Protein aggregation is one of the major challenges in developing and commercializing protein-based drugs. Aggregation can be observed at all stages of protein product development and production processes, such as protein expression, purification, and lyophilization.
Risk of Protein Aggregation
Biopharmaceuticals are widely used as therapeutic agents for the treatment of various cancers and autoimmune diseases. The production of biopharmaceuticals can be very challenging due to the presence of various chemical degradation and protein aggregation.
Protein aggregation may have a significant negative impact on the quality and efficacy of the product, which often diminishes protein bioactivity. Most importantly, they have cytotoxic effects or cause immunogenic responses in the body. For example, the consequences of an immune response to a therapeutic protein product can go from non-apparent effects to serious adverse events, including life-threatening complications such as allergic reactions. Therefore, prevention and mitigation of protein aggregation are essential to maintain the safety, efficacy, and quality of biopharmaceuticals.
Protein Aggregation Mechanism
Protein aggregation is usually achieved through a series of processes. Changes in the internal structure of the protein lead to the formation of dimers or oligomers, and subsequently, the aggregates grow to form sub-visible or visible particles.
Factors Affecting Protein Aggregation
- Protein structure
- Chemical degradation
- Temperature
- pH
- Pharmaceutical processing steps: purification, churning, freeze-thawing, filling, lyophilization, formulation composition, and shipping pressure may convert natural proteins into aggregates.
- Potential contaminants
Multiple non-native aggregation pathways for a multi-domain protein [1]
Our Protein Aggregation Analysis Services
Creative Proteomics can provide both SEC-HPLC and SV-AUC methods for an orthogonal assessment of the aggregation profile of your biopharmaceutical. Our experienced scientists will discuss your specific requirements and select the most suitable approach for your product development.
Analysis of Protein Aggregates by SEC Methods
SEC is suitable for the analysis of aggregates in the range of 1-50 nm. This specific method does not cause a change in the aggregation state and the non-specific interaction with the column packing, which is considered durable and accurate.
The smallest soluble aggregates are dimers, and the upper limit of soluble aggregates size varies depending on the protein and solution conditions. These soluble protein aggregates, either by physical interaction or chemical modification, can usually be detected by SEC-HPLC.
Sedimentation equilibrium analytical ultracentrifugation (SE-AUC) assesses interaction and dissociation between sample components.
Size Exclusion Chromatography with Multi-Angle Laser Light Scattering (SEC-MALS), the combination of UV Refractive Index and MALS enable a qualitative and quantitative assessment of aggregates (usually up to at least trimer) present.
Using Sedimentation Velocity Analysis Ultracentrifugation (SV-AUC)
SV-AUC is used to obtain sedimentation coefficient values and the relative percentages of monomer/ multimer components and aggregates observed, which is widely used in the biopharmaceutical industry, especially for evaluating soluble antibodies with a diameter of less than 100 nm (i.e., nanoparticles).
Reference
- Christopher J. Roberts, Therapeutic Protein Aggregation: Mechanisms, Design, and Control. Trends Biotechnol. 2014; 32(7): 372–380.
