Differentiation and characterisation of subvisible particulates in therapeutic protein products

Michael Caves, India Business Development Manager, Malvern Instruments, in this article elaborates how Archimedes can be used to detect and quantify the formation of protein sub-visible particles and the introduction of silicone oil droplets, in response to shear stress

The formation of protein aggregates is a particular concern for parenteral administration biopharmaceuticals due to the potential for increased immunogenicity. As a consequence, there is an expectation from regulatory agencies for companies to monitor and, if required, reduce the levels of sub-visible particles present in therapeutic protein from manufacture through their complete shelf life. While immunogenicity can be induced by a variety of mechanisms, contamination by non-protein material is known to be a potential cause. The presence of silicone oil in parenterals has attracted considerable interest, due to its use in syringe-based administration systems and the difficulty in distinguishing oil droplets from protein aggregates. In addition to protein aggregates, non-biological contaminants may act as nucleation points for aggregate growth. Consequently, particle sizing alone is not sufficient.

Guidance from the FDA for the biopharmaceutical industry states, “Naturally sourced products should be evaluated for other components, protein and non-protein” (1). Resonant Mass Measurement (RMM) technology in the Archimedes system measures the buoyant mass of particles passing through a cantilever, allowing detection of particles with a density different to that of the buffer solution. Consequently, the Archimedes provides the ability to distinguish between negatively buoyant particles, such as proteins, and positively buoyant particles, such as silicone oil.

Results

The impact of shear stress of the formation of protein aggregates and the introduction of silicone oil was determined using the Archimedes system.

Formation of sub-visible Protein Aggregates in Response to Shear Stress

Understanding the impact of bioprocessing on protein characteristics is an important aspect of product knowledge and design of manufacturing parameters. To ensure compliance with regulatory requirements, the impact of materials used in the manufacturing process stream should be evaluated to ensure no impact on product quality (2). Biopharmaceuticals may have different compatibilities to materials used in the construction of product contact components, based on individual protein and formulation characteristics, as well as the leachables and extractables. Therefore, the impact of product contact components may need to be compared. Sub-visible particles induced by shear stress of two different syringe manufacturers were compared using RMM. These data are shown in Figure 2A.

1 mL syringes from two different manufacturers were used to induce shear-stress. From the data in Figure 2A, the number of sub-visible particles formed by the syringe from manufacturer 1 (red bars) is much higher than the syringe from manufacturer 2 (blue bars). These data would suggest that the protein is more compatible with syringes from manufacturer 1 than those from manufacturer 2.

Detection and quantification of silicone oil from two different syringes manufacturers

The ability to distinguish silicone from protein aggregates based on buoyant mass is a significant benefit for the Archimedes RMM system. The positive mass data from the previous experiment is shown in Figure 2B, and demonstrates the presence of silicone oil from the two syringes tested. While protein aggregates produce a negative frequency shift, silicone which has a positive buoyant mass in water, produces a positive frequency shift. This technology enables quantification of silicone oil content, which can have a detrimental impact on the protein itself, and/ or an increase in immunogenicity following administration. From the data shown in Figure 2, the syringes from manufacturer 1 (red bars) introduces significantly higher levels of silicone oil into the protein sample, following repeated shear-stress. The increased levels of silicone oil may be the cause of the higher levels of protein aggregates detected with this syringe. However, the ability to detect and quantify silicone oil content provides a more detailed insight into the product, and therefore more knowledge concerning the relevant degradation pathways.

Conclusion

As recommended by the FDA, the ability to distinguish protein aggregates from non-protein material is an important aspect of monitoring and studying biopharmaceutical stability. This article demonstrates the ability of the Archimedes system to quantify protein aggregates and silicone oil in response to shear-stress. Such data is greatly beneficial when understanding product stability, immunogenicity and bioprocessing impact. In addition, the ability to distinguish protein aggregates and silicone oil can enable more efficient resolution of product investigations. By quickly ruling in or out silicone oil as a potential root cause (3), efforts can be focused on finding the most likely causes. Importantly, the Archimedes is able to quantify protein particles in a size range that is currently poorly served by analytical instruments, between 0.2-5 µm, that has been identified as an important size range for understanding immunogenicity (1).

In conclusion, the Archimedes RMM system is able to provide highly valuable information about biopharma products, in terms of both formation of sub-visible particle formation and the presence of non-protein contaminants, such as silicone oil.

References:
1. Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (2014): FDA
2. Code of Federal Regulations; CFR21, Part211.65
3. Characterization and Quantitation of Aggregates and Particles in Interferon-ß Products: Potential Links Between Product Quality Attributes and Immunogenicity (2013): J. Pharm. Sci. (102) p.915

Contact Details:
Malvern Aimil Instruments
Naimex House,
A-8, Mohan Co-op. Indl. Estate,
Mathura Road,
New Delhi – 110044
Tel: 011-30810244
Fax: 011-26950011
Email: [email protected]