Recombinant proteins may be crucial in the development of biologics, yet they face a major problem during formulation and manufacturing: protein aggregation. This phenomenon increases the risk of immunogenicity – where the body’s immune system mistakenly attacks the therapeutic protein.1
Such responses can lead to the production of anti-drug antibodies (ADAs), potentially compromising a drug’s therapeutic effectiveness and triggering harmful side effects. Therefore, addressing these factors early in the drug development process is essential for ensuring patient safety and the success of the treatment.2,3

What is immunogenicity?
In brief, immunogenicity refers to the ability of a substance, such as a vaccine or a therapeutic protein, to provoke an immune response in an organism. This immune response typically involves the production of antibodies or the activation of T-cells, which are important components of the body’s immune system. Immunogenicity is a critical aspect of vaccines, as it determines their effectiveness in eliciting protective immunity against infectious diseases.
Additionally, in the context of therapeutic proteins (such as biologic drugs), immunogenicity can sometimes lead to the development of unwanted immune responses, including the production of neutralizing antibodies, which may reduce the efficacy or cause adverse reactions to the treatment. Therefore, understanding and assessing immunogenicity is essential in the development and evaluation of vaccines and biologic therapies.
Challenges of Assessing Immunogenicity
Traditional methods like ELISA and Surface Plasmon Resonance (SPR) are frequently used to detect ADAs, which are crucial for understanding drug-specific immune responses. However, these methods come with their own set of challenges. For instance, ELISA can produce false positives and suffer from high background noise due to nonspecific binding, while SPR, despite its ability to provide real-time monitoring of antigen-antibody interactions, may not be sensitive enough for certain applications and is not suited for high-throughput analyses.4,5
Subvisible Particle Analysis Key in Assessing for Immunogenicity
It is well established that protein aggregates in therapeutic protein products can enhance immunogenicity, and such an effect is therefore an important risk factor to consider when assessing product quality.6
Protein aggregates and subvisible particles (SVPs) can arise at various stages of the manufacturing process, during storage or upon administration, and their formation is influenced by multiple factors, including physical, chemical, and biological stressors. These aggregates can range in size from nanometers to micrometers and may consist of partially unfolded or misfolded proteins that can be recognized by the immune system as foreign, potentially leading to adverse immunological reactions.
Still, legacy particle analysis methods fail to provide adequate protection, leaving invisible particles unchecked and patient safety in danger. To address these challenges and accurately assess immunogenic risks, new solutions are needed.
Unveiling Protein Aggregates Faster and More Efficiently with Aura
Emerging technologies such as Backgrounded Membrane Imaging (BMI) and Fluorescence Membrane Microscopy (FMM) are rising to the occasion thanks to their ability to provide protein aggregate data without having to clean between measurements. Exclusively available on Aura, researchers can now distinguish between various types of particles, including degraded excipients and protein aggregates with only 5 µL of sample volume.
This robust technology duo enables characterization from single protein aggregates to millions of particles in multi-sample formulations within just two hours, facilitating a deeper understanding of their origins and potential impact on drug stability and immunogenicity.
Conclusion
Subvisible particle analysis is key for immunogenic risk assessment of biologics. By implementing innovative technologies, such as Aura, early in development, researchers can make informed decisions to minimize the risk of SVP formation and aggregation, which induces immunogenicity. This proactive stance is instrumental in preventing potential problems that could lead to regulatory setbacks, additional costs, delays in bringing critical therapies to market, and ensuring patient safety.
Related
- Join our virtual course to master the techniques of imaging, counting, and measuring subvisible particles with Backgrounded Membrane Imaging
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References
1. Pham NB, Meng WS. Protein aggregation and immunogenicity of biotherapeutics. Int J Pharm. 2020 Jul 30;585:119523. doi: 10.1016/j.ijpharm.2020.119523. Epub 2020 Jun 9. PMID: 32531452; PMCID: PMC7362938
2. Sauna, Z. E. (n.d.). Immunogenicity of protein-based therapeutics. https://www.fda.gov/vaccines-blood-biologics/biologics-research-projects/immunogenicity-protein-based-therapeutics
3. U.S. Food and Drug Administration. (2014, August). Immunogenicity assessment for therapeutic protein products. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/immunogenicity-assessment-therapeutic-protein-products
4. Kuriakose A, Chirmule N, Nair P. Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications. J Immunol Res. 2016;2016:1298473. doi: 10.1155/2016/1298473. Epub 2016 Jun 29. PMID: 27437405; PMCID: PMC4942633.
5. Pineda C, Castañeda Hernández G, Jacobs IA, Alvarez DF, Carini C. Assessing the Immunogenicity of Biopharmaceuticals. BioDrugs. 2016 Jun;30(3):195-206. doi: 10.1007/s40259-016-0174-5. PMID: 27097915; PMCID: PMC4875071
6. Carpenter JF, Randolph TW, Jiskoot W, Crommelin DJ, Middaugh CR, Winter G, Fan YX, Kirshner S, Verthelyi D, Kozlowski S, Clouse KA, Swann PG, Rosenberg A, Cherney B. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J Pharm Sci. 2009 Apr;98(4):1201-5. doi: 10.1002/jps.21530. PMID: 18704929; PMCID: PMC3928042
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