Characterization of High-Molecular-Weight Nonnative Aggregates and Aggregation Kinetics by Size Exclusion Chromatography With Inline Multi-Angle Laser Light Scattering

Li, Yi; Weiss, William F.; Roberts, Christopher J.

doi:10.1002/jps.21726

Cited by 92 publications

(107 citation statements)

References 54 publications

Supporting

Mentioning

100

Contrasting

Order By: Relevance

“…36,37 The inset shows that the sizes of aggregates formed at 40 C are similar to those for aggregates formed at the temperatures of 55 C, 60 C, and 65 C within the first half-life for monomer loss. The sizes of aggregates are similar at a given amount of monomer loss for 45 C to 50 C, but decrease as temperature increases from 50 C to 55 C, especially at longer times (smaller m values).…”

Section: Protein-protein Interactions Based On G 22 From Laser Scattementioning

confidence: 70%

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Truncali

Ritchie

et al. 2015

mAbs

View full text Add to dashboard Cite

(2015) Weak protein interactions and pH-and temperature-dependent aggregation of human Fc1, mAbs, 7:6, 1072-1083, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77 C), using a combination of differential scanning calorimetry, laser light scattering, sizeexclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C H 2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C H 2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.

show abstract

Section: Protein-protein Interactions Based On G 22 From Laser Scattementioning

confidence: 70%

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Truncali

Ritchie

et al. 2015

mAbs

View full text Add to dashboard Cite

show abstract

“…Aggregation can be described through models incorporating nucleation and subsequent growth steps such as Lumry-Eyring nucleation polymerization models [1,2,9,50,96]. Nucleation generally refers to the steps prior to formation of the smallest net irreversible aggregate, which can include partial unfolding, reversible association of partially unfolded intermediates, and conformational rearrangements.…”

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

View full text Add to dashboard Cite

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.

show abstract

“…3,7 Aggregation pathways have a number of common features across a range of proteins. 2,5,[8][9][10][11][12][13][14][15][16][17][18] Aggregate formation often involves some or all of the following steps, depending on the protein and conditions of interest: monomer unfolding and/ or misfolding, 9,11,17 reversible self-association, 15,16,19 nucleation or creation of the smallest stable (i.e., net irreversible) aggregates, 8,10,13 subsequent growth of soluble aggregates by monomer addition and/ or aggregate-aggregate coalescence or condensation polymerization, [12][13][14]18 and aggregate precipitation and/or macroscopic particle formation. 12 A number of theoretical and experimental models capture some of these steps in the multistep process of aggregation.…”

Section: Introductionmentioning

confidence: 99%

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions, Part 2: Nucleation and Growth Kinetics with Competing Growth Mechanisms

Brummitt

Nesta

Chang

et al. 2011

Journal of Pharmaceutical Sciences

Self Cite

View full text Add to dashboard Cite

Characterization of High-Molecular-Weight Nonnative Aggregates and Aggregation Kinetics by Size Exclusion Chromatography With Inline Multi-Angle Laser Light Scattering

Cited by 92 publications

References 54 publications

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

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

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions, Part 2: Nucleation and Growth Kinetics with Competing Growth Mechanisms

Contact Info

Product

Resources

About