical shear that occurs within the gap between the rotor and the stator. The components are usefully introduced in a direction parallel to the rotor axis.
An important parameter in the performance of rotor-stator homogenizers is the speed of the rotor tip (peripheral speed). This parameter is a function of both the speed of rotation and the diameter of the rotor. A peak speed of at least 10 ms "1 is useful, and ideally faster, for example> 20 ms'1,> 30 ms'1,> ■ 4 0 ms'1, etc. A peak speed of 4 0 ms'1 can be easily achieved at 10,000 rpm with a small homogenizer or at lower rotation speeds (eg 2000 rpm) with a larger homogenizer, suitable high-shear homogenizers are commercially available.
For commercial scale manufacturing, the homogenizer should ideally have a flow rate of at least 3 00 L / h, for example, _> 400 L / h,> 500 L / h,> 600 L / h,> 700 L / h,> 800 L / h,> 900 L / h,> 1000 L / h,> 2000 L / h,> 5000 L / h, or even> 10000 L / h. Suitable high capacity homogenizers are commercially available.
A preferred homogenizer provides a shear rate between 3x103 and 1x105'1, for example, between 3x105 and 7x105 s "1, between 4x105 and 6x105 s'1, for example, about 5x105 s" 1.
Although rotor-stator homogenizers generate relatively little heat during operation, the homogenizer can be cooled during use. Ideally, the temperature of the first emulsion is kept below 60 ° C during homogenization, for example, below 45 ° C.
In some embodiments, the components of the first emulsion may be homogenized several times (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more). To avoid the need for a long chain of containers and homogenizers, the components of the emulsion can instead be circulated (for example, as shown by feature (11) in Figure 1). In particular, the first emulsion can be formed by circulating the components of the first emulsion through a homogenizer a plurality of times (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 times, etc.). However, many cycles can be undesirable because it can produce recoalescence as described in reference 14. In this way, the oil droplet size can be monitored if homogenizing circulation is used to verify that a desired droplet size is achieved and / or that recoalescence is not occurring.
Circulation through the homogenizer is advantageous because it can reduce the average oil droplet size in the first emulsion. Circulation is also advantageous because it can reduce the number of oil droplets having a size> 1.2 μm in the first emulsion. These reductions in average droplet size and droplet number> 1.2 μm in the first emulsion can provide advantages in the downstream process (ES). In particular, the circulation of the components of the first emulsion through the homogenizer can lead to an improved process than microfluidization which can then result in a reduced number of oil droplets having a size> 1.2 μm in the second emulsion, that is, after microfluidization. . This improvement in the parameters of the second emulsion can provide better filtration performance. Improved filtration performance can lead to less loss of content during filtration, for example, losses of squalene, Tween 80 and Span 85, when the oil-in-water emulsion is MF59.
Two particular types of circulation are referred to herein as "type I" and "type II". Type I circulation is illustrated in Figure 5, while type II circulation is illustrated in Figure 6. ~
The circulation of the components of the first emulsion can comprise a type I circulation of transfer of the components of the first emulsion between a first container of the premix and a homogenizer. The first pre-mix container can be 50-500L in size, for example, 100 to 400 L, 100 to 300 L, 200 to 300 L, 250 L or 280 L. The first pre-mix container can be manufactured from from stainless steel. Type I circulation can be continued for 10 to 60 minutes, for example, TXT at 4 Cr minutes or 20 minutes.
The circulation of the components of the first emulsion can comprise a type II circulation of transfer of the components of the first emulsion from a first pre-mixing container, through a first homogenizer to a second pre-mixing container (optionally having the same properties than the first pre-mix container), and then through a second homogenizer. The second homogenizer will generally be the same as the first homogenizer, however in some arrangements the first and second homogenizers are different. After passing the components of the first emulsion through the second homogenizer, the components of the first emulsion can be transferred back to the first premix container, for example, if the type II circulation process is to be repeated. In this way, the components of the emulsion can travel in a figure of eight paths between the first and second pre-mix containers, through a single homogenizer (see Figure 6). Type II circulation can be performed only once or several times, for example 2, 3, 4, 5 times, etc.
The type II circulation is advantageous compared to the type I circulation, as it can help to ensure that all components of the first emulsion pass through the homogenizer. Emptying the first pre-mix container means that the complete emulsion contents have passed through the homogenizer into the second pre-mix container. Likewise, the contents of the second pre-mix container can be emptied, again ensuring that all pass through the homogenizer. In this way, the type II arrangement can conveniently ensure that all components of the emulsion are homogenized at least twice, which can reduce both the average oil droplet size and the number of oil droplets having a size> 1 , 2 μm in the first emulsion. An ideal type II circulation in this way involves emptying the first pre-mix container and passing substantially all of its contents through the homogenizer into the second pre-mix container, followed by emptying the second pre-mix container and re-passing substantially all of its contents through homogenizer back into the first (empty) premix container. In this way, all particles pass through the homogenizer at least twice, which is difficult to achieve with type I circulation.
In some embodiments, a combination of type I and type II circulations is used, and this combination can provide a first emulsion with good characteristics. In particular, this combination can greatly reduce the number of oil droplets having a size> 1.2 μm in the first emulsion. This combination can comprise any order of circulation type I and II, for example, type I, followed by type II, type II, followed by type I, type I, followed by type II, followed by type I again, etc. In one embodiment, the combination comprises 20 minutes of the type I circulation followed by a single type II circulation, that is, transferring the components of the first emulsion circulated from a first pre-mix container; through a first homogenizer — to — a second pre-mix container and then through a second homogenizer once.
The first and second premix containers can be kept under an inert gas, for example, nitrogen, for example, up to 0.5 bar. This can prevent the components of the emulsion from oxidizing, which is particularly advantageous if one of the components of the emulsion is squalene. This can provide an increase in the stability of the emulsion.
As mentioned above, the initial input to the homogenizer can be a non-homogenized mixture of the components of the first emulsion. This mixture can be prepared by mixing the individual components of the first emulsion individually, but in some embodiments, the multiple components can be combined before this mixing. For example, if the emulsion includes a surfactant with an HLB less than 10, then this surfactant can be combined with an oil before mixing. Similarly, if the emulsion includes a surfactant with an HLB greater than 10, then this surfactant can be combined with an aqueous component before mixing. Buffer salts can be combined with an aqueous component before mixing, or they can be added separately.
The methods of the invention can be used on a large scale. Thus, a method may involve preparing a first emulsion whose volume is greater than 1 liter, for example,> 5 liters, _> 10 liters, _> 20 liters,> 50 liters,> 100 liters,> 250 liters , etc.
After its formation, the product — emulsion — can — be microfluidized, or it can be stored to await microfluidization.
In some embodiments, in particular those where multiple cycles of steps (i) and (ii) are used, the input to the homogenizer will be the output of a microfluidizer, such that the first emulsion is microfluidized and then subjected to homogenization again. Microfluidization
After its formation, the first emulsion is microfluidized in order to reduce its average oil droplet size and / or to reduce the number of oil droplets having a size of> 1.2 μm.
Microfluidization instruments reduce the size of the average oil droplets by propelling incoming component currents through channels geometrically fixed at high pressure and high speed. The pressure at the entrance to the interaction chamber (also called the "first pressure") can be substantially constant (i.e., ± 15%; for example, ± 10%, ± 5%, + 2%) for at least 85% of the time during which the components are fed into the microfluidizer, for example, at least 87%, at least 90%, at least 95%, at least 99% or 100% of the time during which the emulsion is fed into the microfluidizer .
In one embodiment, the pressure is first 13 00 bar + 15% (18 kPSI + 15%), that is, between 1100 bar and 1500 bar (between 15 kPSI and 21 kPSI) for 85% of the time during which the emulsion and ãTimentimented in the microfluidizer ^ Two suitable pressure profiles are shown in Figure 3. In Figure 3A the pressure is substantially constant for at least 85% of the time, while in Figure 3B the pressure continuously remains substantially constant.
A microfluidization apparatus typically comprises at least one intensifier pump (preferably two pumps, which can be synchronous) and an interaction chamber. The intensifier pump, which is ideally electrically-hydraulically driven, delivers the high pressure (ie, the first pressure) to force an emulsion into and through the interaction chamber. The synchronous nature of the intensifier pumps can be used to provide the substantially constant pressure of the emulsion described above, which means that the emulsion droplets are all exposed to substantially the same level of shear forces during microfluidization.
