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.
The technique has known limitations, however. Air and immiscible oils in a product could lead to an artificially high particle
count using light obscuration. And the method sometimes fails to properly size nonspherical particles and those with a refractive
index close to that of the liquid formulation, thus resulting in inaccurate and low counts.
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
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
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."
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).