Large Molecule Quantification

Address the quantification of peptide- and protein-based biotherapeutics and biomarkers.

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Intact mAb LC–MS for drug concentration from pre-clinical studies: bioanalytical method performance and in-life samples
September 2020

Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Results/methodology: Here, an intact protein LC–MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC–MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC–MS/MS. Discussion/conclusion: Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC–MS, modification monitoring and evaluation of data processing parameters is demonstrated.

Comprehending COVID-19: Maximizing LC-MS Detection Dynamic Range for Multiple Reaction Monitoring Based SARS-CoV-2 Analysis
August 2020

Targeted MS-based methods show promise to specifically detect SARS-CoV-2 proteins from various types of biological matrices of COVID-19 patients. Here, the combination of selected chemistries and consumables, using the ACQUITY UPLC I-Class PLUS System and Xevo TQ-XS Tandem Quadrupole Mass Spectrometer, are demonstrated for the detection and quantification of SARS-CoV-2 proteins in nasopharyngeal swabs preserved in Universal Transport Medium. A linear response was achieved across five amount levels for recombinant Spike glycoprotein and Nucleoprotein, using at least two transitions per peptide, whilst maintaining good measurement reproducibility across the detection dynamic range.

Comprehending COVID-19: Multiple Reaction Monitoring Transition Selection and Optimization Strategies for LC-MS Based SARS-CoV-2 Detection
August 2020

The COVID-19 pandemic has resulted in the development of mass spectrometry based methods to characterize, identify, and quantify proteins. These methods are aimed at understanding the structural biology and interaction mechanisms of SARS-CoV-2, or as a complementary method to detect relevant markers. Targeted mass spectrometry, through the detection of viral peptides in proteolytically digested body fluids, has been suggested as a SARS-CoV-2 detection method. The work presented here demonstrates application of the MassLynx Skyline Interface for automated peptide Multiple Reaction Monitoring selection and optimization with a Xevo TQ-XS Tandem Quadrupole Mass Spectrometer.

Improved Oligonucleotide SPE-LC-MS Analysis Using MaxPeak High Performance Technology
September 2020

The demand for highly selective and sensitive LC-MS bioanalytical assays in support of research and development of next-generation oligonucleotide therapies has greatly increased. The work described herein uses SPE, RP-UPLC with the sub-2-μm ACQUITY PREMIER Oligonucleotide C18 Column, and tandem-quadrupole MS for detection and quantification of oligodeoxythymidines and the fully phosphorothioated oligonucleotide antisense therapy, GEM91.

More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a µLC–MS/MS system

Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for thyroglobulin (Tg) are resistant to autoantibody (TgAb) interference, recent studies have demonstrated approximately 40% of TgAb-positive individuals with recurrent thyroid cancer have Tg concentrations below the lower limit of quantification (LLOQ) of the LC-MS/MS assays described to date (i.e., <0.5 ng/mL), resulting in false-negative findings during post-thyroidectomy monitoring. To reduce false negative results due to insufficient analytical sensitivity, a new Tg assay was developed on a commercially available LC-MS/MS system operating at microliter/minute flow-rates (i.e., µLC–MS/MS) to maximize the analytical sensitivity and achieve a LLOQ of 0.02 ng/mL.

Development of an UPLC/MS–MS method for quantification of intact IGF-I from human serum

Developing LC–MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC–MS/MS.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones

Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range.

Development of an UPLC/MS–MS method for quantification of intact IGF-I from human serum

Developing LC–MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC–MS/MS.

Triple Quadrupole Mass Spectrometry (Xevo TQ-XS) for the Quantification of Monoclonal Antibody Light Chains in Plasma

The work herein describes the development and optimization of sample preparation and LC-MS/MS methodology for the sensitive quantification of mAb subunit light chains using selective column chemistry and triple quadrupole mass spectrometry.

Achieving Maximum Protein and Peptide Recovery, Sensitivity, and Reproducibility using QuanRecovery Vials and Plates

Quantitative LC-MS analyses for proteins and peptides are getting more challenging every day, requiring greater sensitivity and reproducibility from smaller amounts of samples. Analyte loss due to non-specific binding in sample containers is a significant problem in quantitation that is often not recognized early enough.