Biopharmaceuticals and biologics, such as monoclonal antibodies (mAbs), fragment antibodies, interleukins, and other recombinant proteins are valuable therapeutics, and thousands of further biologics are in clinical development. Due to their size, complex structure, and potential conjugation with hydrophobic ligands, many products tend to form larger aggregates which can result in patient safety issues upon parenteral administration.
Orthogonal analytical methods, such as Analytical Ultracentrifugation (AUC) are a valuable tool to complement routine assays (electrophoresis, spectroscopy, or chromatography), e.g. for root cause investigations or extended product characterization. Protein aggregation can be analyzed without disturbing the product, since AUC is a neutral observer, testing the product in the original matrix, without e.g. dilution of high-concentration formulations that could put low aggregate results in doubt.
Nanolytics offers AUC as the recognized gold standard for aggregate quantification. Combinations of different detectors allow for discrimination of proteinaceous and non-proteinaceous material, ensuring unambiguous identification of any components, intended or not intended. Challenges for aggregate quantification frequently arise from inhomogeneous solvent effects, typically occurring in complex formulation buffers due to redistribution of sugar osmolytes and other excipients, which are addressed with specific data algorithms. In general, the self-association behaviour is amenable across a wide concentration range from a few µg/mL up to approximately 150 mg/mL. Extreme concentrations are tackled by either exploiting peptide bond absorbance (230 nm) or employing advanced interference detection, respectively.
CASE STUDY: The oligomer distribution of a protein analysed with SV using absorbance detection at 280 nm.
The sedimentation coefficient distribution (SCD) features monomeric protein as the predominant population as well as trimers and larger oligomers.