Label-free Quantification of Host-Cell Protein Impurity in a Recombinant Hemoglobin Reference Material

Quantitative analysis depends on pure-substance primary calibrators with known mass fractions of impurity. The datasets included were generated using mass spectrometry for the evaluation of label-free quantification (LFQ) as a method for determining the mass fraction of host-cell proteins (HCPs) in bioengineered proteins. For this purpose, hemoglobin-A2 (HbA2) was used, as obtained by overexpression in E.coli. Two different materials had been produced: natural, and U-15N-labeled HbA2. To quantify impurity, precursor ion (MS1-) intensities were integrated over all E.coli-proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E.coli-cell lysate, that had been spiked at known mass-ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and were found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of nine proteins.