The Thermal Stability of Immunoglobulin: Unfolding and Aggregation of a Multi-Domain Protein

Abstract

The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the F(ab) and F(c) fragments of the IgG molecule. The F(ab) fragment is most sensitive to heat treatment, whereas the F(c) fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate-determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

“…Above that temperature, the characteristic features of the spectra disappear due to the loss of tertiary structure. 33 antibody is remarkably stable in the incubation temperature interval (52-65 C) applied in this study.…”

“…32 This is a common property of large, complex proteins such as mAbs and has been described earlier. 30,33 However, the use of the denaturation enthalpy is still permissible according to the first law of thermodynamics, even if the process is irreversible. 34 In addition, no distortion, which is sometimes observed when the samples aggregate irreversibly, was seen on the shape of the heat capacity profiles for the Rituximab samples.…”

“…This behavior suggests an amplification on the level of secondary structure before unfolding and has been observed for antibodies previously. 33,36 Above 70 C, the spectra decreased in intensity as the secondary structure was gradually lost (spectra omitted for clarity). Figure 2(b) shows near-UV spectra in the wavelength range from 250 to 350 nm recorded while ramping the sample temperature.…”

Dietary supplementation with ovine serum immunoglobulin attenuates acute effects on growth, organ weights, gut morphology and intestinal mucin production in the growing rat challenged with Salmonella enteritidis

“…A portion of the FDOI was inactivated by autoclaving (1218C, 15 min). The inactivation of Ig was confirmed using circular dichroism (Chirascan, Applied Photophysics, Surrey, UK) and based on the method of Vermeer and Norde (2000).…”

Multi‐criteria manufacturability indices for ranking high‐concentration monoclonal antibody formulations

“…When pH is 5.0, low T h (<50 °C) is expected irrespective of the other factors which means the proteins are unstable. Effects of pH on mAb stability have been reported in previous work (Vermeer and Norde, 2000) and it could be explained by the low temperatures of unfolding that occur at low pHs (Vermeer et al, 2000). …”