Peptide mapping characterization analysis of monoclonal antibody (mAb) using liquid chromatography quadrupole time-of-flight mass spectrometer (LC/Q-TOF)

Name: Ubonwan Chaiyo

Organization : King Mongkut's University of Technology Thonburi. Nation Biopharmaceutical Faculty

Name: Dalad Waropastrakul

Organization : King Mongkut's University of Technology Thonburi. Nation Biopharmaceutical Faculty

Name: Ariya Chaisawadi

Organization : King Mongkut's University of Technology Thonburi. Nation Biopharmaceutical Faculty

Email : Ariya.cha@kmutt.ac.th

keyword: Peptide mapping

LCSH: Monoclonal antibodies -- Analysis

LCSH: Liquid chromatography

LCSH: Proteomics

Abstract: A characterization of monoclonal antibody (mAb) by peptide mapping using liquid chromatography time-of-flight mass spectrometry (LC/QTOF) has been developed and validated. Sample preparation steps and gradient ratio of mobile phase were optimized to improve the protein coverage percentage. The Protein Metrics software was also used for analyzing the coverage percentage of peptide mapping. The samples were prepared by reduced disulfide bond of the mAb with dithiothreitol (DTT) and alkylated free thiol group with Iodoacetamide (IAA). The peptides were then digested with restriction enzyme, trypsin, which specifically cleaves C-terminal to Arginine (R) and Lysine (K). The digestion reaction was performed at 37 ºC and incubated overnight then stopped the reaction using formic acid. LC was performed by using C18 column and 0.1% formic acid and 0.01% TFA in water and 0.1% formic acid and 0.01%TFA in acetonitrile (ACN) as mobile phases. The QTOF was operated using positive mode electrospray ionization using data-dependent acquisition. The m/z values were selected for second fragmentation according to a priority list generated by the calculated m/z of the interested molecule. Other data acquisition processing was performed using an inhouse method that can be adjusted for new molecules of interest. This method gives the coverage percentage over 90% in both heavy chain and light chain of the mAb molecule. The use of LC-MS/MS technique gives the highly efficient and robust approach to confirm the characteristics of the trastuzumab molecule in peptide mapping parameter which can be updated to use with other protein molecules for further characterization

King Mongkut's University of Technology North Bangkok. Central Library

Address: BANGKOK

Email: library@kmutnb.ac.th

Created: 2024

Modified: 2024-11-26

Issued: 2024-11-26

บทความ/Article

application/pdf

BibliograpyCitation : In Kasetsart University. Faculty of Science. The Pure and Applied Chemistry International Conference 2024 (PACCON 2024) (pp.141-145). Bangkok : Kasetsart University, 2024

eng

©copyrights King Mongkut's University of Technology North Bangkok

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	PACCON 2024pp.141-145.pdf	577.44 KB

ใช้เวลา

0.031166 วินาที