Studying Conformational Changes and Agglomeration in Biopharmaceuticals Using Spectroscopic Imaging in Microfluidics

Lead Research Organisation: Imperial College London
Department Name: Department of Chemical Engineering

Abstract

In 2011, sales of 40 therapeutic monoclonal antibodies (mAbs) accounted for $45 billion, around 5% of the global therapeutic market and this continues to rise annually. These medicines present a range of new challenges for the pharmaceutical industry. For example, mAbs have a propensity to non-specifically aggregate, reducing bioactive yields, affecting drug potency and potentially producing highly undesirable immunological effects such as anaphylactic shock. Prof. Kazarian's research groups have successfully applied ATR-FTIR spectroscopic imaging to the analysis of mAbs. [1-4] This label-free, non-destructive spectroscopic imaging technique can detect both aggregation status and conformational state of a protein sample. mAb aggregation can be triggered by a range of environmental factors including: shear stress, pH and exposure to different surfaces during processing. In recent years, microfluidic chip-based systems have been developed to allow analysis of mABs at different stages during the sample handling process including; sample pre-treatment, reaction, product separation and analyte detection. I will therefore study mAb solutions under flow using ATR-FTIR imaging approaches in microfluidic devices that provide ideal platforms for rapid, high-throughput screening of dynamic systems. The chemical specificity of this technique allows both detection of changes in the secondary structure as a result of unfolding and/or protein degradation both of which can lead to aggregation. Custom microfluidic devices will be designed which can be attached directly to the ATR-FTIR imaging set-up allowing for experiments to be performed in situ as demonstrated previously by our group for other systems. [5] The devices will be constructed to investigate different possible causes of protein aggregation. For example a device containing multiple channels can be built, each of which would provide a different level of shear stress. The solutions would then be passed through the channels multiple times to investigate the onset of subtle conformational changes and eventually aggregation. Other devices can also be used which would expose the mAbs to a range of different surfaces including silicone oil or other materials, or allow the introduction of air bubbles to assess the effects of an air interface on mAb aggregation.
At the start of the project I will begin where previous work in the group had finished, using 12 individual wells to study protein aggregation in a range of pH's and temperatures as well as at different protein concentrations. [1] This established method will allow me to become familiar with the set up and develop expertise in data analysis prior to moving on to the more complex microfluidic experiments. Prof. Kazarian will provide expertise in spectroscopic imaging and access to the world leading facilities in this area, while Prof. Byrne will provide expertise in protein isolation and behaviour. The laboratories in two different Imperial College departments and at BMS provide state-of-the-art facilities and a unique opportunity to combine and inter-relate multidisciplinary expertise. In addition, alignment with the research interests of BMS provides real-world relevance for my studies. The success of the proposed combination of microfluidics with FTIR spectroscopic imaging for assessment of proteins under flow will impact applications in the fields across physical science, biological chemistry and potentially medicine.
1. Boulet-Audet, M., Byrne, B., Kazarian, S. G. High-throughput thermal stability analysis of a monoclonal antibody by ATR-FTIR spectroscopic imaging Analytical Chemistry (2014) 86(19), 9786-9793. (Open Access article)
2. Boulet-Audet M., Byrne B., Kazarian S. G. Cleaning-in-place for immunoaffinity resin monitored by in situ ATR-FTIR spectroscopy Analytical and Bioanalytical Chemistry (2015) 407, 7111-7122. (Open Access)
3. Boulet-Audet M., Kazarian S. G, Byrne B., In-column ATR-FTIR spectroscopy to

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011178/1 01/10/2015 30/09/2023
1958876 Studentship BB/M011178/1 30/09/2017 30/09/2021 Hannah Daisy Tiernan