Protein G, Protein A and Protein A-Derived Peptides Inhibit the Agitation Induced Aggregation of IgG

Abstract

Controlling and preventing aggregation is critical to the development of safe and effective antibody drug products. The studies presented here test the hypothesis that Protein A and Protein G inhibit the agitation-induced aggregation of IgG. The hypothesis is motivated by the enhanced conformational stability of proteins upon ligand binding and the specific binding affinity of Protein A and Protein G to the Fc region of IgG. The aggregation of mixed human IgG from pooled human plasma was induced by agitation alone or in the presence of: (i) Protein A, (ii) Protein G or (iii) a library of 24 peptides derived from the IgG-binding domain of Protein A. Aggregation was assessed by UV spectroscopy, SDS-PAGE, high performance size-exclusion chromatography (HP-SEC), dynamic light scattering (DLS) and fluorescence spectroscopy. Additional information on IgG-ligand interactions was obtained using differential scanning fluorimetry (DSF) and competitive binding studies. The results demonstrate that Protein A provides near-complete inhibition of agitation-induced aggregation, while Protein G and two peptides from the peptide library show partial inhibition. The findings indicate that the IgG Protein A binding site is involved in the agitation-induced aggregation of IgG, and suggest a dominant role of colloidal interactions.

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

“…Consistent with these results, two studies from different labs have reported similar regions within the C H 2 domain to be least stable83 and more prone to aggregation 83,84. For example, the binding of protein A to residues in the C H 2 and C H 3 domain of an intact IgG resulted in inhibition of aggregation 84. In a separate study, this region (HC 243–247) of the C H 2 domain in both oxidized and deglycosylated IgG1s was found to be associated with increased deuterium uptake 83.…”

“…On the basis of the results from this work, combined with previous literature results with IgGs as described above, key regions within the C H 2 domain may dictate the overall physical stability of the mAb‐B. The physical stability of mAb‐B itself may therefore be further improved by either mutation,86 truncation,89 or masking the hydrophobic segments84 of these key regions of the C H 2 domain. There are two phenylalanine residues in this region (Phe241 and Phe243) that pack against the glycans as observed in the crystal structure 42.…”

Structure and Aggregation Mechanisms in Amyloids

“…[175][176][177][178][179][180][181] Mechanical stress (agitation, stirring, pumping, or shaking) has been associated with cavitation which generates air bubbles and, consequently, the formation of an air-water interface which facilitates protein denaturation and aggregation. [176,[182][183][184][185][186][187][188][189] The use of beads during agitation accelerates the aggregation process by enhancing cavitation. [190] Solid-liquid interface Solid-liquid interfaces may facilitate monomer encounters and initial monomer to monomer association and later further aggregation.…”

Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

“…Conformational flexibility as well as electrostatic and hydrophobic properties of proteins have been described to be of major importance for protein aggregation propensity Sousa, 1995;Valerio et al, 2005). However, different studies disagree about electrostatics (Yadav et al, 2011(Yadav et al, , 2012 or hydrophobicity (Chennamsetty et al, 2009;Kumar et al, 2011;Zhang and Topp, 2012) being the main determinant. According to Valerio et al (2005), a combination of conformational flexibility and of electrostatic and hydrophobic protein surface properties is supposed to be best suited for a predictive approach for protein aggregation propensity.…”