Near-Infrared Spectroscopy to Determine Residual Moisture in Freeze-Dried Products: Model Generation by Statistical Design of Experiments.

Moisture content (MC) is a critical quality attribute of lyophilized biopharmaceuticals and can be determined by near-infrared (NIR) spectroscopy as nondestructive alternative to Karl-Fischer titration. In this study, we create NIR models to determine MC in mAb lyophilisates by use of statistical design of experiments (DoE) and multivariate data analysis. We varied the composition of the formulation as well as lyophilization parameters covering a large range of representative conditions, which is commonly referred to as "robustness testing" according to quality-by-design concepts. We applied principles of chemometrics with partial least squares and principal component analysis. The NIR model excluded samples with complete collapse and MC > 6%. The 2 main components in the principal component analysis were MC (91%) and protein:sugar ratio (6%). The third component amounted to only 3% and remained unspecified but may include variations in process parameters and cake structure. In contrast to traditional approaches for NIR model creation, the DoE-based model can be used to monitor MC during drug product development work including scale-up, and transfer without the need to update the NIR model if protein:sugar ratio and MC stays within the tested limits and cake structure remains macroscopically intact. The use of the DoE approach and multivariate data analysis ensures product consistency and improves understanding of the manufacturing process.

Authentication of pharmaceutical vials.

Packaging analysis is an important step in the authentication of medicines. All types of medicines are nowadays targeted by the counterfeiters, and among them, vials of injectable medicines. This type of pharmaceutical product is often a combination of fake parts and genuine but stolen, expired, reused and/or manipulated parts. For this reason, while the visual comparison with references is necessary to identify the packaging, it is sometimes difficult to see visual differences for each of the parts that compose a pharmaceutical vial. Several analytical tools were successfully tested to support the investigation of 31 counterfeited vials. Moreover eight counterfeited vials originating from different seizures could be linked based on the chemical or elemental composition of the different vial parts. Raman spectroscopy and microscopy, X-ray fluorescence spectroscopy, Infrared spectroscopy and optical microscopy proved especially efficient to support the visual comparison for the authentication of counterfeited flip off caps, aluminium crimping caps, stoppers, glass vials and vial labels and to detect links between the counterfeits at each of these levels. This last information can be especially useful for on-going investigations. The understanding of how the packaging was faked and manipulated could also be provided, which is also very valuable for prevention purposes, for example for the design of a more robust - and therefore more difficult to counterfeit - packaging.

Packaging analysis of counterfeit medicines.

The authentication of the packaging represents an important step in the investigation of suspected counterfeits of pharmaceutical products. The visual comparison with a retained sample is the first step to detect a counterfeited packaging. Due to the improvement of technologies like printing, the emergence of counterfeits with a better appearance can be observed. Moreover counterfeits are nowadays often a combination of fake and genuine parts that have been manipulated. Authenticating each part of the product is important in the frame of the investigation to understand how the counterfeiters proceed, and which prevention measures should be taken. Lab instruments like spectrometers can help confirm counterfeits of packaging for packaging components on which visually, no difference with a reference would be observed. In this study several analytical tools were evaluated to help support the authentication of the primary and secondary packaging of one medicinal product as an example using seven reference materials and five counterfeits. In some cases, visual examination of the packaging already enabled to detect counterfeits of the studied features. Also the boxes, leaflets and vials have been analysed with Infrared (IR) spectroscopy, Raman microspectroscopy, X-ray fluorescence (XRF) spectroscopy, Scanning Electron Microscopy (SEM) and microcomputed tomography (microCT). According to the obtained results, IR and XRF could support the visual examination of the different packaging components. Despite the small amount of counterfeits, relevant links could also be detected between the studied cases based on the packaging characteristics.

Cerebellar compartmentation of prion pathogenesis.

