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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.

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy is a label-free, non-destructive technique that can be applied to a vast range of biological applications, from imaging cancer tissues and live cells, to determining protein content and protein secondary structure composition. This review summarises the recent advances in applications of ATR-FTIR spectroscopy to biopharmaceuticals, the application of this technique to biosimilars, and the current uses of FTIR spectroscopy in biopharmaceutical production. We discuss the use of ATR-FTIR spectroscopic imaging to investigate biopharmaceuticals, and finally, give an outlook on the possible future developments and applications of ATR-FTIR spectroscopy and spectroscopic imaging to this field. Throughout the review comparisons will be made between FTIR spectroscopy and alternative analytical techniques, and areas will be identified where FTIR spectroscopy could perhaps offer a better alternative in future studies. This review focuses on the most recent advances in the field of using ATR-FTIR spectroscopy and spectroscopic imaging to characterise and evaluate biopharmaceuticals, both in industrial and academic research based environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Monoclonal antibodies (mAbs) have been used as therapeutics for the last few decades. It is necessary to investigate the stability of these mAbs under stress conditions and to elucidate aggregation mechanisms as a means of developing approaches which minimize the problem. Attenuated total reflection (ATR)-FTIR spectroscopic imaging allows probing of a sample at a depth of penetration of around 0.5-5 μm, which makes it suitable for the study of aggregated proteins when accumulated as a layer close to the surface of the ZnSe internal reflection element (IRE). Here, macro ATR-FTIR spectroscopic imaging, along with a variable angle of incidence accessory, have been used to differentiate between the secondary structure of proteins in bulk solution and those that have precipitated onto or near the ZnSe IRE surface. IgG spectra obtained from protein samples in individual wells have been averaged, extracted, and preprocessed, and the Amide I bands of the protein samples were compared and further analyzed to reveal protein distribution at the ZnSe IRE surface. These findings show depth profiling of IgG aggregates at the ZnSe IRE surface (0.5-5 μm) and do not follow a trend of decreasing protein presence with an increasing angle of incidence or increasing depth of penetration, suggesting an irregular distribution of aggregates in the z -direction.