Overcoming Challenges in the Reconstitution of a High-Concentration Protein Drug Product

ABSTRACT

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

Lyophilization

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.

Reconstitution

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.

Annealing

FDS at a protein concentration of 40 mg/mL was diluted 1.6-fold with WFI, filtered as above, and 8.8 mL was filled in 20-mL vial, stoppered, and weighed. Shelf temperature ramp rate was constant at 0.5 °C/min in all steps below.

There were five vials per group. Each group was separately cooled at 5 °C for 1 h and frozen at –40 °C for 2 h prior to initiating the annealing described below. The control group without annealing was transferred to a –40 °C freezer at this point.

To determine the effects of annealing temperature on the primary drying rate, each of the four sample groups was annealed for 3 h at either –3 °C, –8 °C, –13 °C, or –18 °C. Samples were resolidified at –40 °C for 1 h and transferred to the –40 °C freezer.

At the completion of the annealing for all the groups, the vials including the controls were transferred from the –40 °C freezer to the –40 °C shelf in the lyophilizer, surrounded by two layers of placebo-containing vials, and held at –40 °C for 1 h. Primary drying was initiated by first evacuating to 100 mTorr and then raising the shelf temperature to –5 °C. Vials were stoppered after 3.5–4.5 h when 20–40% of the crystalline water had sublimed. Samples were reweighed, and the primary drying rate was calculated using the weight loss during the partial drying.

To determine the effects of annealing time on the primary drying rate, each of the three sample groups was annealed at –8 °C for 1 h, 2.5 h, or 4 h. Samples were resolidified at –40 °C for 1 h and transferred to the –40 °C freezer, then the lyophization procedure above was followed.

Size exclusion-high performance liquid chromatography (SE-HPLC)

SE-HPLC was performed using an Agilent 1100 system and detection was at 215 nm. A TSK-Gel G4000SWXL column (TOSOH Bioscience) was used with mobile phase consisting of 0.5 N NaCl, 10 mM sodium phosphate, pH 7.4, at a flow rate of 0.4 mL/min. The mass load of the protein was 25 µg. In preliminary experiments with multiangle light scattering and refractive index detectors connected in series, the molecular weight (MW) of eluted species was determined. The main peak eluted at ~21 min was assigned to native monomeric protein. Higher MW species eluted at ~19 min were defined as soluble aggregates, and lower MW species eluted at ~23 min as degradation products. Empower 2 software (Waters) quantifies each as percent of the total protein.

Fourier-transform infrared (FTIR) spectroscopy

Samples were analyzed on an ABB FTLA 2000 spectrometer equipped with a DuraSampleIR II ATR. Prota (Grams/32) software was used for data acquisition, subtraction of buffer and vapor signals, protein spectra normalization, and secondary derivative analysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

All supplies were from Invitrogen. Samples were diluted with NuPAGE LDS nonreducing 4x sampled buffer to a protein concentration of 0.05 mg/mL and heated at 70 °C for 10 min. Samples (1 µg per lane) and SeeBlue Plus 2 molecular weight marker were applied onto 4–12% Bis-Tris precast 1.0 mm x 10 well gel with 1x MOPS running buffer in a Mini-Cell. Electrophoresis was performed at 125 volts for 1.75 h. The gel was stained with Simply Blue Safestain, scanned via Amersham Biosciences Image scanner, and quantified using ImageQuant software.

RESULTS

Vial size and FDS dilution

Initial attempts to shorten the reconstitution by adding salt or surfactant to WFI were unsuccessful (data not shown), hence other strategies were contemplated. First, using a larger vial (and therefore, a larger surface area) should reduce the reconstitution time. Second, diluting the FDS before freeze-drying should reduce the reconstitution time, because Shire et al. showed that decreasing protein loading concentration resulted in a less dense cake that reconstituted more readily (1). When six groups of samples with various vial sizes and FDS dilutions were freeze-dried in one cycle, the cake from the undiluted FDS reconstituted faster when placed in a 50 mL vial (4.6 min) than it did in a 20 mL vial (6.4 min) (see Table II). Furthermore, the cake from the diluted FDS reconstituted significantly faster than that from the undiluted FDS, regardless of the vial size employed.

Table II: Effects of vial size and dilution on reconstitution. FDS is formulated drug substance, WFI is water for injection.

However, combining both the larger vial with the FDS dilution provided no additive effect in reducing the reconstitution time. The reconstitution time (~3 min) is essentially the same with either a 20-mL or a 50-mL vial at 2-fold dilution of the FDS. Because of the higher production cost associated with a bigger (50 mL) vial (because fewer vials can be produced in a given lyophilizer), the smaller (20 mL) vial with the diluted FDS was a viable scenario. The reconstituted time was reduced 37% and 52% when the FDS was diluted 1.5-fold and 2-fold, respectively, in a 20-mL vial. To minimize the cycle length, a 1.6-fold dilution of the FDS was chosen. This dilution would yield a solution containing 25 mg/mL FDS, which would substantially reduce the original reconstitution time without necessitating a lengthy freeze-drying cycle.

To minimize changes in the manufacturing process of 40-mg/mL FDS at 5.5 mL-fill that had previously passed FDA inspection, the 25-mg/mL FDS was manufactured by performing a 1.6-fold dilution of the existing 40-mg/mL FDS instead of being formulated from scratch. Each vial was filled with 1.6-fold volume (5.5 x 1.6 = 8.8 mL) of the diluted FDS. After lyophilization, the DP was reconstituted with the same volume of 2.3-mL WFI to yield the identical 80-mg/mL solution as the original process. The DP from the 8.8-mL fill was referred as the tall cake, and that from the original 5.5-mL fill as the short cake (see Figure 1). The lyophilization cycle for the 40-mg/mL FDS was previously described. Following is the cycle development for the 25-mg/mL diluted FDS.

Figure 1: Representative lyophilized vials from Table II.

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.

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.

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