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 manufacture of a squalene-containing oil in water emulsion, comprising steps of: (i) forming a first emulsion having a first average oil droplet size; (ii) microfluidizing the first emulsion to form a second emulsion having a second average oil droplet size which is less than the first average oil droplet size; and (iii) filtering the second emulsion using a hydrophilic double-layer filter which has 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 vaccine adjuvant.

2. The method according to claim 1, wherein the average oil droplet size of the emulsion prepared in step (i) is 5000 nm or less.

3. The method according to claim 1, wherein the number of oil droplets having a size of >1.2 m in the first emulsion is 510.sup.11/ml or less.

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

5. The method according to claim 1, wherein the number of oil droplets having a size of >1.2 m in the second emulsion is 510.sup.10/ml or less.

6. The method according to claim 1, wherein the average oil droplet size after filtration is less than 220 nm.

7. The method according to claim 1, wherein the number of oil droplets having a size of >1.2 m after filtration is 510.sup.8/ml or less.

8. The method according to 1, wherein the first hydrophilic polyethersulfone membrane layer is asymmetric and/or porous.

9. The method according to claim 1, wherein the second hydrophilic polyethersulfone membrane layer is asymmetric and/or porous.

10. The method according to claim 1, wherein the second hydrophilic polyethersulfone membrane layer, and optionally the first hydrophilic polyethersulfone membrane layer, comprises a polymeric support material.

11. A method for preparing a vaccine composition, comprising preparing an emulsion adjuvant according to claim 1 and combining the emulsion adjuvant with an antigen.

12. A method for preparing a vaccine kit comprising preparing an emulsion adjuvant according to claim 1 and packaging the emulsion adjuvant into a kit as a kit component together with an antigen component.

13. The method of claim 12, wherein the kit components are in separate vials.

14. The method claim 12, wherein the emulsion adjuvant is a bulk adjuvant and the method comprises extracting unit doses from the bulk adjuvant for packaging as kit components.

15. The method of claim 12, wherein the antigen is an influenza virus antigen.

16. The method of claim 15, wherein the combination of the emulsion and the antigen forms a vaccine composition and wherein the vaccine composition includes about 15 g, about 10 g, about 7.5 g, about 5 g, about 3.8 g, about 3.75 g, about 1.9 g, or about 1.5 g of hemagglutinin per influenza virus strain.

17. The method of claim 15, wherein the combination of the emulsion and the antigen forms a vaccine composition and wherein the vaccine composition includes a thiomersal or 2 phenoxyethanol preservative.

18. The method of claim 11, wherein the antigen is an influenza virus antigen.

19. A method of preparing an oil-in-water emulsion vaccine adjuvant which comprises using a hydrophilic double-layer filter which has 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.

20. A method for manufacture of a squalene-containing oil in water emulsion, comprising steps of: (i) forming a first emulsion having a first average oil droplet size; (ii) microfluidizing the first emulsion to form a second emulsion having a second average oil droplet size which is less than the first average oil droplet size; and (iii) filtering the second emulsion using a hydrophilic double-layer filter which has 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 vaccine adjuvant wherein the squalene-containing oil in water emulsion has a volume that is equal to or greater than about 50 liters.

21. The method according to claim 20, wherein the volume of the squalene-containing emulsion is equal to or greater than about 100 liters.

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. 3A shows a pressure profile for a synchronous intensifier pump mode wherein the pressure is substantially constant for at least 85% of the time.

(4) FIG. 3B shows a pressure profile for a synchronous intensifier pump mode wherein the pressure continuously remains constant.

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

(6) 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.

(7) 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

(8) 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.

(9) 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.

(10) 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.

(11) 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

(12) 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

(13) 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.

(14) 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

(15) 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.

(16) 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

(17) 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.

(18) 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.

(19) 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

(20) 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).

(21) 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:

(22) 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

(23) 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.

(24) 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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