Hydrophilic filtration during manufacture of vaccine adjuvants

Abstract

An improved method for the manufacture of an oil-in-water emulsion involves three procedures: (i) preparation of a preliminary emulsion; (ii) micro fluidization of the preliminary emulsion to reduce its droplet size; and (iii) filtration of the microfluidized emulsion through a hydrophilic membrane. The emulsions are useful as vaccine adjuvants.

Claims

1. A method for a large-scale manufacture of a squalene-containing oil-in-water emulsion, comprising steps of: (i) forming providing a first emulsion having a first average oil droplet size, wherein the first average oil droplet size is 5000 nm or less and/or wherein the number of oil droplets having a size of >1.2 m in the first emulsion is 510.sup.11/ml or less; (ii) microfluidizing the first emulsion to form a second emulsion having a second average oil droplet size which is less than the first average oil droplet size, wherein the number of oil droplets having a size of >1.2 m in the second emulsion is 510.sup.10/ml or less; and (iii) filtrating the second emulsion using a first hydrophilic polyethersulfone membrane layer having a pore size >0.3 m and a second hydrophilic polyethersulfone membrane layer having a pore size <0.3 m, thereby providing a squalene-containing oil-in-water emulsion, wherein at least one of the first and the second hydrophilic polyethersulfone membrane layers is an asymmetric membrane, wherein the squalene-containing oil-in-water emulsion has a volume of more than or equal to 50 liters; and, (iv) filling the oil-in-water emulsion into one or more sterile closed systems.

2. The method according to claim 1, wherein the second average oil droplet size is 500 nm or less.

3. The method according to claim 1, wherein the second emulsion is prefiltered through a membrane prior to filtration.

4. The method of claim 3, wherein the prefiltration and filtration steps are carried out using a double-layer filter.

5. The method of claim 3, wherein the filtration membrane, and optionally the prefiltration membrane, comprises a polymeric support material.

6. The method of claim 1, wherein the squalene-containing oil-in-water emulsion obtained after step (iii) is characterized in that the number of oil droplets having a size of >1.2 m is 510.sup.8/ml or less.

7. The method of claim 1, wherein the filtering step (iii) comprises a double layer polyethersulfone membrane.

8. The method of claim 1, wherein the squalene-containing oil-in-water emulsion comprises, by volume, about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate.

9. The method of claim 1, wherein the squalene-containing oil-in-water emulsion contains between 2-20% of oil by volume.

10. The method of claim 1, wherein the sterile closed system is selected from the group consisting of: glass bottles, flexible, bags, vials and disposable syringes.

11. A method for a large-scale manufacture of a squalene-containing oil-in-water emulsion, comprising steps of: (i) subjecting a first emulsion having a first average oil droplet size, wherein the first average oil droplet size is 5000 nm or less and/or wherein the number of oil droplets having a size of >1.2 m in the first emulsion is 510.sup.11/ml or less to microfluidization to form a second emulsion having a second average oil droplet size which is less than the first average oil droplet size, wherein the number of oil droplets having a size of >1.2 m in the second emulsion is 510.sup.10/ml or less; (ii) filtrating the second emulsion using a first hydrophilic polyethersulfone membrane layer having a pore size >0.3 m and a second hydrophilic polyethersulfone membrane layer having a pore size <0.3 m, thereby providing a squalene-containing oil-in-water emulsion, wherein at least one of the first and the second hydrophilic polyethersulfone membrane layers is an asymmetric membrane wherein the squalene-containing oil-in-water emulsion has a volume of more than or equal to 50 liters; and (iv) filling the oil-in-water emulsion into one or more sterile closed systems.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a specific example of a homogenizer that can be used to form a first emulsion.

(2) FIG. 2 shows detail of a rotor and stator that can be used in such a homogenizer.

(3) FIG. 3 shows two pressure profiles for a synchronous intensifier pump mode.

(4) FIG. 4 shows a Z-type channel interaction chamber.

(5) FIG. 5 shows a type I circulation, whereas FIG. 6 shows a type II circulation. Containers are labeled as C whereas a homogenizer is labeled as H. Direction and order of fluid movements are shown.

(6) In FIG. 6 the homogenizer has two input arrows and two output arrows but in reality the homogenizer has a single input channel and a single output channel.

MODES FOR CARRYING OUT THE INVENTION

Example 1

(7) A microfluidized emulsion comprising squalene, polysorbate 80, sorbitan trioleate and sodium citrate buffer was prepared according to the present invention. The emulsion was microfluidized until it had an average oil droplet size of 165 nm or less and a number of oil droplets having a size >1.2 m of 510.sup.8/ml or less.