An advantage of using a substantially constant pressure is that it can reduce fatigue failures in the microfluidization device, which can lead to a longer life of the device. An additional advantage of using a substantially constant pressure is that the parameters of the second emulsion can be improved. In particular, the number of oil droplets having a size> 1.2 μm present in the second emulsion can be reduced. In addition, the average oil droplet size of the second emulsion can be reduced when a substantially constant pressure is used. Reducing the average oil droplet size and the number of oil droplets having a size> 1.2 μm in the second emulsion can provide improved filtration performance. The improved filtration performance can lead to less loss of content during filtration, for example, the losses of squalene, Tween 80 and Span 85, when the emulsion is MF59.
The interaction chamber can contain a plurality, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., of fixed geometry channels through which the emulsion passes. The emulsion enters the interaction chamber through an entry line that can have a diameter between 200 to 250 μm. The emulsion divides into currents as it enters the interaction chamber and, under high pressure, accelerates at high speed. As it passes through the channels, the forces produced by the high pressure can act to reduce the size of the emulsion oil droplets and reduce the number of oil droplets having a size> 1.2 μm. These forces may include: shear forces, by means of deformation of the emulsion flow occurring from contact with the channel walls; impact forces, through collisions that occur when high-speed emulsion flows collide with each other, and cavitation forces, through the formation of cavities within the flow. The interaction chamber generally does not include moving parts. It can include ceramic (eg alumina) or diamond (eg polycrystalline diamond) channel surfaces. Other surfaces can be made of stainless steel.
The fixed geometry of the plurality of channels in the interaction chamber can be "Y" type geometry or "z" type geometry.
In a Y-type geometry interaction chamber, a single emulsion inlet stream ^ is divided into first and second emulsion streams, which are then recombined into a single emulsion outflow. Before recombination, each of the first and second emulsion streams can independently be divided into a first and second plurality (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), of sub- flows. When the emulsion streams are recombined, the first and second emulsion streams (or their sub-streams) are ideally flowing in substantially opposite directions (for example, the first and second emulsion streams, or their sub-streams, are flowing substantially in the same plane (± 20 °) and the flow direction of the first emulsion stream is 180 ± 20 ° different from the flow direction of the second emulsion stream). The forces produced when the emulsion streams are recombined can act to reduce the size of the emulsion oil droplets and reduce the number of oil droplets having a size> 1.2 μm.
In an interaction chamber of type Z geometry the emulsion flow passes around a plurality (for example, 2, 3, 4, 5, 6, 7, 8, 9,10, etc.), of substantially right-angled corners (i.e., 90 + 20 °). Figure 4 illustrates an interaction chamber with Z-type geometry and two right-angled corners in the flow direction. During its passage around the corners, an incoming emulsion flow can be divided into a plurality (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), of sub-flows and then recombined in a single outgoing emulsion stream (for example, as shown in Figure 4, with four sub-streams (32)). Division and then recombination (31) can occur at any point between entry and exit. The forces produced when the emulsion contacts the channel walls as it passes around the corners can act to reduce the size of the emulsion oil droplets and reduce the number of oil droplets having a size> 1.2 μm. An example of a Z-type interaction chamber is the E230Z interaction chamber from Microfluids.
In one embodiment, the emulsion flow passes around two corners at a substantially right angle. At the point when the incoming emulsion stream passes around the first substantially right angle, it is divided into five sub-streams. At the point when the subflows pass around the second corner at a substantially right angle, they are recombined in a single emulsion outlet stream.
In the prior art, it has been customary to use Y-type interaction chambers for oil-in-water emulsions such as those of the present invention. However, we have found it advantageous to use a Z-channel geometry interaction chamber for oil-in-water emulsions because this can lead to a further reduction in the number of oil droplets having a size of> 1.2 μm present in the second emulsion compared to a Y-type geometry interaction chamber. Reducing the number of oil droplets having a size> 1.2 μm in the second emulsion can provide improved filtration performance. Improved filtration performance can lead to less loss of content during filtration, for example, losses of squalene, Tween 80 and Span 85, when the emulsion is MF5 9.
A preferred microfluidization apparatus operates at a pressure between 170 bar and ~ 27HCrújar (about ~ 2,500 = 40,000 ps ± - psi), for example at about 345 bar, at about 690 bar, at about 1380 bar, at about 2070 bar, etc.
A preferred microfluidization device operates at a flow rate of up to 20 L / min, for example, up to 14 L / min, up to 7 L / min, up to 3.5 L / min, etc.
A preferred microfluidization apparatus has an interaction chamber that provides a shear rate in excess of 1x106 s'1, for example,> 2.5x106 -s'1,> 5X106 s'x,> 107 s'1, etc.
A microfluidization device can include multiple interaction chambers that are used in parallel, for example, 2, 3, 4, 5 or more, but it is more useful to include a single interaction chamber.
The microfluidization device may comprise an auxiliary processing module (APM), comprising at least one channel. The APM contributes to the reduction of the average size of the oil droplets in the emulsion being passed through the microfluidization device, although most of the reduction occurs in the interaction chamber. As mentioned above, the emulsion components are introduced into the interaction chamber by the intensifier pump (s), under a first pressure. The components of the emulsion generally leave the APM at a second pressure that is less than the first pressure (atmospheric pressure, for example). In general, between 80 and 95% of the pressure difference between the first and second pressures is reduced through the interaction chamber (for example, from Pi to P2 in Figure 4) and 5 to 20% of the pressure difference between the first and second second pressures are reduced through the auxiliary processing module, for example, the interaction chamber can provide approximately 90% of the pressure drop while the APM can provide approximately 10% of the pressure drop. If the pressure that dropped through the interaction chamber and the pressure that dropped through the auxiliary processing module are not responsible for the entire pressure difference between the first and the second pressure, this could be due to a finite pressure drop across the connectors between the interaction chamber and the auxiliary processing module.
APM generally does not include moving parts. It can include ceramic channel surfaces (for example, alumina) or diamond (for example, polycrystalline diamond). Other surfaces can be made of stainless steel.
The APM is generally positioned downstream from the interaction chamber and can also be positioned sequentially to the interaction chamber. In the prior art, APMs are generally positioned downstream of the interaction chambers comprising Y-type channels to suppress cavitation and thereby increase the flow rate in the Y-type chamber by up to 30%. In addition, in the prior art APMs are generally positioned upstream of the interaction chambers comprising Z-type channels to reduce the size of large clusters. In the latter case, APM only decreases the flow rate in type Z chambers by up to 3%. However, it has been found that the downstream APM positioning of an interaction chamber comprising a plurality of Z-type channels is advantageous in the present invention-because it can lead to a greater reduction in the size of medium oil droplets and a greater reduction in the number of oil droplets having a size of> 1.2 μm present in the second emulsion. As described above, the reduction in the number of oil droplets having a size> 1.2 μm in the second emulsion can provide improved filtration performance. Improved filtration performance can lead to less loss of content during filtration, losses, for example, of squalene, Tween 80 and Span 85, when the oil-in-water emulsion is MF59. Another advantage of this placement of a Z-type interaction chamber and a downstream APM is that it can lead to a slower decrease in pressure after the interaction chamber. The slower decrease in pressure can lead to an increase in the stability of the product, because there is less gas added to the emulsion.
An APM contains at least one channel of fixed geometry within which the emulsion passes. The APM can contain a plurality, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., of fixed geometry channels within which the emülsaõ passes. The APM channel or channels can be linear or non-linear. Suitable non-linear channels are of "Z" type geometry or "Y" type geometry, which are the same as those described above for the interaction chamber. In one embodiment, the APM channel or channels are of Z-type geometry. A plurality of Z-type channels divide the emulsion into flows that enter the APM.
In contrast to the manufacturer's recommendations, the use of an APM comprising a plurality of channels of fixed geometry is advantageous over unr ^ APM-de — canair ~ de ~ simple fixed geometry, because this can lead to a greater reduction in the number of oil droplets having a size> 1.2 μm present in the second emulsion. As described above, the reduction in the number of oil droplets having a size> 1.2 μm in the second emulsion can provide improved filtration performance. Improved filtration performance can lead to less loss of content during filtration, for example, losses of squalene, Tween 80 and Span 85, when the oil-in-water emulsion is MF59.
