High-Resolution Ultrasonic Spectroscopy Turbo-Charges PAT Installation

Technology Can Have a Profound Impact on the Efficiency and Profitability of Pharmaceutical Processes

Pharmaceutical manufacturing is a complex process often utilizing complex scientific and engineering principles. The new FDA initiative considers process analytical technology (PAT) to be a system for designing, analyzing and controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality.

The key to the success of PAT is applying the process monitoring tools needed to carry out online analysis of each of the critical product attributes. Process analysis has advanced significantly during the past several decades, due to an increasing appreciation for the value of collecting process data. Available tools have evolved from those that predominantly take univariate process measurements, such as pH, temperature and pressure, to those that measure different biological, chemical and physical attributes. Process monitoring is rapidly moving towards non-destructive methods such as new spectroscopic methods. These measurements may be taken off-line, at-line or online, before, during or after the steps of the manufacturing process. Most spectroscopic techniques utilize electromagnetic waves propagating through materials such as optical spectroscopy and its related derivations, such as FTIR, NIR and fluorescence methods. These methods play an essential role in the analysis of chemical attributes of the material (e.g., identity and purity) and in defining parametric end-points for utilized chemical processes. However, despite these obvious advantages, the applications of traditional "electro-magnetic" spectroscopy methods are limited, in particular, for process analysis in opaque samples and concentrated dispersions as well as samples without required optical activity (absorption spectrum). Therefore, certain physical and mechanical attributes of pharmaceutical ingredients that are critical to product quality are not easily achievable with standard spectroscopy methods. Consequently, the inherent, undetected variability of raw materials may be manifested in the final product. Such attributes (e.g. particle size, concentration and their variations within a sample) of raw and in-process materials may pose a significant challenge because of their complexities and difficulties related to collecting representative samples. For example, it is well known that powder sampling procedures can be erroneous.

Several new technologies are now available that can acquire information on multiple attributes non-destructively with minimal or no sample preparation. High-resolution ultrasonic spectroscopy (HRUS) is a novel technique with enormous potential for analysis of a wide range of samples and processes in PAT. This technique is based on precision measurements of the parameters of acoustical waves propagating through materials. It allows fast at-/online analysis of formulation consistency of raw materials, ingredients and intermediates, process impurity analysis, particle sizing, batch-to-batch variation, stability assessment, etc. Unlike traditional analytical spectroscopy, optical transparency is not required as ultrasonic waves propagate through most types of samples. Moreover, in opaque samples it allows analysis of the interior (bulk properties) of the samples in contrary to many optical techniques, which collect the signal reflected at the surface. HRUS generates product quality information in real process time and for a wide range of samples and dynamic processes. It is possible to monitor the manufacturing stages continuously and make adjustments to ensure that the finished product will meet the desired quality and specification.

This article describes some key features of the HRUS technique, which are beneficial for PAT installation, as well as specific advantages of the method in comparison to more traditional spectroscopy methods. This is illustrated using several examples of HRUS applications including real-time monitoring of the sedimentation and particle size evolution in drug suspensions, the effect of drug coating, and drug concentration on these processes.

Benefits of HRUS for PAT

HRUS is a non-destructive analytical tool based on precision measurements of the velocity and attenuation of acoustical waves at high frequencies propagating through materials. It allows the analysis of composition, aggregation, gelation, micelle formation, crystallization, dissolution, sedimentation, enzyme activity, conformational transitions in polymers, ligand binding, antigen-antibody interactions and many other processes that play a key role in drug production. Capable of dealing with a wide range of samples and dynamic processes, HRUS provides real time product quality information.

Figure 1. Evolution of drug particles size (a) and particle volume fraction, j, (b) obtained from the ultrasonic velocity and attenuation data in undiluted and 1:4 diluted drug suspensions with different polymer coating at 25 degrees C.

Two independent parameters, ultrasonic attenuation and ultrasonic velocity are measured in HR-US. Ultrasonic attenuation is determined by the energy losses in ultrasonic waves and can be expressed in terms of the high-frequency viscosity of the medium or its longitudinal loss modulus. This allows analysis of kinetics of fast chemical reactions and microstructure of materials including particle sizing, aggregation, gelation, crystallization and other processes and characteristics. Ultrasonic velocity is determined by the density and the elastic response of the sample to the oscillating pressure in the ultrasonic wave and thus can be expressed in terms of compressibility or storage modulus. This parameter is extremely sensitive to the molecular organization, composition and intermolecular interactions in the analyzed medium and is responsible for the major portion of applications of High-Resolution Ultrasonic Spectroscopy for analysis of chemical properties of materials.

