Introduction to Fc Site Specific Labeling


Columbia Biosciences offers Fc Site Specific Labeling for SureLight® R-phycoerythrin (PE) and SureLight® Allophycocyanin (APC) in collaboration with AlphaThera.

Fc Site Specific labeling offers much greater control for creating explicitly defined immunoconjugates by targeting the Fc region of the antibody. This method ensures that the antibody labeling occurs specifically at the Fc site, which can improve the homogeneity and functionality of the resulting antibody conjugates. Typically, this approach allows for the addition of 2-4 labels per antibody, providing a balance between effective labeling and preserving the antibody’s biological activity. By focusing on the Fc region, this technique minimizes the risk of interfering with the antigen-binding sites, thereby maintaining the antibody’s specificity and efficacy.  This labeling method has the following benefits:

  • Improved homogeneity in conjugate population, enhancing reproducibility and efficacy
  • Preservation of Antigen Binding
  • Minimized Immunogenicity by minimizing the risk of eliciting immune responses against the conjugated antibodies
  • Facilitated Multifunctionalization: attachment of multiple molecules or payloads at defined locations
  • Simplified Manufacturing by avoiding purification steps
Without Fc Site Specific Labeling:
With Fc Site Specific Labeling:

 

 

 

 

 

R-PE
APC

 

Flow Cytometry Analysis: 

The flow cytometry data shows an example of an antibody conjugated to SureLight® R-phycoerythrin (PE) and SureLight® Allophycocyanin (APC). Human EGFR-expressing A431 cells were stained with Cetuximab (humanized anti-EGFR MAb) conjugated to R-PE or APC. The stained cells were analyzed on BD FACS CANTO II flow cytometer. The fluorescence intensity of cells stained with Cetuximab conjugates shown as shaded histogram. Isotype control-stained cells – open histogram.

 

 

Cetuximab Conjugates
SPR and BD FACS Canto II, A431 Cells, All conjugates tested at 100 ng/test

Samplekon (M-1s-1)koff (s-1)KD(M)
Anti-EGFR – Cetuximan (humanized anti-EGFR Ab)3.82E+064.18E-041.09E-10
Aglycosylated Cetuximab6.44E+061.05E-031.63E-10
Cetuximab-RPE2.09E+064.80E-042.29E-10
Cetuximab-APC3.62E+063.54E-039.76E-10

 

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