Identification and quantification of host cell protein impurities in biotherapeutics using mass spectrometry

doi:10.1016/j.ab.2012.05.018

Analytical Biochemistry

Volume 428, Issue 2, 15 September 2012, Pages 150-157

https://doi.org/10.1016/j.ab.2012.05.018 Get rights and content

Abstract

Residual host cell proteins (HCPs) in biotherapeutics can present potential safety risks to patients or compromise product stability. As such, their levels are typically monitored using a multicomponent HCP enzyme-linked immunosorbent assay (ELISA) to ensure adequate removal. However, it is not possible to guarantee ELISA coverage of every possible HCP impurity, and the specific HCPs remaining following purification are rarely identified. In the current study, we characterized the ability of an advanced two-dimensional liquid chromatography/mass spectrometry platform (2D–LC/MS^E) to identify and quantify known low-level spiked protein impurities in a therapeutic peptide Fc fusion protein. The label-free quantification procedure based on the “top 3” intensity tryptic peptides per protein was applied and improved on for this application. Limits of detection for unknown HCPs were approximated from the spiked protein data along with estimates for the quantitative accuracy of the method. In all, we established that most protein impurities present at 13 ± 4 ppm can be identified with a quantitative error of less than 2-fold using the more sensitive of two tested method formats. To conclude the study, we characterized all detectable Escherichia coli proteins present in this Fc fusion protein drug substance and discuss future applications of the method.

Section snippets

Reagents and consumables

The yeast proteins hexokinase (HXKB), glucose-6-phosphate dehydrogenase (G6PD), glucose-6-phosphate isomerase (G6PI), and inorganic pyrophosphatase (IPYR) were purchased from Roche Diagnostics (Mannheim, Germany). Additional yeast proteins enolase (ENO1), triosephosphate isomerase (TPIS), and alcohol dehydrogenase (ADH1) and bovine proteins carbonic anhydrase (CAH2), lactotransferrin (TRFL), α-lactalbumin (LALBA), serum albumin (ALBU), and serotransferrin (TRFE) were purchased from

Results and discussion

In this study, we sought to establish the percentage of known low-level protein impurities that could be identified in peptibody DS and understand whether those proteins could be accurately quantified.

Conclusions

In this study, we investigated the performance characteristics of an advanced LC/MS method, 2D–LC/MS^E, in detecting and quantifying protein impurities in therapeutic DS. We established approximate detection limits for unknown HCPs in DS, and we identified and subsequently remedied factors contributing to top 3 peptide-based quantification error. Taken together, our data indicated that most a priori unknown HCPs should be quantifiable within 2-fold of their actual value, a level of accuracy that

Acknowledgments

We thank Martha Stapels, Keith Fadgen, and Catalin Doneanu of Waters (Milford, MA, USA) for 2D–LC/MS^E training and carrying out an initial feasibility study of ECP detection in DS. We also thank the members of the Material and Metabolic Sciences and Small Molecule Process and Product Development groups at Amgen for nearly uninterrupted use of the Synapt mass spectrometer.

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