Therapeutic protein aggregation: mechanisms, design, and control

Abstract

While 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.

Protein aggregation and its impact on product quality

“…Nucleation is usually a kinetically limited process that can require extremely long time scales (from minutes to years) when compared to typical timescales for protein folding (~ μs to s) under native-favoring conditions where most proteins are stored and delivered. In many cases, partial unfolding of protein monomers appears to be the first step, followed by slow association of two or more chains to create the nuclei [37], although at high protein concentration it has been inferred that association occurs before unfolding [38,39], and in some cases unfolding is clearly rate-limiting. If growth occurs by monomer addition, then the (partially) unfolded monomers can add more easily to the existing aggregates [34,35,40].…”

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

“…15 These protein-protein interactions can be either reversible or irreversible. For example, reversible dimerization of rhuMAb VEGF was described as an equilibrium reaction between native monomer and dimer.…”

“…18,19 Upon formation of aggregates of various size and shape, these aggregation-prone areas are shielded from the aqueous environment by inter-molecular interactions between monomer molecules. 15,20 Dimeric forms of IgG molecules and mAbs have been described previously in literature. Recently, dimers of IgG1 and IgG2 have been found to occur naturally in human serum, although in low amounts.…”

Editorial: Protein stabilization – crossroad for protein‐based processes and products

“…Computational and bioinformatics methods have also emerged as fundamental tools for the understanding of protein aggregation and for designing mutants less prone to aggregate [13]. Protein aggregation is often an obstacle in the production of recombinant proteins, because of the formation of intracellular inclusion bodies.…”

“…Kinetic analysis based on mass balance equations, combined with biophysical and biochemical experimental characterization, might allow the quantification of protein-protein intermolecular interactions and knowledge of aggregation mechanisms [14]. Such data can address operative conditions in industrial processing and formulation, as well as shed light on the aggregation of proteins in the biological context [13]. See the article by Nicoud et al [15].…”