Protein Structural Conformation and Not Second Virial Coefficient Relates to Long-Term Irreversible Aggregation of a Monoclonal Antibody and Ovalbumin in Solution

Bajaj, Harminder; Sharma, Vikas; Badkar, Advait; Zeng, David L.; Nema, Sandeep; Kalonia, Devendra S.

doi:10.1007/s11095-006-0018-y

Cited by 68 publications

(48 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A similar conclusion was reached by Kalonia and coworkers 50 in a study of irreversible aggregation of a mAb under nonstressed conditions. In our study, the CH2 domain is the least stable domain and the overall irreversible aggregation process is most likely determined by the activation barrier for unfolding of this particular domain.…”

Section: Coagulation Governs Aggregate Growthsupporting

confidence: 82%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

View full text Add to dashboard Cite

Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lagtime behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

show abstract

Section: Coagulation Governs Aggregate Growthsupporting

confidence: 82%

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

View full text Add to dashboard Cite

show abstract

“…More importantly, we have provided examples where aggregation does not correlate with conformational stability and native-state protein-protein interactions. As such, there will be limited utility of using k D values as a predictor for aggregate growth rates in the absence of strong electrostatic interactions [7,78].…”

Section: Discussionmentioning

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

“…Although valuable information on the protein colloidal stability can be extracted from the measurement of the second virial coefficient, the correlation between B 22 values and the aggregation propensity is not straightforward, and is still a matter of debate in the literature [35,42,44,47,50,51]. A possible reason for this discrepancy can be rooted in the heterogeneity of the protein surface, especially at pH values close to the isoelectric point of the protein, where electrostatic attraction arises.…”

Section: Colloidal Stability and Conformational Stabilitymentioning

confidence: 99%

A multiscale view of therapeutic protein aggregation: A colloid science perspective

Nicoud

Owczarz

Arosio

et al. 2015

Biotechnology Journal

View full text Add to dashboard Cite

The formation of aggregates in protein-based pharmaceuticals is a major issue that can compromise drug safety and drug efficacy. With a view to improving protein stability, considerable effort is put forth to unravel the fundamental mechanisms underlying the aggregation process. However, therapeutic protein aggregation is a complex multistep phenomenon that involves time and length scales spanning several orders of magnitude, and strategies addressing protein aggregation inhibition are currently still largely empirical in practice. Here, we review how key concepts developed in the frame of colloid science can be applied to gain knowledge on the kinetics and thermodynamics of therapeutic protein aggregation across different length scales. In particular, we discuss the use of coarse-grained molecular interaction potentials to quantify protein colloidal stability. We then show how population balance equations simulations can provide insights into the mechanisms of aggregate formation at the mesoscale, and we highlight the strength of the concept of fractal scaling to quantify irregular aggregate morphologies. Finally, we correlate the macroscopic rheological properties of protein solutions with the occupied volume fraction and the aggregate structure. Overall, this work illustrates the power and limitations of colloidal approaches in the multiscale description of the aggregation of therapeutic proteins.

show abstract

Protein Structural Conformation and Not Second Virial Coefficient Relates to Long-Term Irreversible Aggregation of a Monoclonal Antibody and Ovalbumin in Solution

Cited by 68 publications

References 42 publications

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

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

A multiscale view of therapeutic protein aggregation: A colloid science perspective

Contact Info

Product

Resources

About