Kazuo Nagase

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

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°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°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. Proteins 2007.

In-vivo processing of the initiator methionine from recombinant methionyl human interleukin-6 synthesized in Escherichia coli overproducing aminopeptidase-P.

SummaryHuman interleukin 6 (hIL-6) overproduced in Escherichia coli HB101 was found to partially retain the initiator methionine (Met) residue (Met-hIL-6). In order to remove the residual N-terminal Met in vivo, an attempt was made to express hIL-6 in aminopeptidase-P (Ap-P)-hyperproducing strains, since the N-terminus Met-Pro- structure of nascent recombinant hIL-6 has been shown to be a favoured substrate of the enzyme in vitro. Using a mutant with duplicated Ap-P genes (pepP) on a chromosome or some recombinant strains overproducing Ap-P, we have succeeded in removing the initiator Met form Met-hIL-6 in vivo. The content of the mature product without the initiator Met in the pepP recombinant strains could be increased to approximately 99% from 85%.