The appropriate methods

Compendial methods. Measuring and analyzing particles smaller than 10 µm is challenging for quality-control personnel because the standard tests on parenteral products don't examine particles in this size range. But the required technology is relatively straightforward and readily available, according to industry sources.

The preferred method for the first round of testing is light obscuration, which is described in USP <788> (1). Light obscuration detects particle sizes between 2 µm and hundreds of micrometers. Companies commonly calibrate the instruments to 2 µm, yet most firms outside the biopharmaceutical industry generally don't examine the region of particles smaller than 10 µm, says Aldrich.

Scientists can use the membrane-microscopic technique as an alternate or secondary method. This method is a more direct revelation of particle content than light obscuration is because it isolates solids or semisolids from the sample liquid onto microporous media and counts particles in size thresholds. Membrane microscopy has a wide detection range (i.e., from 5 µm to many millimeters).

The technique also has shortcomings, though. "Because it is [an] optical-microscopy evaluation at 100 × [magnification] with reflected illumination, particles below 10 µm are difficult to resolve on the porous membrane surface," thus making accurate counting difficult, says Aldrich. Inherent proteinaceous particles also may be difficult for the microscopist to count or size. Air and immiscible oils are not problematic, however, because particles are observed in a dry state and may appear as stains.

In addition, the membrane microscopic method is tedious, and no company will resort to it "unless they have a real need to, or if they had to use that methodology to verify the counts they received during light-obscuration counting," says Cherris. Some equipment automatically counts and identifies particles on a filter surface, but it is much more expensive than light-obscuration particle counters are.

The most economical way to test for particles smaller than 10 µm is to use automated particle counting first, then to use microscopic and spectroscopic techniques to identify the particles on a membrane surface, says Cherris. "Most companies that have recently purchased new light-obscuration particle counters have the sensor technology now to start counting down in that range."

Noncompendial methods. Many biopharmaceutical companies use alternate, noncompendial methods to understand the subvisible particle population. These techniques are reliable, but have a measurement gap from 0.1 to 1 µm, says Aldrich.

One such approach is the Coulter principle, which uses a dilution of a product in conducting liquids to measure particles between 0.4 and 50 µm, depending on instrument setup. Formulation buffering and preparation affect the measurements, but the technique measures particles and aggregates in a solution to reveal a relevant particle–product state.

The flow-imaging approach is an extension of optical microscopy that uses cameras to detect and record in situ particles in product fluid. The method detects particles from 0.7 µm to 1 mm, depending on their shape. This technique's ability to capture particle images for subsequent analysis allows the evaluation of the particle population under various shape and size contexts, says Aldrich.

Investigating particles in this size range is important and requires more than one method. Monitoring particles smaller than 10 µm "can provide valuable information on changes in product stability, protein aggregation, and product attributes that affect patient safety," says Dan Berdovich, manager of quality assurance and regulatory affairs at Micro Measurement Laboratories.

The next steps

Certain drugmakers are collecting information about particles smaller than 10 µm at FDA's request. Although its goal is sound, FDA must agree with industry on the most appropriate standardized testing methodology, according to Cherris. "If we don't collect data in the 2–10-µm range in a comparable way, it will be a long road, and probably the wrong road to follow in understanding these particle populations," he says.

The industry, through large nonprofit organizations focused on bettering pharmaceutical quality, should sponsor this data-collection program, not the government, according to Cherris. The Parenteral Drug Association or the US Pharmacopeia should create a scientific committee to collect and study the data, and possibly draft a new standard, he says. "Without that type of industry rally in an organized manner, the requests from FDA to collect this information will not be widely effective."

References

1. USP 23–NF 18 (US Pharmacopeial Convention, Rockville, MD, 1995), pp. 1813–1819

2. J.F. Carpenter et al., J. Pharm. Sci. 98 (4), 1201–1205 (2009).