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Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process‐related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up‐to 50‐fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI‐Toyopearl resin was finally employed in a two‐step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis. [ABSTRACT FROM AUTHOR]

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time‐consuming and cost‐prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF‐1. The RGG domain can undergo liquid–liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme‐RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme‐RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes—a kinase, a phosphorylase, and an ATP‐dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co‐purification of these two enzymes by co‐phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis. [ABSTRACT FROM AUTHOR]

BackgroundIntracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis.ResultsTo overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed.ConclusionsOur strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol-gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol-gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol-gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications.

Abstract Background Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. Results To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed. Conclusions Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

Bio‐oils such as conventional soybean, high‐oleic soybean, canola, and sunflower are valuable as bio‐solvents for removing hydrogen sulfide (H2S) from natural gas. Preceding bench‐scale studies indicate that more than 90% of H2S can be removed from a gas stream; economic analysis of such a process is necessary to determine the solvent regenerative power required and cost constraints on a to‐be‐determined solvent regeneration scheme. With the goal of processing 1000 kmol·h–1 of sour gas and removing 99.9% of H2S from gas streams with various feed concentrations, the design of an absorption unit to process natural gas using bio‐oils as the absorbing solvent was carried out through equilibrium stage analysis. A graphical method combined with the Kremser method found that a trayed tower with 14 stages, a 2 m diameter, and 8.5 m height could meet these goals successfully with a bio‐solvent flow rate of 120 kmol·h–1. Capital costs were centered on the price of an extraction column designed to meet the desired throughput. Comparison with conventional amine gas treatment was used to set a limit for the cost of treating a unit of gas, and sensitivity analysis explored the relationship between solvent recycle and cost of treating the gas. This study found that the economic viability of using bio‐oils as gas‐sweetening agents depended on developing a solvent regeneration scheme capable of recycling more than 98% of the bio‐oil bio‐solvent. The development of such a scheme is unlikely, and the overall process of using bio‐oils to sweeten sour gas is likely not economically viable. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N‐terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion‐tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N‐terminus. RESULTS: Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed‐batch cultivations. The protein of interest (POI) carried an N‐terminal affinity fusion‐tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion‐tag with a circularly permuted human caspase‐2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N‐terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 μg mL−1 dsDNA and ~ 1000 EU mL−1 endotoxin. Only a small number of E. coli host cell proteins were co‐purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off‐target cleavage could be observed. CONCLUSION: Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusion‐protein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2‐based platform fusion protein process (CASPON technology) purification platform. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In the rapidly advancing realms of gene therapy and biotechnology, the efficient purification of viral vectors is pivotal for ensuring the safety and efficacy of gene therapies. This study focuses on optimizing membrane selection for viral vector purification by evaluating key properties, including porosity, thickness, pore structure, and hydrophilicity. Notably, we employed adeno-associated virus (AAV)-sized nanoparticles (20 nm), 200 nm particles, and bovine serum albumin (BSA) to model viral vector harvesting. Experimental data from constant pressure normal flow filtration (NFF) at 1 and 2 bar using four commercial flat sheet membranes revealed distinct fouling behaviors. Symmetric membranes predominantly showed internal and external pore blockage, while asymmetric membranes formed a cake layer on the surface. Hydrophilicity exhibited a positive correlation with recovery, demonstrating an enhanced recovery with increased hydrophilicity. Membranes with higher porosity and interpore connectivity showcased superior throughput, reduced operating time, and increased recovery. Asymmetric polyether sulfone (PES) membranes emerged as the optimal choice, achieving ∼100% recovery of AAV-sized particles, an ∼44% reduction in model cell debris (200 nm particles), an ∼35% decrease in BSA, and the fastest operating time of all membranes tested. This systematic investigation into fouling behaviors and membrane properties not only informs optimal conditions for viral vector recovery but also lays the groundwork for advancing membrane-based strategies in bioprocessing.

