High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• High-throughput characterization of intact antibodies is crucial for biopharmaceutical development.
• Conventional liquid chromatography-mass spectrometry (LC-MS) methods present throughput limitations.

Purpose of the Study:

• To develop a novel, rapid assay for high-throughput mass characterization of intact antibodies.
• To improve the speed and accuracy of antibody species analysis, including bispecific antibodies.

Main Methods:

• Development of a RapidFire-mass spectrometry (MS) assay utilizing electrospray ionization.
• Application of RapidFire-time-of-flight MS for screening and characterization of antibody samples.
• Utilized affinity-RapidFire-MS for analysis of antibodies in complex plasma samples.

Main Results:

• Achieved unprecedented screening throughput of 15 seconds per sample, an order of magnitude faster than LC-MS.
• Obtained intact mass determination accuracy as high as 7 ppm with baseline resolution at the glycoform level.
• Successfully characterized and quantified antibody species from 248 samples, enabling selection of high-purity bispecific antibody clones.
• Demonstrated correlation with conventional LC-MS results and qualified analysis in complex plasma samples.

Conclusions:

• The developed RapidFire-MS assay provides a high-throughput platform for analyzing intact antibodies, including bispecific variants and potential mispaired products.
• This method significantly enhances the efficiency of antibody characterization in complex matrices like cell culture media and plasma.
• The assay facilitates rapid clone selection and quality control in biopharmaceutical research and development.