Pharmaceutical Research

, Volume 32, Issue 4, pp 1383–1394 | Cite as

Small Amounts of Sub-Visible Aggregates Enhance the Immunogenic Potential of Monoclonal Antibody Therapeutics

  • Maryam Ahmadi
  • Christine J. Bryson
  • Edward A. Cloake
  • Katie Welch
  • Vasco Filipe
  • Stefan Romeijn
  • Andrea Hawe
  • Wim Jiskoot
  • Matthew P. Baker
  • Mark H. FoggEmail author
Research Paper



Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics.


Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4+ T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy.


Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4+ T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC.


These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.


anti-drug antibodies biotherapeutics CD4+ T cell responses humanized antibodies immunogenicity 



Anti-drug antibody


B cell receptor


Cytometric bead array


Dendritic cell


Dynamic light scattering


Monocolonal antibodies


Major histocompatibility complex


Monocyte derived dendritic cell


Follicular helper T cell


Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

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Supplementary Fig. 1

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High Resolution Image (TIFF 42 kb)
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Supplementary Fig. 2 (PPTX 117 kb)
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Supplementary Table 1 (DOCX 14 kb)
11095_2014_1541_MOESM4_ESM.docx (14 kb)
Supplementary Table 2 (DOCX 13 kb)


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Maryam Ahmadi
    • 1
  • Christine J. Bryson
    • 1
    • 4
  • Edward A. Cloake
    • 1
  • Katie Welch
    • 1
  • Vasco Filipe
    • 2
    • 5
  • Stefan Romeijn
    • 2
  • Andrea Hawe
    • 3
  • Wim Jiskoot
    • 2
  • Matthew P. Baker
    • 1
  • Mark H. Fogg
    • 1
    Email author
  1. 1.Antitope Ltd, Babraham Research CampusCambridgeUK
  2. 2.Division of Drug Delivery Technology, Leiden Academic Centre for Drug ResearchLeiden UniversityLeidenThe Netherlands
  3. 3.Coriolis PharmaMartinsriedGermany
  4. 4.Department of HaematologyCambridge University HospitalsCambridgeUK
  5. 5.AdociaLyonFrance

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