METHODS FOR THE MANUFACTURE OF AN OIL-IN-WATER EMULSION, TO PREPARE A VACCINE COMPOSITION AND TO PRE

专利摘要:
Arrangement of return and interaction pressure chambers for microfluidization The present invention relates to a methods for manufacturing an oil-in-water emulsion comprising the use of a microfluidization device whose interaction chamber comprises a plurality of flows upstream of Z-type channels of a return pressure chamber.
公开号:BR112012013426B1
申请号:R112012013426-4
申请日:2010-12-03
公开日:2021-04-13
发明作者:Harald Rueckl；Hanno Scheffczik
申请人:Novartis Ag；
IPC主号:

专利说明:

This application claims the benefit of the provisional patent application US 61 / 283,548 filed on December 3, 2009, the entire content of which is incorporated into this document by reference for all purposes. TECHNICAL FIELD
This invention is in the field of manufacturing oil-in-water emulsion adjuvants for microfluidization vaccines. TECHNICAL FUNDAMENTALS
The vaccine adjuvant known as 'MF59' [1-3] is a submicrometric oil-in-water emulsion of squalene, 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 the reference 4, chapter 12 of reference 5 and chapter 19 of reference 6.
As described in reference 7, MF59 is manufactured on a commercial scale by dispersing Span 85 in the squalene phase and Tween 80 in the aqueous phase, followed by mixing at high speed to form a coarse emulsion. This coarse emulsion is then passed through a microfluidizer several times to produce an emulsion with 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 ° C. 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 more and better 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 better microfluidization to provide emulsions with fewer large particles. . DISCLOSURE OF THE INVENTION
The invention provides a method for making an oil-in-water emulsion comprising: passing a first emulsion with a first average oil droplet size through a microfluidization device to form a second emulsion with a second average droplet size of oil that is smaller than the first average oil droplet size. The microfluidization device comprises an interaction chamber comprising a plurality of Z-type channels and an auxiliary processing module comprising at least one channel, wherein the auxiliary processing module is positioned downstream of the interaction chamber.
The first emulsion can be introduced into the interaction chamber at a first pressure and the second emulsion can exit the auxiliary processing module at a second pressure which is less than the first pressure. In a 5 modality, between 80 and 95% of the pressure difference between the first and the second pressure is eliminated through the interaction chamber and 5 to 20% of the pressure difference between the first and the second pressure is eliminated through the pressure module. auxiliary processing.
The present invention also provides a method for making an oil-in-water emulsion comprising the step of passing a first emulsion with a first average oil droplet size through a microfluidization device to form a second emulsion with a second average oil droplet size which is smaller than the first average oil droplet size. The microfluidization device comprises an interaction chamber comprising a plurality of channels and an auxiliary processing module comprising a plurality of channels.
The first emulsion can (i) be introduced into the interaction chamber at a first pressure and the second emulsion can leave the auxiliary processing module at a second pressure below the first pressure; or the first emulsion 25 can (ii) be introduced into the auxiliary processing module at a first pressure and the second emulsion can leave the interaction chamber at a second pressure below the first pressure. In one embodiment, between 80 and 95% of the pressure difference between the first and the second pressure is eliminated through the interaction chamber and 5 to 20% of the pressure difference between the first and the second pressure is eliminated through the module. auxiliary processing.
As described in more detail below, the first emulsion can have an average oil droplet size of 5 50 00 nm or less, for example, an average size between 3 00 nm "and ~ 80 ~ 0 nm. The number of oil droplets in the first emulsion with a size> 1.2 μm can be 5 x 10 ^ / ml or less, as described below. Oil droplets with a size> 1.2 μm are disadvantageous, since they can cause 10 the instability of the emulsion due to the agglomeration and coalescence of the droplets [14].
After formation, the first emulsion can then be subjected to at least one microfluidization pass to form the second emulsion with a reduced average oil droplet size. As described below, the average size of — geb-yertlas — of — oil — of — the second emulsion is 5 00 hm or less. The number of oil droplets in the second emulsion with a size> 1.2 μm can be 5 x 1010 / 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 can then be filtered, for example, through a hydrophilic polyethersulfone 25 membrane, to give 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 with a size> 1.2 μm present in the oil-in-water emulsion produced after filtration can be 5 x 108 / ml or less, for example, 5 x 107 / ml or less, 5 x 10 6 / ml or less, 2 x 10 6 / ml or less or 5 x 10 5 / ml or less.
The final oil-in-water emulsion formed after filtration can have at least 102 times less oil droplets with a size> 1.2 μm compared to the first emulsion, and ideally at least 103 times less (for example, 104 times any less).
In some embodiments, more than one cycle of steps (i) and (ii) is used before step (iii). Likewise, several repetitions of individual steps (i) and (ii) can be used.
In general, the method is carried out at 20-60 ° C, and ideally at 40 ± 5 ° C. Although the components of the first and the second emulsion can be relatively stable even at higher temperatures, thermal degradation of some components can still occur and therefore lower temperatures are preferred.
Emulsion components The average oil droplet size (ie the average numerical diameter of the emulsion oil droplets) can be measured using a dynamic light scattering technique, as described in reference 13. An example of a 25 Dynamic light scattering measurement is the Nicomp 380 submicrometric particle size analyzer (from Particle Sizing Systems).
The number of particles with a diameter> 1.2 μm can be measured using a particle counter, such as the 30 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 main ingredients: an oil; an aqueous component; and a surfactant.
Because the emulsions are intended for pharmaceutical use, the oil will typically be biodegradable (metabolizable) and biocompatible.
The oil used can comprise squalene, a shark liver oil, which is a branched, unsaturated 10 (C3OH5o; [(CH3) 2C [= CHCH2CH2C (CH3)] 2 = CHCH2-] 2; 2,6,10, 15,19,23-hexamethyl-2,6,10,14,18,22 -tetracosa-hexaene; CAS RN 7683-64-9). Squalene is particularly preferred for use in the present invention.
The oil of the present invention may comprise a mixture (or combination) of oils, for example,
Instead of (or in addition to) using squalene, an emulsion may comprise oil (s), including, for example, those from an animal (such as fish) or from a plant source. Sources of 20 vegetable oils include nuts, seeds and grains. Peanut oil, soy oil, coconut oil and olive oil, the most commonly available, exemplify chestnut oils. Jojoba oil can be used, for example, obtained from the jojoba grain. Seed oils 25 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 30 and the like can also be used. The 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 from the oils of chestnuts and seeds. Mammalian milk fats and oils are metabolizable and thus can be used. The procedures for separation, purification, saponification and other means necessary to obtain pure oils of animal origin are well known in the art.
Most fish contain metabolizable oils, which can be easily recovered. For example, cod liver oil, shark liver oils and whale oil, such as spermaceti, exemplify several of the fish oils, which can be used in this document. A series of branched-chain oils is synthesized biochemically into 5-carbon isoprene units and is generally referred to as terpenoids. Squalane, the saturated analogue of 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 o, j3, Y / δ, ε, or tocopherols can be used, but α-tocopherols are preferred. D-α-tocopherol and DL-a-tocopherol can both 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, nicotinate, etc. If a salt of this tocopherol is to be used, the preferred salt is succinate. A combination of oil comprising squalene and a tocopherol (for example, DL-α-tocopherol) can be used.
The aqueous component can be pure 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 Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20 mM range.
