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Structure and Function of Purified Monoclonal Antibody Dimers Induced by Different Stress Conditions

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ABSTRACT

Purpose

To investigate structure and function of different monoclonal antibody (MAb) dimers.

Methods

MAb dimers were induced by process-related, low pH and UV light stress. Dimers were isolated and purified by chromatography and extensively characterized by biochemical, structural and functional methods.

Results

Highly purified dimer forms were obtained which enabled detailed characterization. Dimers induced by process stress were associated by a single non-covalent interaction site between two Fab domains in a characteristic “bone-like” structure observed in Transmission Electron Microscopy (TEM). These dimers showed reduced potency and antigen binding affinity. Low pH stress generated more stable but also non-covalently associated dimers without chemical alterations in a typical “closed” conformation according to TEM. These dimer species were more compact and more hydrophobic as dimers induced by process stress. They showed bioactivity and antigen binding affinity similar to the native monomer. Light-induced dimers, exhibiting various different conformations, were the most stable dimers with various chemical modifications leading to a broad range in size, charge and hydrophobicity. These dimers fully lost bioactivity and antigen binding affinity.

Conclusion

The use of highly purified MAb dimers and a panel of characterizations methods enabled to obtain a clear picture about molecular architecture and function of dimers.

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Abbreviations

AU:

absorption units

AUC:

analytical ultracentrifugation

CDR:

complementarity-determining regions

CE-SDS-NGS:

capillary electrophoresis sodium dodecyl sulfate-non gel sieving

ESI-TOF-MS:

electrospray time-of-flight mass spectrometry

FTIR:

Fourier transform infrared

HBS-EP buffer:

hEPES-buffered saline EDTA polysorbate buffer

HC:

heavy chain

HIC:

hydrophobic interaction chromatography

HUVEC:

human umbilical vein endothelial cell

IEC:

ion exchange chromatography

LC:

light chain

MAb:

monoclonal antibody

SDS:

sodium dodecyl sulfate

SEC-MALLS:

size exclusion chromatography with multi angle laser light scattering

SPR:

surface plasmon resonance

TEM:

transmission electron microscopy

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ACKNOWLEDGMENTS & DISCLOSURES

We thank Thomas von Hirschheydt, Marc Pompiati, Gordan Graf (all Roche Penzberg) and Marco Sonderegger for their support in isolation of aggregates. We sincerely acknowledge Rita Grübel, Gaby Walch, Jürgen May, Bernd Moritz, Stephen Hyland, Philipp Metzger, Jörg Hoernschemeyer and Eric Kusznir for their contributions to analytical characterizations of the dimers. Thanks to Jochem Alsenz and Michael Hennig for supporting the TEM experiments. We acknowledge Thomas Schreitmueller for sponsoring and supporting the project. This work was in part supported by the Swiss initiative for systems biology (SystemsX.ch).

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Authors and Affiliations

  1. Pharma Technical Development Europe (Biologics) Analytics, F. Hoffmann-La Roche Ltd., 4070, Basel, Switzerland

    Rajsekhar Paul, Hermann Beck, Alexandre Briguet, Volker Schnaible, Thomas Buckel & Sabine Boeckle

  2. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, 4085, Switzerland

    Alexandra Graff-Meyer & Henning Stahlberg

  3. Discovery Technologies, F. Hoffmann-La Roche Ltd., Basel, 4070, Switzerland

    Matthias E. Lauer & Arne C. Rufer

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  1. Rajsekhar Paul
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Correspondence to Sabine Boeckle.

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Supplementary Table I

Summary of the determined chemical modifications in light stress dimers in comparison to the monomer by LC/MS peptide map (DOC 33 kb)

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Paul, R., Graff-Meyer, A., Stahlberg, H. et al. Structure and Function of Purified Monoclonal Antibody Dimers Induced by Different Stress Conditions. Pharm Res 29, 2047–2059 (2012). https://doi.org/10.1007/s11095-012-0732-6

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  • DOI: https://doi.org/10.1007/s11095-012-0732-6

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