Phase Transitions in Frozen Systems and During Freeze–Drying: Quantification
Using Synchrotron X-Ray Diffractometry
Dushyant B. Varshney,
Evgenyi Y. Shalaev,
Larry A. Gatlin,
and Raj Suryanarayanan
Received December 23, 2008; accepted February 26, 2009; published online March 27, 2009
Purpose. (1) To develop a synchrotron X-ray diffraction (SXRD) method to monitor phase transitions
during the entire freeze–drying cycle. Aqueous sodium phosphate buffered glycine solutions with initial
glycine to buffer molar ratios of 1:3 (17:50 mM), 1:1 (50 mM) and 3:1 were utilized as model systems. (2)
To investigate the effect of initial solute concentration on the crystallization of glycine and phosphate
buffer salt during lyophilization.
Methods. Phosphate buffered glycine solutions were placed in a custom-designed sample cell for freeze–
drying. The sample cell, covered with a stainless steel dome with a beryllium window, was placed on a
stage capable of controlled cooling and vacuum drying. The samples were cooled to −50°C and annealed
at −20°C. They underwent primary drying at −25°C under vacuum until ice sublimation was complete
and secondary drying from 0 to 25°C. At different stages of the freeze–drying cycle, the samples were
periodically exposed to synchrotron X-ray radiation. An image plate detector was used to obtain time-
resolved two-dimensional SXRD patterns. The ice, β-glycine and DHPD phases were identiﬁed based on
their unique X-ray peaks.
Results. When the solutions were cooled and annealed, ice formation was followed by crystallization of
disodium hydrogen phosphate dodecahydrate (DHPD). In the primary drying stage, a signiﬁcant increase
in DHPD crystallization followed by incomplete dehydration to amorphous disodium hydrogen
phosphate was evident. Complete dehydration of DHPD occurred during secondary drying. Glycine
crystallization was inhibited throughout freeze–drying when the initial buffer concentration (1:3 glycine
to buffer) was higher than that of glycine.
Conclusion. A high-intensity X-ray diffraction method was developed to monitor the phase transitions
during the entire freeze–drying cycle. The high sensitivity of SXRD allowed us to monitor all the
crystalline phases simultaneously. While DHPD crystallizes in frozen solution, it dehydrates incompletely
during primary drying and completely during secondary drying. The impact of initial solute concentration
on the phase composition during the entire freeze–drying cycle was quantiﬁed.
KEY WORDS: disodium hydrogen phosphate dodecahydrate; glycine; in situ freeze–drying; phase
transitions; phosphate buffer; synchrotron X-ray diffraction.
Lyophilization (freeze–drying) is widely utilized for
manufacturing pharmaceutical proteins, diagnostic agents
and other thermolabile agents. Freeze–dried formulations
are multi-component systems containing the active pharma-
ceutical ingredient (API) and excipients such as bulking
agents, lyoprotectants and buffers. Lyophilization of aqueous
solutions typically involves freezing, annealing and drying
stages. During these stages, the API as well as the excipient
can undergo phase transformations. The physical form of the
formulation components in the ﬁnal lyophiles can impact the
product stability (chemical as well as physical) and perfor-
mance (e.g., reconstitution time) (1–4). In protein formula-
tions, a major challenge is to minimize the damage to API
from the stresses (e.g., pH changes, increased solute concen-
tration brought about by ice crystallization, dehydration)
experienced during the freeze–drying. Amorphous sugars
(e.g., sucrose, trehalose) provide lyoprotection during
freeze–drying and subsequent storage. Crystalline bulking
agents (e.g., glycine) enable primary drying at elevated
temperatures and therefore decrease the cycle time and also
result in elegant lyophiles (1,3–8).
When the prelyophilization solution is cooled, crystalli-
zation of ice is often the ﬁrst event, accompanied by freeze
concentration of solutes (3,8–10). Although solute (e.g.,
buffer salt) crystallization is possible, in many cases, it is
2009 Springer Science + Business Media, LLC
Pharmaceutical Research, Vol. 26, No. 7, July 2009 (
Department of Pharmaceutics, College of Pharmacy, University of
Minnesota, Minneapolis, Minnesota 55455, USA.
Present address: sanoﬁ-aventis, Pharmaceutical Sciences Depart-
ment, Bridgewater, New Jersey 08807, USA.
Department of Physics, Kent State University, Kent, Ohio 44242,
Pﬁzer Groton Laboratories, Groton, Connecticut 06340, USA.
To whom correspondence should be addressed. (e-mail: dushamaya@