Related Experiment Video
Updated: Sep 16, 2025
Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Published on: February 27, 2020
Two-Cycle Immunoaffinity Enrichment Strategy with Acid Treatment to Enhance Biotherapeutics Assay Sensitivity in
Yipei Zhang1, Mark G Qian1, Xuemei Yang1
1Department of Drug Metabolism and Pharmacokinetics & Modeling, Takeda Development Center Americas, Inc., 35 Landsdowne Street, Cambridge, Massachusetts 02139, United States.
Accurate and sensitive bioanalysis of therapeutic antibodies and proteins is crucial for ensuring reliable pharmacokinetic and toxicokinetic assessments. Immunoaffinity (IA) LC/MS/MS plays an important role in quantifying therapeutic antibodies in biological matrices due to its high specificity and less stringent reagent requirements compared to ligand binding assays. However, when analyzing complex tissue matrices, a single immunoaffinity enrichment cycle often leaves unwanted nonspecifically bound proteins in the final samples, resulting in high background noise levels and decreased assay sensitivity. To overcome this challenge, a novel two-cycle immunoaffinity LC-MS/MS method was evaluated for quantifying a mouse IgG2a antibody in mouse tumor, liver, and lung tissues. This method involves an additional automated immunoenrichment cycle after the standard initial cycle, effectively reducing nonspecific protein binding by 7.7-24-fold across three types of tissues. This straightforward high-throughput process successfully addresses the common issue of nonspecific binding in tissues and exhibits a significant 5-fold sensitivity improvement for the mIgG2a in mouse tumor and liver homogenates, achieved through a substantial decrease in background noise. Interestingly, no sensitivity improvement was observed among spiked lung homogenates, likely due to their inherently lower interference responses in the extracted MS/MS chromatograms. Calibration curve and quality control analyses have confirmed the method's precision and accuracy, demonstrating its potential to enhance tissue assay sensitivity without compromising intended exposure measurements. The simplicity and effectiveness of this method make it a valuable tool for studying tumor exposure and tissue distribution, requiring minimal effort for maximum impact.
Accurate and sensitive bioanalysis of therapeutic antibodies and proteins is crucial for ensuring reliable pharmacokinetic and toxicokinetic assessments. Immunoaffinity (IA) LC/MS/MS plays an important role in quantifying therapeutic antibodies in biological matrices due to its high specificity and less stringent reagent requirements compared to ligand binding assays. However, when analyzing complex tissue matrices, a single immunoaffinity enrichment cycle often leaves unwanted nonspecifically bound proteins in the final samples, resulting in high background noise levels and decreased assay sensitivity. To overcome this challenge, a novel two-cycle immunoaffinity LC-MS/MS method was evaluated for quantifying a mouse IgG2a antibody in mouse tumor, liver, and lung tissues. This method involves an additional automated immunoenrichment cycle after the standard initial cycle, effectively reducing nonspecific protein binding by 7.7-24-fold across three types of tissues. This straightforward high-throughput process successfully addresses the common issue of nonspecific binding in tissues and exhibits a significant 5-fold sensitivity improvement for the mIgG2a in mouse tumor and liver homogenates, achieved through a substantial decrease in background noise. Interestingly, no sensitivity improvement was observed among spiked lung homogenates, likely due to their inherently lower interference responses in the extracted MS/MS chromatograms. Calibration curve and quality control analyses have confirmed the method's precision and accuracy, demonstrating its potential to enhance tissue assay sensitivity without compromising intended exposure measurements. The simplicity and effectiveness of this method make it a valuable tool for studying tumor exposure and tissue distribution, requiring minimal effort for maximum impact.
