In-Line Multipoint Near-Infrared Spectroscopy for Moisture Content Quantification during Freeze-Drying

During the past decade, near-infrared (NIR) spectroscopy has been applied for in-line moisture content quantification during a freeze-drying process. However, NIR has been used as a single-vial technique and thus is not representative of the entire batch. This has been considered as one of the main barriers for NIR spectroscopy becoming widely used in process analytical technology (PAT) for freeze-drying. Clearly it would be essential to monitor samples that reliably represent the whole batch. The present study evaluated multipoint NIR spectroscopy for in-line moisture content quantification during a freeze-drying process. Aqueous sucrose solutions were used as model formulations. NIR data was calibrated to predict the moisture content using partial least-squares (PLS) regression with Karl Fischer titration being used as a reference method. PLS calibrations resulted in root-mean-square error of prediction (RMSEP) values lower than 0.13%. Three noncontact, diffuse reflectance NIR probe heads were positioned on the freeze-dryer shelf to measure the moisture content in a noninvasive manner, through the side of the glass vials. The results showed that the detection of unequal sublimation rates within a freeze-dryer shelf was possible with the multipoint NIR system in use. Furthermore, in-line moisture content quantification was reliable especially toward the end of the process. These findings indicate that the use of multipoint NIR spectroscopy can achieve representative quantification of moisture content and hence a drying end point determination to a desired residual moisture level

Microscale Freeze-Drying with Raman Spectroscopy as a Tool for Process Development

Until recently, the freeze-drying process and formulation development have suffered from a lack of microscale analytical tools. Using such an analytical tool should decrease the required sample volume and also shorten the duration of the experiment compared to a laboratory scale setup. This study evaluated the applicability of Raman spectroscopy for in-line monitoring of a microscale freeze-drying process. The effect of cooling rate and annealing step on the solid-state formation of mannitol was studied. Raman spectra were subjected to principal component analysis to gain a qualitative understanding of the process behavior. In addition, mannitol solid-state form ratios were semiquantitatively analyzed during the process with a classical least-squares regression. A standard cooling rate of 1 °C/min with or without an annealing step at −10 °C resulted in a mixture of α, β, δ, and amorphous forms of mannitol. However, a standard cooling rate induced the formation of mannitol hemihydrate, and a secondary drying temperature of +60 °C was required to transform the hemihydrate form to the more stable anhydrous polymorphs. A fast cooling rate of 10 °C/min mainly produced δ and amorphous forms of mannitol, regardless of annealing. These results are consistent with those from larger scale equipment. In-line monitoring the solid-state form of a sample is feasible with a Raman spectrometer coupled microscale freeze-drying stage. These results demonstrate the utility of a rapid, in-line, low sample volume method for the semiquantitative analysis of the process and formulation development of freeze-dried products on the microscale

The Univariate and multivariate results for individual risk factors and the generalized additive model risk ratios with the spatial term of a waterborne outbreak in Vuorela, July 2012.

<p>The Univariate and multivariate results for individual risk factors and the generalized additive model risk ratios with the spatial term of a waterborne outbreak in Vuorela, July 2012.</p

Categorized distance and proportion of cases within those groups in a waterborne outbreak in Vuorela, July 2012.

<p>Categorized distance and proportion of cases within those groups in a waterborne outbreak in Vuorela, July 2012.</p

Epidemic curve of a waterborne outbreak in Vuorela, July 2012 based on the reported onset date of illness of the cases, and <i>E. coli</i> bacteria counts and chlorine levels in the point 7 (See Fig. 1) of the water distribution network.

<p>Epidemic curve of a waterborne outbreak in Vuorela, July 2012 based on the reported onset date of illness of the cases, and <i>E. coli</i> bacteria counts and chlorine levels in the point 7 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104713#pone-0104713-g001" target="_blank">Fig. 1</a>) of the water distribution network.</p

Microbiological results of faecal samples from symptomatic patients of a waterborne outbreak in Vuorela, July 2012.

<p>Methods used were cultivation¹, PCR/RT-PCR² (polymerase chain reaction/reverse transcripition-PCR), EM³ (electron microscopy) and/or EIA⁴ (Enzyme Immunoassay). ⁵<i>Campylobacter jejuni</i> and ⁶EHEC O157:H7 (from one sample) and ⁷sapovirus GII.P3 were isolated from the samples.</p

Phylogenetic relationships among OTUs (•) of the genus <i>Arcobacter</i> in a waterborne outbreak in Vuorela, July 2012.

<p>The tree was inferred from a maximum likelihood analysis of aligned 16S rRNA gene sequence (≈255 bp) and nodes with a bootstrap value ≥50% of 1 000 replicates are identified. <i>Sulfurospirillum deleyianum</i> (NR_074378) and <i>Campylobacter fetus</i> (L04314) were used as outgroup. Number in bracket represents the total amount of DNA/RNA reads identified in samples from A) the upper storage reservoir before cleaning, B) tap water during contamination and C) the upper storage reservoir after cleaning. *<i>A. butzleri</i>, <i>A. cryaerophilus</i> and <i>A. skirrowii</i> have been associated with gastrointestinal diseases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104713#pone.0104713-Collado1" target="_blank">[76]</a>.</p