“…Moreover, we found the IgG 4 Fc domain was responsible for nivolumab's tendency to aggregate in acidic conditions. These findings are generally consistent with previous reports that IgG 4 is prone to aggregation in acidic condition, 27,28 and IgG 4 aggregation is dependent on the Fc region, which has some hydrophobic aggregation motifs. 29 To provide context for the results of our experiments with nivolumab, we examined the pH-dependent stability of 3 other IgG subclass antibodies and their Fcs.…”
Nivolumab, an anti-programmed death (PD)1 IgG 4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG 1 ), panitumumab (IgG 2 ) and atezolizumab (aglyco-IgG 1 ) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG 1 < aglyco-IgG 1 < IgG 2 < IgG 4 . This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG 4 backbone.
“…Moreover, we found the IgG 4 Fc domain was responsible for nivolumab's tendency to aggregate in acidic conditions. These findings are generally consistent with previous reports that IgG 4 is prone to aggregation in acidic condition, 27,28 and IgG 4 aggregation is dependent on the Fc region, which has some hydrophobic aggregation motifs. 29 To provide context for the results of our experiments with nivolumab, we examined the pH-dependent stability of 3 other IgG subclass antibodies and their Fcs.…”
Nivolumab, an anti-programmed death (PD)1 IgG 4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG 1 ), panitumumab (IgG 2 ) and atezolizumab (aglyco-IgG 1 ) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG 1 < aglyco-IgG 1 < IgG 2 < IgG 4 . This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG 4 backbone.
“…The higher deamidation rates for Asn325 in IgG1 mAbs were consistent with results from a recent published study, which demonstrated that the overall deamidation rates for IgG1 mAbs were faster than IgG4 mAbs with identical variable regions. 22 10.1080/19420862.2018.1478646-F0005Figure 5.Comparison of Asn deamidation between IgG1 and IgG4 mAbs. A) Deamidation rate constants of Asn325 (left) and Asn384/389 (right) from five IgG4 and three IgG1 mAbs under heat stress and acidic formulation buffer storage conditions.…”
Section: Resultsmentioning
confidence: 99%
“…The starting structure for mAb2 was taken from Protein Data Bank (PDB) entry 4G3Y, 22 with all solvent molecules removed. Based on the deposited electron density maps, a refined model was built by flipping the original Asn57 backbone conformation.…”
Identification of asparagine (Asn) sites that are prone to deamidation is critical for the development of therapeutic monoclonal antibodies (mAbs). Despite a common chemical degradation pathway, the rates of Asn deamidation can vary dramatically among different sites, and prediction of the sensitive deamidation sites is still challenging. In this study, characterization of Asn deamidation for five IgG1 and five IgG4 mAbs under both normal and stressed conditions revealed dramatic differences in the Asn deamidation rates. A comprehensive analysis of the deamidation sites indicated that the deamidation rate differences could be explained by differences in the local structure conformation, structure flexibility and solvent accessibility. A decision tree was developed to predict the deamidation propensity for all Asn sites in IgG mAbs based on the analysis of these three structural parameters. This decision tree will allow potential Asn deamidation hot spots to be identified early in development.
“…8). One potential drawback of the IgG4 form is that it frequently exchanges IgG half-molecules, resulting in the generation of bispecific IgG4 molecules in plasma (Neergaard et al, 2014). Therefore, we created a mutant human chimeric IgG4 construct by substituting serine at position 228 with proline; this point mutation generates inter-H-chain binding in the hinge region, resulting in the formation of stable divalent IgG4 (Aalberse and Schuurman, 2002).…”
Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed four clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest antiviral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcg receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibodydependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcg receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that the IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy.
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