Implications of uncontrolled nucleation
The nucleation behavior can affect several lyophilization process steps and product attributes. "The implications of uncontrolled
nucleation are several-fold," says Shon. "First, since every vial can nucleate at a slightly different time and temperature,
true vial-to-vial uniformity is really not achievable. We have seen a vial nucleate at -7 °C and another vial (of the same
type with the same product) nucleate an hour later at -18 °C. These vials are now very different. They will dry at different
rates and have a different pore structure, different cake structure, and different specific surface area."
Nucleation of freezing strongly influences the size of the resulting ice crystals. Studies have demonstrated that colder nucleation
(i.e., higher degree of supercooling) generally produces smaller and more numerous ice crystals, which leave behind smaller
pores upon sublimation in primary drying. "These smaller pores present a greater resistance to subsequent sublimation of the
remaining ice. As a result, primary drying is slowed by cold nucleation temperatures," explains Thierfelder. "The longer cycles
stemming from uncontrolled nucleation require increased investment in lyophilization capacity and higher operating costs.
It also means greater risk as the product sits in a vulnerable multiday batch operation."
On the other hand, a lower degree of supercooling produces larger ice crystals that result in large pores during drying, reduced
resistance to mass flow, and shorter drying times. "Studies have shown that primary drying times were extended 1–3% for every
1 °C change in ice-nucleation temperature (5, 6). Therefore, by reducing the degree of supercooling from, for example, 15
°C to 5 °C for uncontrolled and controlled nucleation respectively, the primary drying time can potentially be decreased by
10–30%, which is significant given the fact that primary drying may take days," says Brower.
"Reduction in primary drying time is one of the significant benefits of controlled nucleation," Shon adds. "A number of research
groups have reported as much as a 40% reduction in primary drying times (7–9). This can have a significant financial impact
on production as throughput can be significantly increased without increasing the number of expensive production dryers."
Besides influencing the size of ice crystals, the nucleation behavior can also affect product yield in various ways. For example,
previous research has shown that proteins tend to aggregate on the surface of ice (10–14). Colder nucleation tends to produce
smaller ice crystals, which possess larger surface areas; therefore, colder nucleation creates increased aggregation stress
on sensitive proteins.
Another potential source of yield loss in lyophilization arises from vial cracking. "The exact mechanisms for vial cracking
are not well understood, but the problem seems to occur when certain formulation components are improperly frozen into metastable
states that rearrange upon heating in primary drying, and sometimes this rearrangement creates sufficient force to crack the
glass container," Thierfelder explains. "The temperature at which nucleation occurs affects the kinetics of the freezing process
and can influence vial-cracking phenomena."
Uncontrolled nucleation can adversely affect product quality. "No matter how well engineered your process controls or how
uniform your freeze-dryer's heat-transfer environment might be, vial-to-vial uniformity is impossible to achieve in the absence
of controlled nucleation," Thierfelder notes. "The vials will nucleate at random temperatures and times, and therefore, have
separate temperature histories, which will impart different properties to the freeze-dried product." Nucleation and freezing
also affect the cosmetic properties of cakes with effects such as glazing, cake cracking, and stratification often resulting
from problems during the freezing step. "Overall, it should be recognized that the traditional lack of control over nucleation
behavior is poorly aligned with FDA's current emphasis on quality by design," Thierfelder adds.