Conformational changes of antibodies upon adsorption onto hydrophobic interaction chromatography surfaces

Abstract

Differential scanning calorimetry was established for in-situ measurement of the transition temperatures of antibodies when adsorbed on hydrophobic interaction chromatography media. This method is also suitable for non-transparent media, which is an advantage over spectroscopic methods that cannot be used in many cases due to large background signals. The three transition temperatures of an antibody were lowered when the molecule was adsorbed onto Phenyl and Butyl functionalized Toyopearl particles as well as on Phenyl Sepharose 6 Fast Flow when bound at moderate to high salt concentration compared to the values in free solution. The first two melting points, representing the CH2 domain and the Fab fragment, are more affected than the highest melting point, which corresponds to the CH3 domain. It is obvious that domains which are less stable are more likely to undergo conformational change upon adsorption. It could be shown that the conformational changes occurring in antibodies upon adsorption to HIC media are directly proportional to the hydrophobicity of the stationary phase and that they are reversible. Upon elution, the protein returns to its original conformation. For all four tested resins, a negative value for both ΔH as well as ΔS was calculated, leading to opposing contributions to ΔG.

QSAR Implementation for HIC Retention Time Prediction of mAbs Using Fab Structure: A Comparison between Structural Representations

“…The free energy of conformational entropy, G c (F), which describes the stability of the protein with regards to the hydrophobic interactions in the protein core was selected in the CDRs, framework regions, and the constant domains. This is supported by published literature where protein stability has been reported to play a pivotal role in HIC binding [50]. Other important energy descriptors of note were the number of estimated water molecules surrounding the surface, W(F), and the entropy energy from the first shell of water, ∆G w , representing the energy contribution from interactions between polar residues and surrounding water molecules.…”

Antibody Binding Heterogeneity of Protein A Resins

“…Therefore, the heterogeneous binding nature of the resins has to be a key element for the greater molar enthalpy values at low concentration. Conformational changes of the antibody upon adsorption are also probable at low surface coverage, as founded by Bayer and Jungbauer, [35] but they depend on the hydrophobicity of the ligands. TP PA has a greater enthalpy change possibly because of the greater number of binding possibilities when compared to MSS.…”

Role of harvest depth filtration in controlling product-related impurities for a bispecific antibody

“…Furthermore, it has been reported that antibodies can go through conformational changes upon their adsorption on colloidal particles or hydrophobic surfaces, depending on protein structure and surface characteristics [ 24 , 25 ]. In particular, antibodies can experience reversible conformational changes upon adsorption on HIC resins with likely more significant changes for less stable domains (e.g., Fab and CH2) [ 24 ].…”