The surfactant is preferably biodegradable (metabolizable) and biocompatible. Surfactants can be classified by their 'HLB' (hydrophilic / 1ypolytic balance) ~ where a HUB 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 10-2 0 which 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 ™, as linear EO / PO block copolymers; octoxynols, which may vary in the number of ethoxy repeating groups (oxy-1,2-ethanediyl), 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 alcohols - lauryl, cetyl, earyl and oleyl esters (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether, and sorbitan esters (commonly known as the 10 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 — BOySpan 85, or Tween 80 / Triton-X100 mixtures. A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol. Useful mixtures can comprise a surfactant with an HLB value in the range of 10-20 (for example, Tween 80, with an HLB of 15.0) and a surfactant with an HLB value in the range of 1-10 (for example , 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.
The oil droplets in the first emulsion may have an average size of 5000 nm or less, for example, 4000 nm or - ---- less, —3000 nm or less, 2,000 mm or less, 12 0 0 nm or less than 100 nm or less, for example, an average size between 800 and 1200 nm or between 300 nm and 800 nm.
In the first emulsion, the number of oil droplets 10> 1.2 µm in size can be 5 x 10 10 / ml, or less, for example, 5 x 10 10 / ml or less, or 5 x 109 / ml or less.
The first emulsion can then be microfluidized to form a second emulsion with a lower average oil droplet size than the first emulsion and / or 15 less oil droplets> 1.2 µm in size.
The “average” size of the first oil emulsion droplets can be achieved by mixing components of the first 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 operate vertically and / or horizontally. For convenience, in a commercial setting, in-line homogenizers are preferred.
The components are introduced in a rotor-stator homogenizer and serve a fast rotating rotor containing cracks or holes. The components are thrown out by centrifugation like a pump and pass through the slits / holes. In some embodiments, the homogenizer includes multiple combinations of rotors and stators, for example, a concentric array of comb-tooth rings, as shown by characteristics (3) & (4), (5) & (6) and 5 (7 ) & (8) in Figure 1 and Figure 2. Rotors in useful large-scale homogenizers may have comb-tooth rings on the edge of an impeller with multiple horizontally oriented blades (for example, feature (9) in Figure 1) aligned in close tolerance for 10 teeth matching in a static coating. The first emulsion is formed through a combination of turbulence, cavitation and mechanical shear that occur within the gap between rotor and stator. The components are usefully introduced in a direction parallel to the axis of the rotor.
An important performance parameter in rotor-stator homogenizers is the tip speed of the rotor (peripheral speed). This parameter is a function of both the rotational speed 20 and the diameter of the rotor. A peripheral speed of at least 10 ms'1 is useful and ideally faster, for example, at 20 ms'1,> 30 ms'1, at 40 ms'1, etc. A peripheral speed of 40 ms'1 can be easily achieved at 10,000 rpm with a small homogenizer or at lower speeds of 25 rpm (eg 2,000 rpm) with a larger homogenizer. Suitable high-shear homogenizers are commercially available.
For commercial scale production, the homogenizer should ideally have a flow rate of at least 3 00 L / h, for example,> 400 L / h, at 500 L / h,> 600 L / h,> 700 L / h, > 800L / h, at 900 L / h, at 1000 L / h, at 2000 L / h, at 5000 L / h, or even at 10,000 L / h. Suitable high capacity homogenizers are commercially available.
A preferred homogenizer provides a shear rate of between 3 x 105 and 1 x 106 s'1, for example, between 3 x 105 and 7 — x — 10 ^ ~ s 'between 4 x ~ 10 5 ~ ê' 6 x 10 & ~ s "1", for example, about 5 x 105 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 15 emulsion may be homogenized several times (for example, 2, 3, 4, 5, 6., 7, 8, —9, —10, —20, —30, —40, - 50 or more ~ times).
To avoid the need for a long sequence of containers and homogenizers, the components of the emulsion can instead circulate (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, 25, etc.). However, too many cycles may be undesirable, as they can produce recoalescence, as described in reference 14. Thus, the size of the oil droplets can be monitored if the homogenizer circulation is used to verify that a desired droplet size has been reached. 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 with a size> 5 1.2 μm in the first emulsion. These reductions in the average droplet size and the number of droplets> 1.2 μm in the first emulsion may provide advantages in downstream processes. In particular, the circulation of the components of the first emulsion through the homogenizer can lead to an improved microfluidization process 10 which then can result in a reduced number of oil droplets with a size> 1.2 μm in the second emulsion, or that is, after microfluidization. This improvement in the parameters of the second emulsion can provide improved filtration performance. The best filtration performance can lead to less content loss during — filtration, for example —, - losses — of squalene, Tween 80 and Span 85 when the oil-in-water emulsion is MF5 9.
Two particular types of circulation are referred to 20 in this document as "type I" and "type II". The type I circulation is illustrated in Figure 5, while the type II circulation is illustrated in Figure 6.
The circulation of the components of the first emulsion may comprise a type I circulation of transferring the 25 components of the first emulsion between a first premix container and a homogenizer. The first pre-mix container can be 50 to 500 L 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 30 can be made of stainless steel. Type I circulation can be continued for 10 to 60 minutes, for example, 10 to 40 minutes or 20 minutes.
The circulation of the components of the first emulsion can comprise a type II circulation of transferring the 5 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 of the first pre-mix container) and then through a second homogenizer. The second homogenizer will generally be the same as the first homogenizer, but in some arrangements the first and second homogenizers are different. After passing the components of the first emulsion through the second homogenizer, the components 15 of the first emulsion can be transferred back to the first — pre-mixed — container — for example, if the type II circulation process is to be repeated. Thus, the components of the emulsion can travel in an eight-way path between the first and the second premix container through a single homogenizer (see Figure 6). Type II circulation can be performed only once or a plurality of times, for example, 2, 3, 4, 5, etc. times.
The type II circulation is advantageous over the type I circulation, because 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 entire content of the emulsion has passed through the homogenizer to the second pre-mix container. Likewise, the contents of the second pre-mix container can be emptied, again, ensuring that all of it passes through the homogenizer. Thus, the type II arrangement can conveniently ensure that all components of the emulsion are homogenized 5 at least twice, which can reduce both the average oil droplet size and the number of oil droplets with a size> 1.2 μm in the first emulsion. An ideal type II circulation thus involves emptying the first pre-mix container and passing considerably all of its contents through the homogenizer to the second pre-mixing container, followed by emptying the second pre-mixing container and re-passing. from considerably all of its content through the rear of the homogenizer to the first pre-mix container. Thus, all particles pass through the homogenizer at least twice, while this is difficult to achieve with the type I circulation.
In some embodiments, a combination of type I and type II circulations 20 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 with a size> 1.2 μm in the first emulsion. This combination can comprise any circulation order 25 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 30 components of the first circulated emulsion from a first premix container, through a first homogenizer, to a second premix container and then through a second homogenizer once.
The first and the second premix container can be kept under inert gas, for example nitrogen, for example, up to 50 kPa. This can prevent the components of the emulsion from being oxidized, 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 rπθQ.α.-L IQαClc S t v d-L 10O GQlUpOXltillL.and fXXlcill ScI CQLII.Ü ± IICXQXTO crilLco of this mixture. For example, if the emulsion includes a surfactant with an HLB below 10, then this surfactant can be combined with an oil before mixing. Likewise, if the emulsion includes a surfactant with an HLB above 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 first emulsion can be microfluidized, or it can be stored to await microfluidization.
