Differentiation and Characterization of Protein Aggregates and Oil Droplets in Therapeutic Products

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Article submitted: Dec. 17, 2014; Article accepted: Feb. 12, 2015.

AbstractAs naturally unstable molecules, proteins are prone to denature, self assemble, and agglomerate with little external stimulus required. Protein aggregation presents a key challenge in the production of  biopharmaceuticals for parenteral administration. There is an expectation from the regulators that companies will monitor and, where necessary, reduce, levels of subvisible particles in biotherapeutic formulations. A related issue is the presence of aggregates in formulations of silicone oil, a widely used lubricant in prefilled syringes. Silicone oil droplets have the capacity to act as nucleation points for aggregate growth. This article demonstrates how resonant mass measurement can be used not only to detect and quantify the formation of protein subvisible particles in a critically important size range but also to detect and quantify any silicone oil droplets in the formulation.

The formation of protein aggregates is of crucial concern in biopharmaceuticals for parenteral administration. Protein aggregates can elicit an immune response in the recipient and there is an expectation from the regulators that companies will monitor and, where necessary, reduce, levels of subvisible particles of all kinds in biotherapeutic formulations. A related issue is the presence of aggregates in formulations of silicone oil, a widely used lubricant in prefilled syringes. Silicone oil droplets have the capacity to act as nucleation points for aggregate growth.

The ability to distinguish between and quantify aggregates and oil droplets is challenging for most analytical systems. This article demonstrates how the technique of resonant mass measurement (RMM) can be used not only to detect and quantify the formation of protein subvisible particles in a critically important size range but also to detect and quantify any silicone oil droplets in the formulation.

Bridging the subvisible gap
Ensuring protein stability throughout the lifecycle of a biopharmaceutical product, from formulation right through to patient administration, is a complex challenge. Proteins are naturally unstable and are prone to denature, self assemble, and agglomerate with little external stimulus required. Quality control (QC) and in-use process studies that comprehensively characterize the presence of subvisible aggregates within biopharmaceuticals are, therefore, essential to ensuring product performance and safety.

Subvisible particles are usually defined as particles that are not visible to the naked eye and have a size of <100 µm. They can further be defined into a micron (1–100 µm) and submicron (1 µm) size range. United States Pharmacopeia <788> requires the quantification of subvisible particles that are ≥10 µm and ≥25 µm in size (1). Particles at this size are typically characterized using light obscuration and/or imaging techniques. Meanwhile smaller aggregates (<0.1 µm), typically caused by oligomerization, are characterized using size exclusion chromatography (SEC). It is, however, also recognized that particles in the 0.1 to 10 µm size range have a strong potential to be immunogenic. This is a measurement range that is becoming increasingly important to FDA and there are very few characterization techniques that can provide quantitative sizing details in this measurement space.

The application of RMM to detect and count particles in the size range 50 nm–5 µm and to measure particle mass and size provides a system that is uniquely positioned to quantitatively measure protein aggregates within the range of 250 nm-5 µm. This technology is also capable of providing information on sample concentration, viscosity, polydispersity, density, and volume. Moreover, it is able to distinguish between negatively buoyant proteinaceous particles and positively buoyant contaminating silicone oil droplets. The technology allows users to determine how much silicone oil is injected along with the protein, whether this amount impacts aggregation, and whether the intended administration method is fit for purpose.

Resonant mass measurement
RMM exploits the relationship between the buoyancy of a particle within solution and its size. The technique works by passing the sample through a microfluidic channel embedded within a resonating cantilever (see Figure 1). The frequency of cantilever resonance shifts either up or down as particles with densities that are different from that of the carrier solution flow through it. By precisely measuring the magnitude in excursion from the cantilever’s base line frequency, an accurate measurement of particle size can be derived for the particles within solution.

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Applying resonant mass measurement to particles that have very small mass and whose size falls within the submicron region requires the use of low mass resonators. These can be engineered using micro electric-mechanical system (MEMS) sensors. Each sensor chip comprises a microfluidic network and a minute cantilever that resonates with a particular frequency. As the instrument’s fluidics system pushes sample through this channel, the resonant frequency of the cantilever alters. This shift in resonance frequency is measured by a laser focused on the tip of the cantilever, which is then transmitted to a split photodiode detector.