A microfluidization device generates heat during operation, which can raise the temperature of an emulsion by 15-20 ° C compared to the first emulsion. Advantageously, therefore, the microfluidized emulsion is cooled as quickly as possible. The temperature of the second emulsion can be kept below 60 ° C, for example, below 45 ° C. In this way, the output from an interaction chamber and / or the output of an APM can feed on a cooling mechanism, such as a heat exchanger or cooling coil. The distance between the outlet and the cooling mechanism should be kept as short as possible1 to shorten the overall time, reducing cooling delays. In one embodiment, the distance between the outlet of the microfluidizer and the cooling mechanism is between 20-30cm. A cooling mechanism is particularly useful when an emulsion is subjected to multiple microfluidization steps, to avoid overheating the emulsion.
The result of microfluidization is an oil-in-water emulsion, the second emulsion, in which the average oil droplet size is 500 nm or less. This average size is particularly useful as it facilitates the sterilization of the emulsion filter. Emulsions in which at least 80% by number of oil droplets have an average size of 500 nm or less, for example, 400 nm or less, 300 nm less, 200 nm or less, or 165 nm or less, are particularly useful. In addition, the number of oil droplets in the second emulsion having a size of> 1.2 µm is 5x109 / ml or less, for example, 5x109 / ml or less, 5x108 / ml or less, or 2x108 / ml or less.
The initial entry into microfluidization can be the first emulsion. In some embodiments, however, the microfluidized emulsion is subjected to microfluidization again, in such a way that multiple microfluidization cycles occur. In particular, the second emulsion can be formed by circulating the components of the second emulsion through a microfluidization device a plurality of times, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. The second emulsion can be formed by circulating the components of the second emulsion through a microfluidization device 4 to 7 times.
The circulation of the components of the second emulsion can comprise a type I circulation of transferring the components of the second emulsion between a first emulsion container (optionally having the same properties as the first premix container) and the microfluidization device.
The circulation of the components of the second emulsion may comprise a type II circulation of transferring the components of the second emulsion from a first emulsion container, through a first emulsion device (to — a — second — container — of the emulsion (optionally) having the same properties as the first premix container), and then through a second microfluidization device.
The second microfluidization device can be the same as the first microfluidization device. Alternatively, the second microfluidization device can be different from the first microfluidization device.
The first container of the emulsion can be the same as the first container of premixture. Alternatively, the first emulsion container can be the same as the second premix container.
The second container of the emulsion can be the same as the first container of premixture. Alternatively, the second emulsion container may be the same as the second premix container.
The first container of the emulsion may be the same as the first container of premixture and the second container of the emulsion may be the same as the second container of premixture. Alternatively, the first emulsion container may be the same as the second premix container and the second emulsion container may be the same as the first premix container.
As an alternative, the first and second emulsion containers may be different for the first and second premix containers.
After passing the components of the second emulsion through the second microfluidization device, the components of the second emulsion can be transferred back to the first container of the ~ emuisãcr, —for — for example-, if the type of circulation process II has to be repeated. Type II circulation can be performed only once or a plurality of times, for example 2, 3, 4, 5 times, etc.
Type II circulation is advantageous as it ensures that all components of the second emulsion have passed through the microfluidization device at least 2 times, which reduces the average size of the oil droplets and the number of oil droplets having a size> 1 , 2 μm in the second emulsion.
A combination of type I circulation and type II circulation can be used during microfluidization. This combination can comprise any circulation order of type I and II, for example, type I, followed by type II, type II, followed by type I, type I, followed by type II, followed by type I again, etc.
The first and second emulsion containers can be kept under an inert gas, for example, up to 0.5 bar of nitrogen. This prevents the components of the emulsion from oxidizing, which is particularly advantageous if one of the components of the emulsion is squalene. This leads to an increase in the stability of the emulsion.
The methods of the invention can be used on a large scale. Thus, a method may involve microfluidization of a volume greater than 1 liter, for example,> 5 liters, 10 liters,> 20 liters,> 50 liters,> 100 liters,> 250 liters, etc. Filtration
After microfluidization, the second emulsion is filtered. This filtration removes large oil droplets that have survived homogenization and microfluidization procedures. Although small in terms of number, these oil droplets can be large in terms of volume and can act as nucleation sites for aggregation, leading to degradation of the emulsion during storage. In addition, this filtration step can achieve filter sterilization.
The particular filtration membrane suitable for filter sterilization depends on the fluid characteristics of the second emulsion and the degree of filtration required. Characteristics of a filter can affect its ability to filter the microfluidized emulsion. For example, its pore size and surface characteristics can be important, particularly when filtering a squalene based emulsion.
The pore size of the membranes used with the invention should allow for the passage of the desired droplets while retaining the unwanted droplets. For example, it should retain droplets that are> Iμm in size while allowing <200nm drops to pass through. A 0.2 μm or 0.22 μm filter is ideal, and you can also achieve sterilization of the filter.
The emulsion can be pre-filtered, for example, through a 0.45 μm filter. Pre-filtration and filtration can be achieved in a single step by the use of known double layer filters that include a first membrane layer with larger pores and a second membrane layer with smaller pores. Double-layer filters are particularly useful with the invention ^ The first layer- ideally has a pore size> 0.3 μm, such as between 0.3-2 μm or between 0.3-1 μm, or between 0.4 -0.8 μm, or between 0.5-0.7 μm. A pore size of <0.75 μm in the first layer is preferred. Thus, the first layer can have a pore size of 0.6 μm or 0.45 μm, for example. The second layer ideally has a pore size that is less than 75% of (and, ideally less than half) the pore size of the first layer, such as between 25-70%, or between 25-49%, for example. example, of the first layer of pore size between 30-45%, such as 1/3 or 4/9, of the pore size of the first layer. Thus, the second layer can have a pore size <0.3 μm, such as between 0.15-0.28 μm or between 0.18-0.24 μm, for example, a pore size of the second layer 0.2 μm or 0.22 μm. In one example, the first membrane layer with larger pores provides a 0.45 μm filter, while the second membrane layer with smaller pores provides a 0.22 μm filter.
The filtration membrane and / or the pre-filtration membrane can be asymmetrical. An asymmetric membrane is one in which the pore size varies from one side of the membrane to the other, for example, in which the pore size is larger on the inlet side than on the outlet side. One side of the asymmetric membrane can be referred to as the "coarse pore surface", while the other side of the asymmetric membrane can be referred to as the "fine pore surface". In a double-layer filter, one or (ideally) both layers can be asymmetrical.
The filtration membrane can be porous or homogeneous. A homogeneous membrane is generally a "film" dense, ranging from 10 to 200 μm. A porous membrane has a porous structure. In one embodiment, the filtration membrane is porous. In a double layer filter, both layers can be porous, both layers can be homogeneous, or they can have a porous and homogeneous layer. A preferred double layer filter is one with both layers being porous.
In one embodiment, the second emulsion is pre-filtered through an asymmetric, porous hydrophilic membrane and then filtered through another asymmetric porous hydrophilic membrane having smaller pores than the pre-filtration membrane. This can use a double layer filter.
The membrane filter (s) can be autoclaved before use to ensure that it is sterile.
Filtration membranes are typically made of polymeric support materials such as PTFE (poly-tetra-fluoro-ethylene), PES (polyethersulfone), PVP (polyvinylpyridine), PVDF (polyvinylidene fluoride), nylon (polyamides), PP ( polypropylene), celluloses (including cellulose esters), PEEK (polyetheretherketone), nitrocellulose, etc. These have varied characteristics, with some support being intrinsically hydrophobic (eg, PTFE) and others being intrinsically hydrophilic (eg, cellulose acetates). However, these intrinsic characteristics can be modified by surface treatment of the membrane. For example, it is known to prepare hydrophilized membranes or other polymers, graphite, silicone, etc.) to coat the membrane surface, for example, see section 2.1 of reference 15. In a double layer filter the two membranes can be made different materials or (ideally) the same material.