In some modalities, in particular those where 5 several 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 again subjected to homogenization.
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 with a size> 1.2 μm.
Microfluidization instruments reduce the average oil droplet size by boosting incoming component streams through geometrically fixed channels at high pressure and high speed. The pressure at the entrance to the interaction chamber (also called "first pressure") can be considerably constant (ie, + 15%; for example, ± 10%, ± 5%, ± 2%) for at least 8 5 % 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 first pressure is 13 0 0 bar (130 Mpa)) ± 15% (18 kPSI ± 15%), that is, between 1100 bar (110 MPa) and 1500 bar (150 MPa) (between 15 kPSI) and 21 kPSI) 30 for 85% of the time the emulsion is fed into the microfluidizer. Two profiles of suitable pressure are shown in Figure 3. In Figure 3A, the pressure is considerably constant for at least 85% of the time, whereas in Figure 3B the pressure continuously remains considerably 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 driven electrically and hydraulically, provides high pressure (ie the first pressure) to force an emulsion into and through the interaction chamber. The synchronous nature of intensifier pumps can be used to provide the considerably constant pressure of the emulsion discussed above, which means that the droplets of the emulsion are all exposed considerably to the — same — level — of — shear forces — during microfluidization.
An advantage of using a considerably constant pressure is that it can reduce fatigue failures in the microfluidization device, which can lead to longer device life. An additional advantage of using a considerably constant pressure is that the parameters of the second emulsion can be improved. In particular, the number of oil droplets with 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 considerably constant pressure is used. Reducing the average oil droplet size and the number of oil droplets with a size> 1.2 μm in the second emulsion can provide better filtration performance. The best filtration performance can lead to less content losses during filtration, for example, squalene, Tween 80 and Span 85 losses when the emulsion is MF59.
The interaction chamber can contain a plurality, for example, 2, 3, 4, ~ 5, 6, 7, 8,9, 10, etc., channels of fixed geometry through which the emulsion passes. The emulsion enters the interaction chamber through an inlet line, which can have a diameter between 200 and 250 μm. The emulsion divides into currents as it enters the interaction chamber and, under high pressure, accelerates to high speed. As it passes through the channels, the forces produced by the high pressure can act to reduce the oil droplet size of the emulsion and to reduce the number of oil droplets with a size> 1.2 μm. These forces may include: shear-forces, -through-deformation-of-the-current of the emulsion that occurs from contact with the channel walls; impact forces, through collisions that occur high-speed emulsion currents collide with each other; and 20 cavitation forces, through the formation and collapse of cavities within the current. The interaction chamber does not generally include moving parts. It can include ceramic (for example, alumina) or diamond (for example, polycrystalline diamond) channel surfaces. Other surfaces 25 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 an interaction chamber with type Y geometry, a single inlet emulsion stream is divided into first and second emulsion stream, which are then recombined into a single outgoing emulsion stream. Before recombination, each of the first and second emulsion streams can be divided independently into a first 5 and a second plurality (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of undercurrents. When the emulsion streams are recombined, the first and second emulsion streams (or their subcurrents) are ideally flowing in considerably opposite directions (for example, the first and second emulsion streams, or their subcurrents, are flowing considerably 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 droplet size by 6 ± and that of the emulsion and to reduce the number of oil droplets with a size> 1.2 μm.
In an interaction chamber of type Z geometry, the current of the emulsion passes around a plurality (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of angled corners considerably straight (ie 90 ± 20 °). THE
Figure 4 illustrates an interaction chamber with type Z geometry and two right-angled corners in the flow direction.
During its passage around the corners, a stream of the incoming emulsion can be divided into a plurality (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) of undercurrents and, in then recombined in a single emulsion output stream (for example, as shown in Figure 4, with four subcurrents (32)). The division and then the recombination (31) can occur at any point between the input and the output. The forces produced when the emulsion comes into contact with the channel walls as it passes around the corners can act to reduce the emulsion's oil droplet size and to reduce the number of oil droplets with a size> 1.2 μm. An example of a type Z interaction chamber is the Microfluidics E230Z interaction chamber.
In one embodiment, the emulsion current passes around two corners at a considerably right angle. At the point where the incoming emulsion current passes around the first corner at a considerably right angle, it is divided into five subcurrents. At the point where the subcurrents pass around the second corner at a considerably right angle, they are recombined in a single emulsion output stream.
In the state of the 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, as this can lead to a greater reduction in the number of oil droplets with a size> 1.2 μm present in the second emulsion compared to a Y-type geometry interaction chamber. Reducing the number of oil droplets with a size> 1.2 μm in the second emulsion can provide better filtration performance. The best filtration performance can lead to less content losses during filtration, for example, squalene, Tween 80 and Span 85 losses when the emulsion is MF59.
A preferred microfluidization device operates at a pressure between 170 bar (17 MPa) and 2750 bar (275 MPa) (approximately 2500 psi to 40,000 psi), for example, at about 345 bar (34.5 MPa), about 690 bar (69 MPa), about 1380 bar (138 MPa), about 2070 bar (207 MPa), 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 greater than 1 x 106 s'1, for example,> 2.5 x 10s s'1, at 5 x 106 s'1, at 107 s' 1, etc.
A microfluidization apparatus may include several interaction chambers that are used in parallel, for example, 2, 3, 4, ET, or more, but it is more useful to include a single interaction chamber.
The microfluidization device may comprise an auxiliary processing module (APM; also known as microfluidizers such as a back pressure chamber - these terms are used interchangeably throughout this document) that comprise at least one channel. The APM contributes to the reduction of the average size of the oil droplets in the emulsion that passes through the microfluidization device, although most of the reduction occurs in the interaction chamber. As mentioned above, the components of the emulsion are introduced into the interaction chamber by the intensifier pump (s) under a first pressure. Generally, the components of the emulsion leave the APM at a second pressure that is less than the first pressure (for example, atmospheric pressure). In general, between 80 and 95% of the pressure difference between the first and the second pressure is reduced through the interaction chamber (for example, from Px to P2 in Figure 4) and 5 to 20% of the pressure difference between the first and the second pressure is 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 has dropped across the interaction chamber and the pressure has dropped across the auxiliary processing module, it does not count towards the total pressure difference between the first and the second pressure, this may be due to a finite pressure drop in all connectors between the interaction chamber and the auxiliary processing module.
The APM '' does not generally include moving parts. It may include ceramic (eg, alumina) or diamond (eg, polycrystalline diamond) channel surfaces. Other surfaces may be made of stainless steel.
The APM is usually positioned downstream of the interaction chamber and can also be positioned sequentially to the interaction chamber. In the state of the art, APMs are generally positioned downstream of the interaction chambers comprising Y-type channels to suppress cavitation and thereby increase flow in the Y-type chamber by up to 30%. Furthermore, in the state of the art, APMs are generally positioned upstream of interaction chambers comprising type Z channels to reduce the size of large clusters. In the latter case, the APM only decreases the flow in the Z-type chambers by up to 3%. However, it has been found that the positioning of the APM downstream of an interaction chamber comprising a plurality of type Z channels is advantageous in the present invention, because it can lead to a greater reduction in the average size of oil droplets and a greater reduction in the number of oil droplets with a size> 1.2 μm present in the second emulsion. As discussed above, the reduction in the number of oil droplets with a size> 1.2 μm in the second emulsion can provide better filtration performance. Better filtration performance can lead to less content loss during filtration, for example, squalene, Tween 80 and Span 85 losses when the oil-in-water emulsion is MF59. Another advantage of this positioning of a type Z interaction chamber and a downstream APM is that it can lead to a slower pressure decrease after the interaction chamber. The slower pressure decrease can lead to an increase in the stability of the product, because there is less gas inside the emulsion.
