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(BBSRC DTP CASE) Native Mass Spectrometry approaches for Electron and Photon mediated Top Down Sequencing of Biotherapeutics


Project Description

In this project we propose to develop electron and photon mediated top-down sequencing methods to analyse recombinant biological therapeutics including monoclonal antibodies (mAbs) and adeno-associated viral (AAV) vectors.

This project has synergies with but is distinctive to the currently funded BBSRC case with Allergan and Barran, and represents maturation of their strategic relationship. mAbs are the fastest growing class of therapeutic and currently 8/10 of new drugs are mAbs or mAb based, these complex biomolecules with structural heterogeneity are complex to analyse and determining the consistency of a given mAb product in its discovery, development and production is a substantial analytical challenge. AAV vectors have emerged at the forefront of gene therapy due to their lack of pathogenicity, relatively low immunogenicity and persistent gene expression in different tissue types. Allergan is particularly interested in using AAV vectors for Ocular drug delivery as it purchased Retrosense in 2016 that is a biotechnology company that is developing a game-changing gene therapy (AAV) to restore vision in patients suffering from blindness due to retinitis pigmentosa (RP) and advanced dry age-related macular degeneration (advanced dry-AMD). There are currently no FDA approved therapies to improve or restore vision in patients with these retinal degenerative conditions. The analytics to support AAV production are also very limited and the hope is with this project that rapid top down sequencing will significantly advance Allergan’s capabilities in the development and manufacture of biological therapeutics.

The student will learn how to prepare the samples supplied by Allegan and standards obtained from other sources such that they are suitable for analysis by direct infusion mass spectrometry. They will then learn native mass spectrometry techniques to get the viruses and mAbs into the mass spectrometer intact such that their mass and stoichiometry can be determined.
Using Ion mobility mass spectrometry they will then be able to determine the shape and size of the molecules. The student will also be trained in data analysis and in data curation.

As well as this training at UoM the student will spend time at Allergan, learning about the production of the biological therapeutics and how production is improved to maximise the product yield and batch to batch consistency is achieved. At Allergan they will also learn more classical biophysical methods of sample analysis, including bottom up mass spectrometry methods and spectroscopic methods. After this initial training period the student will embark on the main aim of this project to fully characterise the sequence and interactions in the intact biotherapeutic with top down sequencing methods. We will install a new electron trap onto our Q_Exactive Orbitrap mass spectrometer such that electron capture dissociation (ECD) can be performed. This is a non-ergodic dissociation method, which primarily cleaves the polypetide backbone to produce distinctive fragments that report on the tertiary and quaternary fold which can then be mapped back to the native structure. These ECD measurement will be complemented by measurements taken using photons to dissociate covalent bonds on our customised ion mobility mass spectrometer. The student will develop advanced data analysis to compare data from both methods.

Barran is an international expert in the development of mass spectrometry instrumentation. Her team have requisite expertise in this area. It falls in the BBSRC themes in ’new ways of working’, industrial biotechnology’ and ‘bioscience for health.’

http://www.mbc.manchester.ac.uk

Entry Requirements
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Programme. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP websitewww.manchester.ac.uk/bbsrcdtpstudentships

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

[1] K.L. Fort, C.N. Cramer, V.G. Voinov, Y. V Vasil’ev, N.I. Lopez, J.S. Beckman, A.J.R. Heck, Exploring ECD on a Benchtop Q Exactive Orbitrap Mass Spectrometer., J. Proteome Res. 17 (2018) 926–933. doi:10.1021/acs.jproteome.7b00622.
[2] B. Bellina, J.M. Brown, J. Ujma, P. Murray, K. Giles, M. Morris, I. Compagnon, P.E. Barran, UV photodissociation of trapped ions following ion mobility separation in a Q-ToF mass spectrometer, Analyst. 139 (2014) 6348–6351. doi:10.1039/C4AN01656D.
[3] U.H. Mistarz, B. Bellina, P.F. Jensen, J.M. Brown, P.E. Barran, K.D. Rand, UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides, Anal. Chem. 90 (2018) 1077–1080. doi:10.1021/acs.analchem.7b04683.
[4] C.J. Gray, B. Thomas, R. Upton, L.G. Migas, C.E. Eyers, P.E. Barran, S.L. Flitsch, Applications of ion mobility mass spectrometry for high throughput, high resolution glycan analysis, Biochim. Biophys. Acta - Gen. Subj. 1860 (2016). doi:10.1016/j.bbagen.2016.02.003.
[5] S.R. Harvey, M. Porrini, A. Konijnenberg, D.J. Clarke, R.C. Tyler, P.R.R. Langridge-Smith, C.E. Macphee, B.F. Volkman, P.E. Barran, Dissecting the dynamic conformations of the metamorphic protein lymphotactin, J. Phys. Chem. B. 118 (2014) 12348–12359. doi:10.1021/jp504997k.

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