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Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

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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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    Authors and Affiliations

    1. Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark

      Thomas Skamris, Xinsheng Tian, Annette E. Langkilde & Bente Vestergaard

    2. Global Research Unit, Novo Nordisk A/S, Novo Nordisk Park 1, 2760, Måløv, Denmark

      Matthias Thorolfsson, Hanne Sophie Karkov & Hanne B. Rasmussen

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    1. Thomas Skamris
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    2. Xinsheng Tian
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    3. Matthias Thorolfsson
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    4. Hanne Sophie Karkov
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    5. Hanne B. Rasmussen
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    6. Annette E. Langkilde
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    7. Bente Vestergaard
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    Correspondence to Bente Vestergaard.

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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

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