ATR-FTIR spectroscopy and spectroscopic imaging to investigate the behaviour of proteins subjected to freeze-thaw cycles in droplets, wells, and under flow.
File(s)
Published version
Author(s)
Tiernan, Hannah
Byrne, Bernadette
Kazarian, Sergei G
Type
Journal Article
Abstract
Biopharmaceuticals are used to treat a range of diseases from arthritis to cancer, however, since the advent of these highly specific, effective drugs, there have been challenges involved in their production. The most common biopharmaceuticals, monoclonal antibodies (mAbs), are vulnerable to aggregation and precipitation during processing. Freeze thaw cycles (FTCs), which can be required for storage and transportation, can lead to a substantial loss of product, and contributes to the high cost of antibody production. It is therefore necessary to monitor aggregation levels at susceptible points in the production pathway, such as during purification and transportation, thus contributing to a fuller understanding of mAb aggregation and providing a basis for rational optimisation of the production process. This paper uses attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and spectroscopic imaging to investigate the effect of these potentially detrimental FTCs on protein secondary structure in both static wells and under flowing conditions, using lysozyme as a model protein. The results revealed that the amount of protein close to the surface of the ATR crystal, and hence level of aggregates, increased with increasing FTCs. This was observed both within wells and under flow conditions, using conventional ATR-FTIR spectroscopy and ATR-FTIR spectroscopic imaging. Interestingly, we also observed changes in the Amide I band shape indicating an increase in β-sheet contribution, and therefore an increase in aggregates, with increasing number of FTCs. These results show for the first time how ATR-FTIR spectroscopy can be successfully applied to study the effect of FTC cycles on protein samples. This could have numerous broader applications, such as in biopharmaceutical production and rapid diagnostic testing.
Date Issued
2021-05-07
Date Acceptance
2021-03-07
Citation
The Analyst, 2021, 146 (9), pp.2902-2909
ISSN
0003-2654
Publisher
Royal Society of Chemistry
Start Page
2902
End Page
2909
Journal / Book Title
The Analyst
Volume
146
Issue
9
Copyright Statement
© The Royal Society of Chemistry 2021. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/33724288
Subjects
0301 Analytical Chemistry
0399 Other Chemical Sciences
Analytical Chemistry
Publication Status
Published
Coverage Spatial
England
Date Publish Online
2021-03-08