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One CQA that has drawn an increasing amount of attention in recent years is aggregation, the process by which proteins stick to each other, often accompanied by the loss of their natural structure and function. The induction of aggregation in mAbs is influenced by a range of factors, from sequence specific liabilities inherent to individual clones, to exposure to physical and chemical stressors during development and manufacturing such as fluctuations in pH or temperature, mechanical stressors during development and improper buffer formulations for storage2.
Aggregates pose several risks to product quality. When aggregates form, they can mask key residues on the mAb that allow it to bind to its intended target. Aggregates can also impact patient safety by triggering immune reactions3. To lessen these risks, developers monitor aggregation levels to ensure they meet regulatory CQA expectations.
Traditionally, aggregation screening starts in the latter stages of process development, when researchers have already screened thousands of clones for binding affinity to their target and have narrowed their pool to 100s or 10s of clones. They then screen for the highest quality clones that are the least likely to aggregate. However, this is a risky process as they may have already eliminated their most stable clone. If their lead clones turn out to be suboptimal, they’ll have to begin their screening process for a safer clone all over again. This is a time-consuming and expensive process.
Due to recent technological advances, there are now new methods that can be used to detect aggregation earlier in the development process. Screening for aggregates at an earlier stage can help developers select the clones that are most likely to retain an aggregation level of below 5% when they move to process development, cutting development timelines and helping to speed mAbs to patients in need of new therapeutic options.
Detecting aggregates early in development
mAb developers have traditionally used a combination of approaches to measure and monitor aggregation. They include high-performance liquid chromatography size exclusion chromatography (HPLC-SEC) and dynamic light scattering (DLS) technologies. These techniques require dedicated tools and many hours of hands-on time from specially trained scientists. Because of the manpower required to perform HPLC-SEC and DLS, and analyze the resulting data effectively, mAb developers often outsource aggregate monitoring to specialized analytics labs, which can lead to delays in development timelines and force scientists to screen smaller number of clones than is optimal.
Emerging technology, such as plate-based assays, is enabling the detection of aggregation earlier in the development process. One such option is a high-throughput, plate-based screening tool that allows developers to measure aggregation in 96 different samples simultaneously. Each well of the plate is coated with a fluorescent small molecule that binds to protein aggregates. Using fluorescent polarization, the technology excites the fluorescent molecule with light, then detects and measures changes in rotational speed. The concept behind this technology is that unbound proteins rotate more rapidly than aggregates do. As the bound aggregates rotate slowly, they retain polarized light.
With this tool, mAb developers can generate aggregation data for 96 samples in as little as 15 minutes. With no washing required and a five-minute incubation period, the aggregation assay is significantly more efficient than DLS, which typically takes one to two hours, and HPLC-SEC, which can take more than six hours.
Comparable results to existing methods
In a study of the new aggregation assay, the technology was compared to HPLC-SEC using samples of IgG4 with a total protein concentration of 2 mg/mL. Despite differences in plate reader and analyst, both technologies performed comparably. The relative standard deviation (RSD) for all replicates across both tools was under 2%. When three preparations of 15 samples were tested using a standard curve, the RSD was under 3.5%, indicating that variation was low. This demonstrated that the 96-plate assay is highly reproducible4. Importantly, the results from both technologies ranked the samples’ levels of aggregation from lowest to highest effectively, allowing scientists to easily select their most stable molecules.
With a plate-based technology, screening for aggregation can start before process development, potentially saving developers time and costs. If a clone is selected and it unexpectedly demonstrates high levels of aggregation, it can be dropped well before a significant amount of time and resources are invested in developing it further.
In the future, adding automation to plate-based assays will make them even more useful, preventing errors from manual pipetting and potentially reducing the time required to set up and run aggregation assays.
Managing aggregation earlier in the development process will ultimately benefit patients as well. mAb development timelines can be frustratingly long—17 years on average from discovery to marketability5. Any improvements that can be made in managing CQAs have the potential to shorten those timelines, bringing lifesaving therapies to patients sooner.
About the author: Dr. Elisa Nent has worked in the field of life sciences for more than 11 years and has a PhD and master‘s degree in neuroscience. As the global product manager for Cell Health at Beckman Coulter Life Sciences, she leverages her expertise in cellular biology to support customer needs in cell line development.
- Grand View Research. Biologics Market Size & Trends. https://www.grandviewresearch.com/industry-analysis/biologics-market. Accessed October 2024
- Pang, KT, Yang, YS, et al: Understanding and Controlling the Molecular Mechanisms of Protein Aggregation in mAb Therapeutics. Biotechnology Advances, Volume 67, 2023, 108192, ISSN 0734-9750, https://doi.org/10.1016/j.biotechadv.2023.108192. See also https://www.biopharminternational.com/view/aggregation-monoclonal-antibody-products-formation-and-removal
- Swanson, M, Rios, S, et al: Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates. Front. Immunol., 26 July 2022, Sec. Vaccines and Molecular Therapeutics, Volume 13 – 2022 | https://doi.org/10.3389/fimmu.2022.915412. See also https://pubmed.ncbi.nlm.nih.gov/29874996/
- Beckman Coulter Life Sciences. Performance of the Valita Aggregation Pure assay vs HPLC-SEC. Available here as a PDF. Accessed October 2024
- Morris ZS, Wooding S, Grant, J: The answer is 17 years, what is the question: understanding time lags in translational research. JR Soc Med. 2011 Dec;104(12):510–520
Filed Under: Biologics