An APM contains at least one channel of fixed geometry, through 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 through which the emulsion 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 currents as it enters the APM.
In contrast to the manufacturer's recommendations, the use of an APM comprising a plurality of fixed geometry channels is advantageous in comparison to a single fixed geometry channel APM because it can lead to a greater reduction in the number of oil droplets with a size> 1.2 μm present in the second emulsion. As discussed above, the reduction in the number of oil droplets with a size> 1.2 μm in the second emulsion can provide better filtration performance. The best filtration performance can lead to 10 less losses 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 15-20 ° C in relation to the first emulsion. Advantageously, therefore; the — emulsion — microfluidized — is — cooled — as soon as possible. The temperature of the second emulsion can be kept below 60 ° C, for example, below 45 ° C. Thus, the output of an interaction chamber and / or the output of an APM can be fed to a cooling mechanism, such as a heat exchanger or cooling coil. The distance between the outlet and the cooling mechanism should be as short as possible, to reduce the total time, by reducing cooling delays. In one embodiment, the distance 25 between the outlet of the microfluidizer and the cooling mechanism is between 20-30 cm. A cooling mechanism is particularly useful when an emulsion is subjected to several microfluidization steps, to prevent the emulsion from overheating.
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 medium size is particularly useful, as it facilitates filter sterilization of the emulsion. Emulsions, in which at least 80% of the number of oil droplets have an average size of 500 nm or less, for example 400 nm or less, 300 nm or less, 200 nm or less or 165 nm or less, are particularly Useful. In addition, the number of oil droplets in the second emulsion with a size> 1.2 μm is 5 x 10 / ml or less, for example, 5 x 10 9 / ml or less, 5 x 10 8 / ml or less or 2 x 108 µg / ml or less.
The initial entry into microfluidization can be the first emulsion. In some embodiments, however, the microfluidized emulsion is again subjected to
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 microfluidization device to a second emulsion container (optionally having the same properties as the first emulsion container). premix) and then through a second microfluidization device.
The second microfluidization device may be the same as that of the first microfluidization device. Alternatively, the second microfluidization device can be different from the first microfluidization device.
The first emulsion container can be the same as the first premix container. Alternatively, the first emulsion container may be the same as the second premix container. :
The second emulsion container can be the same as the first premix container. Alternatively, the second emulsion container may be the same as the second premix container.
The first emulsion container can be the same as the first pre-mixing container and the second emulsion container can be the same as the second pre-mixing container. Alternatively, the first emulsion container may be the same as that of the second premix container and the second emulsion container may be the same as the first premix container.
Alternatively, the first and the second emulsion container may be different from the first and second premix container.
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 emulsion container, for example, if the type II circulation process is to be repeated. Type II circulation can be performed only once or a plurality of times, for example, 2, 3, 4, 5, etc. times.
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 oil droplet size and the number of oil droplets with 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 type I and II circulation order, 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 inert gas, for example, up to 0.50 bar (50 kPa) of nitrogen. This prevents the components of the emulsion from being oxidized, 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, one method may involve microfluidizing 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 any large oil droplets that have survived the homogenization and microfluidization procedures. Although small in numerical terms, 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. The characteristics of a filter can affect its suitability for filtration of the microfluidized emulsion. For example, their pore size and surface characteristics can be important, particularly when a squalene-based emulsion is filtered.
The size of the membrane pores used with the invention should allow the passage of desired droplets while retaining unwanted droplets. For example, it should retain droplets that are 1 µm in size, allowing droplets <200nm to pass through. A 0.2 μm or 0.22 μm filter is ideal and can also achieve filter sterilization.
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 using 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 <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% (and ideally less than half) of the pore size of the first layer, such as between 25-70% or between 25-49% of the pore size of the first layer, for example, 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 second layer with a pore size of 0 , 2 μm or ~ CT / 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 dense film, 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 there can be a porous and a homogeneous layer. A preferred double layer filter is one in which both layers are porous.
In one embodiment, the second emulsion is pre-filtered through an asymmetric, porous hydrophilic membrane and then filtered through another asymmetric hydrophilic porous membrane with smaller pores than the pre-filtration membrane. This can use a double layer filter.
The filter membrane (s) can / can be autoclaved prior to use to ensure that it is sterile.
Filtration membranes are typically made of polymeric support materials such as PTFE (poly-tetra-20 fluorethylene), PES (polyethersulfone), PVP (polyvinyl pyrrolidone), PVDF (polyvinylidene fluoride), nylon (polyamides), PP ( polypropylene), celluloses (including cellulose esters), PEEK (polyetheretherketone), nitrocellulose, etc. These have different characteristics, 25 with some supports being intrinsically hydrophobic (for example, PTFE) and others being intrinsically hydrophilic (for example, cellulose acetates). However, these intrinsic characteristics can be modified by treating the membrane surface. In a double-layer filter, the two membranes can be made of 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 the 5 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 methods are known to transform hydrophobic PES membranes into hydrophilic PES membranes. This is often achieved by coating the membrane with a hydrophilic polymer. To provide a permanent fixation of the hydrophilic polymer to the PES, a hydrophilic coating layer is usually subjected to a crosslinking or grafting reaction. Reference 15 discloses a process for modifying the surface properties of a hydrophobic polymer with functionalizable chain ends, 20 comprising contacting the polymer with a solution of an active binding unit to form a covalent bond, and then stopping. contact the hydrophobic polymer that reacted with a solution of a modifying agent. Reference 16 discloses a method of PES membrane hydrophilization by direct membrane coating, involving pre-wetting with alcohol and then incorporation into an aqueous solution containing a hydrophilic monomer, a polyfunctional monomer (crosslinker) and a polymerization initiator. The monomer and crosslinker are then polymerized using thermal polymerization or UV-initiated to form a crosslinked hydrophilic polymer coating on the membrane surface. Likewise, references 17 and 18 disclose the coating of a PES membrane by incorporating in an aqueous solution of hydrophilic polymer (polyalkylene oxide) and at least one polyfunctional monomer (crosslinker) and then polymerization of a monomer to provide a non-extractable hydrophilic coating. Reference 19 describes the hydrophilization of the PES membrane by a grafting reaction in which a PES membrane is subjected to low temperature helium plasma treatment followed by grafting of the hydrophilic monomer N-vinyl-2-pyrrolidone (NVP) onto the surface of the membrane. Additionally, these processes are disclosed in the 15 references 20 to 26.
In non-coating methods, PES can be dissolved in a solvent, mixed with a soluble hydrophilic additive, and then the combined solution is used to mold a hydrophilic membrane, for example, by precipitation or by initiating copolymerization. disclosed in references 27 to 33. For example, reference 33 discloses a method of preparing a membrane modified by hydrophilic charge that has low membrane extractables and allows for the rapid recovery of ultrapure water resistivity, having an interpenetrating polymeric network structure. reticulate formed by making a polymeric solution of a mixture of PES, PVP, polyethyleneimine and aliphatic diglycidyl ether, forming a thin film of the solution and precipitating the film as a membrane. A similar process is disclosed 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 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 CO2 plasma at low temperature.
