Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Abstract

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.

Optimizing pH Response of Affinity between Protein G and IgG Fc

“…However, the T m of GB0920 (N22H in GB0919) decreased by almost 5 K as compared with that of GB01. In the crystal structure of the protein G B1 domain, Asn 22 closely contacts with neighboring Thr 25 and supports the local conformation. Avoiding the loss of this local contact, GB0919 could maintain the thermal stability.…”

“…Protein G has an acidic pH optimum for binding relative to another bacterial Fc receptor, Staphylococcus aureus protein A. The harsh elution conditions are likely to induce acidic conformational changes in antibodies (25,26) during the purification procedure, causing aggregation that is problematic for pharmaceutical applications. The usefulness of the histidine-mediated electrostatic repulsion for antibody purification was examined by constructing affinity chromatography columns.…”

Considerations in producing preferentially reduced half-antibody fragments

“…as it results in only limited changes in the conformation and stability of the antibody molecules (Ejima et al 2007, Latypov et al 2012, and still falls into the pH interval that strongly suppresses the reoxidation. In the case of sheep anti-digoxin IgG reduction (Figure 3 A), no increase in the intensity of the HL band was observed with increased 2-MEA concentrations at pH 7.4.…”

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

“…36,37 Antibodies may be exposed to pH as low as 3.5 for a few hours at ambient temperature, which can affect stability of the protein structure and induce aggregation. 36,38 In particular, the C H 2 domains of Fc are likely to be sufficiently destabilized or unfolded to initiate the aggregation process.…”