To reduce the lengthy reconstitution time of a lyophilized high-concentration protein drug product (DP), three approaches
were tested. First, the DP was produced by diluting the formulated drug substance (FDS) and placing a larger volume in each
vial to produce a less dense cake that reconstituted more rapidly. Second, an annealing step was inserted into the lyophilization
cycle that reduced the reconstitution time. Third, the DP was reconstituted more forcefully by vial-shaking instead of the
traditional gentle swirling method. The combination of these changes reduced the reconstitution time from 4–15 min to less
than one minute. The lyophilization cycle was also optimized to accommodate the larger fill volume by adding an annealing
step and by increasing the primary and secondary drying temperatures. The duration of the optimized cycle was essentially
the same as the original cycle. These changes did not compromise the quality of the reconstituted DP as evidenced from aggregate
analysis via stability-indicating size exclusion–high performance liquid chromatography (SE–HPLC) and secondary structure
analysis via Fourier-transform infrared (FTIR) spectroscopy. In addition, reconstitution by the shaking method had no adverse
effect on the integrity of the protein as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE),
forced degradation study, and a functional bioassay.
The protein discussed here is a proprietary pharmaceutical recombinant protein with a molecular weight of approximately 210
kDa. Consisting of 40 mg/mL protein and certain common excipients, the formulated drug substance (FDS) was lyophilized as
the solid drug product (DP) (see Table I). For subcutaneous administration of the 80 mg/mL protein dosage form, end users are required to reconstitute the DP with
water for injection (WFI) to half the original fill volume. With the recommended reconstitution method of continuous swirling,
complete reconstitution of the DP can be variable and lengthy, taking between 4–15 min.
Table I: The formulation.
During the Phase II clinical trial for rheumatoid arthritis, patients complained that the reconstitution of the DP was too
time-consuming and hence painful. Consequently, the lengthy reconstitution needed to be shortened to facilitate patient compliance
and ease-of-use. Because the contract manufacturing for the Phase III clinical trial DP would start in four months, any modifications
to the formulation or the process had to be implemented within that time frame. If the problem remained by then, a mechanical
orbital shaker would be supplied to each patient to replace the reconstitution by hand.
The work to reduce the reconstitution time reported here may provide useful information and practical guidelines for the development
of other high-concentration protein drug products.
MATERIALS AND METHODS
FDS at a protein concentration of 40 mg/mL was filtered through a 0.22-µm filter (Millipore), and 5.5 mL was filled in a 20-mL
vial (West Company). Chamber pressure held at 100 mTorr and controlled with a capacitance manometer. Chamber moisture was
monitored with a Pirani gauge. Product temperature was monitored with thermocouples placed at the bottom center of the vial.
Lyophilization was carried out in a LyoStar II freeze-dryer (FTS Systems). Unless otherwise specified, the cycle consisted
of cooling at 5 °C for 60 min and freezing by lowering the temperature by 0.5 °C/min to –45 °C for 90 min. Primary drying
was initiated by first evacuating to 100 mTorr followed by raising the shelf temperature by 0.5 °C/min to –5 °C. The primary
drying was continued until the Pirani pressure deceased to 100 mTorr. For the secondary drying, the shelf temperature was
increased by 0.7 °C/min to 25 °C for 6 h. Vials were stoppered under 608,000 mTorr anhydrous nitrogen gas pressure.
For reconstitution, 2.3-mL WFI was injected into the vial with a 3-mL BD syringe. Unless otherwise stated, the dissolution
was observed visually as the vial was swirled by hand.