Techniques for Quantitative LC–MS/MS Analysis of Protein Therapeutics: Advances in Enzyme Digestion and Immunocapture

Abstract

LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.

Immunocapture sample clean-up in determination of low abundant protein biomarkers – a feasibility study of peptide capture by anti-protein antibodies

“…1 Despite its unique mass selectivity, sensitive detection of intact proteins by MS is limited. The protein biomarker is thus usually digested by a proteolytic enzyme to generate proteotypic peptides that are analyzed by LC-MS. 2,3 The choice of the surrogate peptide, also known as the signature peptide or proteotypic peptide, used for the quantitation of the entire protein is crucial for the quality of the assay and should thus be unique to the targeted protein. [2][3][4] Among the most promising methods to ensure high sensitivity and selectivity in targeted analysis of low-abundant protein biomarkers is a combination of immunoaffinity enrichment (immunocapture), protein digestion and LC-MS/MS detection of signature peptides.…”

“…[1][2][3][4][5][6] Immunocapture can be performed both prior to and aer the protein digestion step, and provides unique selectivity through the unique interaction of the target protein or signature peptide and the capture antibody. 2 The antibodies used for immunocapture target either linear or conformational epitopes. Linear epitopes consist of continuously neighbouring amino acid residues along the protein sequence, while conformational epitopes consist of amino acid residues that are discontinuously arranged along the protein sequence, that are brought together through folding of the polypeptide chain.…”

Sequential immunoaffinity-LC/MS assay for quantitation of a therapeutic protein in monkey plasma

“…The combination of immunocapture with LC/MS not only can address the selectivity issues affecting LBA, but also add significant benefits such as multiplexing (multiple analytes in one assay; same assay for multiple species/matrices), fast method development, wide dynamic range (up to 4 orders of magnitude), and good robustness and reproducibility. For most immunocapture-LC/MS analysis of biotherapeutics, the immunocapture step is performed at the protein level [17]. It takes advantage of the unique immunoaffinity of the biotherapeutic and the capture agent, and thus provides unique selectivity.…”

2D-LC–MS/MS to Measure Cleaved High-Molecular-Weight Kininogen in Human Plasma as A Biomarker for C1-Inh-Hae

“…To improve assay sensitivity for target protein, IP in conjunction with LC-MS/MS is widely used [29][30][31]41]. A variety of commercially available anti-HMWK, antilight chain and antiheavy chain antibodies were assessed as IP reagents but none was able to efficiently pull down the targeted proteins (data not shown).…”