Lyophilized, or freeze-dried, materials are challenging samples for quality assurance and quality control (QA/QC) measurement
because of the inability to open the container without corrupting the product. Near-infrared analysis presents itself as the
QC method of choice for lyophilized materials due to its ability to penetrate glass or plastic containers to analyze the sample
in a non-destructive manner. This study demonstrates the performance of a Fourier transform near-infrared (FT-NIR) spectrometer
used in analyzing lyophilized samples of thrombin, a topical coagulant commonly used in the medical and dental fields. Key
stability parameters for lyophilized thrombin include moisture and potency, which can be predicted simultaneously from a single
spectrum using multivariate analysis.
Lyophilization is a common process in both the food and pharmaceutical industries that eliminates the need for sample refrigeration
while dramatically increasing shelf life. Products that normally spoil after a few months of refrigeration can be rendered
stable, in some instances, for years at room temperature. Lyophilization works by removing residual moisture in a sample through
sublimation (the process of transitioning water from the solid to the vapor phase without passing through a liquid phase).
Sublimation is the process at the core of lyophilization; if one attempted to remove water from a sample simply by heating
to send the water into a vapor phase, the sample would be destroyed.
The sublimation process begins with a solution of target compound, along with various buffers and bulking agents, which are
injected into a serum vial. The vial is then partially stoppered and the solution is frozen below its glass temperature (Tg) or, in the case of crystalline compounds, its eutectic temperature. The glass temperature is the temperature below which
the material is essentially solid, so removal of water can proceed efficiently. The pressure is then reduced and the freeze-drying
process begins with the primary drying stage. After bulk moisture is removed in this phase, the residual moisture (sometimes
as great as 8% by weight) is removed in the secondary drying phase. The temperature is slowly increased as the pressure is
reduced further until the desired degree of dryness is achieved. The serum vials are then stoppered and sealed.
The most fundamentally challenging issue with lyophilized materials is how to analyze them after they are sealed. Lyophilized
fine chemicals or proteins have many chemical and physical properties associated with them, but without proper analytical
techniques, there is no way to measure them to ensure that the product will be safe and effective. Currently, lyophilized
materials are analyzed by batch sampling, where a small number of samples are pulled from a lot, opened, and analyzed for
parameters such as moisture, concentration of the active pharmaceutical ingredient (API), or efficacy. Batch testing of lyophilized
materials is ineffective for several reasons. Sample subsets are never guaranteed to be representative of the whole lot, and
they are destructive. Protocols like titrations, polyacrylamide gel electrophoresis (PAGE) or enzyme-linked immunosorbent
assay (ELISA) are laborious, complicated, and expensive.
Figure 1. The Water Overtone Region for the Ten Standards.
FT-NIR spectroscopy is an easy-to-use technique that uses low- absorbing vibrational overtones and combination bands for analysis.
FT-NIR analysis allows the user to scan through packing materials like polyethylene bags or glass vials to gain information
about the sample. In the case of lyophilized materials, low-energy light can readily penetrate a serum vial to reach the analyte
without damaging it. Once the spectral information is collected, multivariate analysis techniques can be used to retrieve
information about multiple chemical or physical parameters, all from the same single spectrum. Typical scan times for a lyophilized
material sample by FT-NIR are 15–30 sec which, when compared with the 30–60 min necessary for a Karl Fischer titration, is