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34 BioPharm International eBook July 2021 www.biopharminternational.com to establish a secondary structure base- line fingerprint for a biomolecule and then use it to assess the impact of change in formulation conditions due to pro- cess variability from batch-to-batch and between test and reference products. To further illustrate the advantages of MMS over FTIR and far-UV CD, it is useful to evaluate a set of experi- mental data showing how the perfor- mance of MMS compares with the two historical analytical techniques. In the study shown in Figure 2, 20 mg/ mL immunoglobulin (IgG1) samples were spiked with 20 mg/mL bovine serum albumin (BSA) for a series of 0% to 10% volume per volume (v/v) samples and analyzed using conven- tional FTIR technology and MMS to assess the ability of the techniques to detect and quantify structural impuri- ties within each sample (5). Analogous measurements were carried out using far-UV CD but at a much-reduced sample concentration of 1 mg/mL for each spiked sample in keeping within the limitations of the technique. The M MS results show greater linear- ity versus the F TIR data, and much greater sensitivity with a limit of quan- titation (LOQ ) of 0.76% vs 22.7%, respectively. Furthermore, these results are achieved with a direct labor prepa- ration time for all 10 samples of around 15 minutes for walk-away automation and analysis by MMS, compared with more than five hours by FTIR. Traditional FTIR spectroscopy has been difficult to establish as a useful technique within the commonly used biopharmaceutical characterization tool kit for many reasons. Optimal instru- ments in this tool kit must provide sample flexibility, wide measurable con- centration ranges, automation, and above all, the ability to work with complex formulation conditions found in clini- cally relevant samples. These are features that traditional secondary structure tools such as CD and FTIR simply do not possess. With the introduction of MMS, this tool kit has now evolved. Secondary structure characterization and the ability to detect unpredicted changes in sec- ondary structure for almost all protein biomolecule processes makes MMS a technique that can easily be deployed at any stage of the development process, from the study of protein–ligand inter- actions in early stage development to evaluating formulation conditions, by determining optimal process conditions, and performing batch comparisons post fill/finish. With MMS, it is now pos- sible to observe detrimental effects of these processes at the most fundamental level of protein stability, the secondary structure level, and at the earliest possi- ble stage of development. REFERENCES 1. H. Fabian and W. Mantele, Handbook of Vibrational Spectroscopy, J.M. Chalmers and P.R. Griffiths, Eds., pp. 3399–3425 (John Wiley & Sons, Chichester, UK, 2002). 2. J.K. Koenig and D.L. Tabb, Analytical Applications of FTIR to Molecular and Biological Systems, J.R. Durig, Ed., pp. 241–255 (D. Reidel, Boston, 1980). 3. A. Dong, P. Huang, and W.S. Caughey, Biochemistry 29, 3303–3308 (1990). 4. W. Wang and C.J. Roberts, Aggregation of Therapeutic Proteins (Wiley, Hoboken, NJ, 2010). 5. B.S. Kendrick, et al., J. Pharm Sci. 109 (1) 933–936 (2020). BP Biopharmaceutical Analysis Protein Characterization Figure 2. Microfluidic Modulation Spectroscopy (MMS), shown in the middle, offers reduced labor times relative to Fourier transform infrared (FTIR) spectroscopy (left panel) and a lower limit of quantitation (LOQ) than either of the incumbent technologies, including far ultraviolet circular dichroism (Far UV CD) (right panel) for measuring secondary structure from the same in complex buffer solution.