Adjustment of specific residual moisture levels in completely freeze-dried protein formulations by controlled spiking of small water volumes

The residual moisture (RM) level strongly impacts the stability of freeze-dried biopharmaceuticals. On the one hand, the RM should not be too high to keep the reaction potential of water molecules low and to avoid a decrease in the glass transition temperature through the plasticizing effect of water. On the other hand, over -drying has been described to negatively impact protein stability. Consequently, an optimal RM has to be established and justified for approval by authorities. Therefore, end products with different RM are analyzed over storage. The different RM levels are typically obtained by slightly varying the lyophilization process itself; but deviations from the original process are critical. Additionally, samples can be taken during lyophilization runs, e. g. at the end of primary drying or after the ramp into secondary drying. This, however, does not allow for good control over the RM. Here we present the adaption of a headspace water spiking technique using a microliter syringe to precisely increase and adjust the RM. This technique is suitable to reproducibly introduce water in the microliter scale to achieve RM of less than 1% [w/w] with high precision depending on the dry cake weight. In addition, we show that by using different fill volumes and cake densities, an equal three-dimensional water distribution throughout the whole cake can be achieved. Furthermore, potential limitations and risks in manual RM introduction are discussed.