Integrated Process for Capture and Purification of Virus-Like Particles: Enhancing Process Performance by Cross-Flow Filtration.

Virus-like particles (VLPs) are emerging nanoscale protein assemblies applied as prophylactic vaccines and in development as therapeutic vaccines or cargo delivery systems. Downstream processing (DSP) of VLPs comes both with challenges and opportunities, depending on the complexity and size of the structures. Filtration, precipitation/re-dissolution and size-exclusion chromatography (SEC) are potent technologies exploiting the size difference between product and impurities. In this study, we therefore investigated the integration of these technologies within a single unit operation, resulting in three different processes, one of which integrates all three technologies. VLPs, contained in clarified lysate from Escherichia coli, were precipitated by ammonium sulfate, washed, and re-dissolved in a commercial cross-flow filtration (CFF) unit. Processes were analyzed for yield, purity, as well as productivity and were found to be largely superior to a reference centrifugation process. Productivity was increased 2.6-fold by transfer of the wash and re-dissolution process to the CFF unit. Installation of a multimodal SEC column in the permeate line increased purity to 96% while maintaining a high productivity and high yield of 86%. In addition to these advantages, CFF-based capture and purification allows for scalable and disposable DSP. In summary, the developed set-up resulted in high yields and purities, bearing the potential to be applied as an integrated process step for capture and purification of in vivo-assembled VLPs and other protein nanoparticles.

High-throughput computational pipeline for 3-D structure preparation and in silico protein surface property screening: A case study on HBcAg dimer structures.

Knowledge-based experimental design can aid biopharmaceutical high-throughput screening (HTS) experiments needed to identify critical manufacturability parameters. Prior knowledge can be obtained via computational methods such as protein property extraction from 3-D protein structures. This study presents a high-throughput 3-D structure preparation and refinement pipeline that supports structure screenings with an automated and data-dependent workflow. As a case study, three chimeric virus-like particle (VLP) building blocks, hepatitis B core antigen (HBcAg) dimers, were constructed. Molecular dynamics (MD) refinement quality, speed, stability, and correlation to zeta potential data was evaluated using different MD simulation settings. Settings included 2 force fields (YASARA2 and AMBER03) and 2 pKa computation methods (YASARA and H++). MD simulations contained a data-dependent termination via identification of a 2 ns Window of Stability, which was also used for robust descriptor extraction. MD simulation with YASARA2, independent of pKa computation method, was found to be most stable and computationally efficient. These settings resulted in a fast refinement (6.6-37.5 h), a good structure quality (-1.17--1.13) and a strong linear dependence between dimer surface charge and complete chimeric HBcAg VLP zeta potential. These results indicate the computational pipeline's applicability for early-stage candidate assessment and design optimization of HTS manufacturability or formulability experiments.

Impact of Polymer Bioconjugation on Protein Stability and Activity Investigated with Discrete Conjugates: Alternatives to PEGylation.

Covalent attachment of synthetic polymers to proteins, known as protein-polymer conjugation, is currently one of the main approaches for improving the physicochemical properties of these biomolecules. The most commonly employed polymer is polyethylene glycol (PEG), as evidenced by extensive research and clinical track records for its use in biopharmaceuticals. However, the occurrence of allergic reactions or hypersensitivity and the discovery of PEG antibodies, on the one hand, and the rise of controlled polymerization techniques and novel monomers, on the other hand, have been driving the search for alternative polymers for bioconjugation. The present study describes the synthesis, purification, and properties of conjugates of lysozyme with poly( N-acryloylmorpholine) (PNAM) and poly(oligoethylene glycol methyl ether methacrylate) (POEGMA). Particularly, conjugate species with distinct conjugation degrees are investigated for their residual activity, aggregation behavior, and solubility, by using a high-throughput screening approach. Our study showcases the importance of evaluating conjugates obtained by nonsite-specific modification through isolated species with discrete degrees of conjugation rather than on the batch level. Monovalent conjugates with relatively low molar mass polymers displayed equal or even higher activity than the native protein, while all conjugates showed an improved protein solubility. To achieve a comparable effect on solubility as with PEG, PNAM and POEGMA of higher molar masses were required.

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins.

Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag™ technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag™ was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced.