In Depth Analysis of Host Cell Proteins from Antibody Preparations using PASEF

Importance of the Topic

In biotherapeutic development and quality control, the detection and characterization of host cell proteins (HCPs) at low parts-per-million levels is essential to ensure product safety and efficacy. While enzyme-linked immunosorbent assays (ELISAs) remain the standard for routine monitoring, mass spectrometry (MS) offers a non-targeted alternative that can rapidly identify and quantify a broad range of impurities without bespoke assay development.

Objectives and Study Overview

This study demonstrates the application of parallel accumulation and serial fragmentation (PASEF) on the timsTOF Pro ion mobility QTOF platform to enhance the sensitivity, speed, and depth of HCP analysis in antibody preparations. Two workflows are explored: a routine analytical-flow method and a high-sensitivity nanoflow approach, each benchmarked using NISTmAb and the UPS1 standard.

Methodology

Peptide samples were prepared by reduction with DTT, alkylation with iodoacetamide, and overnight tryptic digestion. For analytical-flow experiments, peptides from NISTmAb and UPS1 were separated on a 2.1 mm × 100 mm C18 column with a 150-minute gradient on an Elute UHPLC. Nanoflow analyses employed a 75 µm × 250 mm C18 column on a nanoElute UHPLC with a 210-minute gradient. PASEF scans were acquired on the timsTOF Pro, and MS/MS spectra were searched against a mouse SwissProt database via Mascot at 1% FDR.

Instrumentation Used

• Bruker Elute UHPLC and nanoElute UHPLC systems
• Intensity Solo 2 C18 analytical column (1.8 µm, 100 × 2.1 mm)
• IonOpticks C18 nanoflow column (1.6 µm, 25 cm × 75 µm)
• timsTOF Pro ion mobility QTOF mass spectrometer with CaptiveSpray source

Main Results and Discussion

Using a UPS1 dilution series in a fixed NISTmAb background (25 µg per injection), PASEF enabled linear quantitation of spiked proteins down to low single-digit ppm, demonstrating high sensitivity in the analytical-flow configuration.
In routine 1D UHPLC-MS/MS, eight HCPs were identified with three or more peptides and ten additional proteins with two peptides (1% FDR), including key enzymes such as glucose-6-phosphate isomerase.
Nanoflow analyses of 1.5 µg NISTmAb yielded over 200 HCP identifications at 1% FDR, with 78 proteins detected by at least two peptides. This deeper coverage revealed both expected and previously unreported trace-level HCPs.
TIMS separated ions by collisional cross section, and PASEF selected precursors in a two-dimensional space (m/z and mobility), achieving sequencing speeds above 100 Hz and high-quality MS/MS spectra for confident protein identification.

Benefits and Practical Applications

PASEF-enhanced workflows offer rapid method development, comprehensive impurity profiling, and sensitive detection suitable for sub-100 ppm HCP monitoring. High-confidence protein IDs can be achieved even from low-abundance peptides, supporting process fingerprinting and enabling swift detection of biomanufacturing changes.

Future Trends and Applications

Advances in ion mobility and PASEF are expected to drive further sensitivity and depth in HCP profiling. Integration with automation and real-time data analysis, as well as coupling to quality-by-design strategies, will expand the role of MS-based impurity monitoring in regulatory environments and process development.

Conclusion

The combination of TIMS-PASEF on the timsTOF Pro with both analytical- and nanoflow LC establishes a robust platform for sensitive, high-throughput HCP analysis. This approach meets and exceeds current impurity monitoring requirements, delivering deep proteome coverage and rapid turnaround for biotherapeutic quality control.

References

• Meier et al.; Journal of Proteome Research. 2015 Dec 4;14(12):5378-5387.
• Lubeck et al.; Bruker Application Note LCMS 131, PASEF™ on timsTOF Pro, September 2017.