The safety requirements for vaccines are extremely high since they are administered to healthy people. For that reason, vaccine development is time-consuming and very expensive. Reducing time-to-market is key for pharmaceutical companies, saving lives and money. Therefore the need is raised for systematic, general and efficient process development strategies to shorten development times and enhance process understanding. High throughput technologies tremendously increased the volume of process-related data available and, combined with statistical and mechanistic modeling, new high throughput process development (HTPD) approaches evolved. The introduction of model-based HTPD enabled faster and broader screening of conditions, and furthermore increased knowledge. Model-based HTPD has particularly been important for chromatography, which is a crucial separation technique to attain high purities. This review provides an overview of downstream process development strategies and tools used within the (bio)pharmaceutical industry, focusing attention on (protein subunit) vaccine purification processes. Subsequently high throughput process development and other combinatorial approaches are discussed and compared according to their experimental effort and understanding. Within a growing sea of information, novel modeling tools and artificial intelligence (AI) gain importance for finding patterns behind the data and thereby acquiring a deeper process understanding.@en

Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E. coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E. coli K12 strain.@en

An optimal purification process for biopharmaceutical products is important to meet strict safety regulations, and for economic benefits. To find the global optimum, it is desirable to screen the overall design space. Advanced model-based approaches enable to screen a broad range of the design-space, in contrast to traditional statistical or heuristic-based approaches. Though, chromatographic mechanistic modeling (MM), one of the advanced model-based approaches, can be speed-limiting for flowsheet optimization, which evaluates every purification possibility (e.g., type and order of purification techniques, and their operating conditions). Therefore, we propose to use artificial neural networks (ANNs) during global optimization to select the most optimal flowsheets. So, the number of flowsheets for final local optimization is reduced and consequently the overall optimization time. Employing ANNs during global optimization proved to reduce the number of flowsheets from 15 to only 3. From these three, one flowsheet was optimized locally and similar final results were found when using the global outcome of either the ANN or MM as starting condition. Moreover, the overall flowsheet optimization time was reduced by 50% when using ANNs during global optimization. This approach accelerates the early purification process design; moreover, it is generic, flexible, and regardless of sample material's type.@en

Protein-based biopharmaceuticals require high purity before final formulation to ensure product safety, making process development time consuming. Implementation of computational approaches at the initial stages of process development offers a significant reduction in development efforts. By preselecting process conditions, experimental screening can be limited to only a subset. One such computational selection approach is the application of Quantitative Structure Property Relationship (QSPR) models that describe the properties exploited during purification. This work presents a novel open-source Python tool capable of extracting a range of features from protein 3D models on a local computer allowing total transparency of the calculations. As open-source tool, it also impacts initial investments in constructing a QSPR workflow for protein property prediction for third parties, making it widely applicable within the field of bioprocess development. The focus of current calculated molecular features is projection onto the protein surface by constructing surface grid representations. Linear regression models were trained with the calculated features to predict chromatographic retention times/volumes. Model validation shows a high accuracy for anion and cation exchange chromatography data (cross-validated R2 of 0.87 and 0.95). Hence, these models demonstrate the potential of the use of QSPR to accelerate process design.@en