One commonly used adjuvant formulation is derived from Freund’s incomplete adjuvant (without mycobacterium). It comprises 10% v/v mannide monooleate, 40% paraffin oil, and an aqueous phase. Mannitol monooleate is a pasty yellow viscous liquid that needs to be mixed with mineral oil and used only after complete solubilization and sterilization. This adjuvant is highly potent and generates a strong immune response, but it is unstable, and the mixture separates quickly, making it a challenge to sustain such a formulation on a commercial scale. In the UK, this adjuvant was used in human vaccines, but it was banned after studies showed tumors in rats and strong local reactions in patients receiving the vaccine.
Today, more stable formulations use surfactants other than mannide monooleate, such as Polysorbate and Sorbitan Monooleate. This compounded emulsion can be customized for viscosity and water content, with more surfactant leading to a more potent immune response, and below, you can find some examples of this formulation and its variants:
Preparation method:
I. Preparation oil phase:
- Weigh the amount of Mineral Oil
- Add the required amount of Span 80 in the oil phase
- Stir until it is a homogeneous mixture
- Add the required amount of Tween 80 in the water phase
- Stir until it is a homogeneous mixture
- Sterilize this mixture by filtration – use a 0.22 micrometer filter of Nylon or Teflon (PTFE) material
II. Preparation emulsion
- The water phase should be added to the oil phase:
- Start the colloid mill and add the water phase slowly to the oil phase while emulsifying.
- Continue the emulsification process until a temperature of 37 °C has been reached.
- Cool the emulsion to room temperature, under continuous stirring.
| Name | Amount (g) |
|---|---|
| Oil Phase | |
| Liquid paraffin | 470 |
| Sorbitan Mono-oleaat | 60.76 |
| Polysorbat 80 | 19.24 |
| WaterPhase | |
| Water phase including antigen material | 450 |
| TOTAL: | 1000 |
Preparation method:
I. Sterilisation Polysorbate 80
- Weigh an amount of 2.1g of WFI in the vessel.
- Add the Polysorbate 80 (7g) to the vessel.
- Stir until homogeneous.
- Autoclave the vessel for 30 minutes at 121°C.
II. Preparation water phase
- Weigh the required amount of autoclaved Polysorbate 80/WFI mixture
- Add the antigen material and residual WFI aseptically into the sterile vessel. Stir until homogeneous.
III. Preparation oil phase
- Weigh the liquid paraffin in a preparation vessel.
- Add the Span 80 and homogenize the oil phase by stirring.
- Filter the oil phase by a 0.22 – micrometer filter into a sterile vessel connected to a colloid mill or Silverson.
IV. Preparation of emulsion
- Heat the oil phase to between 30 – 34°C.
- Start the colloid mill and add the water phase to the oil phase while emulsifying (using the stirrer and the colloid mill).
- The emulsification process is continued until the temperature of the emulsion is between 37 – 39 °C.
- Take a sample to check the homogeneity under the microscope. If the sample is not homogeneous, cool the emulsion to 20°C and emulsify using the colloid mill until the temperature is between 37 – 39°C. Repeat this procedure until the emulsion is homogeneous.
- If homogeneous, cool the emulsion under stirring as quickly as possible to a temperature of 2 – 8°C.
- Store the final bulk at 2-8°C.
| Name | Amount (g) |
|---|---|
| Oil Phase | |
| Liquid Parafin | 469 |
| Sorbitan Monooleaate | 25 |
| Water Phase | |
| Polysorbat 80 | 7 |
| Water | 411 |
| Total | 912 |
Preparation method:
I. Sterilisation Polysorbate 80
- Weigh an amount of 18.7 g of WFI in the vessel.
- Add the Polysorbate 80 (6 g) to the vessel.
- Stir until homogeneous.
- Autoclave the vessel for 30 minutes at 121°C.
II. Preparation water phase
- Weigh the required amount of autoclaved Polysorbate 80/WFI mixture
- Add the antigen material and residual WFI aseptically into the sterile vessel. Stir until homogeneous.
III. Preparation oil phase
- Weigh the liquid paraffin in a preparation vessel.
- Add the Span 80 and homogenize the oil phase by stirring.
- Filter the oil phase by a 0.22 – micrometer filter into a sterile vessel connected to a colloid mill or Silverson.
IV. Preparation emulsion
- Heat the oil phase to between 30 – 34°C.
- Start the colloid mill and add the water phase to the oil phase while emulsifying (using the stirrer and the colloid mill).
- The emulsification process is continued until the temperature of the emulsion is between 37 – 39 °C.
- Take a sample to check the homogeneity under the microscope. If the sample is not homogeneous, cool the emulsion to 20°C and emulsify using the colloid mill until the temperature is between 37 – 39°C. Repeat this procedure until the emulsion is homogeneous.
- If homogeneous, cool the emulsion under stirring as quickly as possible to a temperature of 2 – 8°C.
- Store the final bulk at 2-8°C.
Note: Check the microscopic view (100 x 10): water globuli should be 100 % smaller than 10 µm and mainly about 1 µm.
| Name | Amount (g) |
|---|---|
| Oil Phase | |
| Liquid paraffin | 552 |
| Sorbitan Mono-oleaat | 51 |
| Polysorbat 80 | 6 |
| WaterPhase | |
| Water phase including antigen material | 275 |
| Total | 884.17 |
The emulsification process mentioned in the examples involves using a colloid mill, operating until the temperature reaches 37°C; however, this method may be challenging in cases where the antigen is sensitive to high temperatures. Finding a balanced formulation that combines immune response, cost, local reactions, antigen compatibility, and process feasibility is important and can be time-consuming, costly, and often artisanal, hence it is important to test more than one formulation and explore different paths to reduce product time to marketing.