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. 2024 Sep 7;25(17):9687.
doi: 10.3390/ijms25179687.

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs

Reiko Kiyonami  1 , Rafael Melani  2 , Ying Chen  3 , A I De Leon  3 , Min Du  1
Affiliations

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs

Reiko Kiyonami et al. Int J Mol Sci. .

Abstract

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Keywords: HPLC MS/MS method; host cell proteins; process related HCP profiling.

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Conflict of interest statement

All authors were employed by the company Thermo Fisher Scientific. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Workflow of the developed UHPLC MS/MS method for HCP analysis.
Figure 2
Figure 2
TIC of triplicate LC-MS runs of NISTmAb digest mixture.
Figure 3
Figure 3
NISTmAb HCP id number comparison using one and two search algorithms.
Figure 4
Figure 4
Identified HCPs with the concentration information shown in [29]. Most of them (>65%) were less than 1 ppm.
Figure 5
Figure 5
Information on the trastuzumab samples.
Figure 6
Figure 6
Comparison of the identified HCPs and unique peptides in the two samples collected before and after polish purification.
Figure 7
Figure 7
Identified high-risk HCPs in the two samples.
Figure 8
Figure 8
Identified difficult-to-remove HCPs in the two samples.
Figure 9
Figure 9
Representative high-risk HCP clearance trends.
See this image and copyright information in PMC

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