Effect of freezing on the acidity of pharmaceutical excipients

     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

Buffers are routinely used to maintain pH constant, not only in the liquid state but also during
cryopreservation or freeze-drying. However, the pH of the buffer solution can change significantly
upon freezing due to the sequential precipitation of buffer components. Such large changes in
acidity can dramatically affect the stability of the biomolecules ^[1, 2]. Therefore, studying the
acidity change upon freezing is a vital step for designing freeze-dried products.

In this work, the acidity of frozen excipients (buffers and sugars), which are frequently used in
pharmaceutical formulations, was determined spectrophotometrically by studying the extent of
protonation of the sulfonephthalein indicators, which are used as acid-base probes and is accessed
by the Hammett acidity function ^[3]. These indicators are present in the freeze concentrated
solution giving us precise knowledge about the change in proton concentration.

Following this approach, freezing-induced acidity change of a series of the biologically used
buffers (carboxylic acid, amino acid, Good’s, and phosphate buffer) is reported for varied
concentrations and freezing rates which helped to optimize the suitable buffer for freezing of
sensitive formulations. In addition, a more detailed investigation of the crystallization of
citrate buffer and PBS buffer is done via differential scanning calorimetry and optical
cryomicroscopy techniques. Using this knowledge, we also found a solution to reduce the
freezing-induced acidity change by introducing a buffer blend of Good’s buffer and sodium phosphate
buffer ^[4]. The comprehensive studies of these projects will be presented in detail in the
presentation.


1.            Pikal-Cleland, K.A., et al., Protein Denaturation during Freezing and Thawing in
Phosphate Buffer Systems: Monomeric and Tetrameric β-Galactosidase. Archives of Biochemistry and
Biophysics, 2000. 384(2): p. 398-406.

2.            Pikal-Cleland, K.A. and J.F. Carpenter, Lyophilization-induced protein denaturation
in phosphate buffer systems: Monomeric and tetrameric beta-galactosidase. Journal of Pharmaceutical
Sciences, 2001. 90(9): p. 1255-1268.

3.            Vetráková, Ľ., V. Vykoukal, and D. Heger, Comparing the acidities of aqueous, frozen,
and freeze-dried phosphate buffers: Is there a “pH memory” effect? International Journal of
Pharmaceutics, 2017. 530(1-2): p. 316-325.

4.            Vesely, L., B. Susrisweta, and D. Heger, Making Good's Buffers Good for Freezing: the
Acidity Changes and their Elimination via Mixing with Sodium Phosphate. Int J Pharm, 2020: p.
120128.