Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation

Abstract

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing “fit-for-purpose” bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma.

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

“…First, in silico trypsin digestion of IgG1-4 was performed to generate a list of potential peptide candidates. Peptide size was limited to 6 to 20 amino acid resides which was found to be the right balance between assay specificity/selectivity and sensitivity [22]. The surrogate tryptic peptide must also be a universal peptide and present in all IgG1-4 isotypes, so that it could be used to detect the total of IgG1-4.…”

“…It was thus evident that the mouse mAb was indeed able to capture Protein XYZ regardless of whether Protein XYZ was in ADA-Protein XYZ complexes or free form. The primary goal of this study was to explore the use of LC/MS for detecting monkey ADA, an effort continued from our previous work on LC/MS for detection of pre-existing ADA in human plasma [22]. Assay validation such as accuracy and precision assessment was not part of this preliminary assay development.…”

“…In this study, the immunocapture-LC/MS assay overcame drug interference and thus presents the potential as an alternative technology platform for ADA detection in the presence of high drug levels. The monkey assay was similar to the human ADA assay we have reported [22] but using a monkey-specific peptide for the detection of monkey ADA. Therefore, we demonstrated that LC/MS approach can be used for evaluation of immunogenicity in different species.…”

“…Resultant peptide from the target therapeutic protein was quantified by LC coupled with matrix-assisted laser desorption MS and the results were correlated to the binding capacity of total ADA. Recently, we reported an immunocapture-LC/MS methodology for simultaneous ADA isotyping and semi-quantitation in human plasma [22]. Briefly, ADA or ADA-drug complex was captured by biotinylated drug or anti-drug Ab on streptavidin magnetic beads and separated from human plasma by a magnet.…”

“…The LC/MS assay and a new ECL assay were developed in parallel to overcome the drug interference issue and results from the two assays were compared. Similar to the human ADA assay [22], a monkey-specific universal peptide was identified and used as the surrogate for the detection of monkey ADA Ig1-4. To our knowledge, this is the first case study using immunocapture-LC/MS for direct ADA detection in support of clinical or nonclinical studies.…”