To reduce the reconstitution times encountered with highly concentrated protein pharmaceuticals, Shire et al. illustrated dilution as a viable approach provided the accompanying longer cycle time can also be reduced (1). Two key approaches
were employed here to reduce the cycle time. One approach was to raise the drying temperatures, and the other was to add an
annealing step (2). While raising the drying temperatures is relatively straightforward, adding an annealing step is not,
especially because published results on the effects of annealing are diverse at best.
Annealing involves holding the frozen product above the Tg' of the formulation before the initiation of primary drying. Such
a step potentially accomplishes two things: it allows for crystal growth of excipients that did not fully crystallize during
the initial freezing step and it potentially reduces the freezing-induced heterogeneity in drying rate through Oswald ripening.
By raising the temperature of a vial above the Tg', ice crystals are allowed to reorganize to a lower energy state, thereby
increasing the size of large crystals at the expense of eliminating smaller ones. In this case, formation of larger crystals
minimizes the surface area, which reduces the total energy of the system.
Depending on the formulation and process variables, the above two consequences of annealing have opposite effects on the primary
drying rate. Searles et al. noted that annealing could increase the primary drying rate for a model compound hydroxyethyl starch by allowing ice crystals
grow bigger (through an Ostward ripening process), hence leaving larger pores or channels for water vapor flow in the already-dried
layer (3). Similarly, Webb et al. also noted that annealing reduced the overall length of the lyophilization cycle for the recombinant human interferon-β (4).
However, Lu and Pikal showed that annealing could slow down the primary drying when annealing caused crystal growth of certain
excipients that blocked the water vapor flow and hence increased the dry layer resistance (5).
To accurately determine the effects of annealing on the rate of drying in the present study, all samples needed to undergo
primary drying in the same cycle. This was accomplished by storing the samples in a –40 °C freezer after the individual annealing
step had been performed. Upon completion of the various annealing treatments, the vials including the controls were transferred
to the –40 °C shelf in the lyophilizer before initiating primary drying.
Besides influencing primary drying rate on a case-by-case basis, annealing has been reported to either decrease or increase
dissolution rate on a case-by-case basis. Searles et al. reported the annealed samples dissolved slightly faster than their unannealed counterparts (3). On the contrary, Webb et al. noted annealing caused an 18-fold reduction in the dissolution rate of lyophilized interferon-r through a reduction in the
surface area of the cake available for wetting (4).
In the current report, the insertion of the optimized annealing step significantly increased not only the primary drying rate,
but also the dissolution rate of the lyophilized DP (see Figure 5).
In fewer than three months and before the Phase III clinical trial DP was being produced, the recommended changes as reported
here were sent to the contract manufacturing site for implementation.
Implementing dilution, annealing, and shaking method not only reduced the reconstitution time to less than one min, but also
reduced reconstitution time heterogeneity among vials. The lyophilization cycle was modified to accommodate the large fill
volumes by adding the annealing step and by increasing the primary and secondary drying temperatures. These changes did not
significantly compromise the DP quality nor the cycle duration. Hence, these changes have been incorporated into the manufacture
and reconstitution of the Phase III clinical trial material, and eventually the commercial DP.
The authors thank Drs. Katherine Bowers and Karen Bossert for suggesting the shaking reconstitution, and Abby Thummals for
excellent technical support.
Leu-Fen H. Lin, PhD*, is a senior manager of formulation development and Richard Bunnell, PhD, is a general manager, both at SGS Life Sciences Services, Lincolnshire, IL.
* To whom correspondence should be addressed, firstname.lastname@example.org
Article submitted: Nov. 12, 2012.
Article accepted: Jan. 2, 2013.
1. S.J. Shire, Z. Shahrokh and J. Liu, J. Pharm. Sci. 93, 1390-1402 (2004).
2. M.J. Pikal, BioPharm. 3, 18-27 (1990).
3. J.A. Searles, J.F. Carpenter and T.W. Randolph, J. Pharm. Sci. 90, 872-887 (2001).
4. S.D Webb et al., J. Pharm. Sci. 92, 715-729 (2003).
5. X. Lu and M.J. Pikal, Pharm. Dev. Tech. 9, 85-95 (2004).