Early-stage researchers

15 early-stage researchers (PhD students) participate in the A4B consortium with the aim to produce the much-needed specialists in the field of therapeutic proteins (TP). Within their respective project, each PhD student is receiving a background in production, analysis and economic exploitation of TPs, and training in relevant soft skills. This training is provided in dedicated Training Schools, soft skills training sessions and a number of workshops hosted at the different partner institutions.


ESR positions in the A4B project

ESR1: Fast quantification of intact therapeutic protein species

Universitätsklinikum Hamburg-Eppendorf
Universitätsklinikum Hamburg-Eppendorf, Germany

Manasi Gaikwad

Project description

Manasi is working on the development of fast “top-down” LC-MS/MS based approaches for a relative label-free, unambiguous quantification of intact therapeutic protein species (TP-S). She will apply mass spectrometric methods to identify and quantify TP-S via LC-MS/MS employing rational targeted fragmentation techniques.

ESR2: Improvement of liquid chromatography for analysis and purification of therapeutic protein species via rational protein purification parameter screening and sample displacement chromatography

Universitätsklinikum Hamburg-Eppendorf
Universitätsklinikum Hamburg-Eppendorf, Germany

Siti Hidayah

Project description

SIti aims the improvement of liquid chromatography methods for analysis and purification of therapeutic protein species (TP-S). Therefore, she will rationally screen parameters for liquid chromatography including sample displacement chromatography. Siti is working closely together with Manasi, since the read-out for the chromatographic separations (ESR2) will be done by those TD-MS based quantification methods developed by ESR1.


ESR3: Bioinformatics method development for quantitative therapeutic protein species analytics

Eberhard Karls Universität Tübingen
Eberhard Karls Universität Tübingen, Germany

Jihyung Kim

Project description

Kim is developing new algorithmic techniques to identify and quantify protein species. Recent advances in top-down mass spectrometry enable new options for the characterization of protein species. We will develop novel algorithms integrating top-down data with bottom-up data and thus permit a more comprehensive view on protein species. Integrating the data in a probabilistic framework will support in depth characterization of post-translational modifications. Inspired by algorithmic ideas from label-free quantification, this data will also be used to obtain accurate estimates of relative concentrations of protein species.
We are prod to announce that an ultrafast deconvolution algorithm has been developed. A beta version of FLASHDeconv is available on https://www.openms.de/comp/flashdeconv/.

ESR4: Development and use of monoclonal affinity proteins

Kungliga Tekniska Högskolan
Kungliga Tekniska Högskolan, Sweden

Andreas Wisniewski

Project description

At KTH Andreas is developing efficient affinity capture tools to be used for selective purification and detection. This will be accomplished through the selection of novel binding proteins, monoclonal affinity proteins, from randomized protein libraries. By the use of different display methods, protein domains with high selectivity and affinity will be achieved. The new affinity proteins will be analyzed regarding stability and affinity to the target molecules. Furthermore, the binders will be optimized for analytical and preparative purification of the target proteins.

Andreas is one of our elected student representatives in the A4B supervisory board.

ESR5: New methods for the mass spectrometric glycosylation analysis of therapeutic protein species

Leiden University Medical Center
Leiden University Medical Center, Netherlands

Steffen Lippold

Steffen is developing a site-specific glycosylation analysis of rh EPO using MALDI-FTICR- MS, training various carboxygroup derivatization strategies and applying affinity liquid chromatography hyphenated to native-like mass spectrometry is investigated for characterization of intact mAbs.

Steffen’s goal is it to develop MALDI- TOF-MS based approach for glycosylation analysis on glycopeptide level so substitute the tedious and instrumentally demanding LC-MS approach. This approach will provide in depth site and sialic acid-specific information on N and O- glycosylation.

ESR6: Separation and mass spectrometric characterization of intact therapeutic protein species including conformers and complexes

Leiden University Medical Center
Leiden University Medical Center, Netherlands

Christoph Gstoettner

Christoph is applying CE for separation and analysis of intact mAbs in denaturating and native conditions with respective data analysis. He is training MALDI-FTICR-MS and applying ISD fragmentation as a top-down approach on antibodies and small proteins as well as the measurement of antibody chains in a middle-down approach. The developed method will be used to analyze samples with elevated glycosylation levels and its application for other PTMs will be evaluated.

ESR7: Comprehensive characterization of therapeutic protein species by top-down mass spectrometry (TDMS) and its expansion to large proteins

Syddansk Universitet
Syddansk Universitet, Denmark

Masa Bubovic

Project description

Masa’s project entails the analysis and quantification of intact therapeutic proteins and their post-translational modifications (PTMs, e.g. disulfide bonds, phosphorylation, glycosylation). Masa will test, apply and benchmark chromatographic and electrophoretic techniques for separation of intact protein species (proteoforms) prior to detailed protein structure analysis mass spectrometry. She is using advanced tandem mass spectrometry technologies, including HCD, ETD, UVPD and combinations thereof to obtain comprehensive amino acid sequence information and PTM maps of selected therapeutic proteins. The main focus is the integration of experimental methods (LC, CE, MS/MS) with advanced bioinformatics/statistics workflows for detailed intact protein structure analysis and quantification of protein biologics in the mass range 20 kDa to 200 kDa.

