Adjuvants such as Aluminium Salt (Alum) are often added to vaccines to enhance their immune responses. These adjuvants can aggregate and settle over time due to their electrical charges.
This can result in a compact sediment that can be difficult to redisperse depending on the strength of the bonds between the particles. If this occurs within the shelf life, the following problems present themselves:
• Knowing whether the injected dose remains the same, i.e., do all active ingredients pass through the needle of the syringe despite the large, compacted aggregates?
• Or is it the case that the therapeutic efficacy and the immunogenicity are reduced (i.e., the masked antigen in the aggregate does not get injected).
How does the Turbiscan technology in this study work?
Based on Static Multiple Light Scattering, the Turbiscan technology works by sending a light source into a sample and acquiring the backscattered and transmitted signal all over its height. By repeating this measurement over time at adapted frequency, the instrument enables us to monitor physical stability. The signal is then linked directly to the particle concentration and size according to the Mie theory, knowing the refractive index of continuous and dispersed phase.
What methods were used in this study?
To prevent the loss of immunogenicity, the solution is subjected to a controlled flocculation (by variation of the pH or ionic strength). This results in weakly-bonded particles that form a loose flocculation and produce a low-density sediment (containing a large amount of water) that is then easy to redisperse.
Conventional method: Sedimentation Volume Ratio (SVR)
The conventional method to determine the flocculation tendency of vaccines is to measure the height of the sediment (SVR) on completion of suspension settling after 24 hours. The SVR is the ratio of the settled sediment height to that of the initial suspension. A higher SVR value should translate to better suspendibility as the sediment is less compact. This ratio can be measured with the Turbiscan from via the evolution of the backscattering signal.
New method: Settling Onset Time
Although the conventional SVR method is reliable, it is unfortunately still too long for quality control or routine testing. The new Settling Onset Time method is more convenient due to its quick response time (less than 30 minutes).
The Settling Onset Time corresponds to the time to reach 50% of the clarification of the suspension (50% of maximum light transmitted through the sample). Settling Onset Time is then determined from the transmission signal by determining the time for which clarification area at 50% of transmission gets higher than a few millimetres.
To validate the new Settling Onset Time as a new screening method, the relationship between SVR at 24 hours and the Settling Onset Time was determined with suspensions of AIPO4, at a different pH (3 to 9), ionic strength (0 to 1000 mM NaCl) and either with or without the model antigen (BSA or lysozyme).
How were measurements carried out?
Turbiscan measurements were made using 20mL of suspension in a flat-bottom cylindrical glass vial, and each data set was collected over a period of 24 hours.
The Settling Onset Time after 30 minutes and the sedimentation volume ratio (SVR) after 24 hours for each sample were obtained from the transmission and the backscattering data, respectively.
What were the results of the study?
The results showed a 2-slope curve depending on whether the system is flocculated or deflocculated.
1. When the suspension is deflocculated, (e.g., low pH and without NaCl) the AIPO4 particles remain as discrete units resulting in a slow sedimentation rate. This then prevents the entrapment of solvent within the sediment. Here, it tends to compact to a hard cake and is difficult to redisperse. This is reflected by a low SVR value.
2. In a flocculated suspension (e.g., medium to high pH and/or high ionic strength), the loose structure of the floccs is difficult to preserve in the sediment that contains a large amount of entrapped water. The volume of final sediment is relatively large, reflected by a larger SVR. The sediment rate is high and is strongly dependent on the flocculation level.
Controlling these properties guarantees a good redispersion of the sediment and by extension its immunogenicity. The lower the Settling Onset Time, the more powerful the therapeutic efficiency.
The Turbiscan can help researchers design a correctly flocculated Alum-containing formulation. By varying the pH, ionic strength, and the quantity of antigens, it is possible to identify the ideal conditions that favour a flocculated system. The use of the settling characterisation methodology outlined above supports this decision.
The Settling Onset Time and SVR data from the Turbiscan analysis identified the transition zone between the flocculated and deflocculated states of AIPO4 formulations.
Finally, since the analytical time is much shorter, the Settling Onset Time values can replace the SVR data, as it is more adapted to formulation screening activities.
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