Each particle that passes through the sensor causes a change in the resonant frequency, giving an accurate and precise measurement of the individual particle’s buoyant mass. In this way, the mass and size can be calculated for each individual particle. As protein aggregates are denser than the solution, they will produce a negative change in resonance. Silicone oil droplets, however, are buoyant within what are typically aqueous solutions, and result in a positive shift in resonance. This difference in buoyancy allows users to distinguish between silicone oil particles and those of proteinaceous origin, and characterize and quantify particles in relation to these discrete populations.

The following case studies illustrate how this innovative analytical set up enables subvisible particle characterization in bioformulation QC and process studies.

Case study 1: Formation of subvisible protein aggregates in response to shear stress
The shear force exerted on a biopharmaceutical formulation during syringing has been identified as having the potential to cause aggregation. Quantifying the presence of aggregate species within formulation, pre-, and post-syringing is, therefore, essential for monitoring the safety of administration.

To illustrate the proficiency of RMM in this area, subvisible particles within a biotherapeutic formulation were quantified before and after being subjected to syringe-induced shear stress. Measurements were made using the Archimedes system from Malvern Instruments. Figure 2 compares the number of subvisible particles within a control sample (blue) with those in a sample that has undergone syringe-induced shear (green). Prior to syringe stress, the number of particles detected is low, demonstrating that the sample is reasonably pure, and contains only a few large aggregates. Following the application of shear stress, the number of particles detected increases significantly, with particle sizes ranging from 500 nm up to 1700 nm. This is important data that indicate a formulation’s response to the type of stress induced. It can be used to compare different stress conditions to provide an overall picture of the degradation profile for the biopharmaceutical of interest.

The study also compared the number of subvisible particles produced following injection of a formulation using syringes from two different manufacturers using RMM. Figure 3 shows that the number of aggregates formed using syringes from manufacturer 1 (red bars) is many orders of magnitude greater than with syringes from manufacturer 2 (blue bars). This finding suggests that the protein is more compatible with the latter administration technology.

The next step in this investigative study was to identify the root cause of aggregation. Here, the ability to distinguish silicone oil particles from proteinaceous material is a significant benefit of employing RMM.

Case study 2: Detection and quantification of silicone oil from two different syringe manufacturers
Silicone oil is non-toxic and its delivery during parenteral administration of a drug is not dangerous in itself. However, the presence of silicone oil particles, together with other inorganic contaminants, has been shown to induce aggregation. RMM enables the quantification of silicone oil content, which can have a detrimental impact on the protein itself, and/or promote an increase in immunogenicity following administration. Figure 4 shows variations from the frequency baseline caused by protein and/or silicone oil particles passing across the cantilever. The positive peaks are associated with the detection of the more buoyant silicone oil particles while the negative excursion indicates the presence of the more dense proteinaceous material.

Figure 5 shows the data from RMM measurement following injection using the different syringe systems. The data indicate that syringes from manufacturer 1 (red) introduce significantly higher levels of the lubricating silicone oil during administration. The increased levels of silicone oil, rather than the induced shear, may be driving the protein aggregation mechanism in formulations delivered with syringes from manufacturer 1.

The ability to detect and quantify silicone oil content using RMM provides additional insight into a biotherapeutic product and more knowledge about possible protein degradation pathways. Developers can, therefore, quickly determine whether or not injected silicone oil is contributing to aggregation, or focus on identifying an alternative cause.

The growing bioformulation toolkit
FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products indicates that “…the use of any single method for assessment of aggregates is not sufficient to provide a robust measure of protein aggregation” (2). As such, FDA recommends an orthogonal approach to biopharmaceutical characterization whereby a variety of different and complementary techniques are used. Comprehensive characterization of the components of a biopharmaceutical formulation that lie within the subvisible size region is currently beyond the capabilities of a single analytical technique.

RMM is one of the few techniques that can bridge the sizing gap between SEC and light obscuration and provide quantitative particle information within this space, making it an important part of a bioformulation toolkit. Moreover, its unique ability to distinguish protein aggregates from silicone oil and to quantify each is greatly beneficial in understanding product stability and immunogenicity.

References
1. USP, United States Pharmacopeia-National Formulary, General Chapter <788>, “Particulate Matter in Injections” (US Pharmacopeial Convention, Rockville, MD, 2011).
2. FDA, Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (Rockville, MD, Aug. 2014).

Article DetailsBioPharm International
Vol. 28, No. 5
Pages: 30-34
Citation: When referring to this article, please cite it as C. Murphy, “Differentiation and Characterization of Protein Aggregates and Oil Droplets in Therapeutic Products,” BioPharm International28 (5) 2015.