Freeze-dry microscopy (FDM).
Collapse temperatures (T
c) were determined using a "FDCS 196" freeze-drying stage (Linkam Scientific Instruments, Surrey, UK) and an "Axio Imager"
microscope (Zeiss, Göttingen, Germany). Images were captured with a 1.3 MPix digital camera and analyzed using "LinkSys" (Linkam)
software. The protocol applied was a standard procedure reported in the literature (7, 8). Onset of collapse for the sucrose
was determined as follows:
50 mg/mL: T
c = –34°C
100 mg/mL: T
c = –32 °C
200 mg/mL: T
c = –32 °C.
Scanning electron microscopy (SEM).
Lyophilized samples were broken into pieces, fixed on aluminum stubs and then carefully gold-sputtered at 20 mA/5 kV (Hummer
JR Technics) for about 30 s. Cake morphology was then examined using an Amray 1810 T scanning electron microscope (SEMTech
Solutions, CITY, MA) at 20 kV.
Results and discussion
Cycle design by manometric temperature measurement.
Figure 1 illustrates the cycle recipe obtained for 50 mg/mL sucrose. The recipe is optimized for the type of excipient, individual
c of the product, and container system. Note that T
s was automatically lowered by the software after about 6 h to account for a continuous increase in T
p toward the end of primary drying, caused by an increase in R
p (see Figure 4). Product temperature at the sublimation interface did not exceed the collapse temperature during primary drying
(see Figure 3 and Table I). A freeze-drying cycle may be denoted as "optimized" when T
p is maintained just below T
c (i.e., 2–3 °C) during primary drying to avoid structural loss (9). For the SMART cycle, total primary drying time during
this run was 1136 min and the cycle recipe in primary drying as follows: –39 °C (60 min), –18 °C (358 min), –23 °C (718 min).
The endpoint of primary drying was determined using MTM and based on the total difference between the calculated Pice value by MTM (nonlinear regression analysis of the pressure rise data) and the chamber pressure, P
c. A representative determinant of the endpoint of primary drying is crucial for testing robustness as described in this article.
Once all ice has been removed, the pressure rise in the chamber during an MTM measurement is based on leaks and additional
heat transfer to the product. Thus, the "fitted" vapor pressure of ice obtained from the MTM procedure equals that of the
chamber pressure (see Figure 1) (3–5).