Elucidation of Acid-induced Unfolding and Aggregation of Human Immunoglobulin IgG1 and IgG2 Fc

Latypov, Ramil F.; Hogan, Sabine; Lau, Hollis; Gadgil, Himanshu S.; Liu, Dingjiang

doi:10.1074/jbc.m111.297697

Cited by 137 publications

(129 citation statements)

References 51 publications

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“…36,37 The inset shows that the sizes of aggregates formed at 40 C are similar to those for aggregates formed at the temperatures of 55 C, 60 C, and 65 C within the first half-life for monomer loss. The sizes of aggregates are similar at a given amount of monomer loss for 45 C to 50 C, but decrease as temperature increases from 50 C to 55 C, especially at longer times (smaller m values). Qualitatively, the results in Fig.…”

Section: Protein-protein Interactions Based On G 22 From Laser Scattementioning

confidence: 68%

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Truncali

Ritchie

et al. 2015

mAbs

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(2015) Weak protein interactions and pH-and temperature-dependent aggregation of human Fc1, mAbs, 7:6, 1072-1083, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77 C), using a combination of differential scanning calorimetry, laser light scattering, sizeexclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C H 2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C H 2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.

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Section: Protein-protein Interactions Based On G 22 From Laser Scattementioning

confidence: 68%

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Truncali

Ritchie

et al. 2015

mAbs

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“…as it results in only limited changes in the conformation and stability of the antibody molecules (Ejima et al 2007, Latypov et al 2012, and still falls into the pH interval that strongly suppresses the reoxidation. In the case of sheep anti-digoxin IgG reduction (Figure 3 A), no increase in the intensity of the HL band was observed with increased 2-MEA concentrations at pH 7.4.…”

Section: Figure 2 Sds-page Analysis Of Sheep Polyclonal Anti-digoxinmentioning

confidence: 99%

Considerations in producing preferentially reduced half-antibody fragments

Makaraviciute

Jackson

Millner

et al. 2016

Journal of Immunological Methods

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Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

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“…It has been demonstrated on several occasions that this N-glycan participates in the stabilization of mAbs against aggregation caused by various stresses. 8,24,26,27,56,57 Natural IgGs present in human serum are all glycosylated in the Fc domain whereas only less than a third are glycosylated in the Fab domain. 55 N-glycans are found attached to the variable region of the LC, the HC or both.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, glycosylation can stabilize the tertiary and quaternary structures of mAbs, and in the case of the Fc domain, hydrophobic regions, which are aggregation prone, are covered by a glycosylation moiety. [24][25][26][27] Glycoengineering has proved to be effective in substantially increasing the solubility of biotherapeutics, 28,29 as well as reducing the aggregation propensity of mAbs. It is well accepted that N-linked carbohydrates participate in the stabilization of mAbs against aggregation by covering aggregationprone motifs and through steric hindrance that disrupts intermolecular interactions.…”

Section: Introductionmentioning

confidence: 99%

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

Courtois

Agrawal

Lauer³

et al. 2015

mAbs

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The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.

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Elucidation of Acid-induced Unfolding and Aggregation of Human Immunoglobulin IgG1 and IgG2 Fc

Cited by 137 publications

References 51 publications

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

Considerations in producing preferentially reduced half-antibody fragments

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

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