Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics

Abstract

Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P mutation (in IgG4 core-hinge region) intentionally designed to prevent Fab-arm exchange. A new scaffolding platform for engineering IgG4 antibodies amenable to bioprocessing and bioanalysis is proposed by introducing an "IgG1-like" single-point mutation in the hinge or CH1 region of IgG4S228P. This work offers insight into the design, discovery, and development of innovative therapeutic antibodies that are well suited for robust biomanufacturing and quality control.

Keywords: bioanalysis; bioprocessing; manufacturability; single-point mutation; therapeutic IgG4.

Figure 1.

S228P mutation in hinge region of IgG4 mAbs led to the two-peak elution (TPE) behavior in CEX. (a) Single-peak elution was observed for IgG1 and (c) wild-type IgG4 mAbs. (b) The TPE behavior was observed for all the IgG4S228P mAbs studied. The small shoulder peak to the lower right of the main peak (e.g. mAb4, mAb12, and mAb12-w) was due to minor charge variant species. The CEX-HPLC column was packed with Propac SCX-10 resin (Thermo Fisher Scientific), with a column volume of 0.8 mL and an average particle size of 10 µm. The salt gradient elution was from 20 mM MES, pH 5.0 to 20 mM MES, 1 M NaCl, pH 5.0 in 60 mins at a flow rate of 0.25 mL/min with a constant injected protein mass of 10 µg.

Figure 2.

Design of next-generation therapeutic IgG4 mAbs using protein engineering. (a) CEX-HPLC chromatogram for mAb11, mAb 11 fragments and a fusion protein containing a fully human IgG4-Fc region and the CPPC core-hinge motif. The two-peak elution (TPE) behavior was observed for mAb11 and mAb11 F(ab’)2, but not for mAb 11 Fab and the Fc-fusion protein, suggesting that the TPE behavior may be primarily attributed to F(ab’)2. (b) Conformational energies for IgG1 (mAb1) and IgG4S228P (mAb12). Two distinct conformational energy levels were observed for IgG4S228P (mAb12), while only one was observed for IgG1 (mAb1). (c) Sequence alignment of the constant heavy chain 1 (CH1) and hinge region for IgG1, IgG4S228P, and two wild-type IgG4 (mAb7-w and mAb12-w) with the CPSC motif in the core-hinge region. Twelve amino acid differences were found between IgG1 and IgG4S228P mAbs. C131 in IgG4 forms the inter-chain disulfide bond between heavy and light chains, thus cysteine was not mutated to the corresponding serine to preserve intact IgG4 structure. The other eleven amino acids in IgG4S228P were mutated individually to the corresponding amino acids in IgG1 to evaluate their CEX behavior. Additionally, K196 was further mutated to K196R and K196P to generate mAb12-4b and mAb12-4c in addition to mAb12-4a.

Figure 3.

Successful design of IgG4 mutations with improved manufacturability and bioanalytical characteristics. (a) Single-peak CEX elution behavior was observed for mAb12-4c, mAb12-7, mAb12-9, mAb12-10, and wild-type mAb12-w (mAb12 without the S228P mutation in the core-hinge region). The TPE behavior was observed for mAb12. (b) The mixture of mAb12-w/mAb7-w was incubated for 24 hrs in the absence of 0.5 mM GSH at 37°C, and subsequently analyzed by LC-MS. The wild type mAb7-w contained various glycosylation species (G0F/G0F, G0F/G1F and G1F/G1F). The mixtures of mAb12-w/mAb7-w (c), mAb12/mAb7-w (d), mAb12-4c/mAb7-w (e), mAb12-7/mAb7-w (f), mAb12-9/mAb7-w (g), mAb12-10/mAb7-w (h), and mAb12-11/mAb7-w (i) were incubated for 24 hrs in the presence of 0.5 mM GSH at 37°C, and subsequently analyzed by LC-MS. (j) ELISA binding potency of the mutants. Absorbance at 450 nm was plotted against log ng/mL of mutant concentration, with error bars obtained from duplicate measurements. The relative potency for each mutant was calculated and shown in SI Appendix Table S2.