Rapid global characterization of immunoglobulin G1 following oxidative stress

ABSTRACT

Although peroxide and leachable metal-induced chemical modifications are among the most important quality attributes in bioprocess development, there is no mainstream characterization method covering all common modifications theoretically possible on therapeutic proteins that also gives consistent results quickly. Here, we describe a method for rapid and consistent global characterization of leachable metals- or peroxide-stressed immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). Using two independent protease digestions, data-independent acquisition and data-dependent acquisition liquid chromatography high-resolution mass spectrometry, we monitored 55 potential chemical modifications on trastuzumab, a humanized IgG1 mAb. Processing templates including all observed peptides were developed on Skyline to consistently monitor all modifications throughout the stress conditions for both enzymatic digestions. The Global Characterization Data Processing Site, a universal automated data processing application, was created to batch process data, plot modification trends for peptides, generate sortable and downloadable modification tables, and produce Jmol code for three-dimensional structural models of the analyzed protein. In total, 53 sites on the mAb were found to be modified. Oxidation rates generally increased with the peroxide concentration, while leachable metals alone resulted in lower rates of modifications but more oxidative degradants. Multiple chemical modifications were found on IgG1 surfaces known to interact with FcɣRIII, complement protein C1q, and FcRn, potentially affecting activity. The combination of Skyline templates and the Global Characterization Data Processing Site results in a universally applicable assay allowing users to batch process numerous modifications. Applying this new method to stability studies will promote a broader and deeper understanding of stress modifications on therapeutic proteins.

KEYWORDS:

• Global characterization
• leachable metals
• peroxides
• oxidative stress
• post-translational modifications (PTM)
• monoclonal antibody (mAb)
• immunoglobulin G1 (IgG1)
• quantitative proteomics
• high-resolution mass spectrometry (HRMS)
• data-independent acquisition (DIA)
• data-dependent acquisition (DDA)
• processing automation
• proteomics

Acknowledgments

The authors greatly appreciate the cell culture and purification teams who supplied the trastuzumab for analytical studies at Catalent Biologics, Bloomington. We appreciate Bettina Kehoe, who graciously proofread, and grammar edited this manuscript. We are also grateful that Brendan MacLean and Nicolas Shulman of the University of Washington upgraded the Skyline maximum number of FASTA imported peptides to 200,000 and the maximum number of transitions to 5,000,000, facilitating data processing.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.