Assessment of stable isotope incorporation into recombinant proteins
Section snippets
Materials
Both the mAb (IgG2 subtype) and the Fc fusion protein were produced and purified at Amgen (Thousand Oaks, CA, USA).
All labeled raw materials, including 13C-labeled glucose (13C6–glucose, 98% 13C6), 15N-labeled amino acids (at all N positions), and 15N-labeled Celtone medium, were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Endoproteinase trypsin and Glu-C were obtained from Roche Diagnostics (Penzberg, Germany). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were
Results
Several analytical tools have been used to assess the quality of the stable isotope-labeled recombinant proteins. Purity methods (SE–HPLC, CEX–HPLC, and rCE–SDS) were applied to assess the chromatographic and/or electrophoretic properties of the stable isotope-labeled proteins. The levels of stable isotope incorporation were assessed by total mass analysis, peptide mapping, and amino acid analysis.
Discussion
There has been an increasing number of reports describing methodologies using stable isotope labeling for different purposes, traditionally for NMR studies [32], [38] and more recently for quantification of protein and protein isoforms [7], [9], [10], [11], [12], [13], [14], [15], [16].
Stable isotopes vary in their utility as labeling agents. In life sciences, the most used stable isotopes include 2H (deuterium), 13C, 15N, and 18O. Although deuterium is inexpensive and relatively easy to
Acknowledgment
We are very grateful to Brad Jordan for the production of the stable isotope-labeled Fc fusion protein sample.
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