As upstream product titers increase, the downstream chromatographic capture step has become a significant "downstream bottleneck." Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever‐increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation‐prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2 to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7–7.5 wt/vol% together with 10 mM ZnCl2 gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties. [ABSTRACT FROM AUTHOR]

The design of molecularly selective interfaces can lead to efficient electrochemically-mediated separation processes. The fast growing development of electroactive materials has resulted in new electroresponsive adsorbents and membranes, with enhanced selectivity, higher uptake capacities, and improved energy performance. Here, we review progress on the interfacial design for electrochemical separations, with a focus on chemical and biological applications. We discuss the development of new electrode materials and the underlying mechanisms for selective molecular binding, highlighting areas of growing interest such as metal recovery, waste recycling, gas purification, and protein separations. Finally, we emphasize the need for integration between molecular level interface design and electrochemical engineering for the development of more efficient separation processes. We envision that electrochemical separations can play a key role towards the electrification of the chemical industry and contribute towards new approaches for process intensification. Image 1 [ABSTRACT FROM AUTHOR]

BACKGROUND l‐Asparaginase (ASNase) is an important biopharmaceutical for the treatment of acute lymphoblastic leukemia (ALL); however, with some restrictions due to its high manufacturing costs. Aqueous biphasic systems (ABS) have been suggested as more economical platforms for the separation/purification of proteins, but a full understanding of the mechanisms behind the ASNase partition is still a major challenge. Polymer/salt‐based ABS with different driving‐forces (salting‐out and hydrophilicity/hydrophobicity effects) were herein applied to control the partition of commercial ASNase. RESULTS: The main results showed the ASNase partition to the salt‐ or polymer‐rich phase depending on the ABS studied, with extraction efficiencies higher than 95%. For systems composed of inorganic salts, the ASNase partition was controlled by the polyethylene glycol (PEG) molecular weight used. Cholinium‐salts‐based ABS were able to promote a preferential ASNase partition to the polymer‐rich phase using PEG‐600 and to the salt‐rich phase using a more hydrophobic polypropylene glycol (PPG)‐400 polymer. It was possible to select the ABS composed of PEG‐2000 + potassium phosphate buffer as the most efficient to separate the ASNase from the main contaminant proteins (purification factor = 2.4 ± 0.2), while it was able to maintain the enzyme activity for posterior application as part of a therapeutic. CONCLUSION: Polymer/salt ABS can be used to control the partition of ASNase and adjust its purification yields, demonstrating the ABS potential as more economic platform for the selective recovery of therapeutic enzymes from complex broths. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Highlights • A new pH recycling ATPS has been developed for demeclocycline extraction. • The recoveries of polymers could reach over 95.0%. • The addition of MgSO 4 to ATPS can improve the extraction of demeclocycline. • Reaction temperature and initiator affected the polymer molecular weight. • Phase formation mechanism have been studied using Low-field NMR. Abstract The application of aqueous two-phase systems (ATPS) to bioseparation and biocatalyst engineering has attracted interest. However, despite the distinct advantages of the technique, the scaling up of ATPS is limited by the cost of the system components. In this study, two novel recyclable pH-responsive polymers were synthesized and tested for the partition of demeclocycline. The recoveries of two recyclable pH-responsive polymers could reach over 95.0% by only adjusting the solution pH to the isoelectric points of the polymers. The control variate method was used to investigate the effects of polymerization conditions on polymer synthesis. Polymer P ADB4.6 was firstly synthesized in 500 l reactor with two-stage heating method (25–40 ℃ and 40–62 ℃), as the heating rate is an important factor in the scale-up process. The polymer characteristics (molecular weight, viscosity, surface tension, and zeta potential) were studied to understand their influences on the synthetic process. Furthermore, the phase formation mechanism was studied with low-field nuclear magnetic resonance (LF-NMR). The optimal partition coefficient and recovery of demeclocycline using the recyclable ATPS were 0.24 and 82.9% at pH 6.30 in presence of 20 mmol/L MgSO 4. [ABSTRACT FROM AUTHOR]

Agar microspheres were prepared by water–oil emulsification and cross‐linked under alkaline condition. The thermoresponsive hydrophobic copolymer, poly(N‐isopropylacrylamide‐co‐lauryl methacrylate‐co‐acrylamide), was grafted on the agar microspheres via atom transfer radical polymerization. The agar microspheres grafted with copolymers were characterized by light microphotography, elemental analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, and X‐ray photoelectron spectroscopy. The chain lengths and hydrophobic monomer ratio of the grafting linear polymer had significant effects on the hydrophobicity and adsorption capacity of agar microspheres at different temperatures. The thermoresponsive microspheres were used for separation of proteins and showed binding and release behavior by change of temperatures without change in mobile phase composition. Thus, we suggest thermoresponsive agar microspheres as an alternative separation media for all‐aqueous bioseparations. [ABSTRACT FROM AUTHOR]

