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DOI: 10.1007/s11095-012-0885-3Pages: 641-654

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

1. Institute for Chemical and Bioengineering, ETH Zurich, Department of Chemistry and Applied Biosciences

Correspondence to:
Massimo Morbidelli
Email: massimo.morbidelli@chem.ethz.ch

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Abstract

Purpose

To identify the aggregation mechanism and the stability characteristics of three different monoclonal antibodies under acidic conditions.

Methods

The aggregation kinetics is analyzed by a combination of light scattering, size exclusion chromatography and fluorescence techniques and the aggregation data are correlated to protein structure, hydrophobicity, charge and antibody subclass.

Results

In the investigated conditions, the antibody aggregation follows a mechanism consisting of two-steps: reversible monomer oligomerization followed by irreversible cluster-cluster aggregation. The kinetics of the two steps is differently affected by the operating conditions: mild destabilizing conditions induce formation of oligomers which are stable within weeks, while stronger denaturing conditions promote aggregation of oligomers to larger aggregates which eventually precipitate. For different antibodies significant differences in both oligomerization and growth rates are found, even for antibodies belonging to the same subclass. For all antibodies the aggregate formation is accompanied by a structure re-organization with an increase in the ordered β-sheet structures. At low pH the aggregation propensity of the investigated antibodies does not correlate with antibody subclass, surface net charge and hydrophobicity of the non-native state.

Conclusions

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

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  • Accepted: Sep 7, 2012
  • Online: Oct 9, 2012

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