A comparative study of the developability of full-length antibodies, fragments, and bispecific formats reveals higher stability risks for engineered constructs

Abstract

Engineered antibody formats, such as antibody fragments and bispecifics, have the potential to offer improved therapeutic efficacy compared to traditional full-length monoclonal antibodies (mAbs). However, the translation of these non-natural molecules into successful therapeutics can be hampered by developability challenges. Here, we systematically analyzed 64 different antibody constructs targeting Tumor Necrosis Factor (TNF) which cover 8 distinct molecular format families, encompassing full-length antibodies, various types of single chain variable fragments, and bispecifics. We measured 15 biophysical properties related to activity, manufacturing, and stability, scoring variants with a flag-based risk approach and a recent in silico developability profiler. Our comparative assessment revealed that overall developability is higher for the natural full-length antibody format. Bispecific antibodies, antibodies with scFv fragments at the C-terminus of the light chain, and single-chain Fv antibody fragments (scFvs) have intermediate developability properties, while more complicated formats, such as scFv- scFv, bispecific mAbs with one Fab exchanged with a scFv, and diabody formats are collectively more challenging. In particular, our study highlights the propensity for fragmentation and aggregation, both in bulk and at interfaces, for many current engineered formats.

Keywords: Antibody; Biophysics; Colloidal stability; Formulation; Fragmentation; Self-association; Shelf-life stability; aggregation; antibody formats; bispecifics; developability; fragments; scFv.

Figure 1.

Schematic representation of the 73 antibody constructs used in this study. Our antibody library is composed of full-IgGs, fragments, bispecifics, and bispecific-fragments. The HzATNP variants (Var1-Var9) have been previously described and they have been used as the control antibody set in this study. The subset of bispecifics (IgG/biAb) includes three IgGs (Var21, Var22 and ar23) which serve as controls and are technically not bispecific antibodies because they target a single epitope. Except for the HzATNPs which binds the ATNP, the rest of the variants target TNF-α.

Figure 2.

Biophysical properties of antibodies. The figure shows the critical properties assessed at the very early-stage of discovery. The lines represent the average values of the molecules within a set. The dashed line represents the average of all molecules. Green, yellow and red background indicate good, intermediate and bad behavior, respectively, based on the cutoff listed in Table 1. No major differences were observed in terms of activity. Bispecifics are characterized by low purity.

Figure 3.

Physical stability properties. Thermal stability expressed in terms of melting temperature (T_m) and temperature of the onset of aggregation (T_agg), and colloidal stability represented by the self-interaction parameter (k_d) for the different molecules. We refer to the first melting temperature of all antibody variants since undesirable product changes start occurring from the first thermal unfolding. Interfacial stability was measured as monomer loss during a classical agitation assay and in a nanoparticle assay that accounts for different types of surface chemical properties. The lines represent the average values of the molecules within a set. The dashed line represents the average of all molecules. Green, yellow and red background indicate good, intermediate and bad behavior, respectively, based on the cutoff listed in Table 1. Overall, fragments underperform in the interfacial stress assays.

Figure 4.

Long-term stability data. Percentage of aggregates (HMWP), fragments (LMWP) and intact monomer (Mon) measured after one year incubation at 30°C and 40°C. The lines represent the average values of the molecules within a set. The dashed line represents the average of all molecules. Green, yellow and red background indicate good, intermediate and bad behavior, respectively, based on the cutoff listed in Table 1. The diAb variants were not characterized for all assays due to their very low yield. Fragments are particularly problematic.

Figure 5.

Distribution of flagged variants within the different antibody formats. Green, yellow and red frames indicate the overall developability risk for the respective variants set.

Figure 6.

Risk-assessment of the developability based on biophysical properties. The assays that take more leverage on the overall developability risk are the long-term stability studies as well as the colloidal and interfacial stability. Bispecific fragments and fragments show the least favorable developability profiles.