Spectroscopic evaluation of a freeze-dried vaccine during an accelerated stability study.

This research evaluates a freeze-dried live, attenuated virus vaccine during an accelerated stability study using Near Infrared (NIR) and Fourier Transform Infrared (FTIR) spectroscopy in addition to the traditional quality tests (i.e., potency assay and residual moisture analysis) and Modulated Differential Scanning Calorimetry (MDSC). Therefore, freeze-dried live, attenuated virus vaccines were stored during four weeks at 4°C (i.e., recommended storage condition) and at 37°C (i.e., accelerated storage condition) and weekly analyzed using these techniques. The potency assay showed that the virus titer decreased in two phases when the samples were stored at 37°C. The highest titer loss occurred during the first week storage at 37°C after which the degradation rate decreased. Both the residual moisture content and the relaxation enthalpy also increased according to this two-phase pattern during storage at 37°C. In order to evaluate the virus and its interaction with the amorphous stabilizer in the formulation (trehalose), the NIR spectra were analyzed via principal component analysis (PCA) using the amide A/II band (5029-4690cm(-1)). The FTIR spectra were also analyzed via PCA using the amide III spectral range (1350-1200cm(-1)). Analysis of the amide A/II band in the NIR spectra revealed that the titer decrease during storage was probably linked to a change of the hydrogen bonds (i.e., interaction) between the virus proteins and the amorphous trehalose. Analyzing the amide III band (FTIR spectra) showed that the virus destabilization was coupled to a decrease of the coated proteins ß turn and an increase of α helix. During storage at 4°C, the titer remained constant, no enthalpic relaxation was observed and neither the Amide A/II band (NIR spectra) nor the Amide III band (FTIR spectra) varied.

FTIR spectroscopy for the detection and evaluation of live attenuated viruses in freeze dried vaccine formulations.

This article examines the applicability of Fourier Transform Infrared (FTIR) spectroscopy to detect the applied virus medium volume (i.e., during sample filling), to evaluate the virus state and to distinguish between different vaccine doses in a freeze dried live, attenuated vaccine formulation. Therefore, different formulations were freeze dried after preparing them with different virus medium volumes (i.e., 30, 100, and 400 µl) or after applying different pre-freeze-drying sample treatments (resulting in different virus states); i.e., (i) as done for the commercial formulation; (ii) samples without virus medium (placebo); (iii) samples with virus medium but free from antigen; (iv) concentrated samples obtained via a centrifugal filter device; and (v) samples stressed by 96h exposure to room temperature; or by using different doses (placebo, 25-dose vials, 50-dose-vials and 125-dose vials). Each freeze-dried product was measured directly after freeze-drying with FTIR spectroscopy. The collected spectra were analyzed using principal component analysis (PCA) and evaluated at three spectral regions, which might provide information on the coated proteins of freeze dried live, attenuated viruses: (i) 1700-1600 cm(-1) (amide I band), 1600-1500 cm(-1) (amide II band) and 1200-1350 cm(-1) (amide III band). The latter spectral band does not overlap with water signals and is hence not influenced by residual moisture in the samples. It was proven that FTIR could distinguish between the freeze-dried samples prepared using different virus medium volumes, containing different doses and using different pre-freeze-drying sample treatments in the amide III region.

Thermoplastic polyurethanes for the manufacturing of highly dosed oral sustained release matrices via hot melt extrusion and injection molding.

This study evaluated thermoplastic polyurethanes (TPUR) as matrix excipients for the production of oral solid dosage forms via hot melt extrusion (HME) in combination with injection molding (IM). We demonstrated that TPURs enable the production of solid dispersions - crystalline API in a crystalline carrier - at an extrusion temperature below the drug melting temperature (Tm) with a drug content up to 65% (wt.%). The release of metoprolol tartrate was controlled over 24h, whereas a complete release of diprophylline was only possible in combination with a drug release modifier: polyethylene glycol 4000 (PEG 4000) or Tween 80. No burst release nor a change in tablet size and geometry was detected for any of the formulations after dissolution testing. The total matrix porosity increased gradually upon drug release. Oral administration of TPUR did not affect the GI ecosystem (pH, bacterial count, short chain fatty acids), monitored via the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The high drug load (65 wt.%) in combination with (in vitro and in vivo) controlled release capacity of the formulations, is noteworthy in the field of formulations produced via HME/IM.

Release characteristics of polyurethane tablets containing dicarboxylic acids as release modifiers - a case study with diprophylline.

The influence of several dicarboxylic acids on the release characteristics of polyurethane tablets with a high drug load was investigated. Mixtures of diprophylline (Dyph) and thermoplastic polyurethane (TPUR) (ratio: 50/50, 65/35 and 75/25 wt.%) were hot-melt extruded and injection molded with the addition of 1, 2.5, 5 and 10% wt.% dicarboxylic acid as release modifier. Incorporating malonic, succinic, maleic and glutaric acid in the TPUR matrices enhanced drug release, proportional to the dicarboxylic acid concentration in the formulation. No correlation was found between the water solubility, melting point, logP and pKa of the acids and their drug release modifying capacity. Succinic and maleic acid had the highest drug release modifying capacity which was linked to more intense molecular interactions with Dyph. A structural fit between the primary and secondary alcohol of Dyph and both carboxylic groups of the acids was at the origin of this enhanced interaction.

Near-infrared spectroscopic evaluation of lyophilized viral vaccine formulations.

This article examines the applicability of near-infrared spectroscopy (NIRS) to evaluate the virus state in a freeze-dried live, attenuated vaccine formulation. Therefore, this formulation was freeze-dried using different virus volumes and after applying different pre-freeze-drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze-dried product was measured directly after freeze-drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300-4000 cm(-1) region containing the amide A/II band which might reflect information on the coated proteins of freeze-dried live, attenuated viruses; and (ii) the C-H vibration overtone regions (10,000-7500 and 6340-5500 cm(-1) ) which might supply information on the lipid layer surrounding the freeze-dried live, attenuated viruses. The different pre-freeze-drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS-DA) were developed and evaluated, allowing classification of the freeze-dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume.

Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations.

This study investigates whether Raman spectroscopy combined with multivariate analysis (MVA) enables a rapid and direct differentiation between two classes of conformational states, i.e., native-like and non-native proteins, in freeze-dried formulations. A data set comprising of 99 spectra, both from native-like and various types of non-native freeze-dried protein formulations, was obtained by freeze-drying lactate dehydrogenase (LDH) as model protein under various conditions. Changes in the secondary structure in the solid freeze-dried proteins were determined through visual interpretation of the blank corrected second derivative amide I band in the ATR-FTIR spectra (further called FTIR spectra) and served as an independent reference to assign class labels. Exploratory analysis and supervised classification, using Principal Components Analysis (PCA) and Partial Least Squares - Linear Discriminant Analysis (PLS-LDA), respectively, revealed that Raman spectroscopy is with 95% accuracy able to correctly discriminate between native-like and non-native states in the tested freeze-dried LDH formulations. Backbone (i.e., amide III) and side chain sensitive spectral regions proved important for making the discrimination between both classes. As discrimination was not influenced by the spectral signals from the tested excipients, there was no need for blank corrections. The Raman model may allow direct and automated analysis of the investigated quality attribute, opening possibilities for a real time and in-line quality indication as a future step. However, the sensitivity of the method should be further investigated and where possible improved.