Immobilization of large biomacromolecules is often required for analytical quantification and physicochemical characterization. However, immobilization can alter the structure and size of the particles being studied. Here, two exosomes (derived from HEK-293 and MDA-MB-231 cells) and three viral particles (Suid herpesvirus 1 (SuHV), xenotropic murine leukemia virus (XmuLV), and porcine parvovirus (PPV)) were immobilized to different covalent chemistries to understand how surface chemistry influences particle deformation during immobilization. The surface chemistries explored were: (i) NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride), and (ii) poly l-lysine (PLL) and glutaraldehyde (GA). Morphological changes in biomolecules following immobilization were quantified by measuring the height-to-diameter (H/D) ratios attained from atomic force microscopy (AFM) topographic images. These observations were further supported by complementary size and morphology analyses using dynamic light scattering (DLS) and liquid phase transmission electron microscopy (TEM). NHS/EDC chemistry consistently resulted in more significant particle flattening than PLL/GA, as evidenced by lower average H/D ratios across all biomacromolecules. Greater flattening effects were observed on the soft lipid envelope of exosomes as compared to viruses, due to differences in structural rigidity. Both immobilization chemistries resulted in a lower H/D ratio in tumor-derived MDA-MB-231 exosomes compared to nontumor-derived HEK-293 exosomes, likely due to the known softer mechanical properties of tumor-derived exosomes. Furthermore, immobilization of the enveloped viruses SuHV and XMuLV with NHS/EDC exhibited flattening effects and lower H/D ratios. Immobilization of nonenveloped PPV resulted in a low H/D ratio on NHS/EDC, which was likely due to particle aggregation rather than deformation. These findings provide valuable guidance for selecting appropriate surface chemistries for nanoscale biointerface studies and offer implications for surface-based diagnostics, high-throughput biosensing, and nanomaterial functionalization.

Virus-based vaccines and therapies require a purification method that is both cost-effective and easily scalable. An aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and citrate salt has been proven to deliver high virus recoveries along with high impurity removal. However, these systems often place the virus into a viscous PEG-rich phase or at the two-phase interface, leading to difficulties in subsequent downstream processes. This study explored a second ATPS to extract the virus product back into the citrate-rich phase by changing the chemical conditions, a required step for future application of ATPS in industrial processes. ATPS performance was tested as a function of phase component concentration, phase component volume ratios, PEG molecular weight, salt type, pH, and glycine addition to identify the most impactful parameters for the extraction of non-enveloped porcine parvovirus (PPV). By shifting the pH, lowering phase component concentrations, and increasing the volume ratio of the citrate-rich phase between the first and second ATPS steps, 66 % of infectious PPV was recovered with 2.0 logs of host cell protein removal and 1.0 logs of host cell DNA removal. Using a PEG molecular weight of 8 kDa enabled a pH shift between the first and second ATPS steps without precipitation. Glycine addition during the first step of ATPS and phosphate salt use during the second step of ATPS did not significantly increase the overall recovery. In future studies, the optimized process will be implemented for multiple viral vector types and continuously to demonstrate continuous and low-cost viral vector manufacturing.

Aqueous two-phase systems (ATPS) have been used to purify a range of biomolecules, including small molecules, monoclonal antibodies, viruses, and whole cells. They are known for selective separations, creating a stabilizing, low-shear environment, and high yields. Recently, as biomanufacturing attempts to adopt continuous processing, attention has shifted to ATPS for its ability to operate fully continuously while incurring lower costs than many chromatographic methods. But despite 60 years of exploration and development, the complex network of interlinked driving forces controlling these separations has prevented robust development of process understanding, and most ATPS separations are still optimized using slow and costly manual screening methods. As a result, industry has been unwilling to adopt ATPS. Fortunately, a growing body of literature is developing statistical and mechanistic models of ATPS to predict liquid-liquid equilibria and separations with reduced experimental burden. This review surveys the application of these models to ATPS, comparing their progress and potential to promote rapid development of bioseparations in the near and long term. The discussion evaluates the adaptability of statistical tools, like response surface methodology and artificial neural networks, and contrasts it with the process understanding generated through application of semi-empirical thermodynamic models. Strategies are explored to automate optimization of separations for new biomolecules using these models to create artificial data. By understanding the landscape of models applied to ATPS, this review will start a discussion about bringing this technology closer to commercialization and enabling continuous processing on a broader scale.

Meeting the increasing demand for vaccines throughout the world is key to decrease the spread of infectious diseases. The switch to a fully continuous vaccine manufacturing process would increase productivity and the supply of crucial vaccines. To aid in this switch, we have developed a novel, continuous downstream purification technique based on an aqueous two-phase system (ATPS). The system has the potential to be used as a platform system for viral product purification. A 12 kDa poly(ethylene glycol) (PEG) and trisodium citrate ATPS was able to purify porcine parvovirus (PPV) and human immunodeficiency virus type-1 group antigen virus-like particles (HIV VLPs) from cell supernatant. PPV was recovered in the PEG-rich phase at 90 +/- 16% with a DNA removal of 96 +/- 3% and >= 89% protein removal. The system was also able to recover 99 +/- 2% of HIV VLPs in the PEG-rich phase with a 73 +/- 1% DNA removal and high protein removal shown by SDS-PAGE. Continuous ATPS recovered virus at the same amount as batch recovery. This demonstrates that continuous ATPS can be scaled up and runrun continuously without a loss in purity or recovery. Mixing and settling time both played an important role in developing a continuous ATPS for viral particles.

Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations. The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system.