Skip to main content Skip to navigation

Johanna Paris

PhD Project

My research will focus on the analysis of monoclonal antibodies using the 12T FT-ICR MS/MS instrument under the supervision of Peter O'Connor.

The project is in collaboration with UCB, under the supervision of John O'Hara.

PhD Abstract

The pharmaceutical industry is a highly regulated domain where all products need to be analysed and characterised to ensure high purity, bioprocess understanding as well as degradation pathway understanding. Recombinant monoclonal antibodies and derivatives are widely used as therapeutic drugs. They are susceptible to post-translational modifications that could occur during the manufacturing process and storage, resulting in product-related impurities. PTM can change the efficacy, toxicity or the clearance of the antibody; therefore they need to be well monitored.


High resolution tandem mass spectrometry permits the identification and localisation of post translational modifications such as N-linked and O-linked glycosylation, misincorporation, oxidation, deamidation, isomerization, glycation, N-pyroglutamate formation C-terminal lysine and leader sequence processing. Two approaches are possible. The bottom up approach consists to reduce, alkylate and cleave the antibody into peptides. This strategy can be time consuming and can result in the loss of labile post-translational modifications as well as inducing modifications related to the sample preparation. The connection between modifications can be lost since the antibody is cleaved into peptides. However, due to the size and the complexity of the antibodies, this is the major approach use in Biopharmaceutical Companies.


In the top down MS/MS approach, the antibody is analysed intact, by measuring the masses of the different proteoforms and their fragments. Different fragmentation techniques can be used, for example an Electron Capture Dissociation (ECD) fragmentation technique will permit the detection of labile modifications. The top down approach is most challenging and requires a more powerful mass spectrometer. It is still in early stage development especially in the analysis of such a complex proteins as antibodies. The Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) has the ability to resolve the high molecular weight of intact antibodies and should allow the detailed characterisation of their micro-heterogeneity.

Bottom up Approach

2D MS/MS analysis of the tryptic digest of IgG1 using different fragmentation techniques.

The 2D MS Approach allows the analysis of the peptides without prior separation, keeping the information of the affiliation of the fragments ions and their precursors.

Top down Approach

The antibody is a challenging molecule due to its size and its disulphide bonds.

Different strategies will be applied in order to get the highest cleavage coverage possible.

Conferences

BMSS Birmingham, 2015

ProteoMMX 4.0 Chester, 2016

Glycobiotech Berlin, 2017

BMSS Manchester, 2017 (Poster)

Uppcon Leeds, 2018 (Poster)

EU FT-ICR 2018, Finland (Poster)

BMSS, Manchester 2018 (Poster)

1rst European Consortium in Top Down, Paris, 2019 (Poster)

Native MS, Oxford, 2019 (Poster)

Personal Profile

I graduated from the E.N.S.T.B.B (Ecole Nationale des technologies des biomolecules de Bordeaux).

My Master degree is specialised in Biotechnlology.

Other Interests

I love travelling.

I horse ride since I am little.

I have a 790cc Bonneville, that I am really proud of.

I am starting bouldering and diving, and absolutely love it.

Work Experience at UCB (3years)

Analysis of antibody therapeutics using UPLC or LC-MS/MS methods to understand their degradation pathway, the impact of cell cultures parameters or process changes, their CQAs as well as basic and acidic species or colouration investigation.

Innovative project: Development and implementation of mass spectrometry methods using isotopic labelling strategy to quantify post-translational modifications.

Glycan Project: Analysis of the N-glycans pattern by N-glycan release methods using different labels (UPLC) and quantitative analysis of sialic acids (UPLC) for comparability studies, clone selection and clone stability studies.

Instruments: Thermo exactive + (Orbitrap), Waters Qtof (Synapt and Xevo), Agilent Qtof (chip-cube), UPLC H Class (QDA and FLR).
Software: Xcalibur, Masslynx, Agilent softwares, Biopharmafinder, Biopharmalynx, Pinpoint, Skyline, Empower.
Techniques: LC-MS/MS peptide mapping, LC-MS/MS disulphide peptide mapping, LC-MS intact mass, HPLC/UPLC (HILIC, RP,CEX, SEC).

Work Placement at UCB (8months)

Development of quantitative mass spectrometry methods to measure Post-Translational Modifications of an antibody, using the stable isotope-labelled internal standard (SILIS) strategy.

Production of the labelled antibody by metabolic incorporation using CHO cells, purification of the labelled material (Protein A affinity chromatography with an AKTA purifier and UNICORN software), calculation of the labellin gefficiency (Agilent 6510 QTof) and use of the SITRS protein as an internal standard in peptide mapping analysis to quantify PTMs (Xevo G2 QTof).

Work Placement at Sanofi Pasteur (6months)

Monitoring tools for High Throughput Process Development Robots :
- Development of RP-UHPLC and UHP-SEC methods for proteins
- Use of Agilent 1290 and ChemStation Software

Work Placement at CHU Toulouse (3months)

Laboratoire de pharmacocinétique et de toxicologie clinique
Development of RP-UPLC method to quantify methotrexate in the blood.

Johanna Paris

MAS-CDT phD (FT-ICR MS)

in collaboration with UCB

johanna.paris@warwick.ac.uk