Protein Aggregate and Subvisible Particles Analysis Information

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Protein aggregation is a biological phenomenon in which proteins accumulate and clump together. Protein aggregation is one of the key challenges in the development of protein biotherapeutics. It is a critical product quality issue as well as a potential safety concern due to the increased immunogenicity potential of protein aggregates. Protein aggregates are commonly measured using liquid chromatography with either UV detection (HPLC-UV) or fluorescence detection (FLD), which utilizes the native fluorescence of some therapeutic proteins (e.g., immunoglobulin) primarily resulting from the presence of tryptophan, tyrosine, and phenylalanine.


Dangers of aggregation of monoclonal antibodies

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.


Analysis of protein aggregates

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.

Method Approximate size range Measuring principle Information obtained
Hydrophobic interaction chromatography (HIC) 5–50 nm Separation based on weakly hydrophobic chromatography particle ligand interaction at varying salt concentration Hydrophobicity and charge variation
Size exclusion Chromatography (SEC) 5–50 nm Separation through porous matrix based on size Hydrodynamic diameter
Analytical ultra-centrifugation 1–100 nm Sedimentation rate in response to centrifugal force Molecular weight and conformation
Field flow fractionation 1–1000 nm Separation by flow retention based on diffusion coefficient Hydrodynamic diameter
Dynamic light scattering 0.5 nm–10 µm Fluctuations of scattered light signals Hydrodynamic diameter
Mass spectrometry Atomic resolution - Da Detection of mass/charge ratio of ionized molecules Mass/charge ratio
SDS-PAGE KDa–MDa Separation of denatured protein in a gel in an electric field according to size Molecular weight
Native-PAGE KDa–MDa Separation of native protein in a gel in an electric field according to size/charge Charge and hydrodynamic size
Optical microscopy 1 µm–mm Visualization of protein particles Size and morphology
Electron microscopy nm–mm Visualization of protein particles and detection of chemical composition at high resolution Size and morphology
Fluorescence spectroscopy N/A Protein fluorescence is induced and detected Conformational changes & folding state
Table adapted from Ratanji et al.

SEC for protein aggregate analysis

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.

Size-exclusion chromatography of a monoclonal antibody sample


HIC for protein aggregate analysis

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.

Hydrophobic interaction chromatography of a monoclonal antibody sample


Protein aggregate analysis technologies

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


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