ABSTRACT
Near-infrared (NIR) and frequency modulated spectroscopy (FMS) were employed, for non-invasive moisture determination of a lyophilized biologic drug product (DP). Development of NIR and FMS provides a rapid non-invasive means of residual moisture measurement, and would be beneficial compared with traditional time consuming, product destructive methods such as Karl Fischer (KF). A model therapeutic enzyme in a sucrose-based formulation was employed for proof of concept studies, and NIR and FMS methods were compared side by side for residual moisture analysis. Moisture models were created using lyophilized vials and comparisons were made between the methods using different moisture preparation approaches:1) direct water droplet addition to the vial headspace, 2) use of elevated temperature (80°C), and 3) using various levels of moisture in stoppers generated during the washing and drying procedures, then lyophilizing using the stoppers and placing the sealed vials on stability. The results for direct water addition gave an average percent error for residual moisture of 5.7% for NIR and 9.4% for FMS when compared to KF. The elevated temperature method resulted in an average percent error for residual moisture of 54% for NIR and 43% for FMS compared to KF. The stopper moisture stability study, for FMS, provided an average percent error for residual moisture of 31% compared to KF. The error was greater for the elevated temperature and stopper methods, due to the low moisture values, which resulted in greater error. At this lower range of moisture (<1%) both NIR and FMS were less accurate, but from 1 to 5% their accuracy increased, based on the models used in this study. NIR and FMS methods can be used to complement KF at these lower moisture levels and models could be further improved with additional data points. NIR and FMS methods have advantages and disadvantages for residual moisture analysis when compared to each other, but both provided an accurate measurement of drug product moisture (depending on the method used for moisture increase), they can be used as process analytical technology (PAT), and both can be used for fast non-invasive moisture determination.
Subject(s)
Biological Products , Spectroscopy, Near-Infrared , Freeze Drying , Sucrose , WaterABSTRACT
An amitochondriate parasite, Entamoeba histolytica, has a bifunctional ADHE enzyme (EhADH2) that contains separate acetaldehyde (ALDH) and alcohol (ADH) dehydrogenase activities. In a cluster of 25 bifunctional enzymes of single cell eukaryotes and bacteria, we present a phylogenetic analysis that suggests a lateral gene transfer event (prokaryotic ancestor to single-cell eukaryotic ancestor) and a complex structure that aligns with key homologs in the ADHE evolutionary history based on their similarity with bacterial alcohol dehydrogenases. We show that the ADHE in Entamoeba lineage diverged independently but shows significant similarities to the structure of ADHE in Fusobacterium, and a complex model that maps its ALDH and ADH domain well with bacteria such as Geobaccillus thermoglucosidasius. Our analyses likely support a lateral acquisition of an EhADH2-like ancestral gene from bacteria. Several evolutionary analyses software programs reveal that the enzyme structure is highly conserved, and maintains a similar function within a diverse set of pathogens, including Escherichia coli and Clostridium spp.
