Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography

Abstract

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the author's laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed.

A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance

“…The workflow developed in this study was based on a fast (1-hour) and robust LC-MS /MS method that used charge-surface hybrid (CSH) 7,24 C18 UHPLC interfaced with a mass spectrometer with fast scanning speed (MS/MS at 20Hz). The average relative standard deviation for retention time of 5 different peptides from 5 LC-MS analyses collected over 2 months was 1.1%, indicating good reproducibility of the LC separation.…”

“…To address these challenges, methods using advanced liquid chromatography (LC) and mass spectrometry (MS) techniques have been developed, most of which focus on detecting low level HCPs in the final monoclonal antibody (mAb) products. [6][7][8][9][10][11] However, there is currently an unmet need to provide high throughput and robust LC-MS/MS analysis of HCPs in various in-process pools to help design and optimize purification processes toward optimal clearance.…”

“…One DIA method, 19 MS E , has been coupled with two dimensional liquid chromatography (2D-LC) to identify and quantify low abundance HCPs, using the "Top 3 00 label-free approach (MS signal response for the three most intense tryptic peptides) for protein quantitation. 7,8,10,20,21 However, 2D-LC analysis may have reproducibility challenges and the nano-LC often used in the second dimension for improved sensitivity is not always robust. In addition, its 10-20 hours analysis time/sample makes it challenging to analyze multiple samples from in-process pools using the 2D-LC-MS/MS approach to provide real time support and quick response for addressing potential HCP concerns.…”

The growing role of two-dimensional LC in the biopharmaceutical industry

“…As part of an effort to help users understand the conditions under which 2D-LC provides more resolving power than 1D-LC separations for peptide analyses, the Heinisch group recently demonstrated that the peak capacity of 2D-LC separations is superior to that of 1D-LC separations for analysis times longer than about five minutes [18]. In the area of Host Cell Protein (HCP) analysis, Donenau and coworkers have demonstrated the use of heartcutting 2D-LC [19,20] and offline comprehensive 2D-LC [21] to detect trace-level peptide markers of HCPs. This is very important to quality assurance for biotherapeutic mAbs.…”

Improved Immuno-Detection of a Low-Abundance Cyclophilin Allows the Confirmation of its Expression in a Protozoan Parasite

“…Efforts have been made in characterizing protein profile of biological samples such as placenta [17], urine [18] plants [19], etc. The techniques applied to this goal are diverse and include protein depletion and mass spectrometry [17,20], improved fractionation and purification methods [18], high-resolution NMR [21] among others.…”