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Pharmaceutical Manufacturing and Packing Sourcer

Size Matters

One means of delivering inhalation drug suspensions to the lungs is via a nebuliser.Widely used, these devices convert the suspension into a fine mist which is breathed in by the patient through a mask or mouthpiece. For effective delivery, the droplet size is controlled to below 10μm, and consequently solid drug particles must be smaller than this. The mode of action of nebulisers ensures that oversized particles are not administered and, additionally, the vibrational energy generated during nebulisation causes loose agglomerates to break up, allowing more effective drug administration.

Large particles may form during the manufacture of an inhaled drug. These might be primary particles, hard aggregates or loose agglomerates that are easier to break apart into smaller agglomerates or individual particles. In order to ensure efficient drug administration to the patient, it is important that oversized particles – especially large primary particles or hard aggregates, which cannot be dispersed by the action of the nebuliser – are eliminated. By characterising any large particles that arise, it is possible to identify the point within the manufacturing process responsible for their presence: large primary particles, for example,may suggest ineffective homogenisation. Here, image analysis has the role of providing the detailed information needed to differentiate between populations of particles, and hence determine their origins.

Image Analysis

Complete characterisation of the particles in a drug suspension requires more than size measurement alone. While manual microscopy has been used in the past, advances in imaging technology mean it is now possible to carry out robust, automated measurements of both the size and shape of particles, providing the detailed information necessary to contribute to process improvements. Automated analysers based on imaging flow cytometry, as used in the work described below, are designed to measure the size and shape of particles in suspension and are well suited to monitoring manufacturing processes. Using a charged coupled device (CCD) camera and strobe illumination, the analyser produces images of suspended particles. Samples pass through a sheath flow cell that transforms the particle suspension into a narrow, flat flow, ensuring that the largest area of the particle is oriented towards the camera and that all particles are in focus. Particle size and shape distributions for each sample measured are supported by scattergrams which visualise the data, and results can be overlaid for easy comparison.

Measurement Process

Where large particles are identified, they can be analysed further and their recorded images can be viewed to distinguish between primary particles, loose agglomerates and hard aggregates. If the large particles are found to be primary particles, it suggests a problem in the process that requires adjustment.

In order to differentiate between agglomerates and aggregates, further measurements can be performed on samples which have first been subjected to ultrasound treatment. Applying ultrasound mimics the action of the nebuliser, which would break up agglomerates. If the post-ultrasound measurement shows that there are no longer large particles present, it can be assumed that the large particles observed in the original samples were loose agglomerates and therefore the sample can pass the test. If large particles remain even after ultrasound treatment, they are likely to be aggregates – another indicator of a processing issue.

Automated analysis takes minutes. Modern systems driven by standard operating procedures (SOPs) enable repeat measurements to be built into the analysis protocol. Repeat measurements of the same sample can also be merged to increase statistical significance if required.

Automated Analyser in Action

Six samples of different batches of a suspended inhalation drug, in which the primary particles were generally less than 10μm in diameter, were measured using a flow particle image analyser. A 10x objective lens and high power field (HPF) configuration were used, resulting in a total magnification of 20x.

Three repeat measurements on each sample were merged to give the final result. Analysis of all six samples, with repeats, took less than one hour. Size results in terms of circular equivalent (CE) diameter, defined as the diameter of a circle with the same area as the particle, for the six samples are shown in Table 1. As shown in Figure 1, results from each sample can then be overlaid for easy comparison.

Circularity is a measure of how close the particle’s 2D projection is to a perfect circle. Agglomerates (and aggregates) are generally found to have a lower circularity and larger particle size than the primary particles. Therefore, by analysing scattergrams depicting size versus shape of particles in the sample, it is possible to determine the relative degree of agglomeration (see Figure 2).

Is the Number of these Particles Significant?

Using the instrument software it is a straightforward procedure to select and define particular areas of a scattergram for more in-depth analysis. By selecting the area of the diagram that contains large particles with low circularity, agglomerates can be separated from the main set of primary particles. In this case study, samples 5 and 6 were found to contain the highest proportion of agglomerates larger than 10μm.

These samples were therefore subdivided further, to numerically quantify the proportion of particles greater than 10μm. While sample 5 showed 2.19 per cent of particles were greater than 10μm, sample 6 contained 7.3 per cent of these potentially damaging agglomerates.

What Type of Large Particle Are They?

Subjecting samples to ultrasound mimics the action of a nebuliser. An ultrasonic probe on the image analyser enables samples to be sonicated immediately before a measurement to avoid further settling effects. Sonication will break up any loosely bound agglomerates, leaving only those that may then be significant to final product quality and performance.

One sample, previously measured and found to have a high proportion of large agglomerates,was treated with ultrasound and measured again. Table 2 summarises the results before and after sonication, indicating the proportion of over-sized particles present.

It is clear from the number of results that hardly any particles with a CE diameter above 10μm remain after sonication. This aligns with the decrease in mean CE diameter and the increase in mean circularity seen after sonication. In this sample therefore, the large particles are loosely bound agglomerates and are likely to be dispersed by the action of the nebuliser.

Process Improvements

The automated analyser enables identification of batches of inhalation drug suspensions that contain a high proportion of large agglomerates. Not only can larger particles be quantified, but further analysis helps determine how tightly bound the agglomerates are and how they may respond during nebuliser delivery.

By drilling down into the data, small sets of those particles that may cause processing problems can be singled out, and individual particle images can be investigated in detail by a human operator (see Figure 3). These investigations can determine what was happening in the process when potentially problematic batches were produced. The process can then be improved to prevent the reoccurrence of large agglomerated particles.


In the manufacture of inhalation drug suspensions, and indeed other drug suspensions such as eye drops, it is important to monitor the process to ensure that there are no oversized particles. The automated particle image analyser allows the measurement of size and shape of particles via simple SOPs in a fast, repeatable, routine analysis ideal for either research or quality control purposes. This means processes can be monitored closely and any problems identified rapidly, which in turn can improve efficiency and ultimately the profitability of the manufacturing process.

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Deborah Huck is Product Technical Specialist for morphological imaging systems at Malvern Instruments. Her focus is on applications development and support, working closely with new and existing users of Malvern systems. Deborah joined Malvern in 2005 from ABB instrumentation where she worked as a sales engineer. In 2004 she completed a PhD jointly supervised by the University of Exeter and the Université Louis Pasteur in Strasbourg and sponsored by the EPSRC and the European Doctoral College (Strasbourg).
Deborah Huck
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