Therapeutic protein aggregation: mechanisms, design, and control

Abstract

Although it is well known that proteins are only marginally stable in their folded states, it is often less well appreciated that most proteins are inherently aggregation-prone in their unfolded or partially unfolded states, and the resulting aggregates can be extremely stable and long-lived. For therapeutic proteins, aggregates are a significant risk factor for deleterious immune responses in patients, and can form via a variety of mechanisms. Controlling aggregation using a mechanistic approach may allow improved design of therapeutic protein stability, as a complement to existing design strategies that target desired protein structures and function. Recent results highlight the importance of balancing protein environment with the inherent aggregation propensities of polypeptide chains.

Keywords: computational design; protein aggregation; protein interactions; protein stability.

Figure 1

Schematic diagram illustrating multiple non-native aggregation pathways for a multi-domain protein such as a monoclonal antibody composed of a single Fc fragment and two identical Fab fragments. Red strands denote “hot spot” sequences that are prone to form strong, effectively irreversible inter-protein contacts that stabilize aggregates, but are primarily hidden or buried in fully folded monomers. Double-arrows denote effectively reversible steps. Single arrows denote irreversible steps. Nuclei are defined as the smallest net-irreversible aggregate size; growth from nuclei to form soluble aggregates spanning length scales on the order of 10 to 10² nm occurs primarily via addition of other partly unfolded monomers (upper right) or by the agglomeration of existing aggregates (lower right) [19,25,39,40]. Aggregates can become visible to the naked eye if they are sufficiently large and/or undergo phase separation [–89]. If unfolding / aggregation is mediated by protein adsorption to bulk interfaces [–92], and/or chemical changes such as deamidation [–95], oxidation and other reactions [96,97], or fragmentation [98,99], then additional steps may also be kinetically important in the possible aggregation mechanism(s).

Figure 2

Schematic illustrating shifts in aggregation mechanisms and examples of aggregates that do not grow easily (see also Box 2), to those grown by monomer addition (lower left image, from [46]), aggregate agglomeration to form globular aggregates (middle image, from [44]), and aggregate phase separation to form macroscopic particles (upper image, from [87]].

Box 2 – Figure I

Aggregation propensity based on net charge and screening length.