Creative Biolabs can offer novel in vitro analysis to assess the protein aggregation level by employing our CreDA™ platform. This technology contributes to helping our customers to select candidates with less aggregation propensity and minimize the risk of drug development.
Protein aggregation is a complicated process, which may occur at different stages of the production cycle, such as expression, purification, formulation, storage, and even administration. Aggregates can be of a diverse nature and are affected by different factors, such as pH, temperature, mechanical stress, and the intrinsic instability of the protein fold. In the simplest form, aggregation may originate from the presence of hydrophobic or charged patches on the protein surface, while more complex forms of aggregation may involve partial or complete unfolding of the protein. Aggregation is one of the major concerns in the development of biotherapeutics, as the resulted high molecular mass species (HMMS) can lead to lower efficacy and exert high immunogenicity risk in vivo. Thus, assessing the aggregation level of drug candidates is a regulatory requirement, which is also an important component of our developability assessment service.
Fig.1 Schematic diagram illustrating multiple aggregation pathways for a monoclonal antibody.
Size-exclusion chromatography (SEC)
SEC is a standard separation technique widely employed for the detailed characterization of protein dimers, trimers, and oligomers. The main advantage of this approach is the mild mobile phase conditions that permit the characterization of proteins with minimal impact on the conformational structure. This method, however, is known to be prone to misleading results. For example, part of the aggregates may be retained by the column or other components of the instrumentation, leading to underestimation of the real degree of aggregation in the sample. For this reason, the use of orthogonal techniques to validate the SEC results is strongly recommended by our experts.
Sedimentation velocity analytical ultracentrifugation (SV-AUC)
SV-AUC measures the sedimentation rate at which molecules move in response to centrifugal force generated in a centrifuge. Apart from the instrument settings, the sedimentation velocity mainly depends on the molecule properties, such as mass, density, and shape. Accordingly, it allows a reliable determination of protein aggregate content and the molecular weight. An important strength of SV-AUC is its ability to detect aggregates without any change in solution condition as long as the formulation components do not interfere with the detection. For this reason, SV-AUC is the ultimate standard for determining soluble aggregate content in biopharmaceutical solutions. Often, SV-AUC acts as an orthogonal method to SEC to ensure that this routine method is not altering the true aggregate population prior to detection.
Dynamic light scattering (DLS)
DLS is a widely used technology in the pharmaceutical industry for size distribution characterization and aggregate detection of protein drugs. It determines protein particle size distribution by measuring the fluctuations of light scattered from particles in solution over time scales from ~0.1 μs to ~0.1 s. Larger particles move slower than smaller particles and thus result in distinctive fluctuations of scattered light. A great strength of DLS is that when used in batch mode, it is matrix-free and hence is not subject to erroneous conclusions regarding aggregation due to nonspecific adsorption of the aggregates. Besides, DLS often requires no dilution, thus could prevent potential dissociation of reversible aggregates.
Fig.2 Different analysis technics to assess the protein aggregation level.
Aggregation propensity is a critical quality attribute that must be carefully checked during the drug developability assessment campaign. Creative Biolabs is equipped with all kinds of advanced analytical instrument, such as SEC, SV-AUC, and DLS, which could help evaluate the aggregation level of your drug candidates by a variety of means. If you are interested in this service, please contact us for more information and a detailed quote.