Formulation: SAMPLE ANALYSIS

ANDREW PARKER, PHD

Figure 1. X-ray transmission image for a granulate recorded at a single rotational step.

ALL IMAGES COURTESY OF MOLECULAR PROFILES

Get to Know Your Samples

A new technique better analyzes pharmaceutical formulations

I nformed sample analysis at critical time points during pharmaceutical research and development is an effective strategy for clarifying decisions and reducing both research time and overall cost. Comprehensive understanding of the physicochemical properties of pharmaceutical formulations can be used to accelerate regulatory submissions and reduce late-stage problems.

This information also facilitates greater design space in accord with International Conference on Harmonisation (ICH) Q8 guidelines and increases flexibility in making manufacturing process improvements within the approved design space and without further regulatory review.

Several imaging and analysis techniques can rapidly provide greater insight into the physicochemical properties of active pharmaceutical ingredients (APIs), product design, development hurdles, and dosage form stability. One such powerful technique is micro X-ray computed tomography (CT), which achieves nondestructive 3-D analysis of challenging samples. This method can be used for a variety of applications ranging from quality control to product optimization to formulation analysis in counterfeiting litigation cases. In addition, it can be used for diverse samples that run the gamut from granules to tablets to formulated polymer implants.

Resolving the internal microstructure enables greater understanding of the results of in vitro performance, can identify manufacturing inconsistencies, and can evaluate product stability. This technique readily facilitates the link between drug product structure and drug product function with up to sub-micron scale resolution.

How It Works

A pharmaceutical sample is held in the field of view of a micro X-ray source, and its position is optimized with respect to the detector. The sample is then rotated in a finite series of angular steps encompassing 180^o or 360^o; the transmission of X-rays is recorded as an image for each step (see Figure 1, left). This generates a series of X-ray images that can then be reconstructed by computer to provide 2-D or 3-D data. Through the generation of hundreds of virtual 2-D slices per sample, it is possible to accurately visualize and quantify many different properties for individual components and a multitude of product attributes.

Using complex algorithms, the primary transmission data are reconstructed to accurately pinpoint, in space, the location of features within the object. Material components such as micronized excipients and coatings and sample features such as pores and cracks will generally have differing relative propensities for attenuating the X-ray beam. This is because of a combination of different elemental compositions, relative atomic densities, and material density grouping of components, and gives rise to the contrast in the reconstructed 2-D greyscale virtual cross-sectional images. Binarized image analysis for all three 2-D axes (x-y, x-z, and y-z) enables 3-D reconstruction and allows volumes of components and porosity of systems to be quantified and visualized.

Using appropriate sizing software and thresholding, features of interest can be quantified while formulation integrity is scrutinized. Coating attributes such as uniformity, occlusions, and interfacial properties can be assessed, and the propagation of structural features and inconsistencies can be evaluated. The resulting information can be used as part of a root-cause analysis for batch-to-batch variation. Because the micro X-ray CT is a non-invasive technique, another advantage it offers the user is that the same sample can be reanalyzed by other techniques-like Raman microscopy-which can provide complementary information.

Granular Microstructures

The micro X-ray CT has also been used for granular microstructures. Granulation is a crucial processing step implemented within the pharmaceutical industry in order to improve the material properties of formulation components-including flowability, compression characteristics, and blend homo-geneity-during manufacture. Poor granulation can lead to significant problems during downstream processing. A detailed knowledge of granule micro-structure is essential for developing a better knowledge of granule behavior during these formulation steps and improving understanding of processing parameters, such as granulation end point/set-point, which are often inferred from empirical measurements such as power uptake.

Figure 2. Reconstructed computed tomography slice for an example of a high molecular weight granule (left) next to an example for a low molecular weight granule (right).

Figure 3. Effect of high temperature and humidity on the core material and outer layer of a controlled-release pellet formulation at zero months (left) and three months (right).

In addition, the technique can be used in conjunction with confocal Raman microscopy to provide detailed granule microstructure information and investigate both process optimization and root-cause analysis in granulation.

The micro X-ray computed tomography achieves non-destructive, 3-D analysis of challenging samples.

Researchers developed an experiment in which granules consisting of lactose and either low viscosity or high viscosity binders (of the same chemical composition) were produced using a dry granulation process. In both instances, the binder was added at 3% weight/weight. Reconstruction of 2-D images showed clear qualitative microstructural differences between the two granule types. (See reconstructed tomography data example in Figure 2, above.)

Components within the micro X-ray CT images were isolated through binary image analysis. Using these binary images, 3-D volume calculations were performed on individual components. The binder occupied 2.5% of the binarized image for the high molecular weight material but was better processed for the granulates formed with low molecular weight material, which occupied only 2% of the detected material volume. The residual porosity was 20% and 45%, respectively. Analytical results demonstrated that micro X-ray CT is capable of resolving the porosity, excipient, and binder volumes.

Cross sections of the granules were prepared using an ultramicrotome. Confocal Raman microscopy was used to investigate the distribution of the components within the granule. Combining micro X-ray CT with chemical imaging provided by confocal Raman microscopy on the prepared cross sections provided further solid state and structural information; the latter confirmed the interpretations of micro X-ray CT.

Controlled Release Formulations

Structural and chemical changes within a controlled release (CR) formulation can affect the desired release profile and performance of a carefully designed formulation. While there are a number of solid state imaging techniques available for the physicochemical characterization of pharmaceutical formulations, there are few capable of non-invasive analysis of the formulated product. Application of micro X-ray CT can highlight structural issues that may impact formulation performance and potentially cause product failure. Used in conjunction with imaging techniques like Raman microscopy and scanning electron microscopy (SEM), micro X-ray CT can generate a detailed map of the physicochemical properties of a formulation on a � 1�m resolution scale.

Researchers performed an investigation highlighting the potential effects of long-term elevated humidity storage conditions on the structural and chemical integrity of a CR formulation. The effects of this conditioning were noninvasively assessed using micro X-ray CT at predefined time points over a three-month period. Micro X-ray CT, in conjunction with confocal Raman microscopy, demonstrated that the storage of the CR pellet formulation at 40^oC/75% relative humidity for three months resulted in the redistribution of the core material and the dramatic breakdown of the outer layer of the pellet. (See Figure 3, p. 36 for tomography data example.) Often, the manifestation of subtle changes in solid-state properties, such as levels of hydration in excipients like citrates, can be manifested by a gross change in other physical properties such as viscosity, which is intrinsically linked to microstructure. The combined use of these techniques can characterize both the resultant changes and help explain their root causes.

Through the generation of hundreds of virtual 2-D slices per sample, it is possible to accurately visualize and quantify many different properties for individual components and a multitude of product attributes.

Micro X-ray CT provides high quality analysis of pharmaceutical systems. The method achieves non-invasive visualization and quantification of material properties of individual components, including size, shape, porosity, coating thickness, and formulation integrity, with sub-micron scale resolution