A major portion of biopharmaceuticals today are produced by recombinant DNA technology using well-selected host cell systems. Even after sophisticated purifications steps, low-levels (1-100 ppm) of host cell proteins (HCPs) remain in the final purified drug substance. Some of the HCPs may cause immunogenic reaction in humans, therefore it is critical for patient safety that HCPs be identified and quantified. The analytical methods typically used for HCP quantification are based on immunoassays (ELISA), but ELISA cannot provide proteome-wide coverage.
In recent years, LC/MS-based assays have been adopted as orthogonal techniques to ELISA for HCP analysis due to their flexibility and sensitivity. Here we describe an efficient analytical scale LC/MS assay that allows the identification and quantification of HCPs during mAb purification in a CHO cell line. An antibody product, purified by Protein A affinity chromatography followed by SCX (strong cation exchange) chromatography using different elution conditions, was denatured, reduced, alkylated and digested with trypsin overnight.
The LC/MS assay described here relies on a novel data-independent acquisition mode recently implemented on a quadrupole/time-of-flight (QTOF) mass spectrometer, namely SONAR data acquisition. Instead of transmitting all peptide ions produced by the electrospray source, in SONAR mode the quadrupole slides over the mass range of interest during the time required for recording a single MS spectrum by the TOF analyzer. The precursor ions produced by co-eluting peptides are separated by the quadrupole and their MS/MS fragmentation spectra are recorded individually. SONAR acquisition offers additional selectivity, by producing fragmentation spectra with minimized background interferences.
Seven HCPs were identified across 5 mAb preparations following a CHO database search using Progenesis QIP 4.0 software. The same software was also used to build a database of CHO HCPs including peptide RT, precursor m/z and MS/MS fragmentation spectra for facilitating subsequent HCP identification and quantification in other mAb preparations.