Aim: The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC–quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody. Results: Following method optimization […]
Aim: LC–MS/MS bottom-up quantitation of proteins has become increasingly popular with trypsin as the most commonly used protease. However, trypsin does not always yield suitable surrogate peptides. An alternative enzyme, Glu-C, was used to generate surrogate peptides for quantifying a bispecific IgG1 biotherapeutic antibody in preclinical matrices. Materials and methods: IgG1 was quantified by pellet
In recent years, immunocapture enrichment coupled with LC–MS technology has seen more applications for the measurement of low abundant protein therapeutics and biomarkers in biological matrices. In this article, several critical considerations for the application of immunocapture enrichment to LC–MS bioanalysis of protein therapeutics and biomarkers, including reagent selection, reagent characterization, designing of capture format,
Aim: Sample extraction using immuno-affinity capture coupled with LC–high-resolution mass spectrometer has recently emerged as a novel approach for the determination of concentrations of large molecules at intact level in biological matrix. Methodology: In the current work, different data processing strategies for intact protein bioanalysis, deconvoluted mass spectra or extracted ion chromatogram, were applied to
Aim: The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC–quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody. Results: Following method optimization
Aim: Protein quantitation by digestion of a biological sample followed by LC–MS analysis of a signature peptide can be a challenge because of the high complexity of the digested matrix. Results/methodology: The use of LC with high-resolution (quadrupole-TOF) MS detection allowed quantitation of the 22-kDa biopharmaceutical somatropin in 60 μl of rat plasma down to
Aim: LC–MS/MS bottom-up quantitation of proteins has become increasingly popular with trypsin as the most commonly used protease. However, trypsin does not always yield suitable surrogate peptides. An alternative enzyme, Glu-C, was used to generate surrogate peptides for quantifying a bispecific IgG1 biotherapeutic antibody in preclinical matrices. Materials and methods: IgG1 was quantified by pellet
In recent years, immunocapture enrichment coupled with LC–MS technology has seen more applications for the measurement of low abundant protein therapeutics and biomarkers in biological matrices. In this article, several critical considerations for the application of immunocapture enrichment to LC–MS bioanalysis of protein therapeutics and biomarkers, including reagent selection, reagent characterization, designing of capture format,
Biomarker assays have brought significant challenges to bioanalytical laboratories that historically have provided pharmacokinetic analytical services to the drug development industry. This has largely been due to two reasons: the lack of regulatory guidance in how to validate biomarker assays and the lack of scientists in bioanalytical laboratories with experience in this clinical arena. Since
There has been an increased use of commercial kits for biomarker measurement, commensurate with the increased demand for biomarkers in drug development. However, in most cases these kits do not meet the quality attributes for use in regulated environment. The process for adaptation of these kits can be frustrating, time consuming and resource intensive. In