Protein Aggregate and Subvisible Particles Analysis Information

Monoclonal antibodies (mAb) and related products are widely used as therapeutic agents to treat various cancers and autoimmune diseases. The production of mAb therapeutics can be highly challenging due to various chemical degradation processes and aggregation. mAbs produced from mammalian cell culture may contain significant amounts of dimers, trimers, and other higher-order aggregates. The formation of aggregates may originate from elevated temperature, shear strain, surface adsorption, high protein concentration, or other unknown reasons.

Aggregates in mAb therapeutics can result in an incorrect drug dosage and/or undesired immune response that affects the safety of the drug. The consequence of such immune responses to the therapeutic protein product can range from no apparent effect to serious adverse events , including life-threatening complications such as anaphylaxis. Consequently, monitoring mAb aggregation is important for safety and quality assurance.

Therapeutic protein aggregates can be classified as soluble or insoluble, and the aggregation process can be reversible (noncovalent) or irreversible (covalent). This makes the analysis of protein aggregates a significant analytical challenge. No single technique is capable of providing complete data on aggregates; orthogonal methods should be used to establish the validity of the primary method. This is especially pertinent as case studies have shown that non-covalent aggregates can be dissociated in analytical buffers  making them invisible to a single method approach. The FDA Guidance  for Industry document “Immunogenicity Assessment for Therapeutic Protein Products” states that “Methods that individually or in combination enhance detection of protein aggregates should be employed to characterize distinct species of aggregates in a product”. There is a regulatory expectation to characterize subvisible particles (ICH Q5C and ICH Q6B ) before first-in-human Phase 1 testing.

For submicron aggregates, investigators routinely use size exclusion chromatography (SEC), which separates particles according to hydrodynamic size. However, the use of both standard hydrophobic interaction chromatography (HIC) and mixed-mode HIC for separation of protein aggregates has also received great interest as an orthogonal technique to SEC. HIC separates proteins based on the hydrophobicity in the native state and can often detect changes in protein structure as well as aggregates.

Size exclusion chromatography (SEC) is used for the analysis and quantitation of soluble aggregates that are noncovalent and irreversible in nature. Separation is based on the hydrodynamic size of the protein in solution. SEC is suitable for the analysis of aggregates in the size range of 1–50 nm and is considered robust and accurate when the method induces no changes to the aggregation state, and nonspecific interactions with the column packing media are eliminated. The analysis of mAbs by SEC is typically performed under non-denaturing conditions at near-physiological pH range (6.8). Using volatile buffers such as 20 mM ammonium formate, SEC columns can also be directly coupled to a high resolution mass spectrometer for native SEC-MS analysis . Separation of mAb dimer aggregates and monomer can be optimized through careful choice of the UHPLC system employed, the column and  optimal instrument setup.

The hydrophobic interaction chromatography (HIC) technique uses high salt concentrations to force hydrophobic interactions between a column and a protein. The reduction of the salt concentration during the gradient allows elution of the protein. This technique allows the separation of proteins, protein aggregates, and protein variants through hydrophobicity in the native form, and so can be used to monitor conformational changes in the protein. Examples include separation of aggregates and fragments.

For more information of the latest chromatography and detection technologies for protein aggregate analysis visit our Protein Aggregate Analysis products page.