ULTRAFILTRATION FOR PREPARING OUTER MEMBRANE VESICLES

Abstract

In place of a step of centrifugation during preparation of outer membrane vesicles (OMVs) from bacteria, the invention utilises ultrafiltration. This allows much larger amounts of OMV-containing supernatant to be processed in a much shorter time. Thus the invention provides a process for preparing bacterial OMVs, comprising a step of ultrafiltration. The ultrafiltration step is performed on an aqueous suspension of OMVs after they have been prepared from bacteria and the OMVs remain in suspension after the filtration step. The invention is particularly useful for preparing OMVs from Neisseria meningitidis.

Claims

1-16. (canceled)

17. Bacterial OMVs obtainable by a process comprising a step of ultrafiltration.

18. A pharmaceutical composition comprising the OMVs of claim 17 and a pharmaceutically acceptable carrier.

19. The composition of claim 18, comprising an aluminium hydroxide adjuvant and a histidine buffer.

20. The composition of claim 18, wherein the composition is substantially free from whole bacteria.

21. A vial containing the composition of claim 18.

22. A syringe containing the composition of claim 18.

23. A method for raising an immune response in a patient, comprising administering an immunogenic dose of the composition of claim 18.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0098] FIG. 1 shows a process including two ultracentrifugation steps without ultrafiltration.

[0099] FIG. 2 shows a process in which one ultracentrifugation step has been replaced by an ultrafiltration step.

MODES FOR CARRYING OUT THE INVENTION

Example 1

OMVs from Meningococcal Serogroup B (Norwegian Strain)

[0100] N. meningitidis serogroup B (strain 44/76, from Norway) was cultured on eight selective medium for Meningococci plates at 35 C. in 5% CO.sub.2/air atmosphere for 24 hours. Cell were harvested into 2 tubes with 12 ml Frantz' medium. Contents of tubes were added to 2500 ml flasks containing Frantz' medium (150 ml) and grown with shaking for 12 hours to obtain the correct growth for transferring into 25000 ml flasks containing Frantz' medium (1500 ml). The flasks were grown with shaking for a further 12 hours to yield the inoculum. One flask was added to a Chemap fermentor with 300 L capacity, containing 110 L of pre-sterilised Frantz' medium and sterile-filtered dialysed yeast extract. The pH after inoculation was 7.1, maintained at 7.0 with 3N NaOH. A surface aeration fermentation was performed, controlling the amount of air O.sub.2 and stirrer applied, and cultivating for 10 hours at 35 C. Growth was terminated at an OD.sub.590 mm of 7.10, the fermentor was cooled under 15 C., the air supply was reduced and stirring continued at 100 rpm overnight.

[0101] Transfer of the bacterial suspension from the fermentor was done by pressure to a Millipore CUF cross flow filtration unit equipped with valves, pumps and a filter module with 4 Pellicon P2B300VO5 polyethersulphone filters (300 kD cutoff). Initial transfer of 30 L bacterial suspension was followed with a constant volume concentration until the fermentor was emptied, and then a further concentration was performed to give a volume of 5.5 L.

[0102] Concentrating the suspension was performed in the CFF unit by circulating the suspension to be passing by the filters, with a transmembrane pressure being continuously monitored and kept less then 0.5 bar (observed: 0.5 bar at the end of concentration).

[0103] Adjustment of pH of the concentrated bacterial suspension from pH 7.0 to 8.2 was done by adding, via a tubing system, 5 L of 0.1 M Tris-HCl buffer of pH 9 with 10 mM EDTA, followed by 15 min stirring in the CFF unit to secure uniform conditions.

[0104] Inactivation/extraction of outer membrane (OM) material was initiated by adding, via tubings 500 ml of an 0.1M Tris-HCl buffer (pH 9) containing 10% deoxycholate (DOC), to give a final concentration of 0.5%. Subsequently the suspension was circulated in the CFF unit for 30 min, and the extracted suspension (9.5 L), checked to be completely without living bacteria, was drained off by pumping into a 25 L bottle.

