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

Abstract

A modular and adaptive mass spectrometry (MS)-based platform was developed to provide fast, robust and sensitive host cell protein (HCP) analytics to support process development. This platform relies on one-dimensional ultra-high performance liquid chromatography (1D UHPLC) combined with several different MS data acquisition strategies to meet the needs of purification process development. The workflow was designed to allow HCP composition and quantitation for up to 20 samples per day, a throughput considered essential for real time bioprocess development support. With data-dependent acquisition (DDA), the 1D UHPLC-MS/MS method had excellent speed and demonstrated robustness in detecting unknown HCPs at ≥ 50 ng/mg (ppm) level. Combining 1D UHPLC with sequential window acquisition of all theoretical spectra (SWATH) MS enabled simultaneous detection and quantitation of all HCPs in single-digit ng/mg range within 1 hour, demonstrating for the first time the benefit of SWATH MS as a technique for HCP analysis. As another alternative, a targeted MS approach can be used to track the clearance of specific known HCP under various process conditions. This study highlights the importance of designing a robust LC-MS/MS workflow that not only allows HCP discovery, but also affords greatly improved process knowledge and capability in HCP removal. As an orthogonal and complementary detection approach to traditional HCP analysis by enzyme-linked immunosorbent assay, the reported LC-MS/MS workflow supports the development of bioprocesses with optimal HCP clearance and the production of safe and high quality therapeutic biopharmaceuticals.

Keywords: Bioprocess development; SWATH; host cell protein; mass spectrometry.

Figure 1.

Typical host cell protein level during purification process for mAb biologics.

Figure 2.

Summary of the modular and adaptive 1D UHPLC-MS-based workflow to support fast bioprocess development for optimal host cell protein clearance.

Figure 3.

Proteins are consistently identified with ≥ 2 peptides above 50 ppm using 1D UHPLC-DDA. (A)UPS-1 (48) standard proteins ranging from 8 to 110 ppm were spiked into mAb-1. Dashed line indicates level of method sensitivity. Results from six replicate analyses showed 80–100% detection of ten proteins spiked in at above 50 ppm level. (B) Analysis of the mAb-2 sample spiked with 50 ppm PLBL2 (method control sample) in 69 test sessions over 18 months.

Figure 4.

The number of HCPs identified by 1D UHPLC-DDA in the Column 1 (Affinity) and Column 2 in-process pools of 16 mAb products. Many of the HCPs were common, as they were found in multiple mAb products. There were also significant number of HCPs that were unique to a specific mAb product.

Figure 5.

Comparison of DDA (5A) and DIA SWATH MS (5B) methods to detect PLBL2 at different spiked levels in mAb2. Green represents successful detection, while red indicates failure to detect. The detection sensitivity of DDA is shown in 5A, and the sensitivity and linearity of DIA SWATH MS TOP 3 method is shown in 5B, with the number of peptide identified shown in parentheses. PLBL2 peak area measurement by SWATH (Top 3 peptides) at ≥ 5 ppm levels had observed RSD values ranging from 2.1–5.8%, based on triplicate injections.

Figure 6.

Simultaneous relative quantitation of three HCPs in mAb-3 by 1D UHPLC-DIA SWATH MS (∼10 ppm sensitivity).