Application and Challenges in Using LC–MS Assays for Absolute Quantitative Analysis of Therapeutic Proteins in Drug Discovery

Abstract

As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC-MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC-MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC-MS to support protein quantification in drug discovery.

Strategies for improving sensitivity and selectivity for the quantitation of biotherapeutics in biological matrix using LC-MS/MS

“…The commercial available sequencing grade trypsin provides additional benefits of preventing autolysis, which could introduce interfering peptides, and retaining the enzymatic activity for a longer period. A few applications of other types of proteolytic enzymes have been summarized and published [18] previously. With the specific cleavage site of certain enzyme, different surrogate peptides could be produced with different sensitivity and selectivity and thereby provide structure information about the protein.…”

“…Tandem MS provides unparalleled sensitivity and better selectivity for the surrogate peptides. A recent review included detailed procedures and addresses the use of triple quadruple mass spectrometers as a detector to quantitate digested surrogate peptides [18] from biologic matrices.…”

The clinical utility of mass spectrometry based protein assays

“…LC-MS has been demonstrated to be a suitable alternative to immunoassays in the pharmacokinetic measurements of biotherapeutics, in large part due to rapid and cost-effective method development, high selectivity, and the capability to analyze multiple analytes in a single analytical run [66][67][68][69]. While these assays may never be used in clinical chemistry laboratories, they are excellent examples of the rationale for using LC-MS in lieu of other platforms.…”

Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry

“…Because the most‐commonly used LBA method, enzyme‐linked immunosorbent assay (ELISA) is often considered as the gold standard for protein quantification and poses features such as high sensitivity and throughput. However, several major limitations are associated with this approach (Ezan & Bitsch, ; Pendley & Shankar, ; Geist et al, ; Zheng et al, ): (i) the method depends on high‐quality critical reagents to obtain selectivity and it is highly challenging to achieve sufficient selectivity that unambiguously identify specific amino acid sequenes; (ii) the development process is often time‐consuming and cost‐prohibitive; (iii) the linear range of the calibration curve is narrow (typically within 100‐fold), which is inadequate to cover the dynamic range required for a regular PK study; (iv) high susceptibility to matrix interference and the method is usually matrix‐selective; for example, a method developed for plasma samples cannot be easily transferred for use in tissue samples. Given these drawbacks, substantial efforts have been devoted in developing alternative methods.…”