The origins of nonideality exhibited by monoclonal antibodies and Fab fragments in human serum

Abstract

The development of therapeutic antibodies remains challenging, time-consuming, and expensive. A key contributing factor is a lack of understanding of how proteins are affected by complex biological environments such as serum and plasma. Nonideality due to attractive or repulsive interactions with cosolutes can alter the stability, aggregation propensity, and binding interactions of proteins in solution. Fluorescence correlation spectroscopy (FCS) can be used to measure apparent second virial coefficient (B_2,app ) values for therapeutic and model monoclonal antibodies (mAbs) that capture the nature and strength of interactions with cosolutes directly in undiluted serum and similar complex biological media. Here, we use FCS-derived B_2,app measurements to identify the components of human serum responsible for nonideal interactions with mAbs and Fab fragments. Most mAbs exhibit neutral or slightly attractive interactions with intact serum. Generally, mAbs display repulsive interactions with albumin and mildly attractive interactions with IgGs in the context of whole serum. Crucially, however, these attractive interactions are much stronger with pooled IgGs isolated from other serum components, indicating that the effects of serum nonideality can only be understood by studying the intact medium (rather than isolated components). Moreover, Fab fragments universally exhibited more attractive interactions than their parental mAbs, potentially rendering them more susceptible to nonideality-driven perturbations. FCS-based B_2,app measurements have the potential to advance our understanding of how physiological environments impact protein-based therapeutics in general. Furthermore, incorporating such assays into preclinical biologics development may help de-risk molecules and make for a faster and more efficient development process.

Keywords: fluorescence correlation spectroscopy; macromolecular crowding; nonideality; second virial coefficient; therapeutic proteins.

FIGURE 1

Model of k _diff (or 2B ₂ M) determination: fluorescently labeled antibodies are diluted into varying concentrations of carrier solution (serum, depleted serum, isolated serum IgG antibodies, or HSA). The diffusion coefficient at each condition is determined via FCS diffusion time measurements, and k _diff or B ₂ values are determined by fitting plots of viscosity‐adjusted diffusion coefficients against carrier concentration to Equation 1. Positive values indicate net repulsion in serum or repulsive interactions with isolated serum proteins, whereas negative values indicate net attraction in serum or attractive interactions with isolated serum proteins. A zero value likely indicates a balance between attraction and repulsion in serum as opposed to no interaction when measuring interactions with isolated proteins such as HSA.

FIGURE 2

Comparison of cross‐term interactions in human serum (black circles), IgG‐depleted serum (blue squares), albumin (purple triangles), and IgG antibodies isolated from human serum (green diamonds) for NIST mAb (a), Tocilizumab (b), anti‐gp120 mAb (c), and anti‐RSV mAb (d). D _adj versus c plots (Figures S1–S4) were fit to Equation 1 to obtain k _diff values, with results presented as mean ± S.D. from three replicates with independent sample preparations.

FIGURE 3

Cross‐term interaction for IgG1 Fc fragment (a) with serum (black circle), IgG‐depleted serum (blue square), and HSA (purple triangle). Comparison of cross‐term interactions in human serum for glycosylated (closed symbols) and deglycosylated (open symbols) NIST mAb, tocilizumab, anti‐gp120 mAb, and anti‐RSV mAb (b). D versus c plots (Figures S5 and S7) were fit to Equation 1 to obtain k _diff values, with results presented as mean ± S.D. from three replicates with independent sample preparations.

FIGURE 4

Comparison of cross‐term interactions in human serum (black circles), IgG‐depleted serum (blue squares), and albumin (purple triangles), for full‐length antibodies (closed symbols) and Fab fragments (open symbols). NIST mAb (a), Tocilizumab (b), anti‐gp120 mAb (c), and anti‐RSV mAb (d) D _adj versus c plots (Figures S8–S11) were fit to Equation 1 to obtain k _diff values, with results presented as mean ± S.D. from three replicates with independent sample preparations.