As one of the world’s premier provider of services related to preclinical development of monoclonal antibodies (mAbs), Creative Biolabs offers comprehensive antibody characterization studies to help get an in-depth understanding of antibody characteristics and the structure-function relationship using orthogonal approaches. Our laboratory is equipped with a wide range of analytical tools and techniques. Here, we give an introduction of hydrophobic interaction chromatography (HIC) method for antibody characterization and purification.
Hydrophobic interaction chromatography (HIC) is an analytical technique that utilizes the hydrophobic properties of molecules to achieve their separation. In this type of chromatography, sample molecules with hydrophobic portions interact with and bind to the hydrophobic groups (e.g., phenyl and propyl groups) attached to the column stationary phase. The main advantage of HIC technique is its ability to perform separations under nondenaturing conditions (i.e. physiological pH conditions, ambient mobile phase temperature, and no need for organic solvents).
Fig.1 Schematic view of protein retention mechanism in HIC. (Mccue, 2009)
Antibody aggregates are commonly encountered challenges during the development, manufacturing, transport, and storage processes. The aggregation is not desired because it can dramatically influence the bioactivity of mAbs and have the potential to cause side effects and increased immunogenicity. Consequently, the detection, characterization, and control of aggregation are important for their production and quality assurance.
To ensure mAbs’ safety and efficacy, different analytical techniques have been applied to analyze and remove aggregates. Size exclusion chromatography (SEC) has been the method of choice for the detection and quantification of mAb aggregates. Besides, HIC can be used as a secondary assay to confirm the aggregation of for removal of aggregates during mAb purification. Moreover, SEC is often coupled with other methods, such as sedimentation velocity analytical ultracentrifugation (SV-AUC) and dynamic light scattering (DLS), to make sure that aggregation is well-characterized.
HIC is commonly used as a polishing step in mAb purification processes. It can be used to remove mAb product-related impurities and process contaminants, such as host cell proteins (HCPs), leached protein A, and endogenous viruses, in both analytical and preparatory scale applications. Moreover, Various types of PTMs of mAbs and ADCs can be monitored in HIC, including degradations, fragmentation, misfolding, oxidation, carboxy-terminal heterogeneity, aspartic acid isomerization, unpaired cysteines. For instance, Valliere-Douglass et al. (2008) reported the separation of mAb species modified by isomerization. Boyd et al. (2011) used HIC as a chromatographic method to monitor the oxidation of tryptophan residues located in recombinant mAb.
Fig.2 HIC separation of modified mAb species (Asp→iso-Asp) developed during storage at 40 °C for 12 weeks. (Valliere-Douglass, 2008)
Creative Biolabs’ services are fully adjustable and custom-specific to meet clients’ requests. By combining a range of analytical tools and functional assays, we can offer integrated services related to preclinical development of mAbs. If you are interested in our service, please contact us to discuss your requirements.
References
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