Elucidation of Two Major Aggregation Pathways in an IgG2 Antibody

Buren, Nicholas Van; Rehder, Douglas S.; Gadgil, Himanshu S.; Matsumura, Masazumi; Jacob, J.

doi:10.1002/jps.21514

Cited by 75 publications

(87 citation statements)

References 34 publications

(40 reference statements)

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“…3 Aggregation is hence most likely initiated by minor structural changes to certain parts of the molecule and possibly to just a small fraction of the sample. In the study of IgG2 aggregation mentioned earlier, Van Buren et al 24 showed that oligomerization and aggregation were mainly driven by unfolding of the CH2 domain, although no changes in farand near-UV spectra were observed. The isolated oligomers and aggregates did, however, exhibit minor changes in far-UV CD spectra, demonstrating Figure 10.…”

Section: Coagulation Governs Aggregate Growthmentioning

confidence: 94%

“…15,16 In this study, we used the monoclonal IgG1 antibody Rituximab 17 as a model system to better understand the mechanisms leading to the formation of large aggregates when incubating a multidomain protein under weak thermal stress. Aggregation of antibodies due to various kinds of stress have been addressed in a number of studies, [18][19][20][21][22][23][24] with a few key observations highlighted later. In this context, we distinguish between the formation of oligomers (dimers, trimers, etc.)…”

Section: Introductionmentioning

confidence: 99%

“…The appearance of large aggregates, if any, was not reported. Finally, Van Buren et al 24 incubated an IgG2 monoclonal antibody (mAb) for 15 weeks at 37 C in two different pH regimes, and found the aggregation pathways to be pH dependent: at high pH, dimers had $75% covalent character, while the formation of large aggregates was largely suppressed; at low pH, on the other hand, both dimers and large aggregates were formed. To generalize these findings, incubation at elevated temperatures may lead to the formation of large aggregates, dimers, or both.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2010

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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.

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Section: Coagulation Governs Aggregate Growthmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2010

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“…These results indicated that the IgG dimers consisted of both non-covalently and covalently associated species. Decrease of the aggregation by reducing agents also indicated the presence of disulphide bonds (8). Furthermore, conformations of the dimers have been studied by CD and UV spectrum (7,8) and confirmed that they had the native conformation.…”

mentioning

confidence: 78%

“…F ab and F ab , F ab and F c and F c and F c ) (7). Intermolecular interactions in IgG dimer have been evaluated by SEC in the presence and absence of SDS or by SDS electrophoresis in the presence and absence of reducing reagent (7,8). It is possible to distinguish covalent and non-covalent interactions by measuring changes in molecular weights in the presence of SDS.…”

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confidence: 99%

Intermolecular interactions and conformation of antibody dimers present in IgG1 biopharmaceuticals

Iwura

Fukuda

Yamazaki

et al. 2013

The Journal of Biochemistry

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Intermolecular interactions and conformation in dimer species of Palivizumab, a monoclonal antibody (IgG1), were investigated to elucidate the physical and chemical properties of the dimerized antibody. Palivizumab solution contains $1% dimer and 99% monomer. The dimer species was isolated by size-exclusion chromatography and analysed by a number of methods including analytical ultracentrifugation-sedimantetion velocity (AUC-SV). AUC-SV in the presence of sodium dodecyl sulphate indicated that approximately half of the dimer fraction was non-covalently associated, whereas the other half was dimerized by covalent bond. Disulphide bond and dityrosine formation were likely to be involved in the covalent dimerization. Limited proteolysis of the isolated dimer by Lys-C and mass spectrometry for the resultant products indicated that the dimer species were formed by F ab F c or F ab F ab interactions, whereas F c F c interactions were not found. It is thus likely that the dimerization occurs mainly via the F ab region. With regard to the conformation of the dimer species, the secondary and tertiary structures were shown to be almost identical to those of the monomer. Furthermore, the thermal stability turned out also to be very similar between the dimer and monomer.Keywords: immunoglobulin G/aggregation/intermolecular interaction/conformation/analytical ultracentrifugation.Abbreviations: Ac-Cys, N-Acetyl-Cysteine; ANS, 8-Anilino-1-naphthalenesulphonic acid ammonium salt; AUC-SV, analytical ultracentrifugation-sedimantetion velocity; CD, circular dichroism; DSC, differential scanning micro-calorimetry; DTT, dithiothreitol; HC, heavy chain; IAM, iodoacetamide; LC, light chain; MALLS, multi-angle laser light scattering; NPM, N-(1-pyrenyl)maleimide; PBS, phosphatebuffered saline; SD, standard deviation; SDSPAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography.Soluble proteins in solution can aggregate under normal conditions with relatively small perturbations such as thermal or physical agitation (13). Pharmaceutical proteins are not the exception; in fact, they may aggregate during production, isolation, purification, storage and delivery (4), and the aggregation may affect their activity and immunogenicity (5, 6). Therefore, characterization of aggregation is of great importance in pharmaceutical industry. In monoclonal antibodies which are the fastest growing class of biopharmaceuticals, many studies have been conducted to acquire physical and chemical information regarding antibody aggregations (710). When a monoclonal antibody is prepared as a biopharmaceutical, it is mostly monomer (complex of two heavy chains (HC) and two light chains (LC), H 2 L 2 ) in solution; however, $1% or lower amount of dimer (dimerized monomer, (H 2 L 2 ) 2 ) is always present. Although the amount of dimer may appear insignificant, in the field of biopharmaceuticals, it is highly important to elucidate the nature of dimerization for the safety and prevention of dimerization or further associat...

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Protein and Peptide Formulation Development

Ohtake¹,

Wang²

2013

Pharmaceutical Sciences Encyclopedia

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Proteins/peptides are generally much more sensitive to process‐ or storage‐induced degradations than small synthetic molecules. Therefore, significant efforts are needed to formulate these protein/peptide drug candidates into viable commercial products.This chapter reviews the degradation processes in proteins/peptides, describes factors affecting protein/peptide stability, and discusses strategies in the successful development and manufacturing of protein/peptide commercial products.

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Elucidation of Two Major Aggregation Pathways in an IgG2 Antibody

Cited by 75 publications

References 34 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

Intermolecular interactions and conformation of antibody dimers present in IgG1 biopharmaceuticals

Protein and Peptide Formulation Development

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