BREAKING EMULSIONS OF VACCINES – EASY AND UNCOMPLICATED PROTOCOLS

For any oil-water/water in our or Water–oil–water emulsion measurement employing liquid-liquid extraction, the laboratory must use emulsion-breaking techniques to complete the phase separation if the emulsion exceeds one-third of the solvent layer’s volume. It is essential to remember that if the emulsion cannot be broken, the test method may not provide accurate results for the problematic sample. The ideal approach to break or reduce the emulsion in such cases depends on the sample matrix.

Detecting the antigen in the emulsion is crucial during vaccine emulsion breakage testing. Also, most regulatory agencies require that emulsion-based vaccines have the antigen extracted from the emulsion and proven to be there. (Guideline on data requirements for adjuvants in vaccines for veterinary use (Europa. eu). While complete recovery of all antigens may not always be possible, detecting the presence of antigens in the emulsion is essential to assess the stability and quality of the vaccine. Confirming the presence of antigens, even if not all are recovered, provides valuable insight into the integrity of the vaccine formulation and its potential effectiveness (Guideline on requirements for adjuvants in veterinary vaccines (Europa. eu)

There are several methods for breaking veterinary vaccine emulsions. We have used techniques such as Chloroform, Isopropilic alcohol, and olive oil. It is essential to have a robust method for recovering the active ingredient formulated in the vaccine. Please remember that the idea is to confirm the presence of antigens and not recuperate all of them because this can be challenging. Emulsion breakers are trial and error in hitting the right product and amount.

Below is a list of possible protocols used to break some emulsions.

Procedures:

1. Acidify the sample. If the emulsion is due to an alkali soap, detergent, or surfactant, lower the pH to 2 with sulfuric acid, H2SO4, or hydrochloric acid, HCl. This (as recommended in the oil and grease methods, mainly for preservation of the sample) will modify the surfactant’s charge so that it no longer acts as an emulsifier.

2. Add table salt (NaCl). If you know that your sample forms emulsions, add salt to it before shaking it with the solvent. If you try to break an already-formed emulsion, you can shake the salt into the sample. Hopefully, you will see the salt dropping to the bottom as the droplets from the emulsion coalesce and the emulsion disappears into cleanly partitioned layers. Sodium sulfate can also be sprinkled into the sample as described above, or a more aggressive approach for sodium sulfate can be described next.

4. Another very effective salt – Potassium pyrophosphate. Use in the same manner as the table salt.

5. Filter through sodium sulfate – Pipette as much solvent/extract as possible into a clean container without taking the water layer. Add one or more grams of anhydrous sodium sulfate, Na2SO4, and stir with a glass stirring rod. The extract should be non turbid but may have coloration. Put an 11 cm No.40 Whatman filter paper into a glass funnel and filter to remove sodium sulfate from the solvent. Alternatively, skip the stirring step and pour the solvent and emulsion through the filter paper with the sodium sulfate. In this case, the sodium sulfate binds the water, leaving the solvent behind so it merges into the solvent layer.

6. Centrifugation. A centrifuge is a surefire way to break the emulsion, but not all labs, especially on-site facilities, have one available.

7. Ultrasonic bath. Put the solvent/emulsion extract into an ultrasonic bath with ice.

8. Heating or Cooling: Depending on the emulsion’s components, heating or cooling can help break the emulsion. Some emulsions are sensitive to temperature changes, which can disrupt the stability of the droplets.

The protocols previously mentioned possess a broader scope. Below, more specific protocols are extensively used practically and efficiently. These protocols have been developed with the specific requirements and characteristics of veterinary vaccines in mind, and they are better suited for their preparation, testing, and administration. Protocol:

Safety First

Wear appropriate PPE: Lab coat, gloves, safety glasses.

Work in a designated area: Use a fume hood or biosafety cabinet if available, to handle volatile components or pathogens safely.

Equipment and Materials Needed

Centrifuge

Pipettes and pipette tips

Tubes

Solvents (e.g., ethanol, isopropanol, Chloroform depending on the emulsion type)

Saline solution or distilled water

pH adjustment chemicals (acids or bases)

Steps to Break a Vaccine Emulsion

Dilute the vaccine emulsion with isopropyl alcohol. The exact ratio can vary, but a starting point could be a 1:1 ratio of emulsion to isopropyl alcohol, methanol, chloroform . This step reduces the emulsion’s viscosity and disrupts the interactions stabilizing the emulsion.

*Adjusting the ratio of isopropyl alcohol, the centrifugation speed, or the duration may be necessary based on preliminary results.

Mixing:

Gently mix the solution to ensure thorough blending of the isopropyl alcohol with the emulsion. This can be done by inverting the container or using a laboratory mixer. Avoid vigorous shaking that might introduce air bubbles or further emulsify the mixture.

Centrifugation:

Transfer the mixture to centrifuge tubes and centrifuge at a high speed for a specified time (e.g., 10 minutes at 3000-5000 rpm, though conditions may vary based on the emulsion’s characteristics). Centrifugation forces the separation of the emulsion into distinct layers.

Phase Separation:

After centrifugation, carefully remove the tubes from the centrifuge. You should observe separation into layers, with the denser aqueous phase at the bottom and the lighter oil phase  at the top.

Collection:

Use a pipette to carefully remove the upper layer, which contains the isopropyl alcohol and the oil phase. Be cautious not to disturb the interface between the layers to ensure clean separation.

Analysis or Further Processing:

The separated components can now be analyzed or processed further, depending on your objectives.

Notes:

The effectiveness of this method can depend on the specific properties of the vaccine emulsion, including the type of oil used and the presence of any stabilizers or surfactants.

**Adjusting the ratio of isopropyl alcohol, the centrifugation speed, or the duration may be necessary based on preliminary results.

Always follow specific safety guidelines and disposal regulations for isopropyl alcohol and any biological materials.

Using isopropyl alcohol, methanol, and chloroform is an effective technique for breaking down vaccine emulsions, facilitating the analysis and processing of the individual components. This method can be employed for water-in-oil and oil-in-water emulsion types and double emulsions of water-oil-water with or without the addition of polymeric substances. Its ability to provide a streamlined approach to separating emulsion components makes it a valuable tool for scientific investigation and experimentation in vaccine development.

Conclusion

Breaking a vaccine emulsion requires careful consideration of the emulsion’s composition and the intended purpose of breaking it. It’s a delicate process that, when done correctly, allows for the analysis and modification of the vaccine’s components. Always prioritize safety and consult specific protocols related to the vaccine you’re working with