Monoclonal measured by means of peptide marker

Skip to Navigation

Ezine

  • Published: Aug 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Monoclonal measured by means of peptide marker

Monoclonal antibodies need accurate serum assays

Therapeutic monoclonal antibodies can be used to treat various types of cancer as well as some autoimmune diseases, such as Crohn’s disease, ulcerative colitis and asthma

Nowadays, therapeutic monoclonal antibodies are used to treat various types of cancer as well as some autoimmune diseases, such as Crohn’s disease, ulcerative colitis and asthma. Accurate measurement of the concentrations of these agents in the body is important in order to optimise treatment and reduce side effects. ELISAs (enzyme-linked immunosorbent assays) are typically used to determine monoclonal antibody concentrations, but the results can vary depending on the kit manufacturer. Where multiple tests are required, the cost of ELISAs can be relatively high.

The Naples researchers aimed to develop a method of assaying the antibody trastuzumab (Herceptin), which is used to treat some forms of breast cancer, by using UHPLC (ultra-high performance liquid chromatography) and tandem mass spectrometry to quantify a particular peptide, CH8, specifically produced on the breakdown of the trastuzumab protein structure by digestion with trypsin. The peptide was not formed by the breakdown of other proteins in blood serum so served as an effective marker for the presence of trastuzumab. It gave strong mass spectrometry signals and also lacked cysteine groups, which are known to be liable to oxidation.

UHPLC and tandem MS on peptide used to assay trastuzumab

The CH8 peptide was produced by solid-phase synthesis and purified using semi-preparative reversed phase HPLC. Its concentration could be determined by UV, making use of the amino acids tryptophan and tyrosine within the peptide.

Peptide UHPLC was carried out using a Waters Acquity UPLC M-Class system, fitted with a Waters nanoAcquity UPLC trap column (used to concentrate the sample and remove salts, etc.) and a Waters nanoAcquity UPLC HSS T3 analytical column. Two eluting solvents were prepared: solvent A was 98% water, 2% acetonitrile and 0.1% formic acid, and solvent B with 2% water, 98% acetonitrile and 0.1% formic acid. The trap column was eluted with 99.5% solvent A and 0.5% solvent B. The analytical column was run with a gradient from 5% to 40% of solvent B over 3 minutes, followed by a second gradient from 40% to 95% of solvent B over 1 minute.

Quadrupole time of flight tandem mass spectrometry was carried out using a Waters Q-TOF SYNAPT G2Si instrument with electrospray ionisation and multiple reaction monitoring (MRM). Quantitation and confirmation ions were selected, and the mass spectrometry conditions were carefully optimised.

The system gave good linearity for the CH8 peptide and no carryover was noted. The LOD (limit of detection) for the peptide was only 0.005 fmol/μl, while the limit of quantification (LOQ) was 0.05 fmol/μl. Six replicate injections gave very similar results, all having a 3.97 minutes retention time and giving an RSD (relative standard deviation) value for their peak areas of 5.7%.

Trastuzumab samples were subjected to disulfide reduction with DTT (dithiothreitol) and alkylation with iodoacetamide, and were then digested with trypsin to break down the protein into peptide fragments, giving the CH8 peptide, which was detected by the UHPLC-MS system. Samples of serum were spiked with known concentrations of trastuzumab and treated in the above manner, giving >80% recovery for all the concentrations tested.

LC-MS can compete with ELISA in monoclonal antibody analysis

The new method suggests that LC-MS could be a viable alternative to ELISA for monoclonal antibody quantification and could be extended to other monoclonal therapeutic agents. The economics will be most favourable in laboratories that already possess underused LC-MS capacity and process numerous serum samples. For studies of this type, it is essential to identify relatively stable peptide fragments that are not formed from other protein species in serum.

Related Links

Rapid Communications in Mass Spectrometry, 2017, 31, 1184-1192. Russo et al. Ultra-performance liquid chromatography/multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions.

Wikipedia, Tandem mass spectrometry

Wikipedia, Trastuzumab

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

Follow us on Twitter!

Social Links

Share This Links

Bookmark and Share

Microsites

Suppliers Selection
Societies Selection

Banner Ad

Click here to see
all job opportunities

Copyright Information

Interested in spectroscopy? Visit our sister site spectroscopyNOW.com

Copyright © 2017 John Wiley & Sons, Inc. All Rights Reserved