Virus‐like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo‐assembled VLPs are typically dis‐ and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high‐pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material‐consuming disassembly experiments. To this end, we developed a low‐volume and high‐resolution disassembly screening that provides time‐resolved insight into the VLP disassembly progress. In this study, two variants of C‐terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high‐throughput screening results, a diafiltration‐based disassembly process step was developed. Compared with mixing‐based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration‐based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high‐molecular‐weight species.
Selective protein crystallization is a trending alternative to preparative chromatography in biotechnological downstream processing. To save time and resources in early-stage process development, fast and reliable analytics are required. This work aimed to develop and assess a low-volume, quantitative, analytical tool for faster development of crystallization processes. The analytical tool was based on ultraviolet–visible spectroscopy and partial least-squares modeling and aimed to selectively quantify protein concentrations in heterogeneous supernatants during crystallization process development. For this purpose, a ternary model protein system consisting of hen-egg-white Lysozyme, bovine Ribonuclease A, and equine Cytochrome C was used for model calibration and subsequent crystallization studies for application. In a high-throughput screening, Lysozyme was selectively crystallized varying pH, precipitant concentration, and Lysozyme concentration at 8 °C for 13 days. During a kinetic study, the composition of two selected conditions was monitored over a time range of 7 days. In both studies, the developed tool quantified the different species in the supernatant with high precision. Crystal yield, purity, and selectivity were evaluated with a sensitivity of 96.23% and a short analysis time of 3 min per sample. The studies were carried out in 96-well plates. This said, the methodology could be easily adapted to higher throughput scales, i.e., 384-well or 1536-well plates.
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. 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.
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