Circulation of components during homogenization of emulsions

Abstract

An improved method for the manufacture of an oil-in-water emulsion involves circulation of emulsion components between a first container and a second container via a homogenizer and/or via a microfluidization device. Usefully, all of the emulsion components from the first container are emptied before being returned.

Claims

1. A method for the manufacture of a squalene-containing oil-in-water emulsion, the method comprising the steps of: (b) microfluidizing a first emulsion formed using a mechanical homogenizer, wherein the first emulsion has a first average oil droplet size, (c) circulating the first emulsion by transferring from a first emulsion container through a first microfluidization device to a second emulsion container, and then again through the same microfluidization device, so as to form a second emulsion having a second average oil droplet size which is less than the first average oil droplet size; and (d) filtering the second emulsion.

2. The method according to claim 1, comprising before step (b): (a) forming a first emulsion having a first average oil droplet size.

3. The method according to claim 1, wherein substantially all of the emulsion components from the first container are passed through the microfluidization device into the second container, and then substantially all of the emulsion components from the second container are passed through the microfluidization device back into the first container.

4. A method for the manufacture of a squalene-containing oil-in-water emulsion, the method comprising the steps of: (i) .Iadd.performing a type II circulation comprising .Iaddend.transferring first emulsion components from a first container to a second container through .[.the.]. .Iadd.a .Iaddend.homogenizer, and then returning them from the second container to the first container through the same homogenizer, so as to form a first emulsion .Iadd.having a first average droplet size.Iaddend., wherein the homogenizer provides a shear rate of up to 110.sup.6 s.sup.1; and (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 microfluidizing occurs in an interaction chamber that provides a shear rate >2.510.sup.6 s.sup.1.

5. The method according to claim 4, wherein substantially all of the emulsion components from the first container are passed through the homogenizer into the second container, and then substantially all of the emulsion components from the second container are passed through the homogenizer back into the first container.

6. The method according to claim 4, further comprising: (iii) filtering the second emulsion.

7. The method of claim 4, wherein step (i) comprises two or more cycles of transferring the first emulsion's components from the first container to the second container and back again.

8. The method of claim 4, wherein during step (ii), the second emulsion is formed by circulating the second emulsion components through a microfluidization device a plurality of times.

9. The method of claim 8, wherein the circulation of the second emulsion components comprises transferring the second emulsion components between a first emulsion container and a microfluidization device.

10. The method of claim 8, wherein the circulation of the second emulsion components comprises transferring the second emulsion components from a first emulsion container, through a first microfluidization device to a second emulsion container, and then through a second microfluidization device, wherein the first and second microfluidization devices are the same.

11. The method of claim 8, wherein after passing through the second microfluidization device, the second emulsion components are returned to the first emulsion container and the circulation of claim 8 is repeated one or more times.

12. A method for the manufacture of a squalene-containing oil-in-water emulsion, the method comprising the steps of: providing a first emulsion having a first average oil droplet size of 5000 nm or less, and wherein the number of oil droplets having a size of >1.2 m in the first emulsion is 510.sup.11/ml or less; circulating the first emulsion by transferring from a first emulsion container through a first microfluidization device to a second emulsion container, and then again through the same microfluidization device, so as to .[.foam.]. .Iadd.form .Iaddend.a second emulsion having a second average oil droplet size which is less than the first average oil droplet size; and filtering the second emulsion.

13. 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.

14. The method of claim 1, wherein the second average oil droplet size is 500 nm or less.

15. The method according to claim 1 or 12, wherein the number of oil droplets having a size of >1.2 m in the second emulsion is 510.sup.10/ml or less.

16. A method for preparing a vaccine composition, comprising preparing the oil-in-water emulsion according to claim 1 or 12 and combining the emulsion with an antigen.

17. A method for preparing a vaccine kit comprising preparing the oil-in-water emulsion according to claim 1 or 12 and packaging the emulsion into a kit as a kit component together with an antigen component.

18. The method of claim 17, wherein the kit components are in separate vials.

19. The method of claim 18, wherein the vials are made from borosilicate glass.

20. The method of claim 17, wherein the oil-in-water emulsion is a bulk adjuvant and the method comprises extracting unit doses from the bulk adjuvant for packaging as kit components.

21. The method of claim 17, wherein the antigen is an influenza virus antigen.

22. The method of claim 21, 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 1.9 g, .Iadd.or .Iaddend.about 1.5 g of hemagglutinin per influenza virus strain.