[0105] In a first experiment (experiment A; FIG. 1), crude OMVs were prepared by distributing the inactivated suspension to centrifuge tubes of 500 ml, and centrifuging in a Beckman centrifuge at 9000 rpm (16650g) for 1 hour at 4 C., cellecting of supernatant, 1.35 L of crude OMVs was purified by two subsequent ultracentrifugations at 19000 rpm, 4 C., for 13.6 hours and 6.8 hours respectively, collecting the pellet. The pellet was suspended in 660 ml of 3% sucrose with magnetic stirring at room temperature until homogeneous, obtaining a concentration of the purified material of 1.52 g/L of total protein.

[0106] In a second experiment (experiment B; FIG. 2), 3 L of crude OMVs were prepared from bacterial suspension using a CUF cross flow filtration unit equipped with valves, pumps and a filter module with 1 Pellicon P2B300V05 polyethersulphone filters (300 kD cutoff), Initial transfer of 1 L crude OMVs was followed with a constant volume concentration until the 3 L was finished, and then diafiltered by adding 5 L of 0.05 M Tris-HCl buffer of pH 8.6 with 2 mM EDTA, 1% of DOC and 20% sucrose. The retentate obtained was purged by ultracentrifugation at 19000 rpm. 4 C., for 6.8 hours, cellecting the pellet. The pellet was suspended in 1 t of 3% sucrose with magnetic stirring at room temperature until homogeneous, obtaining a concentration of 0.85 g/L total protein in the purified material.

[0107] Final purifications of OMV obtained by both experiment A and B, after a dilution with 3% sucrose around 1.2 g/L of total protein, were both performed at 20 C. by filtering through 3 capsule filters (Gelman Science Suporlife DCF) in sequence, first pre filters of 0.8 m and 0.45 m, respectively, then the final sterile filtration (0.22 m), testing 836 ml of purified material for experiment A, with an initial protein concentration of 1.1 mg/ml, and 1 L for experiment B. The OMV protein concentrations after the filtration were 0.12 mg/ml and 0.59 mg/ml respectively.

[0108] OMVs were characterised as follows:

TABLE-US-00001 Experiment A Experiment B Specification Deoxycholate 1.5 0.4 0.1-0.4 (g/g protein) DNA 0.004 0.004 <0.035 (g/g protein) Endotoxin 2.8 10.sup.3 2.6 10.sup.3 <20 10.sup.3 (UI/g protein) LPS 0.05 0.08 0.06-0.12 (g/g protein) SDS page 80 kDa 1.7 2.2 1-4 70 kDa 11.8 12.7 1-12 class I 24.6 25.1 22-32 class III 34.8 32.8 30-43 class IV 12.0 12.2 9-18 class V 15.0 15.1 10-24

[0109] Thus the OMVs prepared using ultrefiltration have a similar composition to those obtained by ultracentrifugation. In comparison to the prior art method, however, the method of the invention is much simpler and quicker.

Example 2

OMVs from Meningococcal Serogroup B (New Zealand Strain)

[0110] N. meningitidis serogroup B (strain NZ 98/254, from New Zealand) was cultured as before, except that; (a) Catlin medium was used in place of Frantz' medium; (b) the initial 150 ml cultures were grown to a level ready for transferring into a Chemap fermentor with 300 L capacity, containing 120 L of pre-sterilized medium; (c) growth in the Chemap fermentor was for 12 hours; (d) growth was terminated at OD.sub.690 mm of 5.90.

[0111] Transfer from the fermentor was as before, except that concentration was performed until 5 L volume. Concentration was performed as before.

[0112] pH was adjusted as before, except that: (a) the final pH was 8.6; (b) the amount of 0.1 M Tris-HCl buffer added was 6 L.

[0113] Inactivation/extraction was as before, except; (a) 600 ml of the Tris-HCl buffer was added; (b) the volume of extracted suspension was 19.5 L.

[0114] Preparation of crude OMVs was as before, except; (a) centrifuge tubes were 1000 ml volume; (b) centrifugation was at 8000 rpm (16650g), to give 17.5 L of supernatant.