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 15 in a liquid with a low surface tension, exposing the wet PES membrane to an “aqueous” solution. of an oxidarrte and then heating.
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). It has been found that treatment with PEG (hydrophilic) instead of PVP gives a hydrophilized PES membrane that is easily blocked (particularly when using an emulsion 25 containing squalene) and also disadvantageously releases formaldehyde during autoclaving.
A preferred double layer filter has a first hydrophilic PES membrane and a second hydrophilic PES membrane.
Well-known hydrophilic membranes include Bioassure (from Cuno); polyethersulfone, EverLUX ™; polyethersulfone, STyLUX ™ (both from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200 and Durapore CVGL01TP3 membranes (from Millipore); Fluorodyne ™ EX FED membrane, Supor ™ EAV; Supor ™ EBV, Supor ™ EKV (all from Pall); Sartopore ™ (from Sartorius); Sterlitech's hydrophilic PES membrane; and Wolftechnik PES WFPES membrane.
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 cannot 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 3" 0 - minutes, of production of the second emulsion because after this time it is not possible to pass the second emulsion through the sterile filter without clogging the filter, as discussed 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, 100 liters, 2 250 liters, etc.
The final emulsion The result of microfluidization and filtration is an oil-in-water emulsion in which the average oil droplet size can be less than 220 nm, for example, 155 ± 20 nm, 155 ± 10 nm or 155 + 5 nm, and where the number of oil droplets with a size> 1.2 μm can be 5 x 108 / ml or less, for example, 5 x 107 / ml or less, 5 x 106 / ml or less , 2 x 10s / ml or less or 5 x 105 / ml or less.
The average oil droplet size of emulsions described in this document (including the first and the second 5 emulsions) is generally not less than 50 nm.
The methods of the invention can be used on a large scale. Thus, a method may involve preparing a final emulsion with a volume greater than 1 liter, for example,> 5 liters,> 10 liters,> 20 liters,> 50 liters, 10> 100 liters,> 250 liters, etc.
Once the oil-in-water emulsion has been formed, it can be transferred to sterile glass bottles. Glass bottles can be 5 L, 8 L or 10 L in size. Alternatively, the oil-in-water can be transferred to a sterile flexible bag (flexible bag). The flex bag. can be 50 L, 100 E or 250 L in size. In addition, the flex bag can be equipped with one or more sterile connectors to connect the flex bag to the system. The use of a flex bag with sterile connectors is advantageous 20 compared to glass bottles because the flex bag is larger than glass bottles, which means that it may not be necessary to change the flex bag to store the entire emulsion manufactured in a single batch. This can provide a sterile closed system for the manufacture of the emulsion, which can reduce the chance 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 30 are between 2-20%, for example, about 10%. A squalene content of about 5% or about 10% is particularly useful. A squalene content (w / v) between 30-50 mg / ml is useful, for example, between 35-45 mg / ml, 36-42 mg / ml, 38-40 mg / ml, etc.
Preferred amounts of surfactants (% by weight) in the final emulsion are: 0.02 to 2% polyoxyethylene sorbitan esters (such as Tween 80), in particular, about 0.5% or about 1%; sorbitan esters (such as Span 85) at 0.02 to 2%, in particular, about 0.5% or about 1%; octyl or nonylphenoxy polyoxyethanols (such as Triton X-100) at 0.001 to 0.1%, in particular, 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) at 0.1 to 20%, preferably 0.1 to 10%, and in particular 0.1 to 1% or about 0.5%. A polysorbate 80 (w / v) content between 4-6 mg / ml is useful, for example, between 4.1-5.3 mg / ml. A sorbitan trioleate content— (w / v) —between — 4-6 — mg / mL — is — useful — for example, between 4.1-5.3 mg / mL.
The process is particularly useful for preparing any of the following oil-in-water emulsions:. An emulsion comprising squalene, polysorbate 80 (Tween 80) and sorbitan trioleate (Span 85). The composition of the volume emulsion 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 a-tocopherol (ideally DL-a-tocopherol) and polysorbate 80. These emulsions can 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 (for example, 0.90), since it provides a more stable emulsion. Squalene and polysorbate 80 can be present in a volume ratio of about 10 to 5: 2, or a weight ratio of about 11: 5. Such an emulsion can be made by dissolving polysorbate 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-a-tocopherol and 5 ml of squalene), then 15 microfluidize the mixture. The resulting emulsion may have ubiquitous and droplet oil droplets, for example, with a size between 100 and 250 nm, preferably about 180 nm. . An emulsion of a Triton detergent (for example, 20 Triton X-100), a tocopherol and squalene. The emulsion can also include a 3-O-deacylated monophosphoryl lipid A (13d-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 mass ratio of about 75:11:10 (for example, 750 μg / mL of polysorbate 80, 110 μg / mL of Triton X-100 and 100 μg / mL of a-tocopherol succinate), and 30 of these concentrations should include any contribution of these components from antigens. The emulsion can also include a 3d-MPL. The emulsion can also include a saponin, such as QS21. The aqueous phase can contain a phosphate buffer. . An emulsion comprising squalene, an aqueous solvent, a hydrophilic non-ionic polyoxyethylene alkyl ether surfactant (for example, polyoxyethylene (12) keto-stearyl ether) and a non-ionic hydrophobic surfactant (for example, a sorbitan ester or manide ester, 10 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. —Also — may — include — an 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 20 percentage terms, can be modified by dilution or concentration (for example, by an integer, like 2 or 3 or by a fraction, like 2/3 or 3/4), in which their proportions remain the same. For example, a MF59 concentrated twice would have about 10% squalene, about 1% 25 polysorbate 80 and about 1% sorbitan trioleate. The concentrated forms can be diluted (for example, with an antigen solution) to obtain a desired final emulsion concentration.
The emulsions of the invention are ideally stored between 2 ° C and 8 ° C. They must not be frozen. Ideally, they should be kept out of direct light. In particular, emulsions containing squalene and vaccines of the invention must be protected to prevent photochemical degradation of squalene. If the emulsions of the invention are stored, then preferably this will be in an inert atmosphere, for example, N2 or argon.
Vaccines Although it is possible to administer oil-in-water emulsion adjuvants alone to patients (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 extemporaneously, 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 step 20 of the process of mixing the emulsion with an antigen component. Alternatively, 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. In case the mixing occurs during manufacture, then the volumes of the antigen and emulsion mass that are mixed will typically be greater than 1 liter, for example, at 5 30 liters, 10 liters, at 20 liters,> 50 liters,> 100 liters, 250 liters, etc. In case the mixing occurs at the time 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 considerably equal volumes of emulsion and antigen solution to be mixed, that is, considerably, 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]. If an excess volume of one 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.
In case antigen and adjuvant are presented as separate components within a — kit, eTes — they will be physically separated from each other within the kit, and this separation can be achieved in several ways. For example, components can be in separate containers, such as bottles. The contents of the 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 by separately removing the contents of both vials and mixing them in a third container.
