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28 BioPharm International eBook July 2021 www.biopharminternational.com high throughput. This high-through- put screening does not need the same levels of sensitivity as upstream analysis, but quantitation is often necessary using robust instrumentation. HCP IDENTIFICATION WITH CONFIDENCE To overcome the ongoing challenge of dynamic range in HCP analysis with MS, many research groups have focused on optimizing sample prepa- ration procedures to increase the num- ber of peptides identified. Huang et al. developed a novel native digest tech- nique that removes monoclonal anti- bodies (mAbs) by centrifugation before MS–MS analysis, allowing the identi- fication of additional HCPs compared with traditional trypsin digest meth- ods (7). Alternative methods to native digest have been established, such as h y d r o p h i l i c i n t e r a c t i o n c h r o - m a t o g r a p h y ( H I L I C ) f r a c t i o n- ation proposed by Wang et al., that have enabled the identif ication of novel HCPs (8). The depth of HCP identif ication provided by PASEF technology with TIMS QTOF-MS has been demon- strated using the native digest sample preparation method, and the HCP identifications have been compared to those reported by Huang et al. (native digest) and Wang et al. (native digest and HILIC fractionation). Following the separation of approximately 600 ng of the NISTmAb native digest sample with the Evosep One using the 88-min gradient, 167 HCPs were identif ied from a single dataset, encompassing 79% (90/114) and 87% (52/60) of the HCPs listed in Wang et al. (8) and Huang et al. (7), respectively. In addition, a further 70 NISTmAb HCPs were identified, which have not been reported elsewhere. In most cases, TIMS with PASEF enabled the detection of more peptides, allowing the identification of low abun- dant HCPs with greater confidence. As an example, Figure 1 shows the HCPs identified with two peptides by Huang et al. (Figure 1A) and Wang et al. (Figure 1B) compared with the number of pep- Biopharmaceutical Analysis Impurity Analysis Figure 1. Number of unique peptides identified for host cell proteins (HCPs) using (A) native digest and UHPLC-MS–MS (yellow) and TIMS QTOF-MS–MS (blue); and (B) HILIC fractionation and UPLC-MS–MS (orange) and TIMS QTOF-MS–MS (blue). Five HCPs reported with two peptides by Huang et al. (n=4) (7) and Wang et al. (n=1) (8) were not detected by TIMS QTOF- MS (data not shown). UHPLC is ultra-high performance liquid chromatography, MS is mass spectrometry, TIMS is trapped ion mobility spectrometry, QTOF is quadrupole time-of-flight, HILIC is hydrophilic interaction chromatography. - confidential - HCP 1 Semaphorin-7A 2 Bifunctional glutamate/proline--tRNA ligase 3 Splicing factor 3A subunit 1 4 Malate dehydrogenase mitochondrial 5 Cleavage and polyadenylation specificity factor subunit 5 6 Non-specific lipid-transfer protein 7 Stress-induced-phosphoprotein 1 8 Cathepsin D 9 Protein LYRIC 10 Serine/arginine-rich splicing factor 1 11 Glutathione S-transferase P 1 12 IgE-binding protein 13 Ataxin-2 14 Cytokine receptor common subunit gamma HCP 1 Bifunctional glutamate/proline--tRNA ligase 2 Elongation factor 1-alpha 1 3 Protein PRRC2C 4 Ribosome-binding protein 1 5 Peroxiredoxin-5 mitochondrial 6 ELAV-like protein 1 7 Heterogeneous nuclear ribonucleoprotein H2 8 Histidine--tRNA ligase cytoplasmic 9 Golgi SNAP receptor complex member 2 10 THO complex subunit 4 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Number of unique peptides HCP Unique pept ides tim sT OF Pro Unique pept ides Huang et al 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 Number of unique peptides HCP Unique pept ides tim sT OF Pro Unique pept ides Wa ng et al A B FIGURES COURTESY OF THE AUTHOR.