HRUS ultrasonic spectrometers with their outstanding resolution, down to 0.00001 percent (thus providing the measurements in samples at concentration level down to 0.3 ppm) are the first commercial instruments that allow the user to enjoy the full potential of ultrasonic analysis. A variety of ultrasonic detection cells are available for different types of the analysis: standard sample cells of 1ml in capacity, flow-through cells that allow the carrying out of the analysis in-flow, cells with automatic sampling, cells with small volume capacities (as low as 0.03ml) and cells that allow semi-solid samples to be analyzed. Measurements are computer controlled and the data can be transformed into the concentration of the components during the processes, rate of the reaction, change in molecular weight, particle size and their volume fraction. The analysis can be made in various environment conditions: at temperatures between -60^oto 120^o C, and under pressure up to 15 bars. This dynamic flexibility of the analysis is critical for on-line process monitoring in drug production.

Some advantages of the HRUS technique in comparison with standard spectroscopy methods include:

• No sample preparation (such as dilution or filtration often used in the analysis with standard spectroscopy);

• No moving parts and minimum calibration. Most processes can be monitored with HR-US method in real time until the end point that allows the quantitative analysis without special calibration;

• No consumables needed. The HRUS measurements probe the reactions or processes directly thus avoiding the use of additional substances;

• Ability to analyze a broad range of samples from very diluted solutions down to 0.3 ppm to semi-solids materials;

• Analysis of processes in bulk opaque materials without the dilution which is unavailable with standard spectroscopy methods;

• Fast measurements for flow-through analysis;

• Direct measurements of chemical processes without need of markers used in optical spectroscopy.

HRUS

Applications analysis of the sedimentation and aggregation in the suspensions of drug prepared using different polymer coatings.

Control particle size in dispersed phase is a critical issue in stability of pharmaceutical formulations. Particle characteristics can affect many different areas including inhalation delivery systems, tablet dissolution characteristics, formulation quality and solubility or absorption. Batch to batch variation in particle size can lead to unpredictable variations in the life span and shelf life and heat stability of a product. Traditionally, the characterization of the particles in dispersion is made by optical methods such as light scattering. This means that the sample must be collected from the production line and diluted to reach optical transparency and avoid multiple scattering effects. On the other hand, the application of new HRUS ultrasonic measurements allows direct analysis of the particle size and their volume fraction in the dispersions even concentrated samples (e.g. 40 percent), thus avoiding the problems associated with dilution (which often affects the particle size and the aggregation rate).

In the current example, HRUS technique was used for real-time analysis of the effect of different polymer coatings on the sedimentation rate and the evolution of the particle size in 20 percent drug suspension. The samples of drug suspensions were prepared using hydroxypropyl cellulose as the drug carrier system. The drug particles in the sample 1 were coated with polymer, which had higher molecular weight (approximately twice) in comparison with the polymer used in the preparation of sample 2. Fresh samples were collected using a HRUS colloid stability analyzer, and sedimentation and aggregation processes were monitored continuously.

Sedimentation is reflected in ultrasonic measurements as a change in velocity and attenuation with time. Aggregation (change in the particle size) also affects the attenuation. HRUS software allows de-convolution of these changes and provides real time change in the particle size and the volume fraction. Evolution of particles size and their volume fraction in two types of drug suspension is compared in Figure 1. Initial particle size in the sample 1 is 0.8 mm, which is 25 percent larger than the size in sample 2. However, the particle size in sample 2 increases faster with time due to the aggregation. The growth of particles is accompanied by a decrease in volume fraction that can be attributed to the sedimentation by about 0.7 percent of original volume concentration in the sample 1 and 1.4 percent of original volume concentration in the sample 2 within 20 minutes. Both processes (sedimentation and aggregation) are more evident in sample 2, thus indicating lower stability of this sample 2 compared with sample 1. One of the possible factors responsible for higher stability of suspension 1 can be a "thicker" polymer layer on drug particles in the sample, which protects the particles against aggregation.

