Reconstitution time: effects of dilution, annealing and reconstitution method
Reconstitution times or dissolution rates depended on reconstitution method, and for the same reconstitution method, the rates
were affected by the processing of the DP. All the 8.8 mL-filled tall cakes dissolved more rapidly than the original 5.5 mL-filled
short cakes. Employing various methods including the swirling method, a mechanical orbital shaker rotating at 200 rpm, or
with the vial remaining stationary after WFI addition (i.e., no agitation), the tall cake reconstituted significantly faster
by 47%, 41%, and 72%, respectively, compared with the short cake (see Figure 4, A–C). If reconstitution was performed more forcefully with shaking by hand, the tall cake reconstituted in less than half a minute,
or one-fourth of the time required for the short cake (see Figure 4D). Most interestingly, the tall cake reduced not only the reconstitution time but also the variability in the reconstitution
times compared with that of the short cake. The reduced variability is observed in a lower standard deviation (i.e., 6 s versus
50 s) and a smaller difference observed between the maximum and minimum reconstitution times (i.e., 25 s versus 151 s) with
n=20 (see Figure 4D).
Figure 4: Comparison of reconstituting the 5.5 mL-filled versus 8.8 mL-filled cakes.
The above comparison revealed the combined effects of dilution and annealing. To explore the effect of the annealing per se on the reconstitution time, two groups of 8.8 mL-filled vials were lyophilized with and without the annealing step. All annealed
tall cakes dissolved more rapidly than their nonannealed counterparts. Insertion of the optimized annealing step significantly
reduced the reconstitution time by 39% and 45% with the swirling and shaking methods, respectively (see Figure 5).
Figure 5: Effect of annealing on reconstitution time. Vials were all 8.8 mL-filled and produced with or without annealing
Characterization of the DP from the revised cycle
Besides reconstitution time, the physical attributes including cake appearance, moisture, and turbidity of reconstituted solution
of the lyophilized DP were examined.
No cracks, no collapse, and no meltback of the cake were observed. The moisture content of the cake was less than 0.1%. Turbidity
measured at 405 nm passed the specification, indicating no significant particulates were produced during the lyophilization
and reconstitution processes.
Concerns on the shaking reconstitution method
Upon the more forceful shaking reconstitution, a significant amount of foam was visible at the top of the product, which subsided
after approximately 10 min. However, this reconstitution characteristic did not affect the quality of the drug product. Several
analytical methods demonstrated that the quality of the DP was maintained when reconstituted by the swirling or shaking methods.
Reconstitution by the shaking method had no detrimental effect on either the recovery of the protein or the purity as determined
by SE-HPLC. Figure 6A shows an overlay of SE-HPLC chromatograms of prelyophilized FDS and DP reconstituted by the swirling and shaking methods.
Reconstitution by the shaking method had no effect on the purity of the protein. FTIR analysis showed no significant alterations
in the secondary structure of the protein caused by a reconstitution method (see Figure 6B).
In addition, the effects of swirling and shaking reconstitution methods on the integrity of the DP were examined by SDS-PAGE,
a forced degradation study, and a functional assay (i.e., potency or % bioassay). Reconstitution by the shaking method had
no adverse effect on the integrity of the protein as determined by these assays. The formation of molecular weight species
is the major degradation pathways of the DP. The DP does not oxidize or deamidate. A scan of the SDS-PAGE analysis confirms
that reconstitution via shaking had no detrimental effect on the stability of the DP (see Figure 6C). A forced degradation study examining the stability of the reconstituted DP after storage at 25 °C (data not shown) and
37 °C (see Figure 6D) showed that the degradation profiles were identical for both methods. The acceptance criteria for the bioassay test are
± 50% of the reference standard (100%). The measured bioactivity was 115% and 91% for the swirling and shaking reconstituted
Figure 6: Quality of reconstituted drug product (DP). A. Size-exclusion high-performance liquid chromatography (SE-HPLC)
overlay. Sample traced in: Red, Prelyophilized formulated drug substance (FDS); Green, DP reconstituted by swirling; Blue,
DP reconstituted by shaking. B. Fourier-transform infrared (FTIR) spectroscopy secondary derivative overlay. Sample traced
in: Purple, Prelyophilized FDS; Green, DP reconstituted by swirling; Red, DP reconstituted by shaking. C. Nonreducing sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Lane1: Prelyophilized FDS; Lane 2: DP reconstituted
by shaking; Lane 3: DP reconstituted by swirling. D. SE-HPLC analysis for % native after storing the reconstituted DP (n=2
for each method) at 37 �C for up to 15 days.