[0115] Cross-flow filtration for purifying OMVs (in place of centrifugation) was as in experiment B above, except: (a) using 17.5 L crude OMVs; bh) using two P2B300V05 polyethersulphone filters (300 kD cutoff); (c) using an initial transfer of 4 L crude OMVs; (d) diafiltration with 30 L Tris-HCl buffer; (e) pellet was resuspended in 1.2 L of 3% sucrose; (f) the homogenous material was further sonicated, and gave a final concentration of purified material of 1.5 g/L total protein.

[0116] Final purification was as before, except: (a) filtration was through two capsule filters (Sartoclean CA, Sartobran P) in sequence, first pre-filters of 0.8+0.65 m, then a final sterile filtration 0.45+0.22 m. The OMV protein concentration after the filtration was 1.0 g/L.

[0117] OMVs were characterised as follows:

TABLE-US-00002 Example 2 Specification Deoxycholate 0.4 0.1-0.4 (g/g protein) DNA 0.0005 <0.035 (g/g protein) Endotoxin 5393 <20 10.sup.3 (UI/g protein) LPS 0.10 0.06-0.12 (g/g protein) SDS page 80 kDa 3.8 1-4 70 kDa 6.4 1-12 class I 18.7 22-32 class III + FbpA 31.3 30-43 class IV 10.7 9-18 class V 2.7 10-24 NspA 3.9 1-7

[0118] Thus the production method provided OMVs with a native antigen mosaic and a strongly reduced level of LPS. In comparison to the prior art method where two ultracentrifugation steps are used, however, the invention is much simpler and quicker.

Example 3

OMVs from Meningococcal Serogroup B (New Zealand Strain)

[0119] Crude OMVs were prepared from the 98/254 strain as described above. The pH was adjusted to between 7.5 and 9.0 (typically between and 8.3 and 8.5) with buffer, and then concentrated up to 20 litres by ultrafiltration for between 3.5 and 4.5 hours using Polysulphone Millipore Pellicon 2 cassettes with a surface area of 3 m..sup.2. The concentrate material was diafiltered against 7 volumes of a solution containing Tris-EDTA, 1% DOC and 20% sucrose (buffer B), and then with 3 volumes of buffer B1 (same as buffer B but with only 0.5% DOC). The retentate was concentrated again up to 4 litres and collected. The ultrafiltration system was washed with buffer B1. The retentate was then washed, and OMVs (retentate+washes) were stored at 2-8 C. The bioburden in the final material was aero, and endotoxin content was <0.05 UI/ml. The process showed excellent lot-to-lot consistency.

[0120] The stored material was centrifuged in a Beckman Coulter Optima XL 100K ultracentrifuge using a type 19 rotor and 250 ml Beckman bottles (22010 ml material per bottle), 19000 rpm for 408 minutes at 2-8 C. Pellets were washed in 10 ml of a sucrose solution, and were then re-suspended in 3% sucrose (60 ml volume added) using a 700 rpm magnetic agitator (2.5 cm bar) in 250 ml Beckman bottles. Re-suspended material was sonicated for 300 minute at <20 C. If necessary, the sonicated material was diluted with 3% sucrose solution to give a final protein concentration of 1.2 mg/ml. The bioburden in the final material was zero, and the process showed excellent lot-to-lot consistency.

[0121] The OMVs were subjected to a final filtration step, first through 0.8-0.65 m filters and then through 0.22 m filters. The sonicated OMVs were passed into a sterile glass container with Sartoclean CA 0.8-0.65 m 0.2 m pre-filters. This pre-filtration was performed for 5-6 minutes with a peristaltic pump using only one set of filters. The filtrate was then passed into a second sterile glass container with Sartobran P 0.45-0.22 m 0.4 m.sup.4 filters. This filtration lasted 7-10 minutes, again with peristaltic pumps. The pre-filters were first rinsed with 500-600 ml of 3% sucrose, and the 0.22 m filters were washed with 200 ml of 5% sucrose after filtration. Final OMV material was stored at 2-8 C., and contained <0.16 g LPS per g of protein and <0.4 g DOC per g of protein. Bioburden was zero. Protein content in the OMVs was between .800 g/ml and .1000 g/ml.

[0122] 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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