ArticleAggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress |
Christian Beyschau Andersen 1 2, Mauro Manno 1, Christian Rischel 2 , Matthías Thórólfsson 2, Vincenzo Martorana 1 * |
1Institute of Biophysics, National Research Council, CNR, Via Ugo La Malfa 153, I-90146 Palermo, Italy
2Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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| email: Vincenzo Martorana (vincenzo.martorana@cnr.it) |
*Correspondence to Vincenzo Martorana, Institute of Biophysics, National Research Council, CNR, Via Ugo La Malfa 153, I-90146 Palermo, Italy
Christian Rischel and Matthías Thórólfsson are employees of Novo Nordisk A/S, which develops, manufactures and markets therapeutic proteins.
Funded by:
Novo Nordisk International Postdoc Fellowship (C.B.A.)
| Multidomain protein aggregation dynamic light scattering coagulation Rituximab monoclonal IgG1 antibody Tween 80 |
| 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 lag-time 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. |
Received: 7 October 2009; Accepted: 30 November 2009
10.1002/pro.309
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