Determination of The Solubility Characteristics of

1
Determination of The Solubility Characteristics
of Tyrosine Adsorbed Subcutaneous Allergy
Vaccines J.P. Salvage 1, A.D. Bullimore 2, M.A.
Skinner 2, G.J. Phillips 1 1 School of Pharmacy
Biomolecular Sciences, University of Brighton,
Brighton, UK 2 Allergy Therapeutics Ltd,
Worthing, West Sussex, UK
Introduction
Results
A series of allergy vaccines have been developed
by adsorbing chemically modified allergens
(allergoid) to tyrosine. The resulting product is
insoluble and when administered as a subcutaneous
dosage form the tyrosine can act as a depot
providing a therapeutically beneficial slow
release of vaccine. It was not known whether the
differing particle sizes would influence the rate
at which the tyrosine particles would dissolve as
there was no pre-existing prior knowledge.
Therefore an in vitro experiment was designed and
developed to examine this.
A pair of equivalent tyrosine concentration
samples with differing mean particle sizes will
not produce equal absorbance readings. The
greater surface area of the larger number of
smaller particles results in a higher level of
light obscuration and thus higher absorbance than
the smaller number of larger particles. This
effect can result in the dilution and
solubilisation data appearing to suggest that
particle size influences solubility, as can be
seen when the steady drop in absorbance of
dilution (Figure 1) is compared with sharper
curving reduction of solubilisation (Figure 2).

Aims
The aim of the experiment was to examine the
potential effects of particle size on the
solubility of MATA (Modified Allergen Tyrosine
Adsorbed) vaccines. The effect of allergoid type
and potency (SU/ml) in relation to dilution and
solubility characteristics were investigated
using Vis spectrophotometry to compare the
absorbance at 680 nm.
Figure 1. Sample Dilution Figure 2. Sample
Solubilisation
However, the data can be normalised by comparing
the dilution assay data with the solubility assay
data to calculate the reduction in absorbance due
to tyrosine solubility (Figure 3). The mean
particle size diameters annotated on Figures 1,
2, 3, were determined previously using a
Malvern Mastersizer MS20 instrument. The
reduction in absorbance seen in Figure 3 revealed
that particle size did not appear to influence
the rate of tyrosine solubility.
Methods
Two MATA vaccines and a control tyrosine blank
sample (sans allergoid) (Table 1) were tested
using two separate assays. In the first assay,
samples were examined for the effects of dilution
alone. The samples were prepared at a tyrosine
concentration of 1 mg.ml-1 in a saturated
tyrosine diluent solution with a 10 ml final
volume. The stock saturated solution having been
previously centrifuged and filtered (0.22 µm) to
remove all non dissolved tyrosine particles. The
sample was mixed and an aliquot of 1 ml was
removed from the 10 ml test volume and the
absorbance measured at 680 nm with a Vis
spectrophotometer. 1 ml of fresh saturated
solution was used to return the volume to 10 ml.
This iterative sampling and testing process was
continued until a concentration of 0.25 mg.ml-1
was attained. The second assay examined the
combined effects of dilution and solubility. The
samples were prepared as before, but substituting
the saturated tyrosine diluent solution with
water for injection (WFI).
Figure 3. Solubility as a percent reduction in
absorbance from the equivalent dilution point
absorbance
Conclusions
By using Vis spectrophotometry to compare the
absorbance at 680 nm of tyrosine containing
samples at differing concentrations it was
possible to demonstrate that the size of the
tyrosine particles in the samples did not appear
to influence the rate at which the tyrosine
dissolved. This work allows a detailed
understanding of MATA product characterisation
and assessment of whether allergoid type and/or
tyrosine content may influence the solubility
profile which in turn provides an in vitro
simulation of product solubilisation.
Table 1. MATA vaccines and tyrosine blank sample
details