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www.biopharminternational.com July 2021 BioPharm International eBook 29 tides identified for each by the timsTOF Pro (Bruker Daltonics, Germany). Of the 70 additional HCPs iden- tified by TIMS QTOF-MS–MS, 46 were detected with two or more pep- tides, illustrating the greater depth of HCP coverage. Collisional cross section (CCS) val- ues are determined using gas-phase separation provided by TIMS. This physical property of molecules can be used as an extra parameter to confirm the presence of low abundant peptides even when the MS–MS spectra are low quality or when the precursor ion intensity is too low to be selected for MS–MS. Because the CCS values are measured in the gas phase, they are more reproducible than LC retention times (RT). Innovative MS instrumen- tation is now enabling users to combine HCP discovery and screening in one workf low, and accurately and repro- ducibly measure CCS values. Highly reproducible CCS values eliminate the need to include RT in confidence measurements, enabling easy changing of LC methods, columns, f lows, and other instrument parameters. Recent research has demonstrated that the sensitivity of HCP detection can be extended by a factor of approx- imately 5–10 by using CCS va lues with accurate mass to identify ultra- low abundant precursors which do not trigger MS–MS (9). In this study, two peptides for collagen α-1(V)—a known HCP—were identif ied when 30 and 10 µg were loaded on column but only one was identif ied by MS– MS when the sample amount was reduced to 2 µg. Trace levels of the unidentif ied precursor were detected in the extracted ion chromatograms (EICs) from the 2 µg sample, and using the combination of accurate mass and CCS, the peptide identity was confirmed. To further illustrate the use of CCS to extend the limit of detection for HCPs, a Chinese hamster ovary (CHO) reference sample consisting of non-transfected CHO cells in medium was analyzed. Using a 40-minute gradient, approxi- mately 11,000 peptides were identified for 3000 CHO HCPs. CCS values were recorded for each peptide, allowing ret- rospective analysis of the mAb 30 µg dataset, which revealed that a third pep- tide for collagen α-1(V) could also be identified by mass-to-charge ratio (m/z) and CCS (Figure 2). A QUANTITATIVE APPROACH In addition to identifying HCPs, TIMS QTOF-MS–MS can quantify proteins across several orders of magnitude. To demonstrate this, a 200 ng NISTmAb sample was spiked with 4 ng Universal Protein Standard (UPS2) mix of 48 pro- teins at six different concentration lev- els, and quantified using nanoLC TIMS QTOF-MS–MS (nanoElute and tim- sTOF Pro, Bruker Daltonics) with a 100 min gradient. Figure 3 shows the linear response of the theoretical abun- dance versus the measured abundance of quantified UPS2 Proteins in NISTmAb background. The high degree of lin- Biopharmaceutical Analysis Impurity Analysis Figure 2. Mass spectrometry (MS)1 (top) and collisional cross section (CCS) (bottom) detection of collagen α-1(V) peptide K.TVLEIDTPK.V in Chinese hamster ovary (CHO) reference sample (red) and monoclonal antibody (mAb) sample 30 µg on column (blue). MS2 data was not available for this peptide in the mAb sample but identification was possible after screening for mass-to-charge ratio (m/z) and CCS values of the peptide observed in the CHO reference sample. Biopharma labs cannot afford downtime, so the robustness of modern mass spectrometers is another significant advantage.