In prion diseases, the brain lesion profile is influenced by the prion "strain" properties, the invasion route to the brain, and still unknown host cell-specific parameters. To gain insight into those endogenous factors, we analyzed the histopathological alterations induced by distinct prion strains in the mouse cerebellum. We show that 22L and ME7 scrapie prion proteins (PrP22L , PrPME7 ), but not bovine spongiform encephalopathy PrP6PB1 , accumulate in a reproducible parasagittal banding pattern in the cerebellar cortex of infected mice. Such banding pattern of PrP22L aggregation did not depend on the neuroinvasion route, but coincided with the parasagittal compartmentation of the cerebellum mostly defined by the expression of zebrins, such as aldolase C and the excitatory amino acid transporter 4, in Purkinje cells. We provide evidence that Purkinje cells display a differential, subtype-specific vulnerability to 22L prions with zebrin-expressing Purkinje cells being more resistant to prion toxicity, while in stripes where PrP22L accumulated most zebrin-deficient Purkinje cells are lost and spongiosis accentuated. In addition, in PrP22L stripes, enhanced reactive astrocyte processes associated with microglia activation support interdependent events between the topographic pattern of Purkinje cell death, reactive gliosis and PrP22L accumulation. Finally, we find that in preclinically-ill mice prion infection promotes at the membrane of astrocytes enveloping Purkinje cell excitatory synapses, upregulation of tumor necrosis factor-α receptor type 1 (TNFR1), a key mediator of the neuroinflammation process. These overall data show that Purkinje cell sensitivity to prion insult is locally restricted by the parasagittal compartmentation of the cerebellum, and that perisynaptic astrocytes may contribute to prion pathogenesis through prion-induced TNFR1 upregulation.

Performance of NIR handheld spectrometers for the detection of counterfeit tablets.

Near Infrared (NIR) spectroscopy is an attractive tool for pharmaceutical analyses. While lab spectrometers are very performant, they are expensive and due to their size, not adapted for field analyses. In this study, two handheld NIR spectrometers have been evaluated for the fast detection of counterfeits of pharmaceutical tablets: one low cost sensor providing a short wavelength NIR range (swNIR) and one handheld spectrometer providing a classical NIR range (cNIR). A large database containing almost all the tablets produced by the firm was created on each spectrometer. A screening for supervised classifications was performed in order to determine the most accurate model for product authentication. A Support Vector Machine (SVM) model was finally chosen for the swNIR, providing 100% of correct identification in calibration and 96.0% in validation, and a Linear Discriminant Analysis (LDA) model was chosen for the cNIR delivering 99.9% of correct identification in calibration and 91.1% in validation. Challenging samples (counterfeits and generics) could be 100% identified by the chosen classifiers combined with a class name check and a correlation distance. Statistical tests were used to compare the performance of selected swNIR and cNIR models. These results demonstrate that both devices can be used for tablet identification and the detection of counterfeits.

Near infrared spectroscopy for counterfeit detection using a large database of pharmaceutical tablets.

Medicine counterfeiting is one of the current burdens of the pharmaceutical world. Reliable technologies have become available for the chemical analysis of suspect medicines. Near infrared spectroscopy (NIRS) allows for instance fast, specific and non-destructive authentication of pharmaceutical products. In this paper, a NIRS method is presented for the identification of 29 different pharmaceutical product families of tablets, one family containing one or more formulation (s), e.g. different dosages. This selection represents the whole tablet portfolio of our firm. The high number of product families constituted a challenge, given that the measurement of the samples, made on two similar instruments, generated a dataset of 7120 spectra. Several chemometric tools proved efficient for the identification of these medicines. The dataset was first investigated with a Principal Component Analysis (PCA) in order to provide an overview of the distribution of the samples. The K-Nearest Neighbors (KNN), the Support Vector Machines (SVM) and the Discriminant Analysis (DA) supervised classification tools were successfully applied and generated an outstanding classification rate of 100% of correct answer. The methods were then fully validated with an independent set of spectra. The DA was selected as the method for the routine analysis of suspect tablets with the Mahalanobis distance as acceptance criterion for identification. Counterfeits, generics and placebos samples, constituting a second validation set, were tested and rejected by the method. NIRS has thus been demonstrated as an efficient tool for the quick identification of a large dataset of pharmaceutical tablets and the detection of counterfeit medicines.