In another arrangement, one component of the kit is in a syringe and 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 mixing bottle, and the mixture can then be withdrawn into the syringe. The mixed contents of the syringe can then be administered to a patient, typically through a new sterile needle. A packaging component in a syringe eliminates the need to use a different 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 two-chamber syringe, such as those disclosed 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 be in liquid form. In some arrangements, one component (typically the antigen component, rather than the emuTs 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 to reactivate the dry component and give a liquid composition for administration to a patient. A lyophilized component will typically be located inside a vial, instead of a syringe. Dry components can include stabilizers, such as lactose, sucrose or mannitol , as well as their mixtures, for example, mixtures of 25 lactose / sucrose, mixtures of sucrose / mannitol, etc. One possible arrangement uses a liquid emulsion component in a filled syringe and a lyophilized antigen component in a vial.
If the vaccines contain components other than the emulsion and 30 of the antigen, then those additional components can be included in one of these two kit components, or they can be part of a third kit component.
Containers suitable for mixed vaccines of the invention, or for individual kit components, include 5 vials and disposable syringes. These containers must be sterilized.
In the event that 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-free 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 the vaccine / components-; or it can include — more — of — a — dose— (a 'multidose' vial), for example, 10 doses. In one embodiment, a vial includes 10 x 0.25 ml doses of the emulsion. Preferred bottles are made of colorless glass.
A vial may have a cap (for example, a "Luer-lock" type) adapted such that a filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (for example, to reconstitute the lyophilized material contained in it), and the contents of vial 25 can be removed back into the syringe. After removing the syringe from the vial, a needle can then be attached to and the composition can be administered to a patient. The lid is preferably located within a seal or cover, such that the seal or cover has to be removed before the cover is accessed.
In case a composition / component is packaged in 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 detachable labels on which the batch number, flu season and expiration date of the content can be printed, to facilitate record keeping. The plunger in the syringe preferably has a lock to prevent the plunger from being accidentally removed during aspiration. Syringes may have a latex rubber cap and / or plunger. Disposable syringes contain a single dose of vaccine. The syringe will usually have an end cap to seal the tip before connecting ”—with a needle, and the end 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 cover.
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 adjuvant components, while retaining their relative proportions, for example, to provide an adjuvant with "half the strength".
The containers can be marked to show a half-dose volume, for example, to facilitate distribution to children. For example, a syringe containing a dose of 0.5 ml may have a mark showing a volume of 0.25 ml.
If a glass container is used (for example, a syringe or a bottle), then it is preferable to use a container made of borosilicate glass, instead of soda-lime glass.
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 protein and / or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. The invention is particularly useful for vaccines against influenza viruses, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cholecomegalovirus S t aphyl ococcus aureus, chlamydia, SARS coronavirus, varicella zoster virus, Streptococcus pneumoniae , Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus, human papillomavirus, etc. For example: . Antigens of the influenza virus. These can take the form of a live virus or an inactivated virus. If an inactivated virus is used, the vaccine may comprise the whole virion, the divided 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, 30 H8, H9, H10, Hll, H12, H13, H14, H15 and / or H16. The vaccine may include the antigen (s) of one or more (for example, 1, 2, 3, 4 or more) strains of the influenza virus, including the influenza A virus and / or the influenza B virus, for example , a monovalent A / H1N1 or A / H5N1 vaccine, or a trivalent A / H1N1 + A / H3N2 + B vaccine. The influenza virus may be a strain that has been rearranged, and may have been obtained by reverse genetics techniques [by example, 51-55]. Thus, the virus can include one or more RNA segments from an A / PR / 8/34 virus (typically 6 segments from A / PR / 8/34, with the 10 HA and N segments being from a vaccine strain, or (ie a 6: 2 rearrangement). The viruses used as the source of the antigens can be grown in eggs (for example, embryonated hen's eggs) or in cell culture. If cell culture is used, the cell substrate will typically be from a mammalian cell line, such as
MDCK; CHO; 293 T’fTBHK; Vero; MRC ^ 5 PER. C6; WI-387 — etCT ^ Preferred mammalian cell lines for cultivating influenza viruses include: MDCK cells [56-59], derived from Madin Darby dog kidney; Vero cells [60-62], 20 derived from African green monkey kidney; or cells
PER.C6 [63], derived from human embryonic retinoblasts. In the event that the virus was cultured in a mammalian cell line, then the composition would advantageously be free of egg proteins (for example, ovalbumin and ovomucoid) and chicken DNA, thereby reducing allergenicity. Unit doses of vaccine are typically standardized by reference to the hemagglutinin (HA) content, typically measured by SRID. Existing vaccines typically contain about 15 μg HA 30 per strain, although smaller doses can be used,
particularly when using an adjuvant. Fractional doses like [1/2] (ie 7.5 μg HA per strain), [1/4] and [1/8] were used [64.65], as they have higher doses (for example , 3x or 9x doses [66,67]). Thus, vaccines may include between 0.1 and 150 μg of HA per influenza strain, preferably between 0.1 and 50 μg, for example, 0.1-20 μg, 0.1-15 μg, 0.1- 10 μg, 0.1-7.5 μg, 0.5-5 μg, etc. Particular doses include, for example, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, 10 about 1.9, about 1.5, etc. per strain. . Human immunodeficiency virus, including HIV-1 and HIV-2. The antigen will typically be an antigen on the envelope. . Surface antigens of the hepatitis B virus. This antigen is obtained preferably by recombinant DNA 15 methods, for example, after expression in a yeast Saccharomyces cerevisiae. Unlike native viral HBsAg, the recombinant antigen expressed in yeast is not glycosylated. It can be in the form of considerably spherical particles (average diameter of about 20-20 nm), including a lipid matrix, comprising phospholipids. Unlike native HBsAg particles, particles expressed in yeast can include phosphatidylinositol. HBsAg can be of any of the subtypes aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq- and 25 adrq +.
Hookworm, particularly as seen in canines (Ancylostoma caninutri). This antigen can be recombinant Ac-MTP-1 (astacin-like metalloprotease) and / or an aspartic hemoglobinase (Ac-APR-1), which can be expressed in an insect / baculovirus cell system as a secreted protein [68, 69].
Herpes simplex virus (HSV) antigens. The preferred HSV antigen for use with the invention is the gD membrane glycoprotein. It is preferable to use the gD of an HSV-2 strain (antigen 'gD2.'). The composition may use a form of gD in which the C-terminal membrane anchor region has been deleted [70], for example, a truncated gD, comprising amino acids 1-306 of natural protein 10 with the addition of aparagine and glutamine at the C-terminal end. This form of the protein includes the signal peptide that is cleaved to produce a mature protein of 283 amino acids. The deletion of the anchorage allows the protein to be prepared in soluble form. . Human papillomavirus (HPV) antigens. The preferred HPV antigens for use with the invention are Ll capsid proteins, which can assemble to form structures known as virus-like particles (VLPs). VLPs can be produced by recombinant expression of Ll 20 in yeast cells (for example, in S. cerevisiae) or in insect cells (for example, in cells of
Spodoptera, such as S. frugiperda, or in Drosophila cells). For yeast cells, plasmid vectors can carry the Ll gene (s); for insect cells, baculovirus vectors can carry the Ll gene (s). More preferably, the composition includes Ll VLPs from 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 Ll
VLPs of the HPV-6 and HPV-11 strains. The use of oncogenic HPV strains is also possible. A vaccine can include between 20-60 μg / mL (for example, about 40 LI per strain of HPV.. Anthrax antigens. Anthrax is caused by Bacillus anthracis. Suitable antigens from B. anthracis include A- 5 components ( lethal factor (LF) and edema factor (EF)), both of which can share a common B-component known as a protective antigen (PA). The antigens can optionally be 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, of 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 antigens— S. pneumoniae are known. Suitable antigens include capsular saccharides (for example, from one or more of the serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and / or 2 F) and 20 proteins (for example, pneumolysin, detoxified pneumolysin, polyhistidine D triad protein (PhtD), etc.). Capsular saccharide antigens are ideally conjugated to a carrier protein. Cancer antigens. A variety of tumor-specific antigens 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, in a volume ratio of 1: 1.
