Protein aggregation analysis and characterization to assess the extent of protein degradation, stability and formation of aggregates in solution

Protein aggregation characterisation is key to a successful biopharmaceutical development and manufacturing. Protein aggregates can form at any stage of the development or manufacturing process including bioprocessing, purification, formulation and packaging and also during storage. 

Protein aggregates can be considered an impurity, or a molecular variant, resulting from changes that take place over time and/or by the action of physical factors such as light, temperature, pH, water, shear-forces, or by reaction with an excipient in the formulation, concentration of buffers or interaction with the container/closure system (e.g. elastomer seals or glass delamination). Aggregates include reversible non-covalent and irreversible covalent bonded species, dimers, oligomers and higher multiples, of the desired protein product and can be present as small soluble particles ranging in size from a few nm to large sub-visible/visible particles up to microns.

Aggregation of a protein therapeutic can have serious implications for the patient safety, biologic product stability, potency, biological activity, quality, and efficacy. As aggregation has been reported to lead to adverse immune reactions in patients, it is important to mitigate health risks during drug development through a comprehensive understanding of your biomolecule’s propensity to aggregate and characterisation of the aggregation state.

Our experts apply a range of protein aggregation analysis techniques to detect and quantify aggregates in solution, supporting formulation development, quality control, stability studies, comparability, release testing and aggregation studies. We can conduct aggregation analysis in accordance with the requirements of the ICH Q6B guideline, offering studies to either Good Laboratory Practice (GLP) or Good Manufacturing Practice (GMP) using techniques such as differential scanning calorimetry (DSC), analytical ultracentrifugation (AUC), and light scattering techniques (DLS, SEC-MALS) which allow the study of non-covalent structural aspects of a protein and also study of aggregates or oligomer formation in solution.

Analytical ultracentrifugation (AUC) allows assessment of homogeneity of proteins/peptide solutions and to qualitatively assess the molecular weight and presence of aggregates over a broad range of molecular weights ranging from a few kDa to MDaltons. AUC therefore, allows a unique analysis of multiple species in the same sample while most of the other methods used for aggregation analysis are limited by either the size range or the resolution between different oligomers. 

For submicron aggregates, size-exclusion chromatography (SEC) is routinely used to detect and quantify irreversible aggregates from oligomers through to ultra-high order aggregates. This technique is used to establish the molecular weight of observed aggregates and is typically coupled with multi-angle static light scattering (SEC–MALS).
Dynamic light scattering (DLS) allows the generation of data (R&D, GLP or cGMP) allowing the measurement of particulate size distribution of proteins in solution, particle size analysis (0.6nm to 6 microns) and zeta potential measurement / electrostatic aggregation. DLS can also be applied to measure and understand nanoparticles in healthcare products including nanoliposome encapsulation. 

Protein Aggregation Analytical Techniques:

  • Analytical ultracentrifugation (AUC)
  • Size exclusion chromatography (SEC, SEC-MALS)
  • Dynamic light scattering (DLS)
  • Light obscuration 
  • Zeta potential
  • Capillary electrophoresis CE (CZE, MECC, Gel CE, cIEF/iCIEF)
  • Gel electrophoresis (Native, SDS-PAGE, IEF)
  • Electron microscopy (TEM and SEM)
  • Circular dichroism (CD)

Through our unique level of expertise and experience we can expedite your protein aggregate characterisation needs to meet your formulation development, quality control, submission, comparability, stability or batch release testing requirements. Bringing quality and safety to life, we offer Total Quality Assurance expertise to help you to meet and exceed quality, safety and regulatory standards concerning aggregation. As a key part of our leading biophysical characterisation portfolio for biologics we deliver efficient and detailed information to drive insight for your biopharmaceutical development programs.

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Intertek Pharmaceutical Services Manchester

Intertek Pharmaceutical Services Laboratory in Manchester, UK, provides cGMP and GLP/GCP compliant biopharma characterization, stability, release testing and bioanalysis supporting drug development, regulatory submission and production.

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