As the largest market for particle size and distribution analysis, the pharmaceutical industry accounted for approximately 15% of the sales of instruments and aftermarket products last year. Particle size is a crucial metric for the design, quality control and manufacturing of pharmaceuticals. In developing new drugs, researchers are increasingly exploring the use of nano-sized particles (1 nm–100 nm). Application areas for such particles in drug development include formulation design and drug delivery. For example, nano-sized particles can improve the solubility of active pharmaceutical ingredients, enabling the reformulation of failed drugs, and they hold promise for the development of new drug delivery mechanisms.

Instruments for the measurement of the size and distribution of particles in the nano-range are the fastest growing segment of the particle size instrumentation market. There have been notable new business developments in this area. Agilent entered the market earlier this year through its acquisition of Collodial Dynamics (see IBO 3/15/08), and Malvern Instruments acquired Viscotek in January (see IBO 1/31/08), adding to its platforms for nanoscale analysis. IBO spoke with three vendors of particle sizing instrumentation about the latest developments in the market for pharmaceutical applications.

No single analytical technique can provide complete information about particle size and distribution. The choice of technique to use depends on the sample type, application and experimental parameters. Techniques for the analysis of particles down to 10 nm include laser diffraction, disc centrifugation and acoustic spectroscopy. Techniques that can analyze the size and distribution of particles smaller than 10 nm include dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy and particle techniques used as detectors for chromatography. Sample types of this size include micelles, proteins and liposomes.

One of the most popular technique for the analysis of the size and distribution of nano-sized particles is DLS. DLS is fast, sensitive and nondestructive. Drawbacks include the potential distortion of the measurement by larger sized particles and the need to adequately dilute the sample. According to Malcolm Connah, Malvern Instruments’ product manager for Nanometrics, these problems have been largely overcome. He told IBO that advancements in particle size analysis for pharmaceutical applications have occurred in two areas: “First, in presenting the sample to the instrument. There is no longer the need to prepare the sample to exacting concentration limits. And the extended sensitivity and size range available through the latest technologies mean that there is virtually no protein or biopolymer sample that cannot be measured.”

Malvern, a Spectris company, provides particle analysis systems for both laboratory and process applications. For the analysis of nano-sized particles, the company offers the Zetasizer Nano Series based on DLS for the measurement of particle sizes from 0.6 nm to 6000 nm. The Zetasizer also measures zeta potential, a calculation of the electronic charge on a particle’s surface in suspension, which is indicative of the particles’ stability in liquid.

The validity of DLS data has also been disputed due to the calculations required to transform the measurements into results, including adjustments made for nonspherical particles. As Mr. Connah explained, advancements in data interpretation have been significant: “Three decades of experience in the subject have led to the development of quality tests, and an ‘expert system’ that looks at the data and trends in the data to give a report on how meaningful the results are and advice about remedies if the measurement could be improved.”

DLS is a so-called “ensemble technique,” as it relies on mathematical calculations based on statistical measurements of the distribution to determine mean particle size. Hence, software developments have been paramount to more accurate and faster analyses.

Such developments have made DLS easier to use, which is a key consideration, according to Mr. Connah: “With a range of techniques being used on a daily basis, there is simply not the time to become an expert in each one of them. Light scattering in the past was seen to be the preserve of specialists hired for the purpose. In the absence of such specialists, systems need to be as simple and intuitive to use as possible, as well as giving confidence that the numbers produced are meaningful.”

Future developments of particle size instrumentation for pharmaceutical R&D are also likely to rely heavily on software development, according to Mr. Connah. “The rise of the use of proteins as drugs, and the advent of nanotechnology techniques to solubilize and deliver these proteins to targeted sites, means that the formulation, storage, quality control and shelf life of proteins are becoming increasingly important for pharmaceutical companies,” he told IBO. “Techniques that can easily, rapidly and accurately provide the parameters to monitor these processes on an absolutely routine basis are required,” he said. “Future software developments will contribute at least as much as hardware improvements to assist with interpreting and understanding data.”