An ideal filter for use with the invention has a hydrophilic surface, in contrast to the teaching of references 9-12 that hydrophobic filters (polysulfone) should be used. Filters with hydrophilic surfaces can be formed from hydrophilic materials, or by hydrophilizing hydrophobic materials, and a preferred filter for use with the invention is a hydrophilic polyethersulfone membrane. Several different methods are known to transform hydrophobic PES membranes into hydrophilic PES membranes. It is usually obtained by coating the membrane with a hydrophilic polymer. To provide permanent fixation of the hydrophilic polymer to the PES a hydrophilic coating layer is usually subjected either to a crosslinking reaction or to the graft. Reference 15 describes a process for modifying the surface properties of a hydrophobic polymer having functionalizable chain ends, comprising contacting the polymer with a solution of a binder portion to form a covalent bond, and then contacting the hydrophobic polymer reacted with a solution of a modification agent. Reference 16 describes a method of hydrophilizing the PES membrane by direct membrane coating, involving pre-wetting with alcohol, and then soaking in an aqueous solution — containing a hydrophilic monomer, —a polyfunctional monomer (crosslinking) and a polymerization initiator. The monomer and crosslinking is then polymerized using UV or thermal initiated polymerization to form a hydrophilic crosslinked polymer coating on the membrane surface. Similarly, references 17 and 18 describe coating a PES membrane by immersion in an aqueous solution of hydrophilic polymer (polyalkylene oxide) and at least one polyfunctional monomer (crosslinking) and then polymerizing a monomer to provide a non-extractable hydrophilic coating . Reference 19 describes the hydrophilization of the PES membrane by a graft reaction and a PES membrane is subjected to treatment with low temperature helium plasma followed by grafting of hydrophilic N-vinyl-2-pyrrolidone (NVP) onto the membrane surface . Other such processes are described in references 20 to 26.
In non-coating methods, PES can be dissolved in a solvent, mixed with a soluble hydrophilic additive, and then the mixed solution is used for melting a hydrophilic membrane, for example, by precipitation or initiating co-polymerization. Such methods are described in references 27 to 33. For example, reference 33, describes a method of preparing a hydrophilic modified charge membrane that has extractable base membranes and allows for the rapid recovery of ultrapure water resistivity, having a network structure. of cross-linked inter-penetrating polymer formed by making a polymer solution of a mixture of PES, PVP, polyethyleneimine, and "diglycidylcoataphatic ether -, - forming a thin film of the solution, and precipitating the film as a membrane. A similar process is described in reference 34.
Hybrid approaches can be used, in which hydrophilic additives are present during the formation of the membrane and are also added later as a coating, for example, see reference 35.
Hydrophilization of the PES membrane can also be achieved by treatment with low temperature plasmas. Reference 36 describes the hydrophilic modification of the PES membrane by treatment with low temperature CO2-plasma.
Hydrophilization of the PES membrane can also be achieved by oxidation, as described in reference 37. This method involves pre-moistening a hydrophobic PES membrane in a liquid with a low surface tension, exposing the wet PES membrane to an aqueous solution of the oxidizer and, then heat.
Phase inversion can also be used, as described in reference 38.
An ideal hydrophilic PES membrane can be obtained by treating PES (hydrophobic) with PVP (hydrophilic). Treatment with PEG (hydrophilic) instead of PVP has been found to produce a hydrophilized PES membrane that is easily soiled (especially when using an emulsion containing squalene) and also disadvantageously releases formaldehyde during autoclaving.
A preferred double layer filter has a first hydrophilic membrane PES and a second hydrophilic membrane "PESl
Well-known hydrophilic membranes include Bioassure (from Cuno); polyethersulfone from EverLUX ™; polyethersulfone from STyLUX ™ (both from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200 and Durapore CVGL01TP3 membranes (from Millipore); Fluorodyne ™ EX EDF membrane, Supor ™ EAV; Supor ™ EBV, Supor ™ EKV (all from Pall); Sartopore ™ (by Sartorius); hydrophilic PES membrane from Sterlitech, and WFPES PES membrane from Wolftechnik.
During filtration, the emulsion can be maintained at a temperature of 40 ° C or less, for example, 30 ° C or less, to facilitate successful sterile filtration. Some emulsions may not pass through a sterile filter when they are above 40 ° C. It is advantageous to carry out the filtration step within 24 hours, for example, within 18 hours, within 12 hours, within 6 hours, within 2 hours, within 30 minutes, of producing the second emulsion, because after this time , it may not be possible to pass the second emulsion through the sterile filter without clogging the filter, as described in reference 39.
The methods of the invention can be used on a large scale. Thus, a method may involve filtering a volume greater than 1 liter, for example, _> 5 liters,> 10 liters,> 20 liters, _> 50 liters, j> 100 liters,> 250 liters, etc. The final emulsion
The result of microfluidization and filtration is an oil-in-water emulsion in which the average size of the glass bottles can be 5L, 8L, or 10L of that size. Alternatively, the oil in water can be transferred to a sterile flexible bag (flexible bag). The flexible bag can be 50L, 100L or 250L in size. In addition, the flexible bag can be equipped with one or more sterile connectors to connect the flexible bag to the system. The use of a flexible bag with a sterile connector is advantageous compared to glass bottles, because the flexible bag is larger than glass bottles meaning that it may not be necessary to change the flexible bag to store all the emulsion manufactured in a single batch. This can provide a closed sterile system for the manufacture of the emulsion which can reduce the possibility of impurities being present in the final emulsion. This can be particularly important if the final emulsion is used for pharmaceutical purposes, for example, - if the final emulsion is the MF59 adjuvant.
Preferred amounts of oil (% by volume) in the final emulsion are between 2-20%, for example, about 10%. A squalene content of about 5% or about 10% is particularly useful. A squalene content (weight / volume) between 30-50mg / ml is useful, for example, between 35-45mg / ml, 36-42mg / ml, 38-40mg / ml, etc.
Preferred amounts of surfactants (% by weight) in the final emulsion are: polyoxyethylene sorbitan esters (such as Tween 80) 0.02 to 2%, in particular about 0.5% or about 1%, sorbitan esters (such as Span 85) 0.02 to 2%, in particular about 0.5% or about 1%; octyl- or nonylphenoxy polyoxyethanols (such as, Triton X-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as lauret 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%. A content of polysorbate 80 (weight / volume) between 4-6mg / ml is useful, for example, between 4.1-5.3mg / ml. A sorbitan trioleate content (weight / volume) between 4-6mg / ml is useful, for example, between 4.1-5.3mg / ml.0 process is particularly useful for the preparation of any of the following oil emulsions in water: • An emulsion comprising squalene, polysorbate 80 (Tween 80), and sorbitan trioleate (Span 85). The emulsion composition, by volume, can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate. In terms of weight, these amounts become 4.3% squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate. This adjuvant is known as 'MF59'. The MF59 ^ emulsion advantageously includes citrate ions, for example, 10 mM sodium citrate buffer. • Emulsions comprising squalene, an α-tocopherol (ideally DL-α-tocopherol), and polysorbate 80. These emulsions may have (by weight) 2 to 10% squalene, 2 to 10% a-tocopherol and 0.3 to 3% polysorbate 80, for example, 4.3% squalene, 4.7% a-tocopherol , 1.9% polysorbate 80. The weight ratio of squalene tocopherol is preferably <1 (e.g. 0.90), as it provides a more stable emulsion. Squalene and polysorbate 80 can be present in a volume ratio of about 5: 2, or in a weight ratio of about 11: 5. Such an emulsion can be made by dissolving polysorbate 80 in PBS to produce a 2% solution, then mixing 90 ml of this solution with a mixture of (5g of DL-α-tocopherol and 5 ml of squalene), then , microfluidizing the mixture. The resulting emulsion may have submicron oil droplets, for example, between 100 and 250 nm in size, preferably about 180 nm. • A squalene emulsion, a tocopherol, 'and a Triton detergent (for example, Triton X- 100). The emulsion may also include a 3-0-deacylated monophosphoryl lipid A ('3d-MPL'). The emulsion may contain a phosphate buffer. • An emulsion comprising squalene, a polysorbate (for example, polysorbate 80), a Triton detergent (for example, Triton X-100) and a tocopherol (for example, a-tocopherol succinate ). The emulsion can include these three components in a 75: 11: 10 surround mass ratio (for example, 750μg / ml — polysorbate 80, 110μg / ml Triton X-100 and 100μg / ml succinate a- tocopherol), and the concentrations used must include any contribution of these antigen components. The emulsion can also include a 3d-MPL. The emulsion can also include a saponin, such as QS21. The aqueous phase may contain a phosphate buffer. • An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (for example, polyoxyethylene keto stearyl ether (12)) and a hydrophobic nonionic surfactant ( for example, a sorbitan ester or maintained ester, such as sorbitan monoleate or 'Span 80'). The emulsion is preferably thermoreversible and / or has at least 90% of the oil droplets (by volume) less than 200 nm in size [40]. The emulsion can also include one or more of: alditol; a cryoprotective agent (for example, a sugar, such as dodecylmaltoside and / or sucrose), and / or an alkyl polyglycoside. It can also include a TLR4 agonist, such as one whose chemical structure does not include a sugar ring [41]. These emulsions can be lyophilized.
