Liposome manufacturing under continuous flow conditions: towards a fully integrated set-up with in-line control of critical quality attributes.

Continuous flow manufacturing (CFM) has shown remarkable advantages in the industrial-scale production of drug-loaded nanomedicines, including mRNA-based COVID-19 vaccines. Thus far, CFM research in nanomedicine has mainly focused on the initial particle formation step, while post-formation production steps are hardly ever integrated. The opportunity to implement in-line quality control of critical quality attributes merits closer investigation. Here, we designed and tested a CFM setup for the manufacturing of liposomal nanomedicines that can potentially encompass all manufacturing steps in an end-to-end system. Our main aim was to elucidate the key composition and process parameters that affect the physicochemical characteristics of the liposomes. Total flow rate, lipid concentration and residence time of the liposomes in a high ethanol environment (i.e., above 20% v/v) emerged as critical parameters to tailor liposome size between 80 and 150 nm. After liposome formation, the pressure and the surface area of the filter in the ultrafiltration unit were critical parameters in the process of clearing the dispersion from residual ethanol. As a final step, we integrated in-line measurement of liposome size and residual ethanol content. Such in-line measurements allow for real-time monitoring and in-process adjustment of key composition and process parameters.

Blend uniformity evaluation during continuous mixing in a twin screw granulator by in-line NIR using a moving F-test.

This study focuses on the twin screw granulator of a continuous from-powder-to-tablet production line. Whereas powder dosing into the granulation unit is possible from a container of preblended material, a truly continuous process uses several feeders (each one dosing an individual ingredient) and relies on a continuous blending step prior to granulation. The aim of the current study was to investigate the in-line blending capacity of this twin screw granulator, equipped with conveying elements only. The feasibility of in-line NIR (SentroPAT, Sentronic GmbH, Dresden, Germany) spectroscopy for evaluating the blend uniformity of powders after the granulator was tested. Anhydrous theophylline was used as a tracer molecule and was blended with lactose monohydrate. Theophylline and lactose were both fed from a different feeder into the twin screw granulator barrel. Both homogeneous mixtures and mixing experiments with induced errors were investigated. The in-line spectroscopic analyses showed that the twin screw granulator is a useful tool for in-line blending in different conditions. The blend homogeneity was evaluated by means of a novel statistical method being the moving F-test method in which the variance between two blocks of collected NIR spectra is evaluated. The α- and ß-error of the moving F-test are controlled by using the appropriate block size of spectra. The moving F-test method showed to be an appropriate calibration and maintenance free method for blend homogeneity evaluation during continuous mixing.

An efficient, maintenance free and approved method for spectroscopic control and monitoring of blend uniformity: The moving F-test.

Dry powder mixing is a wide spread Unit Operation in the Pharmaceutical industry. With the advent of in-line Near Infrared (NIR) Spectroscopy and Quality by Design principles, application of Process Analytical Technology to monitor Blend Uniformity (BU) is taking a more prominent role. Yet routine use of NIR for monitoring, let alone control of blending processes is not common in the industry, despite the improved process understanding and (cost) efficiency that it may offer. Method maintenance, robustness and translation to regulatory requirements have been important barriers to implement the method. This paper presents a qualitative NIR-BU method offering a convenient and compliant approach to apply BU control for routine operation and process understanding, without extensive calibration and method maintenance requirements. The method employs a moving F-test to detect the steady state of measured spectral variances and the endpoint of mixing. The fundamentals and performance characteristics of the method are first presented, followed by a description of the link to regulatory BU criteria, the method sensitivity and practical considerations. Applications in upscaling, tech transfer and commercial production are described, along with evaluation of the method performance by comparison with results from quantitative calibration models. A full application, in which end-point detection via the F-test controls the blending process of a low dose product, was successfully filed in Europe and Australia, implemented in commercial production and routinely used for about five years and more than 100 batches.

Granule characterization during fluid bed drying by development of a near infrared method to determine water content and median granule size.

PURPOSE: Water content and granule size are recognized as critical process and product quality parameters during drying. The purpose of this study was to enlighten the granule behavior during fluid bed drying by monitoring the major events i.e. changes in water content and granule size. METHODS: NIR spectra collected during drying and water content of sampled granules were correlated by principal component analysis (PCA) and partial least squares regression (PLSR). NIR spectra of dried granules were correlated to median granule size in a second PCA and PLSR. RESULTS: The NIR water model discriminates between various stages in fluid-bed drying. The water content can be continuously predicted with errors comparable to the reference method. The four PLS factors of the granule size model are related to primary particle size of lactose, median granule size exceeding primary particle size and amorphous content of granules. The small prediction errors enable size discrimination between fines and granules. CONCLUSION: For product quality reasons, discrimination between drying stages and end-point monitoring is highly important. Together with the possibilities to determine median granule size and to distinguish fines this approach provides a tool to design an optimal drying process.

Near-infrared imaging for studying homogeneity of protein-sugar mixtures.

PURPOSE: To investigate the applicability of near-infrared (NIR) imaging for assessing the homogeneity of dried protein-sugar formulations. METHODS: Physical mixtures of lysozyme and trehalose in different ratios were prepared and analyzed by near-infrared (NIR) imaging with a spatial resolution of 10 or 40 microm. To define and select the best imaging strategy, besides visual inspection of the images, several approaches for data processing were tested: single wavelength intensity, peak/height ratio of two specific wavelengths, correlation coefficient with a reference spectrum and principal component analysis (PCA). In order to relate the contrast directly to concentration differences of lysozyme and trehalose, quantitative models were created based on correlation coefficient and partial least squares (PLS) regression. The selected imaging method was applied to compare the homogeneity of a supercritical fluid (SCF) dried and a freeze-dried lysozyme-trehalose mixture. RESULTS: All tested methods confirmed each other and showed spatial heterogeneity in the lysozyme and trehalose contents of the physical mixtures. However, multivariate data processing methods (correlation coefficient and PCA/PLS) resulted in more distinct contrasts than univariate approaches (single wavelength analysis) and allowed a quantitative estimation of the homogeneity. As shown by NIR imaging in combination with the correlation coefficient or the PLS method, the SCF dried lysozyme-trehalose formulation was at least as homogeneous as its lyophilized counterpart, at 10 microm pixel size resolution. CONCLUSIONS: NIR imaging is a useful tool for studying the homogeneity of dried protein-sugar formulations.