This mixture can be performed by a vaccine manufacturer, before filling, or it can be performed at the time of use, by a healthcare 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 vaccine 10 is considerably free of (that is, less than 5 μg / mL) of mercurial material, for example, without thiomersal [75,76]. Vaccines and components that do not contain mercury are most preferred.
The pH of a composition will generally be between 5.0 and 15 8.1 and more typically between 6.0 and 8.0, for example, between 6 ~ T5 — and ~~ 7.5. —A process of the invention can , —So, —include a step of adjusting the pH of the vaccine before packaging.
The composition is preferably sterile. The composition is preferably pyrogenic, for example, containing <1 EU 20 (endotoxin unit, a standard measure) per dose and preferably <0.1 EU per dose. The composition preferably does not contain gluten.
The composition can include material for a single immunization, or it can include material for several immunizations (i.e., a 'multidose' kit). The inclusion of a preservative is preferred in multiple dose arrangements.
Vaccines are typically administered in a dose volume of about 0.5 ml, although half a dose (ie about 0.25 ml) can be administered to children.
Methods of treatment and administration of the vaccine 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 for inducing an immune response in a patient, comprising the step of administering that composition to the patient.
The invention also provides these kits and compositions for use as medicines.
The invention also provides for the use of: (i) an aqueous preparation of an antigen; and (ii) an oil-in-water emulsion prepared according to the invention, in the manufacture of a medicament to elicit an immune response in a patient.
The immune response elicited by these methods is 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, into 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 both children and adults. The patient can be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. The patient can be elderly (for example,> 50 years old, preferably> 65 years), young (for example, <30 5 years old), hospitalized patients, health professionals, military and armed service personnel, pregnant women , the chronically ill, immunodeficient and people traveling abroad. Vaccines are not only suitable for these groups, however, and can be used 5 more generally in a population. - The vaccines of the invention can be administered to patients considerably at the same time as (for example, during the same medical consultation or visit to a health 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 15 has the characteristics described above.
The invention — also provides for a — a method for the manufacture of an oil-in-water emulsion, comprising the filtration of a second emulsion, that is, a microfluidized emulsion. The microfluidized emulsion has the characteristics described above.
The invention also provides a method for the production of a vaccine, which comprises combining an emulsion with an antigen, where the emulsion has the characteristics described above.
Specific modalities The specific modalities of the present invention include:. A method for producing an oil-in-water emulsion comprising squalene, comprising the steps of (i) forming a first emulsion with a first average oil droplet size; (ii) microfluidization of the first emulsion to form a second emulsion with 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 producing an oil-in-water emulsion, comprising the steps of (i) forming a first emulsion with a first average oil droplet size of 5000 nm or less; (ii) microfluidizing the first emulsion to form a second emulsion with 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 producing an oil-in-water emulsion, comprising the steps of (i) forming a first emulsion with a first average oil droplet size; (ii) microfluidization of the first emulsion to form a second emulsion with 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 polyethersulfone membrane. . A method for producing an oil-in-water emulsion comprising squalene, the method comprising the step of (i) forming a first emulsion with a first average oil droplet size using a homogenizer, wherein the first emulsion it is formed by the circulation of the components of the first emulsion through a homogenizer a plurality of times. . A method for producing an oil-in-water emulsion comprising squalene, the method comprising the step of (b) microfluidizing a first emulsion with a first average size of oil droplets to form a second emulsion with a second average size of oil droplets that is smaller than the average first oil droplet size, where the second emulsion is formed by circulating the components of the second emulsion by transferring the components of the second emulsion from a first emulsion container, through a first microfluidization device for a second emulsion container, and then through a second microfluidization device, where the first and second microfluidization device are the same. . A method for producing an oil-in-water emulsion comprising: passing a —first — emulsifier — with — an average first size of oil droplets through a microfluidization device to form a second emulsion with a second average size oil droplet that is smaller than the first average oil droplet size; wherein the microfluidization device comprises an interaction chamber comprising a plurality of Z-type channels and an auxiliary processing module comprising at least one channel; wherein the auxiliary processing module is positioned downstream of the interaction chamber. . A method for producing an oil-in-water emulsion comprising the step of passing a first emulsion with a first average oil droplet size through a microfluidization device to form a second emulsion with a second average oil droplet size. oil that is smaller than the first average oil droplet size; wherein the microfluidization device comprises an interaction chamber and an auxiliary processing module comprising a plurality of channels. '. A method for producing an oil-in-water emulsion comprising the step of passing a first emulsion with a first average oil droplet size through a microfluidization device to form a second emulsion with a second average droplet size oil that is smaller than the first average oil droplet size, where the microfluidization device comprises an interaction chamber and where the pressure of the 15 emulsion components at the entrance to the interaction chamber is considerably — constant — by— peie — less — 85% —do — temper during which the emulsion is fed to the microfluidizer. . A method for producing an oil-in-water emulsion, comprising the step of forming a first emulsion with a first average oil droplet size, in which the formation of the first emulsion is carried out under an inert gas, for example. eg nitrogen, for example, at a pressure of up to 0.5 bar (50 kPa). . A method for producing an oil-in-water emulsion, comprising the step of passing a first emulsion with a first average oil droplet size through a microfluidization device to form a second emulsion with a second average size of oil droplets that is smaller than the first average size of oil droplets, in which the formation of the second emulsion is performed under an inert gas, for example nitrogen, for example, at a pressure of up to 0.5 bar (50 kPa). . A method for producing an oil-in-water emulsion, comprising the steps of (i) forming a first emulsion with a first average oil droplet size; (ii) microfluidization of the first emulsion to form a second emulsion with 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 to a sterile flex bag.
General The term "comprising" encompasses "including" as well as 15 "consisting", for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example, X + Y.
The word "considerably" does not exclude "completely", for example, a composition that is "considerably free" from Y can be completely free from Y. Where necessary, the word "considerably" 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 + 10%.
Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus, components can be combined in any order. If 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.
In the case of using animal (and particularly bovine) materials in cell culture, they must be obtained from sources that are free of. transmissible spongiform encephalopathies (TSEs) and in particular free of bovine spongiform encephalopathy (BSE). In general, it is preferable to grow cells in the total absence of materials derived from animals.
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 the detail of a rotor and stator that 15 can be used in such a homogenizer. A -Figure — 3 — shows two pressure profiles for a synchronous intensifier pump mode. Figure 4 shows a type Z channel interaction chamber. Figure 5 shows a type I circulation, while Figure 6 shows a type II circulation. The containers are labeled "C", while a homogenizer is labeled "H". The direction and order of fluid movements are shown. 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
A first emulsion comprising squalene, polysorbate 80, sorbitan trioleate and sodium citrate buffer was prepared by homogenization. The first emulsion was homogenized until it had an average oil droplet size of 1200 nm or less and a number of oil droplets with a size> 1.2 μm of 5 x 109 / mL 5 or less.