The compositions of these emulsions, expressed above in percentage terms, can be modified by dilution or concentration (for example, by an integer, such as 2 or 3, or by a fraction, such as 2/3 or 3/4), being that their relationships remain the same. For example, a 2-fold more concentrated MF59 would have about 10% squalene, about 1% polysorbate 80 and about 1% sorbitan trioleate. The concentrated forms can be diluted (for example, with ütnã solution-of anúigenenoj to produce a desired final emulsion concentration.
The emulsions of the invention are preferably stored between 2 ° C and 8 ° C. They must not be frozen. They should ideally be kept out of direct light. In particular, emulsions containing squalene and vaccines of the invention must be protected to prevent photochemical breakdown of squalene. If emulsions of the invention are stored, then it is preferably in an inert atmosphere, for example, N2 or argon. Vaccines
Although it is possible to administer oil-in-water emulsion adjuvants to the patient himself (for example, to provide an adjuvant effect for an antigen that has been administered separately to the patient), it is more common to mix the adjuvant with an antigen before administration, to form an immunogenic composition, for example, a vaccine. The mixture of emulsion and antigen can occur out of time, at the time of use, or it can occur during the manufacture of the vaccine, before filling. The methods of the invention can be applied in both situations.
Thus, a method of the invention may include an additional process step of mixing the emulsion with an antigen component. As an alternative, you can include an additional step of packaging the adjuvant in a kit as a component of the kit, along with an antigen component.
In general, therefore, the invention can be used when preparing mixed vaccines or when preparing kits including antigen and adjuvant ready for mixing; —Where — mixing occurs during manufacture, then the bulk antigen and emulsion volumes that are mixed will typically be greater than 1 liter, for example, 5 liters, _> 10 liters,> 20 liters,> 50 liters,> 100 liters,> 250 liters, etc. Where mixing takes place at the point of use, then the volumes that are mixed will typically be less than 1 milliliter, for example, <0.6 ml, <0.5 ml, <0.4 ml, <0.3 ml, <0.2 ml, etc. In both cases it is usual for substantially equal volumes of emulsion and antigen solution to be mixed, that is, substantially 1: 1 (for example, 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). In some modalities, however, an excess of emulsion or an excess of antigen can be used [42]. Where an excess volume of a component is used, the excess will generally be at least 1.5: 1, for example,> 2: 1,> 2.5: 1,> 3: 1,> 4: 1, 5: 1, etc.
Where the antigen and adjuvant are present as separate components within a kit, they are physically separated from each other within the kit, and this separation can be achieved in several ways. For example, components can be contained in separate containers, such as vials. The contents of two vials can then be mixed when necessary, for example, by removing the contents of one vial and adding it to the other vial, or separately by removing the contents of both vials and mixing them in a third container.
In another arrangement, one component of the kit is non-syringe and <5 the other is in a container such as a vial. The syringe can be used (for example, with a needle) to insert its contents into the bottle for mixing, and the mixture can then be removed from inside the syringe. The mixed contents of the syringe can then be administered to a patient, typically through a new sterile needle. Packing a component in a syringe eliminates the need to use a separate syringe for patient administration.
In another preferred arrangement, the two components of the kit are kept together, but separately in the same syringe, for example, a double-chamber syringe, such as those described in references 43-50 etc. When the syringe is activated (for example, during administration to a patient), then the contents of the two chambers are mixed. This arrangement avoids the need for a separate mixing step at the time of use.
The contents of the various components of the kit will generally all be in liquid form. In some arrangements, one component (typically the antigen component instead of the emulsion component) is in dry form (for example, in a lyophilized form), with the other component being in liquid form. The two components can be mixed in order to reactivate the dry component and produce a liquid composition for administration to a patient. A lyophilized component will typically be located inside a vial instead of a syringe. The dry components can include stabilizers, such as sucrose, lactose or mannitol, as well as mixtures thereof, for example, lactose / sucrose mixtures, sucrose / mannitol mixtures, etc. One possible arrangement uses a liquid -component-of-emulsion in a pre-filled syringe and a lyophilized antigen component in a vial.
If vaccines contain components in addition to the emulsion and antigen, then these additional components can be included in one of these two components of the kit, or they can be part of a component of the third kit.
Suitable containers for mixed vaccines of the invention, or for individual kit components, include disposable vials and syringes. These containers must be sterile.
Where a composition / component is located in a bottle, the bottle is preferably made of glass or plastic material. The bottle is preferably sterilized before the composition is added to it. To avoid problems with latex-sensitive patients, the bottles are preferably sealed with a latex stopper, and the absence of latex in all packaging material is preferred. In one embodiment, a bottle has a butyl rubber stopper. The vial may include a single dose of vaccine / component, or may include more than one dose (a 'multidose' vial), for example, 10 doses. In one embodiment, a bottle includes doses of 10x0.25 ml of emulsion. Preferred bottles are made of colorless glass.
A vial can have a cap (for example, a Luer lock) adapted in such a way that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (for example, to reconstitute the material lyophilized in it), and the contents of the vial can be removed into the syringe. After removing the syringe from the vial -, - a needle can then be attached and the composition can be administered to a patient. The cap is preferably located within a seal or cap, such that the seal or cap must be removed before the cap can be accessed.
Where a composition / component is packaged inside a syringe, the syringe will not normally have a needle attached to it, although a separate needle can be provided with the syringe for assembly and use. Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes can be provided with peelable labels on which the batch number, influenza season and expiration date of the contents can be printed, to facilitate record keeping.
The syringe plunger preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. Syringes may have a latex cap and / or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip before attaching a needle, and the tip cap is preferably made of butyl rubber. If the syringe and needle are packaged separately, then the needle is preferably equipped with a butyl rubber pad.
The emulsion can be diluted with a buffer before packaging in a vial or syringe. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Dilution can reduce the concentration of the components — from the — adjuvant —, - to — the same — while retaining their relative proportions, for example, to provide a "half strength" adjuvant.
The levels can be marked to show a half-dose volume, for example, to facilitate delivery to children. For example, a syringe containing a dose of 0.5 ml may have a mark showing a volume of 0.25 ml.
Whenever a glass container (for example, a syringe or a bottle) is used, then it is preferable to use a container made of borosilicate glass, rather than a glass of soda lime.
