Sample-preparation techniques

All of Genentech's (South San Francisco, CA) lyophilized products are packaged in glass vials. Many configurations, spanning various compositions, fill volumes, and vial sizes, exist, however. The authors considered the techniques as outlined below for preparing samples.

Method A: extraction in the product vial. Mettler-Toledo recommends this technique for handling freeze-dried substances (3). The procedure calls for injecting a known amount of anhydrous solvent (e.g., methanol or other primary alcohols) directly into the unopened sample vial and allowing time for water to be extracted into the solvent. Agitation or sonication can expedite the extraction. An aliquot of the extract is then withdrawn from the sample vial and introduced into the titration cell for analysis.

Method C: direct extraction of crushed sample in titration cell. Genentech has traditionally used this technique. The product vial is uncapped, and the contents are crushed inside the vial using a smooth metal rod. A known amount of powdered sample is then transferred directly into the titration vessel for analysis.

All of the authors' historical data were collected using Method C. The following study was undertaken to determine the comparability between Method A and Method C only. The experimental setup for Method B was not available. Furthermore, many of Genentech's lyophilized products are formulated with large amounts (e.g., as high as 85% wt.) of sugars, which limits drying ovens to low temperatures. Other products contain high amounts of salts and require high oven temperature for efficient moisture desorption. Because it was undesirable to develop a separate method for each product, the authors did not use the moisture-analysis technique that relies on drying ovens.

A Mettler-Toledo DL31 volumetric Karl Fischer titrator with a one-component Hydranal Composite 2 titrant was used in all cases. Sodium tartrate dihydrate was used as the standard to determine the reagent titer at the beginning of each day, as recommended by the instrument manufacturer.

The authors used active lyophilized product configured as a 0.88-mL fill volume in 5-cm³ vials in the study. Two sets of samples, spanning the typical range of moisture normally observed for this product (i.e., 0.5–5% wt.), were tested.

The authors investigated the following two extraction schemes for Method A:

I Introduction of anhydrous methanol into the product vial, 5-min sonication, withdrawal of an aliquot and analysis. This is the instrument manufacturer's recommended procedure.

II Introduction of anhydrous methanol into the product vial, 5-min sonication, 5-min standing, an additional 5-min sonication, withdrawal of an aliquot, and analysis.

Figure 1: Comparison between extraction methods. Squares represent low-moisture samples, and circles represent high-moisture samples. (IMAGE IS COURTESY OF PHILIPPE LAM)

Comparing results from the two methods, it is clear that Scheme II of Method A extracted more water than the recommended Scheme I, suggesting that Scheme I was not able to fully extract the water from the sample. Yet it is unclear whether Scheme II achieved complete extraction.