Engineering an improved IgG4 molecule with reduced disulfide bond heterogeneity and increased Fab domain thermal stability

Abstract

The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (C(H)1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227-230) at the C terminus of C(H)1. An inter-LC-C(H)1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-C(H)1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227-230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.

FIGURE 1.

Schematic representation of the arrangement and number of DSBs of the human IgG isotypes. The dotted and solid lines indicate inter- and intra-DSBs, respectively.

FIGURE 2.

Expression analysis of IgG1 WT, IgG4 WT, and IgG4 mutants as determined by using protein A biosensors in an OCTET detection system (n = 3). Error bars, S.D.

FIGURE 3.

SEC HPLC and immunoblot of IgG4 molecules with altered DSB arrangements toward an IgG1-like DSB, including mutants with a 3-residue spacer within the hinge region. All mutants include C127S in addition to the mutation noted. A, SEC HPLC trace comparison of IgG4 WT and M1 (C127S/G230C). B, comparison of IgG4 WT with mutants that had a Cys introduced at different positions in the C_H1. Samples were run under non-reducing conditions and probed with an anti-human Fc antibody. C, comparison of the same samples, also run under non-reducing conditions but probed with an anti-human κ antibody. For both A and B, thick black lines indicate HC, thick gray lines indicate LC, and red lines indicate DSBs.

FIGURE 4.

Comparison of the thermal stability of the Fab domains of selected mutants determined by the thermofluor assay (n = 3). Error bars, S.D.

FIGURE 5.

Investigating the effect of introducing the S241P mutation within the core hinge of IgG4 WT of selected mutants. A, immunoblot analysis under non-reducing conditions with (+) or without (−) the S241P mutation. The membrane was probed with an anti-human FC antibody. B, the membrane was probed with an anti-human κ antibody. For both A and B, thick black lines indicate HC, thick gray lines indicate LC, and red lines indicate DSBs.

FIGURE 6.

Thermal stability of the Fab domains of IgG4 and mutant IgG4 with (+) or without (−) the S241P mutation as determined by the thermofluor assay (n = 3). Error bars, S.D.