Various antigens can be used with oil-in-water emulsions, including, but not limited to: viral antigens, such as viral surface proteins; bacterial antigens, such as proteins and / or saccharide antigens; fungal antigens; antigens of the parasite; and tumor antigens. The invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomegalovirus, Staphylococcus aureus, chlamydia, SRA coronavirus, varicella zoster virus, Streptococcus pneumoniae , Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr, human papilloma virus, etc. For example: • Influenza virus antigens. These can take the form of a live virus or an inactivated virus. Where an inactivated virus is used, the vaccine may comprise whole virion, fragmented virion, or purified surface antigens (including hemagglutinin and, generally, also including neuraminidase). Influenza antigens can also be presented in — the — form — of virosomes. The antigens can have any hemagglutinin subtype, selected from Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hll, H12, H13, H14, H15 and / or H16. Vaccine may include the antigen (s) from one or more (for example, 1, 2, 3, 4 or more) strains of the influenza virus, including influenza A virus and / or influenza B virus, for example, a monovalent A / H5N1 or A / H1N1 vaccine, or a trivalent A / H1N1 + A / H3N2 + B vaccine. The influenza virus can be a recombinant strain, and it may have been obtained by reverse genetics techniques [for example, 51-55]. Thus, the virus can include one or more RNA segments from an A / PR / 8/34 virus (typically 6 segments of A / PR / 8/34, with the HA and N segments being from one vaccine strain, that is, a 6: 2 redisposition). The viruses used as the source of the antigens can be produced either in eggs (for example, embryonated hen's eggs) or in cell culture. Where cell culture is used, the cell substrate will typically be a mammalian cell line, such as MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38, etc. Preferred mammalian cell lines for influenza virus growth include: MDCK cells [56-59], derived from canine kidney Madin Darby; Vero cells [60-62], derived from African green monkey kidney, or PER.C6 cells [63], derived from embryonic human retinoblasts. Where viruses have been grown in a mammalian cell line, then the composition will advantageously be free of egg proteins (eg, ovalbumin and ovomucoid) and chicken DNA, thus reducing allergenicity: Single doses of vaccine — are —Typically standardized by reference to the hemagglutinin (HA) content, typically measured by SRID. Existing vaccines typically contain about 15μg of HA per strain, although lower doses can be used, particularly when using an adjuvant. Fractional doses such as (ie 7.5 μg HA per strain), 1/4 and 1/8 were used [64.65], as well as higher doses (for example, 3x or 9x doses [66.67 ]). Thus, vaccines can include between 0.1 and 150g of HA per strain of influenza, preferably between 0.1 and 50μg, for example, 0.1-20μg, 0.1-15μg, 0.1-10μg and 0 , l-7.5μg, 0.5-5μg and etc. Particular 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, etc. per strain. • Human immunodeficiency virus, including HIV-1 and HIV-2. The antigen will typically be an envelope antigen. • Hepatitis B virus surface antigens. This antigen is preferably obtained by recombinant DNA methods, for example, after expression in a yeast of Saccharomyces cerevisiae. Unlike native viral HBsAg, the antigen expressed by recombinant yeast is non-glycosylated. It can be in the form of substantially spherical particles (average diameter of about 20nm), including a lipid matrix comprising phospholipids. Unlike particles of native HBsAg, particles expressed by yeast can include phosphatidylinositol. HBsAg can be of any of the subtypes aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq- and adrq + • Amoeba, particularly as seen in canines (Ancylostoma caninum). This antigen can be recombinant Ac-MTP-1 (astacin-like metalloprotease) and / or an aspartic hemoglobinase (Ac-April-1), which can be expressed in an insect cell / Baculovirus system as a secreted protein [68, 69] • Herpes simplex virus (HSV) antigens. A preferred HSV antigen for use with the invention is gD glycoprotein membrane. It is preferred to use gD from an HSV-2 strain ('gD2' antigen). The composition may use a form of gD in which the anchor region of the C-terminal membrane has been deleted [70], for example, a truncated gD comprising amino acids 1-306 of the natural protein, with the addition of aparagine and glutamine in position C-terminal. This form of the protein includes the signal peptide that is cleaved to produce a mature protein 283 amino acids. The deletion of the anchor allows the protein to be prepared in soluble form. • Human papillomavirus (HPV) antigens. Preferred HPV antigens for use with the present invention are LI capsid proteins, which can come together to form structures known as virus-like particles (VLPs). VLPs can be produced by recombinant expression of LI in yeast cells (for example, in S. cerevisiae) or in insect cells (for example, in Spodoptera cells, such as S.frugiperda, or in Drosophila cells). For yeast cells, plasmid vectors can carry the LI (s) gene; for insect cells, baculovirus vectors can carry the LI (s) gene. More preferTer ± a ± ment-e -; - et — composition -includes- VLP LI of both HPV-16 and HPV-18 strains. This bivalent combination has been shown to be highly effective [71]. In addition to the HPV-16 and HPV-18 strains, it is also possible to include LI VLPs from HPV-6 and HPV-11 strains. The use of oncogenic HPV strains is also possible. A vaccine can include between 20-6μg / ml (for example, about 4 μg / ml) of LI per HPV strain. • Anthrax antigens. Anthrax is caused by Bacillus anthracis. Suitable B.anthracis antigens include components A (lethal factor (LF) and edema factor (EF)), both of which may share a common B component, known as protective antigen (PA). Antigens can be optionally detoxified. More details can be found in references [72 to 74]. • S.aureus antigens. A variety of S. aureus antigens are known. Suitable antigens include capsular saccharides (for example, from a type 5 and / or type 8 strain) and proteins (for example, ISDB, Hla, etc.). Capsular saccharide antigens are ideally conjugated to a carrier protein. '• S. pneumoniae antigens. A variety of S.pneumoniae antigens are known. Suitable antigens include capsular saccharides (for example, from one or more of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and / or 23F) and proteins (for example, pneumolysin, detoxified pneumolysin, protein D polyhistidine triad (PhtD), etc.). Capsular saccharide antigens are ideally conjugated to a carrier protein. • Cancer antigens. A variety of specific antigens — from — tumor — are — known. —The — invention — can — be used with antigens that elicit an immunotherapeutic response against lung cancer, melanoma, breast cancer, prostate cancer, etc.
A solution of the antigen will normally be mixed with the emulsion, for example, at a volume ratio of 1: 1. This mixing can be done by a vaccine manufacturer, before filling, or it can be done at the time of use, by a health professional. Pharmaceutical Compositions
Compositions made using the methods of the invention are pharmaceutically acceptable. They can include components in addition to the emulsion and the optional antigen.
The composition can include a preservative such as thiomersal or 2-phenoxyethanol. It is preferable, however, that the vaccine should be substantially free of (ie, less than 5μg / ml) mercurial material, for example, free of thiomersal [75,76]. Vaccines and components that contain no mercury are more preferred.
The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0, for example, between 6.5 and 7.5. A process of the invention can therefore include a step of adjusting the pH of the vaccine prior to packaging.
The composition is preferably sterile. The composition is preferably non-pyrogenic, for example, containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The composition is preferably gluten free.
The composition can include material for a single immunization, or it can include material for multiple immunizations (i.e. — ie ~, —a kit of —mutidosis-M ^, —the —inclusion — of_ a condom is preferred in multidose arrangements.
Vaccines are typically administered in a dosage volume of about 0.5 ml, although a half dose (i.e., about 0.25 ml) can be administered to children.
Vaccine Treatment and Administration Methods
The invention provides kits and compositions prepared using the methods of the invention. Compositions prepared according to the methods of the invention are suitable for administration to human patients, and the invention provides a method of enhancing an immune response in a patient, comprising the step of administering such a composition to the patient.
The invention also provides these "kits" and compositions for use as medicaments.
The invention also provides for the use of: (i) an aqueous antigen preparation, and (ii) an oil-in-water emulsion prepared according to the invention, in the manufacture of a medicament to enhance an immune response in a patient.
The increased immune response by these methods and uses generally includes an antibody response, preferably a protective antibody response.
The compositions can be administered in a number of ways. The most preferred route of immunization is by intramuscular injection (for example, in the arm or leg), but other available routes include subcutaneous, intranasal [77-79], oral [80], intradermal [81.82], transcutaneous injection, transdermal [83], etc.
Vaccines prepared according to the invention can be used "to treat children and adults -.— © patient may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old age, or at least 55. The patient can be an elderly person (for example,> 50 years of age, preferably> 65 years), young people (for example, <5 years), hospitalized patients, health workers health, armed service and military personnel, pregnant women, chronically ill, immunodeficient patients, and people traveling abroad.Vaccines are not only suitable for these groups, however, and can be used more generally in a population.
The vaccines of the invention can be administered to patients at substantially the same time as (for example, during the same medical consultation or visit to a healthcare professional) other vaccines. Intermediate Processes
The invention also provides a method for making an oil-in-water emulsion, comprising microfluidizing a first emulsion to form a second emulsion and then filtering the second emulsion. The first emulsion has the characteristics described above.
The invention also provides a method for making an oil-in-water emulsion, comprising filtration of a second emulsion, i.e., a microfluidized emulsion. The microfiuidized emulsion has the characteristics described above.