The first emulsion was then 'subjected to microfluidization to form a second emulsion. The microfluidization device comprised two synchronous intensifier pumps providing a considerably constant pressure of approximately 700 bar (70 MPa) (i.e., approximately 10,000 psi). The emulsion passed through the microfluidization device five times. The emulsion was maintained at a temperature of 40 + 5 ° C during microfluidization through the use of a cooling mechanism.
Four “test runs” were performed. — In the first pair of test runs a single-channel auxiliary processing module (APM) was positioned upstream of a channel 8, type Z (IXC) interaction chamber, as recommended 20 by the manufacturer and the flow of the emulsion in the microfluidizer device was 10.2 L / min. In the second pair of test runs, the multichannel APM was positioned downstream of an IXC type Z, channel 8 and the emulsion flow rate in the microfluidizer device was 11.6 L / min. 25 Both executions were carried out on a large scale (250 liters). The results of the four test runs are shown in Table 1:

Table 1
As shown in Table 1, the test runs in which the APM was positioned downstream of IXC produced emulsions with a smaller average particle size and fewer particles with a size> 1.2 μm. In addition, the order IXC-APM reached a particle diameter <200 nm after 1 pass, while this size was not reached even after 4 passes with the order APM-IXC. Therefore, the positioning of the APM downstream from the IXC type Z proved to be advantageous for large-scale production.
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 Adjuvant Approach (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 series). ISBN: 1-59259-083-7. Ed. O'Hagan. [6] New Generation Vaccines (eds. Levine et al.). 3rd 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: 1 191- 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 Applied Polymer Science, 72: 1699-171 1. [20] US-4,413,074 [21] US-4,432,875 15 [22] US-4,340,482 F 2 31 π.q-4,473, 474 [24] US- 4,473,475 [25] US-4,673,504 [26] EP-A-0221046. [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 [36] Wavhal & 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) Pharmaceutical Research 9 (7): 860-863. [40] US-20 07/0014 8 05. [41] [42] [43] [44] [45] [46] [47] [48] [49] W02007 / 080308. W02007 / 052155. W02005 / 089837. US 6,692,468. WO00 / 07647. W099 / 17820. US 5,971,953. US 4,060,082. EP-A-0520618. [50] WO98 / 01174. [51] Hoffinann et al. (2002) Vaccine 20: 3165-3170. [52] Subbarao et al. (2003) Virology 305: 192-200. [53] Uu et al. (2003) Virology 314: 580-590. [54] Ozaki et al. (2004) J. Virol. 78: 1851-1857. [55] Webby et al. (2004) Lancet 363: 1099-1 103. [56] W097 / 37000. [57] Brands et al. (1999) Dev Biol Stand 98: 93-100. [58] Halperin et al. (2002) Vaccine 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: 1149-58. [63] Pau et al. (2001) Vaccine 19: 2716-21. [64] WOOl / 22992. [65] Hehme 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 Lab Immunol 3: 507-10. [68] Williamson et al. (2006) Infection and Immunity 74: 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. (1996) J Biotechnol 44: 155- 160. [75] Banzhoff (2000) Immunology Letters 71: 91-96. [76] W002 / 097072. [77] Greenbaum et al. (2004) Vaccine 22: 2566-77. [78] Zurbnggen et al. (2003 ) Expert Rev Vaccines 2 ~: H95— 304. [79] Piascik (2003) J Am Pharm Assoc (Wash DC). 43: 728-30. 20 [80] Mann et al. (2004) Vaccine 22: 2425-9 [81] Halperin et al. (1979) Am J Public Health 69: 1247-50. [82] Herbert s al. (1979) J Infect Dis 140: 234-8. [83] Chen et al. (2003) Vaccine 21: 2830-6.

权利要求:
Claims (15)
[0001]
1. Method for the manufacture of an oil-in-water emulsion, characterized by the fact that it comprises: passing a first emulsion having a first average oil droplet size through a microfluidization device to form a second emulsion having a second average oil droplet size that is smaller than the first average oil droplet size; wherein the microfluidization device comprises an interaction chamber comprising a plurality of Z-type channels and a return pressure chamber comprising at least one channel; and in which the return pressure chamber is positioned downstream of the interaction chamber.
[0002]
2. Method according to claim 1, characterized by the fact that the first emulsion is introduced into the interaction chamber at a first pressure and the second emulsion leaves the pressure chamber returning to a second pressure which is less than at the first pressure; and where between 80 and 95% of the pressure difference between the first and the second pressure are eliminated through the interaction chamber and 5 to 20% of the pressure difference between the first and the second pressure are eliminated through the pressure chamber. return.
[0003]
Method according to claim 1, characterized in that the return pressure chamber comprises a plurality of channels.
[0004]
4. Method according to claim 1, characterized by the fact that it comprises the filtration of the second emulsion.
[0005]
5. Method according to claim 1, characterized by the fact that it comprises the step of forming the first emulsion using a homogenizer.
[0006]
6. Method according to claim 1, characterized by the fact that the first average oil droplet size is 5000 nm or less.
[0007]
7. Method according to claim 1, characterized in that the number of oil droplets having a size of> 1.2 μm in the first emulsion is 5 x 1011 / ml or less.
[0008]
8. Method according to claim 1, characterized by the fact that the second average oil droplet size is 500 nm or less.
[0009]
9. Method according to claim 1, characterized in that the number of oil droplets having a size of> 1.2 μm in the second emulsion is 5 x 1010 / ml or less.
[0010]
10. Method for preparing a vaccine composition, characterized in that it comprises preparing an emulsion as defined in claim 1, and combining the emulsion with an antigen.
[0011]
11. Method for preparing a vaccine kit, characterized in that it comprises preparing an emulsion, as defined in claim 1, and packaging the emulsion in a kit as a component of the kit together with an antigen component.
[0012]
12. Method, according to claim 11, characterized by the fact that the kit components are in separate bottles, such as, for example, in which the bottles are made of borosilicate glass.
[0013]
13. Method according to claim 10, characterized by the fact that the emulsion is a bulk adjuvant and the method comprises extracting unit doses of bulk adjuvant for packaging as components of the kit.
[0014]
14. Method according to claim 10, characterized in that the antigen is an antigen of influenza virus in which, for example, the combination of the emulsion and the antigen forms a vaccine composition and in which the vaccine composition includes 15 μg, 10 μg, 7.5 μg, 5 μg, 3.8 μg, 1.9 μg or 1.5 μg of hemagglutinin by strains of influenza virus.
[0015]
15. Method according to claim 1, characterized by the fact that: the interaction chamber has a plurality of channels of fixed geometry, in which the emulsion passes and in which the emulsion is accelerated under pressure to reduce the droplet size emulsion oil and reduce the number of oil droplets having a size of> 1.2 μm; wherein channels in the interaction chamber include a plurality of substantially right-angle sections; and in which the return pressure chamber has at least one channel of fixed geometry in which the emulsion passes.

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引用文献:

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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| B09X| Republication of the decision to grant [chapter 9.1.3 patent gazette]|

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2021-04-13| B16A| Patent or certificate of addition of invention granted [chapter 16.1 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 MEDIDA CAUTELAR DE 07/04/2021 - ADI 5.529/DF |

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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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US28354809P| true| 2009-12-03|2009-12-03|

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US61/283,548|2009-12-03|

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PCT/IB2010/003390|WO2011067672A2|2009-12-03|2010-12-03|Arranging interaction and back pressure chambers for microfluidization|

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