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
From the two lyocycle runs, eight groups of the 5-mL vials, each contains ~1\6 of the cake with known moisture contents (italicized
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
. 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).
demonstrates that all eight t=0 samples (
) 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
suggest that the moisture specification can be set at 3% for this protein drug product.
Table II: Vials from Table I selected for stability study.
Stability data using the same approach with a second protein drug product is shown in
. The results suggested a moisture specification of 2.7% is appropriate for this drug product.
A few technical points are worth mentioning. First, for easier transfer of the divided cake/powder in the glove bag, vials
with an opening of 20 mm (for a 5-mL vial or a 6-mL oven vial from Metrohm) instead of 13 mm (for 2- or 3-mL vials) are recommended.
Second, before performing the actual experiment, one should test the system suitability by selecting at least two cakes with
high and low moisture levels (at > 3% and < 1%, respectively) and perform moisture analysis on all six vials from each cake.
The percentage of moisture should be identical (or with very low relative standard deviation) for all the replicates.
Third, during a real experiment, one can also include vials/samples with very close moisture content for an accelerated stability
study. An observation that the accelerated stabilities are extremely close for samples with similar moisture levels will reinforce
the confidence that one is on the right track.
Finally, it is possible that, with extensive product knowledge and process understanding, one can, in a single lyocycle run,
achieve cakes with all the desirable spread of moisture contents (between 0.1–7.0%). Therefore, it is not always necessary
to perform two lyocycle runs when using the proposed approach.
There is no need for large amounts of product or sophisticated equipment. In addition, generating residual moisture in situ
offers a more realistic simulation of an actual freeze-drying run in drug production. Furthermore, the residual moisture of
each product vial incubated at accelerated temperatures for predetermined time periods (i.e., in storage stability study)
can be measured directly and, therefore, can be considered more accurate than that inferred from sister vials. Thus, ambiguity
in stability studies can be reduced.
When manufacturing lyophilized drug product, the conventional wisdom is to aim for no more than 1% residual moisture. Based
on the approach outlined here, the moisture specification for two different protein drug products was 3.0% and 2.7%, respectively,
thereby providing for a higher margin than the conventional 1% residual moisture for lyophilized drug products. Information
generated using this approach can help to strengthen one's data package to support specifications for regulatory filings and
Leu-Fen Lin, PhD, is a senior manager, Formulation Development, and Richard Bunnell, PhD, is general manager, both at SGS Life Science Services, 616 Heathrow Drive, Lincolnshire, IL 60069, tel. 847.821.8900, firstname.lastname@example.org
1. E.D. Breen et al., Pharm. Res.
18, 1345–1353 (2001).
2. N.K. Jain and I. Roy, Pharm. Res. 28, 626–639 (2011).