Probabilistic modeling of an injectable aqueous crystalline suspension using influence networks.

Probabilistic modeling using influence networks is an efficient, intuitive, and easy to communicate strategy in the development of complex pharmaceutical products. This study was aimed to use a risk-based approach to explore the complex interactions between product and process design parameters affecting size and shape of the particles in injectable aqueous crystalline suspensions (ACS). Based on a risk assessment, a design of experiments (DOE) was applied to evaluate the most important parameters, i.e., four critical material attributes and two critical process parameters. A model hydrophobic drug (carbamazepine) was milled and homogenized in a multistep process (dispersion and milling steps). The final formulations were characterized with automated at-line image analysis of thousands of individual particles. The particle size and shape distributions were summarized with descriptive parameters, and the relationship of these parameters and the DOE was modeled using influence networks (INs). This approach was compared and contrasted with a classical modeling approach based on multivariate linear regression (MVLR). INs had a superior visual interpretation capability of the complex and multivariate ACS system making the risk-based decision making more accessible. The probability and causality were included in the IN, i.e., the relationships between size and shape. Moreover, IN allowed to incorporate prior knowledge in a systematic way by implementing a 'black and white list'. An IN based model was created with the following model performance: a mean absolute percentage error of 1.7% and 1.1% for the size and 6.2% and 5.0% for the shape, respectively for dispersed and milled ACS. Parameters with the highest and lowest probability to control the critical quality attributes of ACS could be identified. Consequently, the parameter settings giving the optimum particle size and shape could be predicted using a Monte Carlo simulation to calculate the probability of success including the uncertainty of the model. The cubic MVLR model for the size of milled ACS was comparable to the IN in terms of the mean absolute percentage error, i.e., 1.1%. However, IN was more efficient in visualizing the complex and multivariate data set, including all the critical quality attributes and formulation/process parameters of the ACS at the same time. Moreover, the prior knowledge used in probabilistic modeling of IN could be systematically documented.

Simultaneous automated image analysis and Raman spectroscopy of powders at an individual particle level.

Solid form diversity of raw materials can be critical for the performance of the final drug product. In this study, Raman spectroscopy, image analysis and combined Raman and image analysis were utilized to characterize the solid form composition of a particulate raw material. Raman spectroscopy provides chemical information and is complementary to the physical information provided by image analysis. To demonstrate this approach, binary mixtures of two solid forms of carbamazepine with a distinct shape, an anhydrate (prism shaped) and a dihydrate (needle shaped), were characterized at an individual particle level. Partial least squares discriminant analysis classification models were developed and tested with known, gravimetrically mixed test samples, followed by analysis of unknown, commercially supplied carbamazepine raw material samples. Classification of several thousands of particles was performed, and it was observed that with the known binary mixtures, the minimum number of particles needed for the combined Raman spectroscopy - image analysis classification model was approximately 100 particles per solid form. The carbamazepine anhydrate and dihydrate particles were detected and classified with a classification error of 1 % using the combined model. Further, this approach allowed the identification of raw material solid form impurity in unknown raw material samples. Simultaneous automated image analysis and Raman spectroscopy of powders at an individual particle level has its potential in accurate detection of low amounts of unwanted solid forms in particulate raw material samples.

Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR.

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.