Analytical Charcterization of Monoclonal Antibody IgG2
by Sepax SEC, IEX, HIC and RP Products
Vectibix is a fully human mAb IgG2 specific to the epidermal growth factor receptor (EGFR). In this application note, we would like to present the IgG2 Vectibix (panitumumab) characterization in a few different chromatographic areas:  
  • Size Exclusion (SEC)
  • Strong Cation Exchange (SCX)
  • Hydrophobic Interaction (HIC)
  • Reversed Phase (RP)
* Vectibix IgG2 aggregate, monomer and fragment can be analyzed by size exclusion chromatography. With added MALS detector, molecular weight of different species can be determined in the same SEC separation. 

* SCX  provides the charge variants separation which may be due to IgG2's major disulfide-mediated structural isoforms. Different charge variant profiles are generated for IgG1 and IgG2 due to structure difference while targeting same EGFR.
* HIC  provides an orthogonal analysis of Vectibix variants under the native running condition based upon the different species' hydrophobicity. IgG2 Vectibix and IgG1 Erbitux present different HIC profiles.

*With large pore reversed phase chemistry, i ntact IgG2 and DTT reduced subunits can be analyzed by Proteomix RP-1000 with online mass spec analysis capability.

In conclusion, these four chromatographic methods provide a comprehensive characterization of IgG2 Vectibix heterogeneity.
Experimental:

Samples:  EGFR-targeted mAbs

- Vectibix (PanitumumAb) IgG2
- Erbitux (CetuximAb) IgG1

Columns:
Zenix®-C SEC-300-LS  (3 µm, 300 Å, 7.8 x 300 mm,  PN: 233300LS-7830)
Unix™-C SEC-300  (1.8 µm, 300 Å, 4.6 x 300 mm,  PN: 231300-4630)
Proteomix® SCX  (5 µm, NP, 2.1 x 250 mm, PEEK,  PN: 401NP5P-2125)
Proteomix® HIC Butyl-NP5  (5 µm, NP, 4.6 x 50 mm,  PN: 431NP5-4605)
Proteomix® RP-1000  (5 µm, 1000 Å, 2.1 x 100 mm,  PN: 465950-2110) 
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