Quantifying Redispersion Potential of Al Salts in Pharmaceutical Vaccines - A New Method

In order to enhance the immune responses of vaccines, adjuvants like aluminum salt (Alum) are often added. As a result of their electrical charges, these adjuvants can aggregate and then settle over time. Depending on the strength of the bonds between the particles, the sediment which is produced can be more or less compact and difficult to redisperse.

If these phenomena occur within the storage time, problems arise such as:

  • Knowing whether or not the injected dose remains the same (despite compact and large aggregates, do all the active ingredients pass through the syringe’s needle?)
  • Knowing whether or not the therapeutic efficacy and consequently the immunogenicity is reduced (the masked antigen in the aggregate is not injected).

This article outlines a rapid evaluation method of sediment redispersibility which takes less than 30 minutes.

Reminder on the Technique

Based on Static Multiple Light Scattering, Turbiscan technology consists of a light source at around 880 nm being sent onto a sample. Backscattered (BS) and transmitted (T) signal is then acquired across the entire of the sample’s height.

By adapting the frequency and repeating this measurement over a period of time, the monitoring of physical stability is enabled by the instrument.

According to the Mie theory, this technology also facilitates the direct measurement of the mean spherical equivalent diameter (d), with the signal intensity and knowing refractive index of continuous (nf) and dispersed phases (np) and the particle concentration (φ): BS = f(φ,d,np,nf) (BS represents ‘Backscattering Intensity’ and T represents ‘Transmission Intensity’).

Materials and Method

The solution is subjected to a controlled flocculation in order to prevent the loss of immunogenicity. This is achieved by varying pH or ionic strength. This results in particles which are weakly-bonded and which consequently form a loose floc and produce a low-density sediment (where a large amount of water is entrapped) which is easy to redisperse.

Conventional Method: SVR (Sedimentation Volume Ratio)

The conventional method for determining vaccines’ flocculation amounts is by measuring the sediment’s height (SVR) at the completion of suspension settling after 24 hours. SVR is the ratio of the height of the settled sediment against that of the initial suspension.

A larger SVR value (>0.1) usually translates to an improved redispersability or suspendability, as the sediment is less compact. As the figure below indicates, this ratio can be measured with Turbiscan from backscattering evolution.

Delta Backscattering data for SVR measurement after 24 hours measuremen.

Figure 1. Delta Backscattering data for SVR measurement after 24 hours measuremen.

New Method: Sonset (Settling Onset Time)

Even though the SVR method is extremely reliable, it takes too long for quality control or routine. Due to its ability to respond within less than 30 minutes, the Settling Onset Time (Sonset) seems to be quicker and more convenient.

Sonset corresponds to the time it takes to reach 50% of the suspension’s clarification (50% of the maximum amount of light transmitted through the sample). By measuring the time taken for the clarification area at 50% of the transmission to exceed a few millimeters, Sonset is determined (as shown in the Figure below).

(a) Delta transmission, (b) clarification layer at 50% of maximum transmission level.

Figure 2: (a) Delta transmission, (b) clarification layer at 50% of maximum transmission level.

As a means of validating Sonset as a new screening method, the relationship between it and SVR at 24 hours was determined using suspensions of AlPO4 at varying pH levels of between 3 and 9, and varying ionic strength (0 to 1000 mM NaCl) with or without model antigens (BSA, lysozyme).

With the use of 20 ml of suspension in a flat-bottomed cylindrical glass vial, Turbiscan measurements were undertaken. Each set of data was collected over a period of 24 hours. As described above, the SVR after 24 hours and the settling onset time (Sonset) after half an hour were obtained for each sample from the backscattering and transmission data respectively.

Results: Sonset and SVR Relation

As displayed in Figure 3, the results indicate a 2-slope curve which is dependent upon whether the system is flocculated (steep slope) or deflocculated (gentle slope).

Relationship between Settling Onset time (Sonset) and Sediment Volume Ratio (SVR) at 24 hours.

Figure 3. Relationship between Settling Onset time (Sonset) and Sediment Volume Ratio (SVR) at 24 hours.

  1. When the suspension is deflocculated (for example, with a low pH and without NaCl), the AlPO4 particles continue as discrete units (<3 µm). This results in a slow rate of sedimentation (Sonset>60 min), which prevents the entrapment of solvent within the sediment. This is difficult to redisperse as it tends to compact into a hard cake. A low value of SVR (<0,1) reflects this.
  2. In a suspension which is flocculated (having a medium to high pH and/or high ionic strength), it is difficult to preserve the loose structure of flocs in the sediment as it contains a large amount of entrapped water. There is a relatively large volume of final sediment, which is reflected by a larger SVR (>0,1). There is a high sediment rate (Sonset < 30 minutes) which is strongly dependent upon flocculation level (represented by a steep slope).

If these properties are controlled, a positive redispersion of the sediment is guaranteed. By extension, its immunogenicity is also guaranteed. The lower the Sonset, the more powerful the therapeutic efficiency.

Conclusion

TurbiscanLAB can help design a properly flocculated Alum-containing formulation. By changing the ionic strength, pH, and quantity of antigens in the formulation, ideal conditions which favor a flocculated system can be identified.

The decision is facilitated by the use of the settling characterization methodologies outlined above (Sonset and SVR). The transition zone between the flocculated and deflocculated states of AlPO4 formulations was identified by the SVR and Sonset data from the Turbiscan analysis.

In the end, due to the reduced analytical time, Sonset values may be a good candidate to replace the SVR data (a widely used parameter for characterizing vaccine stability) as they are more adapted to formulation screening activities.

Reference

  1. Investigation of the Sedimentation Behavior of Aluminum Phosphate: Influence of pH, Ionic Strength, and Model Antigens; Kevin Muthurania and al.; Pharmaceutical Research and Development; Pfizer Inc.; JOURNAL OF PHARMACEUTICAL SCIENCES 104:3770–3781, 2015).

This information has been sourced, reviewed and adapted from materials provided by Formulaction.

For more information on this source, please visit Formulaction.

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