Overcoming Challenges in the Reconstitution of a High-Concentration Protein Drug Product - The authors present approaches used to reduce reconstitution time of a lyophilized high-concentration protein

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Overcoming Challenges in the Reconstitution of a High-Concentration Protein Drug Product
The authors present approaches used to reduce reconstitution time of a lyophilized high-concentration protein drug product.


BioPharm International
Volume 26, Issue 3, pp. 28-39

Lyocycle development for the diluted FDS

To shorten the cycle despite the increased fill volume, the following studies were carried out to optimize the process including annealing, primary, and secondary dryings. The development of this cycle fixed the chamber pressure at 100 mTorr.

Annealing: The effect of annealing on the primary drying rate is dependent on the formulation and process variables. To increase the primary drying rate for the 25-mg/mL FDS, samples were annealed over a range of temperatures for various durations and partially lyophilized to determine the primary drying rate.


Figure 2: Effect of annealing temperature on the primary drying rate. Annealing hold time was 3 h. n=5, w/o A is without annealing control, *p<0.05 compared to w/o A control.
As shown in Figure 2, cycles with a 3 h annealing at either –3 C or –8 C had a significantly higher drying rate compared with annealing at –13 C or –18 C. However, –8 C was chosen over –3 C as the target annealing temperature to provide for a margin ensuring that the temperature was well below the ice melting point.

As shown in Figure 3, annealing at –8 C for either 2.5 h or 4 h had a significantly higher drying rate compared to the control that had no annealing step. An annealing time of 2.5 h was chosen.


Figure 3: Effect of annealing hold time on the primary drying rate. Annealing at -8 C. n=5, w/o A is without annealing control, *p<0.05 compared to w/o A control.
Primary drying: When a formulation freezes, a phase separation occurs. A pure crystalline phase will separate from a saturated amorphous phase. The crystalline phase includes ice or any other crystallizing excipients (e.g., sodium phosphate). During primary drying, the pure ice phase is removed, leaving behind other crystalline phases and any saturated amorphous phases. The aim of primary drying is to remove this unbound water while maintaining the cake structure and protein stability.

To prevent the cake from collapsing during primary drying, the product temperature must be kept under the collapse temperature (Tc). Since Tc is usually 2–3 C above glass transition temperature (Tg'), using the Tg' to gauge the allowable product temperature represents a more conservative approach. As the Tg' of the 25 mg/mL FDS was –20 C (data not shown), a maximum allowable product temperature of –25 C was selected to provide a 5 C safety margin during primary drying.

Two primary drying temperatures were examined: 10 C and 22 C. Primary drying at 10 C represented the more conservative viable cycle, while primary drying at 22 C was chosen to decrease the drying time. The cycle performed with a shelf temperature of 10 C and 22 C had a product temperature of –26 C and –21 C, respectively. Based on the proximity of the product temperature (–21 C) to the observed Tg' (–20 C), the 22 C primary drying shelf temperature was not appropriate.

Secondary drying: Once the unbound, pure ice phase is removed, primary drying concludes. The next step is to remove water trapped in the amorphous phase during secondary drying. Because the Tg' increases when primary drying is complete, the shelf temperature can be increased while keeping the product temperature below the Tg'. Increasing the shelf temperature increases the heat available to remove the bound water and increases the drying rate.

Two secondary drying temperatures were examined: 25 C and 40 C. The 25 C represented the more conservative viable step, while the 40 C was expected to decrease the drying time. Product stability, moisture content, and cake appearance were used to evaluate the feasibility of the two different process conditions. Secondary drying at both temperatures had no effect on the target moisture content (of less than 1%), reconstitution time, pH, or turbidity. In addition, the stability of the products generated from the two temperatures was not significantly different. DP form both temperatures lost 0.2 % native protein compared with the prelyophilized FDS in the SE-HPLC assay, the most sensitive stability indicator for the DP. Therefore, a secondary drying temperature of 40 C was chosen to shorten the overall length of the cycle.

Comparison of the original and the revised cycle: The optimized revised cycle had a cycle length comparable to the original cycle. The original cycle with 5.5-mL fill at the primary and secondary drying temperatures of –5 C and 25 C, respectively, was completed in approximately 50 h. Despite the larger volume and insertion of the optimized annealing step, the revised cycle with 8.8-mL fill at the primary and secondary drying temperatures of 10 C and 40 C, respectively, could be completed in approximately 52 h.


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