Preparative liquid chromatography has been a staple of purification processes for biopharmaceutical products for decades. Current liquid chromatography processes rely heavily on in-line measurements of UV absorption at 280 nm as a surrogate signal to monitor the separation of the target compound from impurities such as product related species, DNA, host cell proteins and media components. Monitoring the UV signal may indicate deviations from expected process characteristics but does not usually yield any highly specific information. While FTIR is slower and less sensitive, it is more selective than UV/Vis as it can detect molecular vibrations of any heteromolecular bonds. Therefore, FTIR can be utilized for monitoring multiple critical quality attributes of the chromatographic process such as the secondary structure of proteins and process related impurities.<br/>Here, a custom experimental setup was implemented to perform in-line Fourier Transform Infrared Spectroscopy (FTIR) measurements to evaluate the potential of using in-line FTIR as a PAT tool in downstream processing. The experimental setup consisted of a BioATR II flow cell in a Bruker Tensor 27 attached to an AKTApurifier chromatography system and was used to monitor different cation-exchange chromatography experiments. The recorded data was pre-treated to account for the influence of salt gradients on the spectrum. Partial least square regression was employed as a means of multivariate data analysis (MVDA) to correlate offline analytics of collected fractions to the FTIR 3D-fields.<br/>As a first case study, the elution of a protein mixture consisting of lysozyme and IgG 2 was used as a model system to show the two species can be distinguished using the FTIR signal. Second, the separation of PEGylated protein species was monitored, with PEG being an example of a compound widely used in pharmaceuticals that is not UV-active. Finally, Triton X-100 was added to a feed of lysozyme as a simulated process related impurity and it was shown that the species could be observed in the FTIR 3D-field of the effluent.<br/>The proposed PAT tool can potentially be used as an alternative option to monitor downstream processes towards a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques.

In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography

255th National Meeting (2018)

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