Process optimization and protein engineering mitigated manufacturing challenges of a monoclonal antibody with liquid-liquid phase separation issue by disrupting inter-molecule electrostatic interactions

Abstract

We report a case study in which liquid-liquid phase separation (LLPS) negatively impacted the downstream manufacturability of a therapeutic mAb. Process parameter optimization partially mitigated the LLPS, but limitations remained for large-scale manufacturing. Electrostatic interaction driven selfassociations and the resulting formation of high-order complexes are established critical properties that led to LLPS. Through chain swapping substitutions with a well-behaved antibody and subsequent study of their solution behaviors, we found the self-association interactions between the light chains (L C s) of this mAb are responsible for the LLPS behavior. With the aid of in silico homology modeling and charged-patch analysis, seven charged residues in the L C complementarity-determining regions (CDRs) were selected for mutagenesis, then evaluated for self-association and LLPS properties. Two charged residues in the light chain (K30 and D50) were identified as the most significant to the LLPS behaviors and to the antigen-binding affinity. Four adjacent charged residues in the light chain (E49, K52, R53, and R92) also contributed to self-association, and thus to LLPS. Molecular engineering substitution of these charged residues with a neutral or oppositely-charged residue disrupted the electrostatic interactions. A double-mutation in CDR2 and CDR3 resulted in a variant that retained antigen-binding affinity and eliminated LLPS. This study demonstrates the critical nature of surface charged resides on LLPS, and highlights the applied power of in silico protein design when applied to improving physiochemical characteristics of therapeutic antibodies. Our study indicates that in silico design and effective protein engineering may be useful in the development of mAbs that encounter similar LLPS issues.

Stability of a high-concentration monoclonal antibody solution produced by liquid–liquid phase separation

“…To achieve that conditions under which LLPS occurs must first be identified for each individual mAb. While the primary sequence of some mAbs may render them inherently more prone to self-association and LLPS, 25,38 a wide variety of additives, such as salts, macromolecular crowders and polyvalent ions, [39][40][41] can be used alongside temperature and pH to induce LLPS. Selection of these additives and conditions could be achieved using conventional highthroughput screening platforms, for example, through detection of opalescence by light scattering techniques.…”

Mitigation of liquid–liquid phase separation of a monoclonal antibody by mutations of negative charges on the Fab surface

“…Gentiluomo et al [ 34 ] characterized the native reversible self-association of mAbs using Fab and Fc fragments and confirmed that self-associations such as hydrophobic and electrostatic interactions are driven by the Fab fragment. In addition, Du et al [ 24 ] and Chow et al [ 25 ] identified the amino acids responsible for the LLPS propensity of mAbs, located in the Fab region. In this study, we calculated Fab dipole moments to investigate the Fab–dipole-related association.…”

“…The effective in silico design of mAbs has previously been reported to reduce the risk of viscosity escalation [ 19 – 23 ]. In terms of LLPS, Du et al [ 24 ] successfully used in silico homology modelling and charged patch analysis to identify seven amino acids that were potentially related to self-association. They confirmed that mutations of the amino acids resulted in a lower LLPS propensity.…”

Toward Drug-Like Multispecific Antibodies by Design

“…One specific result of increased self-association on formulation stability and manufacturing processes is thermodynamically driven liquid-liquid phase separation (LLPS) [ 146 ]. The formation of a protein-rich bottom layer and protein-depleted top layer, characteristic of LLPS, typically occurs for highly concentrated solutions at low temperatures (2–8 °C), such as refrigerated formulations [ 147 ]. It can also occur during large-scale purification in which intermediates are kept either at room temperature or 2–8 °C for extended periods of time.…”

“…Therefore, it would be desirable to minimize LLPS with early-stage property engineering. Recent work has used protein engineering to investigate the mechanistic foundations of antibody site-specific contributions to LLPS behavior [ 147 ]. One study found that strong self-association between the light chains of a mAb contributed to electrostatic interactions resulting in LLPS.…”