Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC -MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is prom-ising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

Automated summary

Liquid chromatography coupled to mass spectrometry (LC-MS) is utilized for host cell proteins (HCP) identification in antibody drug development, but its application in prokaryotic-derived growth hormone (GH) samples is rare. In this study, a universal workflow combining optimized sample preparation and one-dimensional ultra-high performance LC-MS-based shotgun proteomics was developed to profile HCP in GH samples, providing guidance for purification process development and biosimilar development. A standard-spiking strategy was also developed to enhance HCP identification, enabling trace-level HCP analysis. These protocols open avenues for HCP profiling in biotherapeutics derived from prokaryotic host cells.