PhD defence: Developing New LC-MS Technologies for the Characterization and Quantitation of Biotherapeutics


Jennifer M. Nguyen

PhD thesis

About the thesis

Biotherapeutics are growing at a rapid pace. Recombinant mAbs for therapeutics are one of the fastest growing markets, alongside a rapid rise in interest towards antisense therapies like oligonucleotides. Because of the heterogeneity of mAbs and complexity of antisense oligonucleotides, the characterization and pharmacokinetic monitoring of these drug targets are important to ensure quality and can require much testing.

This thesis focused on optimizing LC or LC-MS consumables and methods for the analysis of proteins and oligonucleotides. Broadly, the thesis aimed to both discover and evaluate modern consumables and reagents such as columns and new surface technologies, emphasizing the development of these methods to improve RPLC-MS.

Applications for the qualitative and quantitative analyses of intact proteins, protein subunits, and oligonucleotides were also investigated, and by optimizing sample preparation approaches, the performance of these applications could be maximized.

In paper 1, reversed-phase chemistries and ion-pairing reagents were evaluated to develop a qualified platform method for subunit profiling using MS friendly separations.

New column potential for low tortuosity superficially porous particles, non-porous polymeric particles, or gigaporous PS-DVB particle to address protein analysis at low nanogram mass loads was assessed in paper 2, with a focus on designing the ideal column technology and describing fundamentals regarding the improved approach. The selectivity of different proteins and integration with affinity enrichment was realized through an aptamer-based immunocapture method.

For oligonucleotide separations, papers 3 and 4 highlight the benefits of a new, inert LC surface technology to improve chromatographic performance and recovery versus conventional column technologies for quantifying antisense oligonucleotides.

In all, with the rapid growth occurring in the biopharmaceutical world, maximizing the characterization and quantitation of proteins and oligonucleotides for efficiency and accuracy is not only important but necessary.

The four papers sought to investigate and improve this through developing new LC-MS technologies for RPLC-based chromatographic separations.

Together, these papers provide a compelling story about the growth of biotherapeutics and how the analytical world can advance to accommodate the demanding diagnostic challenges of these new modalities.

2021, 210 pages.


29 June 2021, 14:00


Digital defence.


Professor Anne Raben, (chair), Department of Nutrition, Exercise and Sports, University of Copenhagen, Denmark.

Professor Govert Willem Somsen, Vrije Universiteit Amsterdam, The Netherlands.

Principal Scientist Kim F. Haselmann, Novo Nordisk A/S, Denmark.

Main supervisor

Professor Lars Ove Dragsted, Department of Nutrition, Exercise and Sports, University of Copenhagen, Denmark.


Professor Cristina Legido-Quigley, Steno Diabetes Center in Copenhagen and King’s College in London.

Doctor Matthew Lauber, Waters Corporation in the United States.

Doctor Erin Chambers, Waters Corporation in the United States.

The thesis is available for inspection at the library, Nørre Allé 51, DK-2200 Copenhagen N.