Aggregation Mechanism of an IgG2 and two IgG1 Monoclonal Antibodies at low pH: From Oligomers to Larger Aggregates

Arosio, Paolo; Rima, Simonetta; Morbidelli, Massimo

doi:10.1007/s11095-012-0885-3

Cited by 106 publications

(84 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This value is much greater than that for the wild type indicating a slower aggregate growth rate, even though 5K exhibits the strongest native-state association out of any mutant (k D = À13.0 mL/g). These examples indicate the association steps involved in aggregate growth are determined by partially unfolded regions of the protein that do not contribute to native state protein-protein interactions [4].…”

Section: Correlations Between K D and Aggregation Propensitymentioning

confidence: 99%

“…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%

See 1 more Smart Citation

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

Section: Correlations Between K D and Aggregation Propensitymentioning

confidence: 99%

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

show abstract

“…Spectroscopic techniques, such as infrared spectroscopy [15], dye binding [16,17], circular dichroism spectroscopy [7,18], and intrinsic fluorescence spectroscopy [19], can be used to monitor changes in protein secondary and/or tertiary structure. However, the signals may or may not correlate with aggregation monitored more directly by techniques such as chromatography, laser scattering, electron microscopy, atomic force microscopy, or small angle neutron/x-ray scattering [20].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Barnett

Wei

Amin

et al. 2015

Biophysical Chemistry

View full text Add to dashboard Cite

Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

show abstract

“…At pH lower than 4.0, addition of salt induces a reversible aggregation to oligomers accompanied by an increase in the content of the b-sheet structure (Arosio et al, 2011). Significant differences in both oligomerization and growth rates were found for different antibodies, even those belonging to the same subclass, as was shown for IgG1 and IgG2 (Arosio et al, 2013;Nicoud et al, 2014a). Cosolutes, such as NaCl or sorbitol, were shown to accelerate and inhibit, respectively, the aggregation kinetics of monoclonal antibodies.…”

Section: Introductionmentioning

confidence: 69%

Formation of multimeric antibodies for self-delivery of active monomers

Dekel

Machluf²,

Gefen

et al. 2017

Drug Delivery

View full text Add to dashboard Cite

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

show abstract

Aggregation Mechanism of an IgG2 and two IgG1 Monoclonal Antibodies at low pH: From Oligomers to Larger Aggregates

Abstract: The aggregation mechanism of three antibodies in acidic conditions as well as differences and analogies in their stability behavior has been characterized.

Cited by 106 publications

References 43 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

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Formation of multimeric antibodies for self-delivery of active monomers

Contact Info

Product

Resources

About