Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Abstract

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.

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses

“…23,46 Characterization of IAB by SEC-UPLC, LC-MS, and competitive ELISA The homogeneity of IAB were verified by size-exclusion chromatography-ultra performance liquid chromatography (SEC-UPLC) and molecular weight was accurately measured by LC-MS, as previously described. 47,48 The CD47/PD-L1 binding activity of IAB was evaluated by competitive ELISA. Briefly, ELISA plates (Costar) were coated overnight at 4 Cwith hPD-L1 Fc (Shanghai Sinomab Biotech Co., ZJ-01-041) or hCD47 Fc (Shanghai Sinomab Biotech Co., ZJ-01-053) in phosphate-buffered saline (PBS) buffer.…”

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

“…The shifts to larger A/R ratios in the stressed samples are indicative of increased flexibility, while the significant increase in CSM0 observed for the 50°C, pH 3 and 5 mlux.hr samples suggests an increase in the average solvent exposure. The detection of unfolding in the pH 3 sample is not surprising, as there is evidence that low pH can cause unfolding of the Fc for IgG4s (40) but see our discussion below. Figure 5C-5F show a range of other approaches used to assess the effect of different physical perturbations on mAb1.…”

Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

“…After investigating the Fab, we then moved on to compare the Fab as a whole to the CH2 and CH3 domains of the Fc, since there are pH-dependent experimental data with which to compare for these constituents (Figure 3). For example, results in the literature suggest that the Fab has a lower melting temperature than the Fc (Welfle et al, 1999;, however the Fc is believed to unfold faster than the Fab in the presence of chemical denaturants (Rowe, 1976), and the Fc is more sensitive than the Fab to aggregation at low pH (Liu et al, 2016;. In terms of ionisable contributions to the folded state stability (Figure 3a), the CH2 domain appears to be more stable than the CH3 domain at neutral pH, but less stable at low pH.…”

“…Our understanding is that the pH instability of the CH2 domain is in part related to its high stability at neutral pH, and the importance of the CH2 domain in acid aggregation has also been observed experimentally. The role of the CH2 domain for driving IgG aggregation in an acidic environment appears to be linked to the glycosylation state of the CH2 domain, with aglycosylated mAbs demonstrating a lower T m (Hari et al, 2010;Liu et al, 2016;Latypov et al, 2012) and fragmentation of the IgG at pH 4 also appears to occur in the CH2 domain (Gaza-Bulseco and Liu, 2008). Also upon exposure to low pH, the CH2 appears to undergo a structural transformation and once renatured by neutral pH titration, leads to an increased thermal stability of 12 − 15 • C for the whole IgG (Martsev et al, 1994), however this conformation change appears to have little effect on the structure of the IgG overall (Ejima et al, 2007).…”

“…Due to the requirements of protein A chromatography and viral inactivation, many reports have investigated antibody pH sensitivity and aggregation pathways (Vázquez-Rey and Lang, 2011;Brummitt et al, 2011a,b;Arosio et al, 2013;Liu et al, 2016;Imamura and Honda, 2016;Skamris et al, 2016;Imamura et al, 2017). To better understand the cause of acid induced protein aggregation, research has focussed on understanding if aggregation is driven by specific domains within the antibody.…”