Fig 1.
Sequence by MALDI and ESI of Herceptin and 5G4 mAb.
The proteolytic degradation of the heavy chain and the light chain for both antibodies is observed. In red: mapped sequence, in black: sequence without mapping and highlighted in red: the complementarity determining regions. A and B: Proteolytic degradation by Trypsin of the heavy chain of Herceptin and 5G4 mAb, respectively. C and D: Proteolytic degradation by Trypsin of the light chain of Herceptin and 5G4 mAb, respectively.
Fig 2.
Molecular exclusion chromatographic profiles of 5G4 mAb and Herceptin.
Absorbance at 280 nm (mAu) and conductivity (mS/cm) against elution time for different batches evaluated are shown. 5G4 mAb batch 1 (black), 5G4 mAb batch 2 (red), 5G4 mAb batch 3 (blue), Herceptin batch 1 (green) and Herceptin batch 2 (pink).
Fig 3.
Far and near-UV CD spectra of 5G4 mAb and Herceptin in PBS solution, respectively.
Different batches of each mAb are shown: 5G4 mAb batch 1 (black), 5G4 mAb batch 2 (red), 5G4 mAb batch 3 (blue), Herceptin batch 1 (green) and Herceptin batch 2 (pink). The mean ellipticity readings per residue (deg.cm2.dmol-1) for each wavelength at 25°C. A: Far-UV CD spectra of 5G4 mAb and Herceptin in PBS solution. The arrows indicate the positive band at about 202 nm and the negative band at about 218 nm. B: Near-UV CD spectra of 5G4 mAb and Herceptin in PBS solution. The arrows indicate the wavelength ranges corresponding to the signals of the side chains of Phe, Tyr and Trp.
Table 1.
Percentage of secondary structures of 5G4 mAb and Herceptin by DichroWeb.
Fig 4.
Thermal stability spectra by far-UV CD for 5G4 mAb and Herceptin in PBS solution.
Batch 1 of 5G4 mAb (A) and batch 2 of Herceptin (B) are shown. Different temperatures of each mAb are shown: Temperature 25°C (black), temperature 65°C (red), temperature 70°C (blue), temperature 75°C (green) and temperature 95°C (pink).
Fig 5.
Thermal denaturation curves for 5G4 mAb and Herceptin in PBS solution.
Different batches of the mAbs are shown. 5G4 mAb batch 1 (black), 5G4 mAb batch 2 (red), 5G4 mAb batch 3 (blue), Herceptin batch 1 (green) and Herceptin batch 2 (pink). The indicated region shows the temperature range corresponding to the Tm values obtained.
Table 2.
The temperature of melting for 5G4 mAb and Herceptin.
Fig 6.
Fluorescence emission spectra for 5G4 mAb and Herceptin in PBS solution.
Different batches of each mAb are shown. 5G4 mAb batch 1 (black), 5G4 mAb batch 2 (red), 5G4 mAb batch 3 (blue), Herceptin batch 1 (green) and Herceptin batch 2 (pink).
Fig 7.
Degree of exposure of Trp residues.
Fluorescence emission spectra of 5G4 mAb (A) and Herceptin (B) attenuated with different concentrations of acrylamide are shown. Black, red, light blue, yellow, dark blue, brown and pink colors spectra correspond to 0, 50, 100, 150, 250, 300, 350 and 400 mmol/L acrylamide. C and D: Stern Volmer representation for acrylamide attenuated 5G4 mAb and Herceptin. Different batches of each mAb are shown. 5G4 mAb batch 1 (black), 5G4 mAb batch 2 (red), 5G4 mAb batch 3 (blue), Herceptin batch 1 (green) and Herceptin batch 2 (pink).
Table 3.
Stern-Volmer constant values (Ksv) for 5G4 mAb and Herceptin.