Lyophilization, or freeze-drying, is a process by which a drug formulation is first frozen and then the ice is removed by
sublimation under a vacuum. For a lyophilized drug product, the residual moisture specification is a crucial component of
the data package for regulatory filing. A defined acceptable range of water content also provides flexibility in the manufacturing
process. Such data are usually generated by assessing product stability at various moisture levels.
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The moisture content of the product vial in stability studies is usually inferred from that determined for sister vials. Such
an approach raises two concerns. First, the moisture content of the vial in the stability study potentially may not be identical
to the reference sister vials due to vial-to-vial variability, and therefore, may be imprecise for resolving stability issues.
Second, moisture levels in the product being analyzed are usually generated by sorption of water (via exposure or equilibration) onto a previously highly dried sample, whereas the moisture in an actual product results from
incomplete desorption (during freeze-drying). Further data must be obtained to justify that each individual product stability
is the same no matter which method is used to introduce moisture, due to the unique properties of each product being evaluated.
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 (1°) 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 (2°) drying
begins. Vials on each shelf are stoppered sequentially at approximately 1–3 h intervals between the end of 1° drying and the
beginning of 2° drying.
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: Moisture content of vials in lyophilizer (capacitance manometer set at 100 mTorr).