ESR8: Automatable and high throughput techniques to determine site specificity and quantification of N and O glycan profiles of EPO and TNF-AB

Ludger Ltd.
Ludger Ltd., United Kingdom

Manuela Amez

Manuela’s focus lies on the development of automatable and high throughput techniques to determine site specificity and quantification of N and O glycan profiles of EPO and TNF-AB. The project builds upon a recently developed product, VTAG, used to analyze and relatively quantify glycan types on the Fc receptor of monoclonal antibodies. Manuela is developing novel tags with fluorescence and MS detection capabilities (VTAGS) that allow rapid glycan characterisation of TPs.


ESR9: Pharmacokinetics: Analysis of therapeutic protein species in complex matrices

Rijksuniversiteit Groningen
Rijksuniversiteit Groningen, Netherlands

Oladapo Olaleye

Oladapo focusses on the enrichment of Trastuzumab and Pertuzumab proteoforms from blood plasma using recombinant Her-2, anti-idiotypic antibodies (only available for Trastuzumab) and custom-made affimers for Pertuzumab. He is evaluating the affimers and notably for their ability to enrich different proteoforms of the targeted TPs. Currently the ESR9’s work focuses on establishing the complementarity of affimers and Her-2 with respect to their binding sites with the goal to cover as many proteoforms as possible.

ESR10: Metabolism: Characterizing protein metabolites from in vitro and in vivo studies

Rijksuniversiteit Groningen
Rijksuniversiteit Groningen, Netherlands

Baubek Spanov

Baubek works on developing novel chromatographic methods to separate proteoforms of Trastuzumab and Pertuzumab.  The separated proteoforms will be analysed by peptide mapping for modifications as well as by a Her-2 receptor binding assay for the capability to bind to the target Her-2. The bioactivity of selected, isolated proteoforms is tested in a cell-based toxicity assay.

ESR11: Protein quantification by isotope labeling

Universitetet i Oslo
Universitetet i Oslo, Norway

Christina Johannsen

Christina is focusing on developing protein quantification methods for therapeutic proteins and their modifications using peptide and protein labeling techniques in combination with liquid chromatography and mass spectrometry.

ESR12: Continuous separation of recombinant antibodies by non-chromatography methods

Universität für Bodenkultur Wien
Universität für Bodenkultur Wien, Austria

Gregory Silva Dutra

Gregory is focusing in the capture of monoclonal antibodies via precipitation methods. Starting with polyethylene glycol (PEG) combined with Zinc Chloride (ZnCl2) as precipitate agents. He is investigating the complete removal the PEG from the process. This would be a great advantage for analytical and environmental reasons. Drawbacks related to increased viscosity, incompatibility with mass spectrometric analysis, process overall cost and environmental footprint would be further reduced.


ESR13: Improvement of the production process

Universität für Bodenkultur Wien
Universität für Bodenkultur Wien, Austria

Daniel Komuczki

Daniel is specializing in the optimization of TP production and downstream processing.  Daniel has implemented a soft sensor in periodic counter current chromatography (PCCC) which functions as on-line monitoring tool for continuous bioreactors, allowing the prediction of the concentration of antibody in the perfusion fermentation. PCCC therefore, generates an information feedback loop rendering titter determination during the fermentation process obsolete.

ESR14: Development of Multiple Reaction Monitoring (MRM) / Data-Independent-Acquisition (DIA) for protein quantification

Alphalyse A/S
Alphalyse A/S, Denmark

Magdalena Sandberg

At Alphalyse, Magdalena has her main research focus on the ability to quantify TP’s through the entire development phase of a TP. Multiple Reaction Monitoring (MRM) is an LC-MS/MS based approach for direct quantification of defined proteins. In addition, data-independent-acquisition (DIA) based quantification will be used. One of the key challenges is to be able to perform quantification on material throughout the upstream and downstream process and in different matrices. Therefore, she is optimizing the protease digest procedures, analyte enrichment and MS/MS analysis.

ESR15: Pharmacokinetics and Metabolism: Analysis of therapeutic species in complex matrices

HEXAL AG, Germany

Sandra Carolina Mena Perez

Sandra is developing an in vitro perfusion system for simulating in vivo conditions for testing and predicting modifications of therapeutic proteins, which might impact their efficacy and safety. Simultaneously, she established a parallel reaction monitoring (PRM) method for the relative quantification of modified peptides. The method is being optimized to analyse peptides of interest resulting from deamidation and oxidation the TP Pertuzumab.