Spectroscopical measurements of freezing-induced acidity jump of pharmaceutical reliable buffers

     Susrisweta Behera^1, Lukáš Veselý ^1, Radim Štůsek ^1, Thomas Loerting ^2, Dominik Heger

                   ^1 Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

      ^2 Department: Institute of Physical Chemistry, University of Innsbruck, Tyrol, Austria

 Freezing biological substances is a crucial stabilization step that extends the viability and
promotes manufacturing flexibility. However, the stability of the products in frozen solutions is
associated with several problems including potential destabilization by the acidity changes ^[1].
Until now, there have been no systematic studies related to the acidity variations in buffers due
to freezing, thus creating a loophole in the freeze-drying process ^[2].

The work explores a method that measures the ability of frozen buffer solutions to protonate the
sulfonephthalein indicators which is used as a spectroscopical acid base probe ^[3]. The indicators
are present in the freeze concentrate solution and give precise knowledge about the change in
proton activity. Most commonly used pharmaceutical buffers like carboxylic acid, amino acid, amine
buffer, Good’s buffer ^[4] and phosphate buffer were frozen and the acidity change was investigated
for various concentrations, temperatures, and rates of cooling. Furthermore, differential scanning
calorimetry and optical cryomicroscopy studies were used to find the crystallisation of the buffer
components leading to acidity change. At last, the results obtained were compared with the results
of the most commonly used low-temperature pH electrode method ^[5].

Keywords: Biological buffers, cryopreservation, freeze-induced acidity change, freezing

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