Abstract
Purpose
Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation.
Methods
We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations.
Results
We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively.
Conclusions
We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.
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Abbreviations
- AUC:
-
Area under the curve
- AUP:
-
Area under the peak
- DLS:
-
Dynamic light scattering
- Fab:
-
Antigen-binding fragment
- Fc:
-
Crystallizable fragment
- HMWS:
-
High molecular weight species
- HPLC:
-
High-performance liquid chromatography
- I0 :
-
Forward scattering intensity
- Ig:
-
Immunoglobulin
- mAb:
-
Monoclonal antibody
- MALS:
-
Multi-angle static light scattering
- MW:
-
Molecular weight
- P(r):
-
Pair distance distribution function
- PBS:
-
Phosphate buffered saline
- PDB:
-
Protein data bank
- pI:
-
Isoelectric point
- q :
-
Length of the scattering vector
- Rg :
-
Radius of gyration
- Rh :
-
Hydrodynamic radius
- SAP:
-
Spatial aggregation propensity
- SASA:
-
Solvent accessible surface area
- SAXS:
-
Small-angle X-ray scattering
- SEC:
-
Size-exclusion chromatography
- Tm :
-
Melting point
- UV:
-
Ultraviolet
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ACKNOWLEDGMENTS AND DISCLOSURES
The authors thank Zhiru Yang for her support with protein production, Mikkel Melchior Rasmussen for his assistance with biophysical analyses. We are grateful for the availability of beamtime and we thank the beamline staff for their great help at P12 EMBL/PETRA III, Hamburg. Funding from the Drug Research Academy, Novo Nordisk A/S, Carlsberg Foundation, Danish Council for Independent Research, Sapere Aude programme and DANSCATT is sincerely acknowledged.
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Thomas Skamris and Xinsheng Tian contributed equally to this work.
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Skamris, T., Tian, X., Thorolfsson, M. et al. Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization. Pharm Res 33, 716–728 (2016). https://doi.org/10.1007/s11095-015-1821-0
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DOI: https://doi.org/10.1007/s11095-015-1821-0