Figure 2 shows the effect of concentration on the size of the drug particles with two different polymer coatings as determined by the HRUS measurements. The particle size was taken after 20 minutes of the sedimentation.

Effect of dilution on the evolution of drug particles size was analyzed by the measurements of the sedimentation profile (similar ones in Figure 1) in diluted suspensions. Figure 1 gives an example of time-dependence of the particles size and volume fraction in diluted drug samples (1:4) monitored with HR-US Colloid Stability Analyzer. Two tendencies, an increase in the particles size with time and decrease in the particle fraction (sedimentation) observed in diluted suspensions are similar to those found in undiluted samples, however the intensity of these processes is different. In the case of the suspension 2, the aggregation and sedimentation decrease significantly with the sample dilution. The relative loss of volume fraction within 20 minutes in diluted suspension 2 is 0.7 percent of the original volume concentration in comparison with 1.4 percent in undiluted suspension. Particle size in the diluted sample 2 increases by 1.3 times within 20 minutes, which is significantly smaller that in the undiluted sample, 1.7. The effect of the dilution on the aggregation and sedimentation rates is less evident for the sample 1. This observation is in the agreement with higher stability of suspension 1 discussed above.

Figure 2 shows that average particle size decreases with dilution. This decrease can be explained by the effect of the particle concentration on aggregation. In undisrupted suspension, the particles grow in size due to an aggregation. However, since the aggregation rate decreases at lower drug concentration the average particle measured after the same time interval (e.g. 20 minutes) is smaller in undiluted suspension. Therefore, some attention needs to be paid when the particle size in concentrated suspension is estimated by the measurement in diluted samples (e.g. light scattering techniques). Figure 2 also demonstrates that the effect of dilution on the particle size depends on the particle coating. In very diluted samples (e.g. 1:20) the drug particles are larger in sample 1, which can be attributed to "thicker" layer formed by higher molecular weight polymer on original particles. The results show that increase in the drug concentration overall enhances the aggregation and that this process is less pronounced in the sample 1 due to a more effective protection polymer layer on drug particles as discussed above. Optimization of drug coating and the improvement of sample stability can be effectively controlled using HRUS measurements.

Figure 3: Ultrasonic velocity and attenuation profiles in aqueous suspension of solid polymer particles in flow. (12 MHz).

Figure 4. Particle size and concentration profile in aqueous suspension of solid polymer particles in flow obtained from HRUS measurements (see Figure 3).

2) Real time HR-US measurements of particle size and concentration profile of suspensions in flow.

A key element of analytical technique for PAT installation is its ability to provide real time analysis of the sample inline during the production processes. This task becomes particularly difficult when the testing must be done in opaque samples such as suspensions or emulsions. In these systems, the control of particle size in dispersed phase in technological line is a critical issue. The HR-US ultrasonic measurements provide a unique opportunity for the real time in-line analysis of practically all processes used in pharmaceutical technologies. In dispersions, these measurements allows simultaneous monitoring of both of particle size and volume fraction (or concentration) of dispersed phase.

The following example is a simple illustration of the capacity of the ultrasonic method for monitoring of the particle size and particle concentration in suspension in flow. The fluid flow rate in the line was 1 mL/min. The fluids in the pipe were water then suspension of solid polymer particles (2 percent) in water and water again. Ultrasonic velocity and attenuation in the line were continuously monitored (Figure 3). These data were translated (using HRUS particle size analysis software) to real time profile of the particle size and their concentration as shown in Figure 4.

The main fraction of particles has average size about 100 nm and it is detected around 20 minutes. Due to the particle size distribution, some minor part of the sample of the suspension is detected at earlier stage, and it corresponds to larger particles (e.g. 110 nm at 16 minutes). The smaller particles are recorded at later time (e.g. 94 nm particles after 24 minutes of the process monitoring).

Conclusion

HRUS provides a powerful tool for the development of new products and optimization of existing products as well as for quality control and process control in PAT installation. It provides real time information on chemical and microstructural characteristics across a broad range of materials. The arrival of a new generation of ultrasonic spectrometers, HRUS analyzers, will have a profound impact on the efficiency and profitability of pharmaceutical processes. �

Evgeny Kudryashov and Breda O'Driscoll, of Ultrasonic Scientific (Dublin, Ireland), can be reached at 353-1-218 0600.