Api electrospray single quadrupole msd

Characterization of PEP40233 was performed to determine purity, isoelectric point (pI) and thermal stability, prior to performing biodistribution studies. The lyophilized molecule was dissolved in Dulbecco’s phosphate-buffered saline (DPBS) to a concentration of 1 mg/mL. To determine the purity, a sample of PEP40233 was loaded on an RP-UPLC column (Agilent Zorbax 300 SB-C8 RRHD; 1.8 µm, 2.1 × 100 mm column) and eluted by a linear gradient of acetonitrile from 25–50% in 0.1% TFA during 8.3 min at a flow rate of 0.5 mL/min, at 40 ℃. The molecular mass of the construct was determined using mass-spectrometry (API electrospray single quadrupole MSD, Agilent Technologies, Santa Clara, CA, USA). Isoelectric focusing was applied to determine the pI of PEP40233 (Novex™ pH 3-10 IEF Protein Gels, ThermoFisher, Manassas, MA, USA). The reversible folding ability of PEP40233 after heating to 90 ℃ was analyzed by circular dichroism spectroscopy (Jasco J-810 spectropolarimeter, Jasco Scandinavia AB, Mölndal, Sweden).

The characterization of ABY-027 and ABY-271 was performed to determine thermal stability, purity and isoelectric point (ABY-271) before performing biodistribution studies. The lyophilized molecules were dissolved in PBS containing 0.5 mM EDTA to a concentration of 1 mg/mL.
The molecules were characterized by Circular Dichroism (Jasco J-810 spectropolarimeter, Jasco Scandinavia AB) for reversibility of structure after heating to 90 °C, and with RP-UPLC (Agilent Zorbax 300 SB-C8 RRHD; 1.8 µm, 2.1 × 100 mm column). Elution by linear gradient of acetonitrile from 25–50% in 0.1% TFA during 8.3 min at a flow rate of 0.5 mL/min and at 40 °C. The correct molecular mass of the construct was confirmed using mass-spectrometry (API electrospray single quadrupole MSD, Agilent Technologies, Santa Clara, CA, USA). Isoelectric focusing was used to determine the isoelectric point for ABY-271 (Novex™ pH 3-10 IEF Protein Gels, ThermoFisher, Manassas, MA, USA).