Determination of immunogenic proteins in biopharmaceuticals by UHPLC–MS amino acid analysis

Abstract

Nowadays, there is a growing interest in innovative and more efficient therapeutics—biopharmaceuticals, based on peptides or proteins. There are increased demands on quality control of such therapeutics. One of the methods usually used for characterization and quantification of biopharmaceuticals is amino acid analysis. In this work, a modern advanced analytical method based on precolumn derivatization and reversed-phase ultra high-performance liquid chromatography in combination with single quadrupole mass spectrometer was developed for amino acid analysis in different protein samples—model sample of bovine serum albumin, sample of strong immunogenic protein keyhole limpet hemocyanin, and sample of drug etanercept present in commercially available biopharmaceutical Enbrel. The method used isotopically labeled internal standards and was validated according to the International Council for Harmonisation guideline. The developed method was characterized by favorable performance and validation parameters, such as time of analysis (6 min), specificity, linearity (r 2 ≥ 0.99), limit of detection (0.009–0.822 µM), limit of quantification (1–2.5 µM), accuracy (recovery in the range 90–102.8%), intra-day (RSD in the range 0.25–11.97%) and inter-day precision (RSD in the range 1.67–11.57%), or stability (RE ≤ 12%). According to these findings, the developed amino acid analysis approach is suitable for routine use in areas of peptide/protein quantification, such as quality control laboratories of biopharmaceutical companies.

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry

“…The chromatographic separation of twenty amino acids was carried out according to our previously developed LC-MS method [36]. Briefly, the separation was carried out using a reversed phase column Cortecs UPLC C18 (2.1 mm × 100 mm, 1.6 µm) obtained from Waters (Prague, Czech Republic).…”

What is the role of current mass spectrometry in pharmaceutical analysis?

“…Typical hydrolysis conditions are elevated temperature, up to 165°C, and a strongly acidic media, such as 6 M hydrochloric acid. The duration of acidic hydrolysis depends on the sequence and can be up to 24 h (Piestansky et al, 2019). The main drawback of this sample preparation procedure is the structural conversion and destruction of asparagine, glutamine, and tryptophan, respectively (Sandra et al, 2014).…”

“…Derivatization in pre‐column or post‐column arrangement is needed due to the absence of a moiety allowing UV, FD, or electrochemical detection in the chemical structure of AA (Piestansky et al, 2019). Rigas compares these tagging strategies (Rigas, 2013).…”

“…The AccQTag Ultra reagent was used to derivatize the biopharmaceutical etanercept for RPLC‐MS analysis. The authors emphasize that the proposed method with derivatization and single quadrupole QDa MS can be implemented in routine assays for QC laboratories and adopted in clinical and biomedical analysis (Piestansky et al, 2019). AA from the complex hydrolyzate can also be analyzed using IEX (Hanko & Rohrer, 2004) or HILIC (Prinsen et al, 2016; Themelis et al, 2017) methods (Vilches et al, 2017).…”

Multifunctional Biopolymers‐Based Composite Materials for Biomedical Applications: A Systematic Review

“…Classification of biopolymers. [59][60][61][62][63][64][65][66][67] reinforce their present bioactivities as well as produce new functional bioactivities. The hydrogels developed through chemical routes involve covalent bonds formation between used polymers through the process of free radical polymerization, [83,84] or graft copolymerization, [85] or radical copolymerization [86] etc.…”