Ice fog as a means to induce uniform vial-to-vial ice nucleation
As discussed above, one approach for reducing supercooling and controlling ice-nucleation temperature is to introduce nucleating
particles into the supercooled solution. A particularly advantageous nucleating particle is microscopic ice (i.e., frozen
water) crystals in the form of a fog introduced into the freezing chamber (3). The concept of temperature-controlled ice nucleation
was suggested by T.W. Rowe in 1990 (4). A cryogenically created fog containing microscopic ice crystals is introduced into
the lyophilization chamber after the vials have reached the temperature at which nucleation is desired. The ice crystals subsequently
make their way into the vials and induce nucleation inside the vial. Although this technique has found success on a laboratory
scale, it has proven difficult to scale up to commercial lyophilizers. The difficulty is not only forming the ice fog and
ensuring it is sterile, but also uniformly distributing the ice fog rapidly throughout the freezing chamber so that all vials
are properly seeded with nucleating ice particles.
This article will describe a means to produce and distribute an aseptic ice fog that nucleates all vials in a short time.
This work has resulted in a novel means to produce and distribute a sterile ice fog that is applicable to laboratory-, pilot-,
and production-scale lyophilizers. This scalable cryogenic ice fog technology could provide a much-needed degree of control
during lyophilization and thus facilitate application of QbD principles in this crucial downstream operation.
Figure 1 is a schematic illustration of a typical lyophilization system employing the scalable cryogenic ice-fog technique.
Creating a uniform dispersion of ice fog, distributing it into the freezing chamber and seeding vials with ice crystals for
nucleation are achieved by a patent-pending technique involving contact between liquid nitrogen and water in a mixing device
such as an ejector, outside the lyophilization chamber (see Figure 1). The ejector circuit is composed of a port for introducing
ice fog into the freezing chamber and another port for recycling fog out of the chamber.
Figure 1: Illustration of a typical lyophilization system employing the scalable cryogenic ice-fog technique. (ALL FIGURES
ARE COURTESY OF THE AUTHORS)
Ice-fog introduction followed the two-step approach shown in Figure 2. The vials containing the product to be freeze dried
were placed on the cold plates inside the freezing chamber. In the initial phase of the freezing process, the vials were cooled
to a suitable temperature at or below their freezing point. When the suitable vial temperature was achieved, a cryogenic ice
fog was introduced into the chamber for about 30–50 s. Detection of ice nucleation in the vials was assessed by a combination
of direct observation and temperature measurements on the outside of select vials. The metal door of the lyophilizer was replaced
with a Plexiglas construction to facilitate visual observation and video recording.
Figure 2: Illustration of the two-step approach for ice-fog introduction.
In control experiments, the normal freezing cycle was run with no ice fog introduction. The goal was to determine the shelf
temperature at which the first vials nucleate and freeze. This shelf temperature in subsequent trials helped determine the
trigger temperature (tg) that indicated when the ice fog should be introduced into the chamber. The experiment also showed the extent of subcooling
and vial-to-vial variability in freezing temperature by recording the range of temperature and time over which all vials nucleated.
Two sets of tests were performed using two lyophilizers. The first set was performed in a MINIFAST 1.0 (IMA Life) with 1.1
m2 of shelf area and represented a laboratory-scale lyophilizer. The second set was performed in a LYOMAX 2.5 (IMA Life), with
2.5 m2 of shelf area and represented a pilot or commercial-scale lyophilizer. Prefilled sterile vials were obtained for the testing,
with between 10–20 vials arranged to be visible from the front of the chamber. Some vials were also strategically placed inside
the lyophilizer on areas of the shelves where distribution of ice fog was expected to be most challenging. One of these vials
was designated as the trigger vial (see Figure 3, Note 1). The temperature of this vial was monitored to determine when ice
fog should be introduced into the chamber.
Figure 3: Temperature measurements obtained in control experiments as a function of time.
Of the total vials, nine were instrumented using K-type thermocouples. Vials without temperature probes were observed visually
(see Figure 3, Note 4). All instrumented vials, with the exception of the trigger vial, had thermal probes mounted on the
outside of the vials and touching the vial wall. The trigger vial contained the temperature probe inside the solution, but
not touching the walls or bottom of the vial. Thus, it gave a truer indication of the solution temperature compared with the
other instrumented vials where the temperature probe was mounted on the outside. However, it was also the vial most likely
to freeze first because the probe itself served as a point of nucleation. tg was set at the temperature at which the trigger vial froze.
Two populations of vials were used in the same test. One population was filled with pure water only (see Figure 3, Note 2),
and the other was filled with a solution comprising 5% glycine and 1% NaCl (see Figure 3, Note 3). All solutions were filter
sterilized through standard 0.22-µm filters before use. A modular cleanroom was constructed around the front side of each
lyophilizer to replicate the particulate-free condition of production-grade environments. A laser counter was used to measure
the particle concentration inside the clean room, and it measured particulate (i.e., > 5 µm) impurity concentration to be
under 15 particles/ft3.