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

Abstract

In recent years, the applicability of using LC-MS/MS as a complementary technique to traditional ligand binding assays in the absolute quantitation of therapeutic proteins in biologic matrix has been demonstrated. Protein quantitation workflow via LC-MS/MS is primarily based on a enzymatic digestion model and recent works seek to improve selectivity and sensitivity. This review focuses on recent innovations in this field and discusses the following in detail: the applicability of two-dimensional liquid chromatography and its use to improve sensitivity and alleviate matrix ion suppression; the use of derivatization agents after digestion to improve extraction and MS ionization efficiency; techniques to reduce excess protein background and their positive effects on sensitivity, selectivity, and extraction consistency; the application of immunoaffinity extraction of proteins to enrich the analyte(s) of interest while improving selectivity and sensitivity.

Technologies to improve the sensitivity of existing chromatographic methods used for bioanalytical studies

“…Affinity enrichment is another effective way to improve the sensitivity of large‐molecule analysis, due to the specific interaction between antigen and antibody. Shen et al () have explained the theory of immunoaffinity (IA) purification in detail, and listed several typical research examples, such as protein‐level IA purification and peptide‐level immunocapture, from 2004 to 2013. Zhang and co‐workers () discovered that the formation of a complex between mAb‐I (capture antibody) and IP‐10 (analyte) led to low recovery in their experiments.…”

“…(2019) achieved sufficient digestion efficiency by improving the incubation temperature to 60 C and optimizing the reaction time to 1.5 h, resulting in an LLOQ value of 250 ng/mL for an anti-CD47 monoclonal antibody (SHR-1603).Affinity enrichment is another effective way to improve the sensitivity of large-molecule analysis, due to the specific interaction between antigen and antibody Shen et al (2015). have explained the theory of immunoaffinity (IA) purification in detail, and listed several typical research examples, such as protein-level IA purification and peptide-level immunocapture, from 2004 to 2013.…”

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

“…Rapid progression of the discovery and development of biotherapeutics necessitates methods for quantification in biomatrices (tissues and bodily fluids) with sufficient specificity, accuracy, sensitivity, reproducibility, and throughput (Leary et al, ; Bults, van de Merbel, & Bischoff, ; Chilewski & Jiang, ; Shen, Liu, & Zhao, ); nonetheless, such method has been challenging to achieve and some key difficulties are discussed below.…”

“…Depletion of high‐abundance matrix components before digestion might considerably increase sensitivity, because such a strategy not only increases the ratio of target protein versus total proteins in sample, but also might benefit downstream sample preparation and LC/SRM‐MS analysis by enabling more‐efficient digestion, higher loading capacity, and decreased chemical noises and ion suppression (Anderson & Hunter, ; Liu et al, ; Shen, Liu, & Zhao, ). Due to technical limitations, such strategies are mainly applied for plasma/serum samples.…”

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

“…lipids and phospholipids) can cause ion suppression. To obtain better sensitivity and robustness, the usual approach is to remove high‐abundance matrix interferences by applying extraction procedures at the protein and/or peptide levels, such as immunodepletion, immunocapture, or solid‐phase extraction [26–32]. Immunoaffinity‐based sample pretreatment techniques are quite expensive, usually not suitable for multiplexed analysis, and there can be cross‐reactivity, as well as difficulties in the transfer of the methodology in‐between different laboratories.…”