Visible and Subvisible Protein Aggregates/Insoluble Particulate Matter in Biopharmaceutical Drugs

Measurement of Protein Aggregates / Insoluble Particulate Matter in Biopharmaceutical Drugs in Visible and Subvisible (SVP) Range

Protein aggregation is a serious immunogenic problem that can be encountered in biopharmaceutical products during clinical trial periods and after market launch. The analytical methods for protein aggregation and insoluble particles are shown in Figure 1 according to measurement ranges. As can be seen in the figure, Size Exclusion Chromatography (SEC) is used for measuring oligomers/aggregates below 0.1-0.2 μm. The SEC method has many advantages over other methods, such as easier application and high repeatability. Due to its working principle (pressure), the SEC method may cause dispersion of protein oligomers and aggregates. However, the FDA and researchers require the SEC method for quantitative evaluation of protein oligomers and aggregates1. Figure 1. Measurement Ranges of Analytical Methods Used for Measuring Protein Aggregates / Insoluble Particulate Matter in Visible and Subvisible (SVP) Range

Figure 2. Light Extinction/Obscuration Method Working Principle and Characteristics

Although the Light Extinction/Obscuration method has been used for years to analyze insoluble particulate matter of 10 μm - 25 μm in size, there are serious problems encountered in the analysis of protein aggregates in biopharmaceutical drugs. • As seen in Figure 3, detection sensitivity depends on the area and intensity of the shadow. When light is applied to particles, protein aggregates [Figure 3a] and silicone oil droplets [Figure 3b] are difficult to detect because these particles transmit light. • Air bubbles, as seen in Figure 3c, are extremely sensitive to the Light Extinction/Obscuration method because they block light. • It is not possible to know what type of particles have been detected.

Figure 3. Images of insoluble protein particles, silicone oil droplets and air bubbles obtained under cell transmission microscopy using a microflow cell

In fact, during measurement with the Light Extinction/Obscuration method, protein aggregates are detected at smaller sizes than their actual particle sizes [depending on the size of protein aggregates, generally detected as approximately one-tenth of their actual size.]. For accurate analysis of insoluble particulate matter, the microflow digital imaging method, which is similar to and has an even wider working range than the Light Extinction/Obscuration method, is used as a supporting/alternative method. The principle of the microflow digital imaging method is extremely simple. Only images of all particles are captured and particle size is determined from the actual image [Figure 4a and 4b]. Therefore, unlike the Light Extinction/Obscuration method, the microflow digital imaging method is often successful in the analysis of transparent protein aggregates such as silicone oil droplets. The use of the microflow digital imaging method instead of the Extinction/Obscuration method in the analysis of insoluble particulate matter is extremely important in protecting patients using biopharmaceutical drugs from large protein aggregates. As shown in Figure 1, protein oligomers/aggregates below 0.1-0.2 μm, insoluble particulate matter above 10 μm and 25 μm, and visible particles can be analyzed and monitored according to regulations. On the other hand, until recently, analysis and monitoring according to regulations of particles in the 0.1-0.2 μm and 10 μm range were not possible. For this reason, these particles are called "subvisible particles" (SVP)3. Recently, it has been confirmed that particles of this size exhibit high immunogenicity [Figure 5]. The reason SVP could not be analyzed was the lack of adequate analytical methods in this area. Archimedes using Resonant Mass measurement, NanoSight operating with nanoparticle tracking analysis method, and Aggregates Sizer based on Laser Diffraction/Scattering method are devices specially developed for SVP measurements [Figure 1].  

At the "2nd Progress and Challenges in Protein Particles and Immunogenicity of Biotherapeutics 2015" conference, it was proposed that the definition of SVP be changed from the current 0.2 – 10 μm range to particles in the 1 – 100 μm range. [Figure 1].

As SVP analysis devices such as Archimedes and NanoSight are insufficient in measuring the newly defined SVP range, the FDA recommends the use of orthogonal methods4. The microflow digital imaging method and Aggregates Sizer, which give excellent results [Figure 6] on quantitative measurements of the same particles, [Figure 7], make it possible to measure SVPs in the newly defined range.  

References:

1. Shire SJ. Assessment of Self-Association of Protein Therapeutics at High Concentration and its Impact on Viscosity. International Light Scattering Colloquium 2006.

2. Thomas D. Particle Characterization Using Flow Microscopy. 2008 BioProcess International Conference & Exhibition: Formulation Strategies for Protein Therapeutics 3. Carpenter JF. et al. 2008. Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May Compromise Product Quality. J Pharm Sci, DOI: 10.1002/jps.21530.

4. Susan L. Kirshner. Regulatory Expectations for Analysis of Aggregates and Particles. 2014 Workshop on Protein Aggregation and Immunogenicity, 2014. July 17th.