Principles, approaches, and challenges for predicting protein aggregation rates and shelf life

Weiss, William F.; Young, Teresa M.; Roberts, Christopher J.

doi:10.1002/jps.21521

Cited by 275 publications

(300 citation statements)

References 155 publications

(440 reference statements)

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“…Once formed, the aggregates can grow through different mechanisms such as via monomer addition (referred to as chain polymerization), by aggregate-aggregate coalescence or condensation, and possibly phase separation or precipitation. Predicting aggregation is not always possible due to the multitude of possible pathways and difficulties in isolating key partially folded intermediates and characterizing their intermolecular interactions [68,70,94,97].…”

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

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Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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“…Several studies have shown that the formation of intermolecular crossbeta-sheets and amyloidogenic substructures is a crucial step for some aggregation pathways. 8,10 This mechanism requires a preceding, at least partial, unfolding step of the protein to allow exposure and alignment of the reactive sequence parts. Protein self-association or aggregate growth is then thought to follow a mechanism similar to polymerization, but this process is reversible up to a certain degree.…”

Section: Introductionmentioning

confidence: 99%

Boosting antibody developability through rational sequence optimization

Seeliger

Schulz

Litzenburger

et al. 2015

mAbs

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(2015) Boosting antibody developability through rational sequence optimization , mAbs, 7:3, 505-515, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and longterm stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

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“…[1][2][3][4] Protein aggregation is generally described as a complex, multi-stage process involving unfolding or misfolding of free monomers followed by one or more assembly steps to form soluble or insoluble oligomers. 5,6 Throughout the manufacturing process, protein therapeutics may be exposed to various stress conditions, and understanding the effects of these factors on protein aggregation is important.…”

Section: Introductionmentioning

confidence: 99%

“…These techniques can be categorized as direct or indirect assays depending on whether the aggregated protein is physically separated from the native (monomeric) protein. 4 Spectroscopy-based (indirect) techniques (CD, fluorescence, derivative UV, IR spectroscopy, etc.) probe changes in protein structure and correlate them with aggregation.…”

Section: Introductionmentioning

confidence: 99%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

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Principles, approaches, and challenges for predicting protein aggregation rates and shelf life

Cited by 275 publications

References 155 publications

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Boosting antibody developability through rational sequence optimization

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

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