The invention also provides a method for making a vaccine, comprising combining an emulsion with an antigen, where the emulsion has the characteristics described above. Specific Modalities
Specific embodiments of the present invention include: • A method for making an oil-in-water emulsion comprising squalene, comprising the steps of (i) forming a first emulsion having a first average oil droplet size; (ii) microfluidization of the first emulsion to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size; and (iii) filtering the second emulsion using a hydrophilic membrane. • A method for making an oil-in-water emulsion, comprising the steps of (i) forming a first emulsion having an average first oil droplet size of 5000 nm or less; (ii) microfluidization of the first emulsion to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size; and (iii) filtration of the second emulsion using a hydrophilic membrane • A method for the manufacture of an oil-in-water emulsion, comprising the steps of (i) forming a first emulsion having a first average oil droplet size; (ii) microfluidizing the first emulsion to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size; and (iii) filtering the second emulsion using a polyethersulfone hydrophilic membrane. • A method for making an oil-in-water emulsion comprising squalene, the method comprising the step of (i) forming a first emulsion having a first average oil droplet size using a homogenizer, the first emulsion being formed by circulating the components of the first emulsion through a homogenizer a plurality of times • A method for making an oil-in-water emulsion comprising squalene, the method comprising the step of (b) microfluidizing a first emulsion having a first size medium oil droplet to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size, the second emulsion being formed by circulating the components of the second emulsion through transfer of the components of the second emulsion of a first emulsion container, through a first microfluidization device for the one s second emulsion container, and then through a second microfluidization device, the first and second microfluidization devices being the same. • A method for making an oil-in-water emulsion comprising: passing a first emulsion having a first size of medium oil droplet through a microfluidization device to form a second emulsion having a second medium oil droplet size that is smaller than the first medium oil droplet size; the microfluidization device comprising — an — interaction — chamber — that- comprising a plurality of Z-type channels and an auxiliary processing module comprising at least one channel; the auxiliary processing module being positioned downstream of the interaction chamber • A method for manufacturing an oil-in-water emulsion comprising the step of passing a first emulsion having a first medium oil droplet size through a device microfluidizing to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size; the microfluidization device comprising an interaction chamber and an auxiliary processing module comprising a plurality of channels. • A method for making an oil-in-water emulsion comprising the step of passing a first emulsion having a first medium oil droplet size through a microfluidization device to form a second emulsion having a second medium oil droplet size which is smaller than the first average oil droplet size, the microfluidization device comprising an interaction chamber and the pressure of the emulsion components at the entrance to the interaction chamber being substantially constant for at least 85% of the time during which the emulsion is fed into the microfluidizer • A method for making an oil-in-water emulsion, comprising the step of forming a first emulsion having a first medium oil droplet size, the — that — the — formation —Of — the first — emulsion — is carried out under an inert gas, for example, nitrogen, for example, at a pressure of up to 0.5 bar. • A method for making an emulsion d and oil in water, comprising the step of passing a first emulsion having a first medium oil droplet size through a microfluidization device to form a second emulsion having a second medium oil droplet size that is smaller than the first size medium oil droplet, the formation of the second emulsion being carried out under an inert gas, for example, nitrogen, for example, at a pressure of up to 0.5 bar. • A method for the manufacture of an oil emulsion in water, comprising the steps of (i) forming a first emulsion having a first medium oil droplet size; (ii) microfluidization of the first emulsion to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size; (iii) filtration of the second emulsion; (iv) transferring the oil-in-water emulsion into a sterile flexible bag. " "----General
The term "comprising" encompasses "including" as well as "consisting", for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example, X + Y.
The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y can be completely free of Y. If necessary, the word "substantially" can be omitted from the definition of the invention
The term "about" in relation to a numerical value x is optional and means, for example, x +%.
Unless otherwise specified, a process comprising a step of mixing two or more components does not require any specific order of mixing. So the components can be mixed in any order. When there are three components then two components can be combined with each other, and then the combination can be combined with the third component, etc.
Where animal (and particularly bovine) materials are used in cell culture, they must be obtained from sources that are free from transmissible spongiform encephalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). In general, cultured cells are preferred in the total absence of derivatives of animal materials. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a specific example of a homogenizer that can be used to form a first emulsion.
Figure 2 shows a detail of a rotor and stator that can be used in such a homogenizer.
Figure 3 shows two pressure profiles for a synchronous intensifier pump mode.
Figure 4 shows a Z-type channel interaction chamber.
Figure 5 shows a type I circulation, while Figure 6 shows a type II circulation. Têdipientes — are labeled — as - “- G’h — at — the same time — enuque_ a homogenizer is labeled" H ". Direction and order of fluid movements are presented. In Figure 6 the homogenizer has two input arrows and two output arrows, but in reality the homogenizer has a single input channel and a single output channel. MODES FOR CARRYING OUT THE INVENTION Example 1:
The components of the squalene emulsion, polysorbate 80, sorbitan trioleate and sodium citrate buffer were introduced in a high speed inline homogenizer, rotor / stator (IKA Super Dispax Reactor DRS 2000/5). Emulsion starting volumes of 280L and 250L were used and the homogenizer speed was set at 5000 ± 1000 rpm. The temperature of the emulsion during homogenization was kept below 60 ° C.
Three test runs were performed. In the first test run, 280L of the emulsion components were subjected to type I circulation, between the homogenizer and a first premix container, for 20 minutes, followed by a single type II circulation, transferring the components of the first emulsion from a first pre-mix stainless steel container, through the homogenizer to a second pre-mix stainless steel container, and then back through the homogenizer. In the second test run, 280L of the emulsion components were subjected to type I circulation, between the homogenizer and a first pre-mix stainless steel container, for 5 minutes, followed by —5 — circulations — of — type — II , transferring the components of the first emulsion from a first pre-mix stainless steel container, through the homogenizer to a second pre-mix stainless steel container, and then back through the homogenizer to the first pre-stainless steel container -Mix. In the third test run, 250L of the emulsion components were subjected to a type I circulation, between the homogenizer and a first pre-mix stainless steel container, for 20 minutes followed by a single type II circulation, transferring the components from the emulsion. first emulsion of a first pre-mix stainless steel container, through the homogenizer to a second pre-mix stainless steel container, and then back through the homogenizer to the first pre-mix stainless steel container.
The first emulsion was homogenized until it had an average oil droplet size of 1200 nm or less and a number of oil droplets having a size> 1.2 µm of 5x109 / ml or less. * ~
The first emulsion was then subjected to microfluidization to form a second emulsion. The emulsion was passed through the microfluidization device five times. The microfluidization device was operated at approximately between 600 and 800 bar (that is, between approximately 9000 and 12000 psi) and the emulsion was maintained at a temperature of 40 ± 5 ° C during microfluidization through the use of a cooling mechanism. .
The second emulsion was then sterile filtered.
The average size of the oil droplets in the filtered emulsions in each test run met the specification for an MF59 adjuvant.
Other parameters of the emulsions during the first, second and third test runs can be found in Table 1.

The results of all three test runs are excellent. However, the results in Table 1 show that test run 1 produced the largest percentage reduction (99.5%) in the number of particles with a size> 1.2 μm in the emulsion after filtration compared to the present number in the second emulsion. — Therefore, the best circulation pattern for homogenization is about 20 minutes of type I circulation, followed by a type II circulation. Example 2:
In additional experiments, a first emulsion was formed by type I (Figure 5) or type II (Figure 6) circulation. For five separate runs the average number of large particles per ml was as follows:

Thus, type II circulation results in less large droplets and less variation from batch to batch.
It will be understood that the invention has been described by way of example only and modifications can be made, while remaining within the scope and spirit of the invention.
