Characterization of Phosphate Buffered Saline (PBS) in Frozen State and after Freeze-Drying

Alpana A. Thorat, Raj Suryanarayanan

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Abstract

Purpose: To study the effect of mannitol or trehalose on the crystallization behavior of solutes in phosphate buffered saline (PBS) when the solutions were frozen and freeze-dried. Methods: PBS (pH 7.5 at RT) either alone, or with trehalose (5% w/v) or mannitol (1% w/v), were frozen and characterized using low temperature differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and pH measurement. Freeze dried lyophiles were characterized by XRD. Results: In the absence of cosolutes, upon freezing PBS, a pH shift of ~ 4 units was observed due to crystallization of Na2HPO4•12H2O. XRD indicated sequential crystallization of Na2HPO4•12H2O, NaCl•2H2O and KCl during cooling. When the frozen solutions were heated, two eutectics were observed – the first at ~ −24°C (ternary, NaCl•2H2O-KCl-ice) and the second at ~ −22°C (binary, NaCl•2H2O-ice). Trehalose completely inhibited buffer salt crystallization, whereas mannitol suppressed it partially thereby attenuating the magnitude of pH shift. The two eutectic meltings were also suppressed by the cosolutes. XRD of final lyophiles from PBS alone revealed peaks of anhydrous Na2HPO4, NaCl, and KCl. Trehalose rendered the lyophiles completely XRD amorphous, whereas in presence of mannitol, all the solutes except KH2PO4 crystallized. Conclusions: Freezing of PBS solution caused a pronounced pH shift due to selective crystallization of Na2HPO4•12H2O. The addition of trehalose or mannitol suppressed the buffer salt crystallization and attenuated the magnitude of pH shift. The potential instability of biologics due to pH shift in PBS, can be potentially mitigated with the cosolutes.

Original languageEnglish (US)
Article number98
JournalPharmaceutical research
Volume36
Issue number7
DOIs
StatePublished - May 13 2019

Bibliographical note

Funding Information:
The project was partially supported by the William and Mildred Peters endowment fund. We thank Jayesh Sonje and Seema Thakral, PhD for numerous stimulating and helpful discussions. Parts of this work were carried out at the Characterization Facility, University of Minnesota, which received partial support from NSF through MRSEC program. We thank Qi Cheng, PhD, and Todd Geders, PhD from Bio-Techne, Minneapolis for providing PBS stock solution, access to the pilot scale freeze-dryer and the helpful discussions.

Publisher Copyright:
© 2019, Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • cosolutes
  • crystallization
  • eutectic melting
  • freeze-drying
  • frozen state
  • phosphate buffered saline

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