International Reid Bioanalytical Forum – Analysis of Large Molecules using Chromatography
Burlington House, 27th September 2018

Christina Jayne Vanhinsbergh
PhD, University of Sheffield

It is a lovely September morning as I walk into the Royal Society of Chemistry in London Piccadilly. I am always taken aback by the building and courtyard, with its figurehead above the entrance and stained glass windows of the stairway up to the library. I am greeted by the organisers of the Reid Bioanalytical Troubleshooting workshop for large molecules, using chromatography.

I am a third year PhD researcher having travelled down from Sheffield to the workshop. My work within the University of Sheffield Chemical and Biological Engineering department focuses on developing two-dimensional high performance liquid chromatography analytical methods to separate and characterise oligonucleotide therapeutics, alongside their manufacturing impurities. Additionally, I use mass spectrometry within my workflows to identify various species within manufacturing batches. Although the molecules I analyse are synthetically made, they fall under the umbrella of Biotherapeutics. My background has been centred on the analysis of biological molecules, such as proteins, oligonucleotides and glycans within both academic research projects and assay development in industry. I regularly optimise chromatographic methods in collaboration with my fellow scientists at the University of Sheffield, as well as train new users of liquid chromatography equipment within the departmental bioanalytical lab.

The international Reid Bioanalytical Forum is a meeting for bioanalysts to network with one another and collectively share ideas and knowledge in relation to biological molecules. Eric Reid of the University of Surrey set the meeting up in 1975, and it is still a flourishing opportunity to learn from peers and more experienced members of the chromatographic world. This is especially of use to early career scientists and research students, such as myself, who are still in the process of developing their own networks and professional relationships. The forum today is focused on large molecules like biotherapeutics, however, the Reid forum considers all aspects of bioanalysis- inclusive of large and small molecules. I am especially pleased that I was able to bring a research group colleague along with me to share the experience of developing chromatographic and spectrometric know-how. My motivation for attending today is to re-cap on some of the current issues in relation to proteomic analysis. As my research focuses on oligonucleotides, I intend to keep my skills in protein analysis up-to-date- by investing time to learn more about proteomic biotherapeutic analysis. I am also curious as to how many scientists will be there that analyse oligonucleotides as I do, hopeful to broaden my own professional network.

Stephen Williams (Charles River Laboratories) introduced the audience to quantitative bioanalysis of biopharmaceuticals. First, Stephen gives a historical overview of biopharmaceutical development since the 19th century, leading into today’s world of Biosimilars as patents expire. He introduces some key factors that influence analysis of biopharmaceuticals, such as the quantification of the drug, its metabolites or catabolites and pharmacodynamics markers. Quality analysis is an essential feedstock for regulatory approval and is influenced heavily by the quality of sample preparation. The room is introduced to the range of different analytical techniques that can be applied, such as ligand binding assays, chromatography and mass spectrometry. Stephen reminds his audience of the challenges associated with the analysis of large molecules, such as their degree of heterogeneity, vast array of post translational modifications and large molecular weight, which can affect how the molecules are able to be analysed. For example, larger proteins suffer from poor ionisation efficiency during mass spectrometry analysis – due to their size- and contain multiple charges that reduce sensitivity. Stephen stresses the importance of chromatographic separation for the removal of matrix effects and how internal standards can reduce variability during different analytical approaches. I am pleasantly surprised that Stephen touched on oligonucleotide analysis during his presentation, in addition to some of the considerations applied during tissue analysis.

Next to present is Mohammed Abrar (BioApp Solutions) who focuses more towards the different chromatographic approaches to biopharmaceuticals. He zooms straight into the chemistry of peptides created by protein digestion in analytical preparation. It makes sense to think about the peptide chemical nature before analysis begins- in order to apply the most functional separation technique. For example, peptides containing a high amount of hydrophobic amino acids are well suited to reverse-phase separations.

Good sample preparation is presented, along with strategies for reducing protein precipitation and loss through adsorption. Mohammed then moves on to best analytical techniques to utilise in bioanalytical workflows. He touches on orthogonal approaches and discusses various techniques of improving sensitivity in mass spectrometry, for example, the use of super chargers (such as Nitrobenzyl alcohol) give higher charge states during mass spectrometry.

During the second session with Mohammed, two-dimensional analysis is discussed as a methodology for sample clean-up, which is fully automatable with modern technology. This enables better recovery and concentration than more manual techniques. The room is provided with a method development route for biomolecules and given some tips and tricks that Mohammed has learnt along his journey as a Bioanalytical scientist.
Session three is a case study presentation from attendees, providing real life examples of method development issues early career researchers have come across. After the presentations, the audience is divided into groups to collaboratively discuss the issue. The groups brainstorm solutions to their case study problem during discussions, and decide on what they would do to circumnavigate it. My group discusses the case of higher limit of detection threshold in urine in comparison to serum during the analysis of insulin. After some discussion about what could be happening to reduce the amount of analyte in urine, my group decides the main problem is sample loss through adsorption to the sample container. Among other strategies, we solve the issue by adding protein to the sample so it mimics serum proteins; that would prevent adsorption processes from occurring.
The meeting is concluded with thanks from the organising committee and a request to pencil in 2019’s planned meeting.

Riding home on the train, I discuss the workshop with my colleague. Personally, I found the day useful to recap on some of the proteomic strategies I have not used since starting my work on oligonucleotide therapeutics. I also learnt a little more about mass spectrometry, which will populate my analytical toolbox to help me in future troubleshooting. I enjoyed the collaborative environment during the meeting and will be aiming to attend the next event.

Reid Bioforum 2019 is planned for September 2019 in Cambridge. More information can be found on the website. I extend my thanks to the Chromatographic Society for funding my attendance to the workshop, and send appreciation to the Royal Society of Chemistry for being exceptional hosts!