For the analysis of irregular shaped nano-sized particles or to validate DLS results, SEM can be employed. SEM is a “counting method,” which, in contrast to ensemble methods, provides higher resolution but lower dynamic range. In addition, SEM provides morphological information about particles, including shape and surface texture

To increase the ease of using multiple techniques, Malvern and FEI have partnered to make Malvern’s Morphologi image analysis software available for use with FEI’s Quanta FEG electron microscope. “Electron microscopy is a well established technique in the pharmaceutical industry, mainly because of its capability to give detailed qualitative information. With the increasing interest in optimizing submicron size distributions in pharmaceutical ingredients, the need for quantitative information is rapidly growing,” explained Ulf Willén, product manager for Malvern’s Morphological Imaging Systems. ”By combining the FEI Quanta FEG Electron Microscope with the Malvern Morphologi image analysis software, the first absolute sizing technique has become available that routinely enables not only qualitative, but also quantitative size and shape analysis of individual nanoparticles in polydisperse products.”

However, according to Philip E. Plantz, PhD, Applications, Compliance and Training manager for Microtrac, a supplier of DLS particle analysis systems, SEM also has its drawbacks, particularly when working with organic molecules. “There’s a certain amount of sample preparation that has to be performed to make these molecules electron dense. Because they are organic molecules, using an electron beam that can actually pass right through some of these [molecules] may even burn them or vaporize them,” he explained.

Microtrac’s Nanotrac Ultra measures particle size down to 1 nm. The company’s Zetatrac, introduced last year, measures both particle size and zeta potential. Asked how this product differs from others, he responded: “First of all, it uses a fiber optic probe to transmit the laser light and also to receive the scattered light.” This not only increases flexibility, but makes the instrument easier to use. As Dr. Platz said, “There is no need for cuvettes or anything like that . . . So the operation and the use of that is not anything different than what a person would experience if they’re using a pH electrode.” The Zetatrac also utilizes a different type of DLS technique. “A typical DLS instrument uses what’s called photon correlation spectroscopy. We use what’s called a frequency spectrum analysis,” he explained. “We believe that is a better approach because we’re really looking very directly at frequency shifts of the light, which is really the basis of DLS, without other types of mathematical models,” he explained.

Dr. Plantz noted that the use of zeta potential is one of the main developments in particle size analysis for pharmaceutical applications. “The charge on the molecule can impact a number of things. To start with, [it can impact] the stability of the molecule—if that charge is disrupted then the molecule can become inactive.” Future developments, he believed, will include the integration of more measurement techniques into one instrument. “I think what you’ll see is probably some repackaging of additional types of measurements, [such as] titration and zeta potential­—things of that nature, where a single instrument can do a number of examinations of these molecules.”

A new approach to the analysis of nano-sized particles utilizes optical microscopy. Traditional optical microscopy cannot view particles in the nano-range. Nanosight’s Nanoparticle Tracking Analysis technology, which utilizes an optical microscopy, digital camera and dedicated software, can view particles in suspension ranging in size from approximately 10 nm (depending on the type of particle) to 1000 nm by tracking the spot of light produced by a scattered particle. According to Jeremy Warren, CEO of NanoSight, in contrast to other systems, the company’s LM 10 and LM 20 instruments offer a low-cost particle sizing solution and real-time analysis. He told IBO that the technique is complimentary to DLS and other particle sizing methods. The company has more than 100 units installed, half of which are with industrial customers, such as pharmaceutical companies. According to Mr. Warren, the company plans to introduce a new version of its system that includes zeta potential analysis.

As established techniques for the analysis of particle size and distribution continue to improve, growing interest in nano-sized particles has encouraged new approaches. Pharmaceutical research is set to continue to be at the forefront of many of the developments for the analysis of size and distribution of particles in the nano range.