Improved identification of host cell proteins in a protein biopharmaceutical by LC-MS/MS using the ProteoMiner™...

Host cell proteins (HCPs) in biotherapeutics can be identified by the use of enzymatic digestion and LC-MS/MS analysis. However, the major challenge is that HCPs are often present at very low levels in relation to the protein drug (low ppm-levels). In this study, the ProteoMiner™ Enrichment Kit (Bio-Rad) was evaluated as a strategy to enable identification of HCPs by LC-MS/MS by enrichment of low-abundant HCPs and a simultaneous depletion of the high-abundant product protein. A recombinant protein produced in Chinese hamster ovary (CHO) cells was spiked with six standard proteins at varying concentrations (10-1000 ppm). The sample was split into two aliquots; one that was prepared with the ProteoMiner™ Enrichment Kit and one control, where the enrichment procedure was omitted. The ProteoMiner™ Enrichment Kit was combined with the ProteoMiner Sequential Elution Large-Capacity Kit (Bio-Rad), eluting the proteins into four fractions. The samples were then digested with trypsin and analyzed with LC-MS/MS. In addition, multiple reaction monitoring (MRM) was applied to obtain an estimate of the protein abundance of HCPs and spiked proteins. The results demonstrated that with the untargeted LC-MS/MS method, 30 HCPs and four of the six spiked standard proteins were identified in the four fractions. The spiked standard proteins were identified down to 30 ppm in the ProteoMiner treated samples, while no HCPs and only the most abundant standard protein (≈1000 ppm) were identified in the non-enriched control sample. MRM assays were developed for 14 out of the 30 identified HCPs. All targeted HCPs and five of the six standard proteins were detected in all fractions as well as in the control sample by MRM. There was an acceptable agreement between estimated concentrations of spiked standard proteins and expected values. An 80-700 fold enrichment of individual HCPs was observed in the fractions. In conclusion, the results clearly demonstrated that the ProteoMiner technology can be used for enriching HCPs in biotherapeutics, enabling their identification by LC-MS/MS.