Calibration-free PAT: Locating selective crystallization or precipitation sweet spot in screenings with multi-way PARAFAC models

When developping selective crystallization or precipitation processes,
biopharmaceutical modalities require empirical screenings and analytics
tailored to the specific needs of the target molecule. The multi-way
chemometric approach called parallel factor analysis (PARAFAC) coupled
with ultraviolet visible light (UV/Vis) spectroscopy is able to predict specific
concentrations and spectra from highly structured data sets without the need
for calibration samples and reference analytics. These calculated models can
provide exploratory information on pure species spectra and concentrations in
all analyzed samples by representing one model component with one species.
In this work, protein mixtures, monoclonal antibodies, and virus-like particles in
chemically defined and complex solutions were investigated in three high-
throughput crystallization or precipitation screenings with the aim to construct
one PARAFAC model per case. Spectroscopic data sets of samples after the
selective crystallization or precipitation, washing, and redissolution were
recorded and arranged into a four-dimensional data set per case study.
Different reference analytics and pure species spectra served as validation.
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Appropriate spectral preprocessing parameters were found for all case studies
allowing even the application of this approach to the third case study in which
quantitative concentration analytics are missing. Regardless of the modality or
the number of species present in complex solutions, all models were able to
estimate the specific concentration and find the optimal process condition
regarding yield and product purity. It was shown that in complex solutions,
species demonstrating similar phase behavior can be clustered as one
component and described in the model. PARAFAC as a calibration-free
approach coupled with UV/Vis spectroscopy provides a fast overview of
species present in complex solution and of their concentration during
selective crystallization or precipitation, washing, and redissolution.