23. The method of claim 21, 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.

24. The method of any one of claims 1, 4 and 12, wherein the squalene-containing oil-in-water emulsion comprises polysorbate 80 and sorbitan trioleate.

25. The method of claim 24, wherein the squalene-containing oil-in-water emulsion comprises about 4.3% squalene, about 0.5% polysorbate 80 and about 0.48% sorbitan trioleate by weight.

26. The method of claim 16, wherein the squalene-containing oil-in-water emulsion comprises polysorbate 80 and sorbitan trioleate.

27. The method of claim 26, wherein the squalene-containing oil-in-water emulsion comprises about 4.3% squalene, about 0.5% polysorbate 80 and about 0.48% sorbitan trioleate by weight.

.Iadd.28. The method of claim 4, wherein step (i) further comprises first performing a type I circulation of transferring the first emulsion components between a first container and a homogenizer before performing the type II circulation..Iaddend.

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. 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

(6) The emulsion components squalene, polysorbate 80, sorbitan trioleate and sodium citrate buffer were introduced into an in-line, high speed, rotor/stator homogenizer (IKA Super Dispax Reactor DRS 2000/5). Emulsion starting volumes of 280 L and 250 L were used and the speed of the homogenizer was set at 50001000 rpm. The temperature of the emulsion during homogenization was maintained at below 60 C.

(7) Three test runs were carried out. In the first test run, 280 L of the emulsion components were subject to type I circulation, between the homogenizer and a first premix container, for 20 minutes followed by a single type II circulation, transferring the first emulsion components from a first premix stainless steel container, through the homogenizer to a second premix stainless steel container, and then back through the homogenizer. In the second test nm, 280 L of the emulsion components were subjected to type I circulation, between the homogenizer and a first premix stainless steel container, for 5 minutes followed by 5 type II circulations, transferring the first emulsion components from a first premix stainless steel container, through the homogenizer to a second premix stainless steel container, and then back through the homogenizer to the first premix stainless steel container. In the third test run, 250 L of the emulsion components were subject to type I circulation, between the homogenizer and a first premix stainless steel container, for 20 minutes followed by a single type II circulations, transferring the first emulsion components from a first premix stainless steel container, through the homogenizer to a second premix stainless steel container, and then back through the homogenizer to the first premix stainless steel container.

(8) The first emulsion was homogenized until it had an average oil droplet size of 1200 nm or less and a number of oil droplets having a size >1.2 m of 510.sup.9/ml or less.

(9) The first emulsion was then subject to microfluidization to form a second emulsion. The emulsion was passed through the microfluidization device five times. The microfluidization device was operated at between approximately 600 and 800 bar (i.e. between approximately 9000 and 12000 psi) and the emulsion was maintained at a temperature of 405 C. during microfluidization through the use of a cooling mechanism.

(10) The second emulsion was then sterile filtered.

(11) The average size of the oil droplets in the filtered emulsions in each test run met the specification for an MF59 adjuvant.

(12) Other parameters of the emulsions during the first, second and third test runs can be found in Table 1.

(13) TABLE-US-00001 TABLE 1 Parameter Unit First run Second run Third run Number of oil droplets with /ml 43.7 10.sup.6 56.4 10.sup.6 45.1 10.sup.6 a size >1.2 m in second emulsion No. of oil droplets with a /ml 0.2 10.sup.6 0.6 10.sup.6 0.5 10.sup.6 size >1.2 m after filtration

(14) The results from all three test runs are excellent. However, the results in Table 1 show that test run 1 produced the largest percentage reduction (99.5%) in the number of particles with a size >1.2 m in the emulsion after filtration compared to the number present in the second emulsion. Therefore, the best homogenization circulation pattern is about 20 minutes of type I circulation followed by a type II circulation.

Example 2

(15) In further experiments a first emulsion was formed by type I (FIG. 5) or .Iadd.a type I (FIG. 5) followed by a .Iaddend.type II (FIG. 6) circulation. For five separate runs the average number of .[.larges.]. .Iadd.larger .Iaddend.particles per ml was as follows:

(16) TABLE-US-00002 Mean Coefficient of variation Type I 1.70 10.sup.9 0.23 .Iadd.Type I followed by.Iaddend. Type II 1.04 10.sup.9 0.13

(17) Thus the .Iadd.inclusion of .Iaddend.type II circulation results in fewer large droplets and less batch-to-batch variation.

(18) 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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