Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates

Brummitt, Rebecca K.; Nesta, Douglas P.; Chang, Liuquan; Chase, Susan F.; Laue, Thomas M.; Roberts, Christopher J.

doi:10.1002/jps.22448

Cited by 106 publications

(153 citation statements)

References 74 publications

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“…With increasing temperature further, a bimodal population distribution develops indicating oligomers grow to form larger aggregates, but the large aggregates do not precipitate even at the highest temperature of 90°C measured during the static light scattering experiment. This behaviour reflects conditions with strong electrostatic repulsion, where nucleation dominated growth followed by chain polymerization occur with increasing time or temperature [10,71,75]. This pattern should be contrasted with what happens when electrostatic repul- sion is weakened by increasing ionic strength.…”

Section: The Patch-charged Mutantsmentioning

confidence: 91%

“…Similar characteristics are observed for the other scFv proteins (WT, 1SB, 2SB, 3SB, and DSV) at all salt concentrations. This behaviour reflect aggregation pathways governed by much weaker electrostatic interactions, where aggregate growth initially occurs through chain polymerization followed by aggregate-aggregate condensation and then precipitation either with increasing time or temperature [11,75].…”

Section: The Patch-charged Mutantsmentioning

confidence: 92%

“…5K and 5R exhibit other characteristics observed in the aggregation behaviour of highly charged proteins such as antibodies [11,43,75] and a-chymotrypsinogen [51] in acidic conditions. The increase in net positive charge at low pH causes an increase in intramolecular charge repulsion and a reduced conformational stability.…”

Section: The Patch-charged Mutantsmentioning

confidence: 94%

“…The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82].…”

Section: Introductionmentioning

confidence: 99%

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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: The Patch-charged Mutantsmentioning

confidence: 91%

Section: The Patch-charged Mutantsmentioning

confidence: 92%

Section: The Patch-charged Mutantsmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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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show abstract

“…Aggregation of several proteins appear to follow the LENP model reasonably well. These include the non-native aggregation of α-chymotrypsinogen (aCgn) and anti-streptavidin IgG1 over a range of pH and (NaCl) conditions (11,30) (Kim et al, under review), a model IgG1 at pH 4.5 and 5.5 (35,36), and gamma-D crystallin (37). This type of aggregation can be unfolding limited, association limited, or nucleation limited (8).…”

Section: Protein Aggregation and Its Temperature Dependencementioning

confidence: 99%

Non-Arrhenius Protein Aggregation

Wang

Roberts

2013

AAPS J

Self Cite

121

109

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Abstract. Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. Therefore, investigations of protein aggregation remain a major focus in pharmaceutical companies and academic institutions. Due to the complexity of the aggregation process and temperature-dependent conformational stability, temperature-induced protein aggregation is often non-Arrhenius over even relatively small temperature windows relevant for product development, and this makes low-temperature extrapolation difficult based simply on accelerated stability studies at high temperatures. This review discusses the non-Arrhenius nature of the temperature dependence of protein aggregation, explores possible causes, and considers inherent hurdles for accurately extrapolating aggregation rates from conventional industrial approaches for selecting accelerated conditions and from conventional or more advanced methods of analyzing the resulting rate data.

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Biophysical Methods Applied in Early Discovery of a Biotherapeutic: Case Study of an Egfr‐Igf1R Bispecific Adnectin

Doyle

Bryson

Lafont

et al. 2014

Biophysical Methods for Biotherapeutics

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Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates

Cited by 106 publications

References 74 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

Non-Arrhenius Protein Aggregation

Biophysical Methods Applied in Early Discovery of a Biotherapeutic: Case Study of an Egfr‐Igf1R Bispecific Adnectin

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