(8) The emulsion was filtered through a sterilizing grade filter cartridge (filter A) having a prefilter membrane of hydrophilic asymmetric porous polyethersulfone having a pore size of 0.45 m and an endfilter membrane of hydrophilic asymmetric porous polyethersulfone having a pore size of 0.2 m. During filtration, the emulsion was maintained at a temperature of 405 C.

(9) The above process was carried out for four separate runs and the characteristics of the filtered emulsions were measured and are shown in Table 1.

(10) TABLE-US-00001 TABLE 1 Actual Value Test parameter Run 1 Run 2 Run 3 Run 4 Average oil droplet size 148 144 144 150 Number of oil droplets 0.08 10.sup.6 0.08 10.sup.6 0.12 10.sup.6 0.20 10.sup.6 having a size > 1.2 m

(11) As shown in Table 1, filter A consistently reduced the average size of the oil droplets in the emulsion. Furthermore, filter A consistently reduced the number of oil droplets having a size >1.2 m in the emulsion by approximately 10.sup.3 fold.

Example 2

(12) The same microfluidized emulsion as used for example 1 was filtered through a different sterilizing grade filter cartridge (filter B). Filter B had a prefilter membrane of hydrophilic asymmetric porous polyethersulfone and an endfilter membrane of hydrophilic asymmetric porous polyethersulfone having a pore size of 0.2 m. During filtration, the emulsion was maintained at a temperature of 405 C. This process was carried out for four separate runs and the characteristics of the filtered emulsions were measured and are shown in Table 2.

(13) TABLE-US-00002 TABLE 2 Measured value Test parameter Run 1 Run 2 Run 3 Run 4 Average oil droplet size 142 143 141 141 Number of oil droplets 0.23 10.sup.6 0.15 10.sup.6 0.20 10.sup.6 0.23 10.sup.6 having a size > 1.2 m

(14) As shown in Table 2, filter B consistently reduced the average size of the oil droplets in the emulsion. Furthermore, filter B consistently reduced the number of oil droplets having a size >1.2 m in the emulsion by approximately 10.sup.3 fold.

(15) From examples 1 and 2, it can be seen that filter B results in a slightly lower oil droplet size but a slightly greater number of oil droplets having a size greater than 1.2 m. However, both filters A and B showed excellent results.

Example 3

(16) The same microfluidized emulsion as used for example 1 was filtered through another different sterilizing grade filter cartridge (filter C). Filter C had a prefilter membrane of hydrophilic asymmetric porous polyethersulfone having a pore size of 0.45 m and an endfilter membrane of hydrophilic asymmetric porous polyethersulfone having a pore size of 0.2 m. During filtration, the emulsion was maintained at a temperature of 405 C.

(17) In addition, the same microfluidized emulsion as used for example 1 was filtered through another different sterilizing grade filter cartridge (filter D). Filter D had a prefilter membrane of hydrophilic asymmetric porous polyethersulfone and an endfilter membrane of hydrophilic porous PVDF. During filtration, the emulsion was maintained at a temperature of 405 C.

(18) Filter C showed excellent filtration results providing a filtered emulsion having an average oil droplet size of 15520 nm and a number of oil droplets having a size >1.2 m of 510.sup.6/ml or less. Although, filter D also provided a filtered emulsion meeting the above criteria, it was found to block more quickly, thus necessitating the replacement of the filter D membrane. Thus, all polyethersulfone filters were superior to this PVDF filter.

Example 4

(19) Ten different hydrophilic membranes, from various manufacturers, were tested for filtering a microfluidized emulsion comprising squalene, polysorbate 80, sorbitan trioleate and sodium citrate buffer. The filters were numbered 1 to 10 as shown in Table 3 (NB: filter 1 is the same as filter C in Example 3 above; filter 2 is filter D; filter 9 is filter A; and filter 10 is filter B).

(20) The yield of emulsion after filtration of 50 liters of emulsion was measured to determine if the filters were suitable for industrial-scale use. The % of input emulsion which was recovered after filtration was as follows:

(21) TABLE-US-00003 TABLE 3 1 2 3 4 5 6 7 8 9 10 % 50 16 8 8 8 13 4 82 88 89

(22) A low recovery % indicates that the filter retains the emulsion, for instance because of blocking. It is clear that only filters 1, 8, 9 and 10 (i.e. filters A, B & C from above, plus one further filter which is similar to filter A but has a larger pore size in the first layer) gave yields of 50%, and that the yields that are most practicable for using at an industrial scale are filters 8-10. Filters 1, 8, 9 and 10 are all double-layer hydrophilic PES membranes, prepared by three different manufacturers. The first layers in these four membranes are either 0.45 m or 0.6 m and the second layer is 0.2 m. The best results were seen when at least one of the two layers was an asymmetric membrane.

(23) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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