FTIR-based structural and fingerprinting analysis
FTIR spectroscopy relies on the fact that most molecules absorb light in the infrared region of the electromagnetic spectrum, converting it to molecular vibrations. It is recognized as a powerful and sensitive tool to record fingerprints of complex mixtures such as biological samples. These fingerprints are robust and account not only for the chemical nature of molecules but also for their conformation and are, in particular, very sensitive to protein secondary structure.
Concerning protein and antibodies, 4 critical quality attributes can be assessed: structure, glycosylation, total protein concentration and the quantification of excipients such as Tween. This analysis is realized in a quick (only a couple of minutes) and direct manner, with limited sample volume (around 50 µg) and without need for extensive sample pre-treatment, physical separation or recurring calibration. Multivariate and comparative statistical tools are used to interpret the fingerprints provided by the FTIR spectra. The three main applications are the following:
- Structural assessment: detailed information related to the secondary structure ca be obtained. It is also very interesting to study amyloid proteins as it allows the distinction of monomers, oligomers and fibrils.
- Stability studies: Comparing a control sample (timepoint zero or unstressed sample) with various stability or stressed conditions allows the detection of minor structural and chemical changes. It can thus be used as a predictive tool to evaluate the stability of biological samples.
- Batch-to-batch consistency check: the unique fingerprints provided by FTIR spectra can also be used to compare several batches of the same product (pure protein or more complex products).