Autoprotease Npro: Analysis of self-cleaving fusion protein

• Analytical method to quantify self-cleaving fusion proteins.
• Preparation of standards of un-cleaved fusion protein.
• Determination of LLOQ and LLOD.
• Resolution on 3 μm particles to resolve all proteins species of fusion proteins.

A reversed phase high pressure liquid chromatography method was developed for determination of in vitro refolding and cleavage kinetics for the Npro autoprotease fusion peptide EDDIE-pep6His using a TSK Super-Octyl column with a segmented acetonitrile gradient. Self-cleaving fusion proteins such as Npro autoprotease fusion proteins consist of the single autoprotease Npro and a target peptide or a target protein as fusion partner. Hence, three protein species are present after self-cleavage: the target peptide or protein, the single Npro autoprotease and, in case of incomplete cleavage, residual Npro fusion protein. Thus, for an accurate analysis the method must be standardized for three components in the presence of host cell impurities. For method validation, protein standards of EDDIE-pep6His and the single Npro autoprotease EDDIE were prepared from inclusion bodies (IBs) by ion exchange, immobilized metal ion affinity, size exclusion, and reversed phase chromatography. A linear correlation was obtained for EDDIE-pep6His and EDDIE in the range from 95 to 730 μg/ml with a lower limit of quantification (LLOQ) and a lower limit of detection (LLOD) of 34.5 and 11.4 μg/ml, respectively, for EDDIE-pep6His and 39.6 and 13.1 μg/ml, respectively, for EDDIE. Finally, a fully automated batch refolding of EDDIE-pep6His from IBs was performed to demonstrate the applicability of this method. It was shown that the initial EDDIE-pep6His concentration in the refolding solution decreased from 194.3 to 83.8 μg/ml over a refolding time of 385 min resulting in a final refolding and cleavage yield of 50%.