Two-Cycle Immunoaffinity Enrichment Strategy with Acid Treatment to Enhance Biotherapeutics Assay Sensitivity in...

Area of Science:

• Bioanalytical Chemistry
• Pharmacokinetics
• Mass Spectrometry

Background:

• Accurate bioanalysis of therapeutic antibodies is vital for pharmacokinetic and toxicokinetic assessments.
• Immunoaffinity LC-MS/MS offers high specificity for antibody quantification in biological matrices.
• Nonspecific protein binding in complex tissue matrices reduces assay sensitivity.

Purpose of the Study:

• To evaluate a novel two-cycle immunoaffinity LC-MS/MS method for quantifying a mouse IgG2a antibody in mouse tissues.
• To reduce nonspecific protein binding and improve assay sensitivity in tissue bioanalysis.

Main Methods:

• A two-cycle immunoaffinity LC-MS/MS method was developed and applied to mouse tumor, liver, and lung tissues.
• The method incorporates an additional automated immunoenrichment cycle to minimize nonspecific binding.

Main Results:

• Nonspecific protein binding was reduced 7.7-24-fold across tumor, liver, and lung tissues.
• A 5-fold sensitivity improvement was observed for mIgG2a in tumor and liver homogenates due to decreased background noise.
• No sensitivity improvement was noted in lung homogenates, likely due to lower inherent interference.

Conclusions:

• The two-cycle immunoaffinity LC-MS/MS method effectively reduces nonspecific binding in tissues.
• This straightforward, high-throughput approach enhances assay sensitivity for therapeutic antibody tissue exposure and distribution studies.
• The method demonstrates precision and accuracy, supporting reliable exposure measurements.