REFERENCES [1]. WO90 / 14837. [2] . Podda & Del Giudice (2003) Expert Rev Vaccines 2: 197-203. [3] . Podda (2001) Vaccine 19: 2673-2680. [4]. Vaccine Design: The Subunit and AdjuvantApproach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0- 306-44867-X). [5] . Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine serfes). ISBN: 1- 59259- 083 - 7. Ed. 01 Hagan .—--———- [6]. New Generation Vaccines (eds. Levine and others). 3- edition, 2004. ISBN 0-8247-4071-8. [7] . O'Hagan (2007) Expert Rev Vaccines 6 (5): 699-710. [8]. EP-B-2029170. [9] . Baudner et al. (2009) Pharm Res. 26 (6): 1477-85. [10] . Dupuis et al. (1999) Vaccine 18: 434-9. [11] . Dupuis et al. (2001) Eur J Immunol 31: 2910-8. [12]. Burke et al. (1994) J Infect Dis 170: 11 10-9. [13] . Light Scattering from Polymer Solutions and Nanoparticle Dispersions (W. Schartl), 2007. ISBN: 978-3- 540-71950-2. [14] . Jafari et al. (2008) Food Hydrocolloids 22: 1191-1202. [15] . W090 / 04609. [16] . US-4,618,533. [17] . US-6,193,077. [18] . US-6,495,050. [19] . Chen et al. (1999) Journal of AppliedPolymer Science, 72: 1699-1711. [20] . US-4,413,074. [21]. US-4,432,875. [22] . US-4,340,482. [23] . US-4,473,474. [24] . US-4,473,475. [25] . US-4,673,504. [26] . EP - A- 0 22104 6. [27]. US-4,943,374. [28] . US-6,071,406. [29] . US-4,705,753. [30]. US-5,178,765. [31] . US-6,495,043. [32] . US-6,039,872. [33] . US-5,277,812. [34] . US-5,531,893. [35] . US-4,964,990. [366 ^ Wavhad— & Fisher— (2002) Journal of Polymer Science Part B: Polymer Physics 40: 2473-88. [37] . W02006 / 044463. [38] . Espinoza-Gomez et al. (2003) Revista de la Sociedad Quimica de Mexico 47: 53-57. [39] . Lidgate et al. (1992) PharmaceuticalResearch 9 (7): 860-863. [40] . US-2007/0014805. [41] . WG2007 / 080308. [42] . W02007 / 052155. [43] . WG2005 / 089837. [44] . US 6,692,468. [45] . WO00 / 07647. [46] . WO99 / 17820. [47] . US 5,971,953. [48]. [49]. [50]. [51]. US 4,060,082.EP-A-0520618 .WO98 / 01174.Hoffmann et al. (2002) Vaccine 20: 3165-3170. [52]. Subbarao et al. (2003) Virology 305: 192-200. [53]. Liu et al. (2003) Virology 314: 580-590. [54]. Ozaki et al. (2004) J. Virol. 78: 1851-1857. [55]. Webby et al. (2004) Lancet 363: 1099-1103. [56]. W097 / 37000. [57]. Brands et al. (1999) Dev Biol Stand 98: 93-100. Halperin — and others — C2-QXL2J — JZaccine 20: 1240-7. [59] . Tree et al. (2001) Vaccine 19: 3444-50. [60], Kistner et al. (1998) Vaccine 16: 960-8. [61] . Kistner et al. (1999) Dev Biol Stand 98: 101-110. [62] . Bruhl et al. (2000) Vaccine 19: 1 149-58. [63] . Pau et al. (2001) Vaccine 19: 2716-21. [64] . WOOl / 22992. [65] . Hebe et al. (2004) Virus Res. 103 (1-2): 163-71. [66] . Treanor et al. (1996) J Infect Dis 173: 1467-70. [67] . Keitel et al. (1996) Clin Diagn LabImmunol 3: 507-10. [68]. Williamson et al. (2006) Infection and Immunity 14: 961-7. [69] . Loukas et al. (2005) PLoS Med 2 (10): e295. [70] . EP-A-0139417. [71] . Harper et al. (2004) Lancet 364 (9447): 1757-65. - _ _ ___ [72]. J Toxicol Clin Toxicol (2001) 39: 85-100. [73] . Demicheli et al. (1998) Vaccine 16: 880-884. [74]. Stepanov et al. (996) J Biotechnol44: 155: 160. [75] . Banzhoff (2000) Immunology Letters 71: 91-96. [76] . W002 / 097072. [77] . Greenbaum et al. (2004) Vaccine 22: 2566-77. [78]. Zttrbriefgen. and others (20 03) Expert RevVaccines 2: 295-304. [79] . Piascik (2003) J Am Pharm Assoc (Wash DC). 43: 728-30. [80] . Mann et al. (2004) Vaccine 22: 2425-9. [81] . Halperin et al. (1979) Am J Public Health 69: 1247-50. [82] . Herbert et al. (1979) J Infect Dis 140: 234-8. [83] . Chen et al. (2003) Vaccine 21: 2830-6.

权利要求:
Claims (27)
[0001]
1. Method for the manufacture of an oil-in-water vaccine adjuvant, characterized by comprising squalene, the method comprising the step of: (a) forming a first emulsion having a first medium oil droplet size; (b) microfluidization of a first emulsion using a mechanical homogenizer in which the first emulsion has a first average oil droplet size; (c) circulating the first emulsion transferring from a first emulsion container through a first microfluidization device to a second container of emulsion and then again through the same microfluidization device, so as to form a second emulsion with a second average oil droplet size that is smaller than the first average oil droplet size, and (d) filter the second emulsion.
[0002]
Method according to claim 1, characterized in that substantially all of the emulsion components of the first container are passed through the microfluidization device to the second container, and then substantially all of the emulsion components of the second container are passed through the microfluidization device back to the first container.
[0003]
3. Method, according to claim 1, characterized in that it comprises the step of: (i) performing a type II circulation by transferring its components from a first container to a second container through a homogenizer and then returning them from the second container to the first container through the same homogenizer, so as to form a first emulsion having a first medium oil droplet size using a homogenizer, where the homogenizer provides a shear rate of up to 1x106 s-1, and the microfluidization takes place in an interaction chamber that provides a shear rate> 2.5x106 s-1.
[0004]
Method according to claim 3, characterized in that substantially all components of the emulsion of the first container are passed through the homogenizer to the second container, and then substantially all components of the emulsion of the second container are passed through passed back through the homogenizer into the first container.
[0005]
Method according to any one of claims 1 to 4, characterized in that it comprises: (iii) filtration of the second emulsion.
[0006]
Method according to any one of claims 1 to 5, characterized in that step (i) comprises two or more cycles of transferring components from the first emulsion from the first container to the second container and back again.
[0007]
Method according to any one of claims 1 to 6, characterized in that during step (ii), the second emulsion is formed by circulating the components of the second emulsion through a microfluidization device a plurality of times .
[0008]
Method according to claim 7, characterized in that the circulation of the components of the second emulsion comprises the transfer of the components of the second emulsion between a first emulsion container and a microfluidization device.
[0009]
Method according to claim 8, characterized in that the circulation of the components of the second emulsion comprises the transfer of the components of the second emulsion from a first emulsion container, through a first microfluidization device to a second container of the emulsion. emulsion, and then through a second microfluidization device, the first and second microfluidization devices being the same.
[0010]
Method according to any one of claims 1 to 9, characterized in that after passing through the second microfluidization device, the components of the second emulsion are returned to the first emulsion container and the circulation as defined in claim 7 is repeated one or more times.
[0011]
Method according to any one of claims 1 to 10, characterized in that the first average oil droplet size is 5000 nm or less.
[0012]
12. Method according to any one of claims 1 to 11, characterized in that the number of oil droplets having a size> 1.2 μm in the first emulsion is 5x1011 / ml or less.
[0013]
13. Method according to any one of claims 1 to 12, characterized in that the second average oil droplet size is 500 nm or less.
[0014]
14. Method according to any one of claims 1 to 13, characterized in that the number of oil droplets having a size> 1.2 μm in the second emulsion is 5x1010 / ml or less.
[0015]
15. Method for the preparation of a vaccine composition, characterized in that it comprises the preparation of an emulsion according to any one of claims 1 to 14 and combining the emulsion with an antigen.
[0016]
16. Method for preparing a vaccine preparation kit, characterized in that it comprises an emulsion according to any one of claims 1 to 15 and packaging the emulsion in a kit as a component of the kit, together with an antigen component.
[0017]
17. Method according to claim 16, characterized by the fact that the kit components are in separate bottles.
[0018]
18. Method according to claim 17, characterized in that the bottles are made of borosilicate glass.
[0019]
19. Method according to any one of claims 20, 21 or 22, characterized in that the adjuvant is a bulk adjuvant and the method comprises extracting unit doses from the bulk adjuvant for packaging as components of the kit.
[0020]
20. Method according to any one of claims 19, 20, 21, 22 or 23, characterized in that the antigen is an antigen of the influenza virus.
[0021]
21. Method according to claim 24, characterized by the fact that the combination of the emulsion and the antigen forms a vaccine composition and the vaccine composition includes about 15μg, about 10μg, about 7 μg, about of 5μg, about 3 μg, about 1 μg, about 1 μg of hemagglutinin per strain of the influenza virus.
[0022]
22. Method according to claim 20, characterized in that the combination of the emulsion and the antigen forms a vaccine composition and the vaccine composition includes a preservative of 2-phenoxyethanol or thiomersal.
[0023]
23. Method according to any one of claims 1 to 12, characterized in that the oil-in-water emulsion containing squalene comprises polysorbate 80 and sorbitan trioleate.
[0024]
24. Method according to claim 23, characterized in that the oil-in-water emulsion containing squalene comprises about 4.3% squalene, about 0.5% polysorbate 80 and about 0.48% of sorbitan trioleate by weight.
[0025]
25. Method according to claim 15, characterized in that the oil-in-water emulsion containing squalene comprises polysorbate 80 and sorbitan trioleate.
[0026]
26. Method according to claim 25, characterized in that the oil-in-water emulsion containing squalene comprises about 4.3% squalene, about 0.5% polysorbate 80 and about 0.48% of sorbitan trioleate by weight.
[0027]
27. Method according to claim 3, characterized by the fact that step (i) further comprises first carrying out a type I circulation of transferring the first emulsion components between a first container and a homogenizer before carrying out the circulation of the type II.

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WO2011067673A3|2012-05-10|

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US20160113870A1|2016-04-28|

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EP2356983B1|2013-02-20|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2021-03-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-30| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 30/03/2021, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/12/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US28351809P| true| 2009-12-03|2009-12-03|

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US61/283,518|2009-12-03|

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