PROPOSED APPROACH FOR MOISTURE GENERATION WHEN FREEZE-DRYING
The approach proposed here addresses both concerns by generating moisture in situ from freeze-drying and by ascertaining the actual moisture content of the vials being analyzed for stability.
Conventional method (ex-situ)
In a conventional method of generating residual moisture (i.e., ex-situ), samples are placed in a humidity chamber at a set temperature and a set relative humidity (RH) (e.g., 40 °C/60% RH) to
allow subject materials to adsorb various amounts of moisture (1). An alternative is to pre-equilibrate samples at discrete
RH values (2). Weighed samples in open vials are placed in desiccators with CaSO4 (Drierite) or saturated salt solutions to
provide various RH at 25 °C (LiCl, 11%; CH3CO2K, 23%; MgCl2, 32%; K2CO3, 43%; Mg(NO3)2, 51%; NaCl, 75%; and K2CrO4, 86%).
Proposed method (in situ)
Two runs of lyocycle (of identical set-up) are proposed. Twelve 20-mL vials are filled with a fixed volume of a protein drug
formulation so that the final lyophilized product (the cake) weighs approximately 0.4–0.6 g. Four vials are placed on each
of the three shelves in the lyophilizer. Lyophilization is performed by freezing the sample and subsequently subliming ice
from the frozen content at a temperature suitable for primary (10 ) drying. Primary drying is continued until the Pirani pressure readout deceases to the pressure level (e.g., 100 mTorr)
of the capacitance manometer (CM) when the secondary (20 ) drying begins. Vials on each shelf are stoppered sequentially at approximately 1–3 h intervals between the end of 10 drying and the beginning of 20 drying.
Table I: Moisture content of vials in lyophilizer (capacitance manometer set at 100 mTorr).
Each cake is split into 6 pre-weighed 5-mL vials in a glove bag where a low RH (< 2%) is maintained by flushing with dry nitrogen.
Cake weight is recorded. One vial from each cake is analyzed for moisture content by Karl Fisher (KF) titrator connected to
a KF Thermoprep (oven, Metrohm) equipped with an adaptor for the 5-mL vials. An example of the analysis for a protein drug
product is shown in Table I.
Table II: Vials from Table I selected for stability study.
Aside from the expected shelf-to-shelf differences in moisture content (since each shelf was stoppered at different times),
there was also substantial vial-to-vial variability among vials on the same shelf, mainly because those vials were stoppered
when sublimation was still in dynamic process and the system was not in equilibrium. These data guide the second lyocycle
run (with additional 12 drug formulation-filled vials) in which one can better gauge when to stopper the vials to obtain cakes
complementary to those from the first run so that the full range of 0.1–7.0% moisture levels can be obtained (see Table I).
From the two lyocycle runs, eight groups of the 5-mL vials, each contains ~1/6 of the cake with known moisture contents (italicized
in Tables I and II), are selected for stability studies. One vial from each group is the t=0 sample, while the remaining 4 vials from each are
incubated at accelerated temperatures for predetermined time periods (e.g., 50 °C for 2 weeks and 4 weeks; 40 °C for 3 months;
and 25 °C for 24 months) and analyzed by the stability-indicating assay upon reconstitution.
Figure 1: Moisture content versus stability via SE-HPLC.
Stability data using the above approach with two different protein drug products are shown in Figure 1. The major degradation route for both proteins upon lyophilization was aggregation; therefore, protein stability was assessed
by size exclusion high-performance liquid chromatography (SE-HPLC) to measure the recovery of intact native protein (% native).
Figure 1A demonstrates that all eight t=0 samples (Table II) with 0.3–6.2% moisture content had identical % native protein indicating
the residual moisture level had no impact on the in-process stability. However, the 50 °C and 40 °C storage stability decreased
when the residual moisture was ≥ 3.6%. Results in Figure 1a suggest that the moisture specification can be set at 3% for this protein drug product.
Stability data using the same approach with a second protein drug product is shown in Figure 1b. The results suggested a moisture specification of 2.7% is appropriate for this drug product.