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

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 Na 2 HPO 4 •12H 2 O. XRD indicated sequential crystallization of Na 2 HPO 4 •12H 2 O, NaCl•2H 2 O and KCl during cooling. When the frozen solutions were heated, two eutectics were observed – the first at ~ −24°C (ternary, NaCl•2H 2 O-KCl-ice) and the second at ~ −22°C (binary, NaCl•2H 2 O-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 Na 2 HPO 4 , NaCl, and KCl. Trehalose rendered the lyophiles completely XRD amorphous, whereas in presence of mannitol, all the solutes except KH 2 PO 4 crystallized. Conclusions Freezing of PBS solution caused a pronounced pH shift due to selective crystallization of Na 2 HPO 4 •12H 2 O. 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.