What Is It?
Other technologies such as light obscuration can count particles, but neither it nor DLS can identify what the particle is.
With newer techniques like resonance mass measurement and flow imaging microscopy, “you can see if it’s a protein particle, or if it’s a silicone oil droplet,” said Andrea Hawe, Ph.D., CSO for research at Coriolis Pharma, and a co-founder of the company.
The Archimedes resonance mass instrument from Affinity Biosensors will query the mass and density of a particle from about 500 nm to about 1–2 µm, while the MFI microflow imaging system from ProteinSimple can analyze properties such as particle size, shape, and transparency from 2 µm to larger micron sizes, Dr. Hawe pointed out. Software algorithms can then determine the makeup of the particles—whether protein or silicone oil, for example. Combining the two techniques allows you to characterize a wide size range of subvisible particles.
This is highly relevant for therapeutic proteins in prefilled syringes because they are siliconized on the stopper and glass barrel, and “as soon as the formulation gets into the cartridge the silicone oil droplets start to form,” she said.
In protein and peptide formulations—especially when highly concentrated—the refractive index of particles is often so similar to that of the formulation itself that there is insufficient contrast to be adequately picked up by light obscuration (which is often required by regulators for batch release).
“What you can do is switch to (or additionally do) microflow imaging where you have better contrast, or you can switch to a different measurement principle like resonance mass measurement,” Dr. Hawe noted. “You can also think about diluting your sample in a different formulation, but this is not recommended because when you change the formulation, this might also change your particles.”
Silicone oil droplets are only one example of extractables and leachables that may be picked up from process equipment, disposable systems, packaging, and delivery materials, including syringes, cartridges, tubing, filters, and gaskets. We have to better understand the interaction with the drug product, “and be careful because these have a strong impact on the product quality and on the safety profile,” said Jöel Richard, Ph.D., vp, peptides, at Ipsen.
Metallic compounds and organic compounds—such as unreacted oligomers and curing agent residues—can interact with protein molecules in the formulation and chemically degrade them, cause them to aggregate, or form composite impurities with them.
Stainless steel, cellulose, glass, and silica have all been implicated in aggregation of protein therapeutics and formation of particulates. Tungsten microparticles, formed during the boring of needle holes, have been seen to produce visible particles with proteins, and soluble tungsten polyanions in slightly acidic buffer can precipitate monoclonal antibodies within seconds.
Dr. Richard recommends first performing a careful evaluation of the manufacturing steps that can generate aggregates and particulates, including an assessment of the packaging system, and gathering and analyzing whatever data can be obtained from suppliers. Then use a quantitative risk-based approach. Derive the daily dose of leachables that would be administered and compare them to toxicity guidelines. Steps with higher risk should be monitored using independent analytical methods.
“The key is to determine what is necessary, because you can generate a lot of data,” he said. “Depending on the type of formulation you have, if you have companion packaging materials, are extraction studies necessary or not?”
Any change of formulation, container closure components, and process steps need to be analyzed and monitored as well. To drive the point home, Dr. Richard cited a famous example in which a company commercializing epoetin alfa in Europe simultaneously changed both the primary container and the formulation composition, and the new formulation extracted some low molecular weight organic compounds—residues of the vulcanizing process used to create the rubber syringe stoppers.
These acted like adjuvants, practically turning the formulation into “a vaccine” that elicited an immune response against administered and endogenous erythropoietin, leading to a depletion of the patient’s red blood cells (i.e., pure red-cell aplasia).
Guidance on exactly what constitutes a safe formulation can be somewhat lacking, Dr. Richard pointed out. “There are a lot of guidelines, a lot of regulations. They tell you basically what to do, but they don’t tell you how to do it, nor when to do it, nor propose clear acceptance criteria.”