Virus filters are essential in bioprocessing for safe therapeutic protein production. Understanding the correlation between virus filter structure and performance is important for efficient process design. By applying a multilayer structural model comprised of theoretical layers having pore size distributions, to published visualization data, we demonstrate in the present study that virus removal performance and filtration behavior can be quantitatively calculated. Virus filter structure can be classified into asymmetric high porosity, symmetric high density and laminated structure types. All types exhibit sufficient virus removal properties when used in normal processes. Laminated structure filters show extremely high virus particle removal performance, achieving the most robust virus removal even in filtration with process pauses, although flux decay tends to occur during filtration of solutions containing even very small amounts of aggregate. Conversely, asymmetric high porosity filters may show lower virus removal in processes involving multiple pauses that exceed practical conditions but shows stable filtration behavior with lower increase in pressure and lower flux decay even for the filtration of solutions containing aggregates. Such filtration behavior and virus removal properties can be quantitatively expressed and predicted by numerical calculations using a model based on the theoretical multilayer structure. [Display omitted] • The pore size distribution structure of the virus filter is theoretically calculated. • Each commercially available virus filter has a unique pore size distribution structure. • Particle retention distribution in the filter is reproduced by the multilayer model. • The pore size distribution in the filtration direction is given by the multilayer model. • The log reduction value (LRV) is able to be calculated by the membrane structure. [ABSTRACT FROM AUTHOR]

BACKGROUND: The proton‐translocating protein F0F1 ATP synthase is a transmembrane protein that catalyzes ATP synthesis and is important in artificial photosynthesis and other life science studies. The objective of this study was to understand the accumulation and ATPase activity of F0F1 ATP synthase in the thermophilic bacteria Bacillus PS3 affected by culture conditions, including the type of nitrogen source and the concentrations of polypeptone and sodium chloride in the culture medium, as well as medium pH and aeration rate. RESULTS: The highest yield and ATPase activity of purified enzymes were obtained when adding polypeptone as the nitrogen source at higher concentrations. Medium pH and aeration rate showed significant effects on the accumulation and activity of F0F1 ATP synthase. Contrarily, sodium chloride concentration had little effect on the growth of PS3 or F0F1 ATP synthase accumulation. The highest yield and specific activity of purified F0F1 ATP synthase achieved were 1.631 g L−1 and 7.99 µmol min−1 mg−1, under the culture conditions of pH 7, aeration of 3 L min−1 and 0.3% NaCl and 1.2% (w/w) polypeptone concentrations. The F0F1 ATP synthase purified was characterized by SDS‐PAGE and contained all expected subunits. CONCLUSION: All of the studied culture conditions except for NaCl concentration significantly affected Bacillus PS3 cell growth and ATP synthase accumulation and reactivity. Polypeptone at higher concentrations in the culture medium along with neutral pH and greater aeration rates were desirable. Cell growth and ATPase activity of F0F1 ATP synthase were highly positively associated. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Highlights • Protein A chromatography can be improved significantly through the use of continuous chromatography and new Protein A stationary phases. • A model was developed in order to optimize Protein A processes with respect to load per cycle and productivity. • Multi-column chromatography reduces product yield loss while increasing load per cycle. • Smaller Protein A resin bead sizes provide performance improvements of up to 50%. Abstract Effect of particle size (85μm vs. 50μm) on the performance of continuous capture chromatography using Protein A affinity was evaluated in combination with varied feed titers, loading flow rates and target breakthrough using a Design of Experiments (DoE) approach. In comparison to previous studies, higher cell culture titers on the order of 5–15 g/L, relevant to current high productivity industrial cell lines, were evaluated. Further, three modes of capture continuous chromatography were included in the DoE: single column batch, 2-column CaptureSMB and 4-column periodic counter-current chromatography (PCC). The breakthrough percentage at the outlet of the first column being loaded showed the most significant impact on process performance, confirming the advantage of multi-column over batch chromatography processes. Out of the two resins, the one with smaller particle size displayed significantly better performance. To verify and generalize these results, a shrinking core model for protein A chromatography has been developed and validated. The model was used to optimize the processes with respect to capacity utilization (load per cycle) and productivity (load per time). The smaller particle size resin (50μm) produced steeper breakthrough curves and allowed for better capacity utilization at any given productivity value. The improvement in loading was around 15% on average in comparison to the 85μm bead size in spite of the ligand density being same. The 50μm resin also allowed for higher maximum productivity values compared to the 85μm resin (improvements of 25–50%, depending on the process), despite lower maximum flow rate due to increased pressure drop. In addition, it is worth noting that recovery and regeneration rather than the maximum flow rate (pressure drop) became the limiting factor for process optimization in almost all considered scenarios. [ABSTRACT FROM AUTHOR]

Abstract: Expanded bed adsorption (EBA) emerged in the early 1990s in an attempt to integrate the clarification, capture and initial product concentration/purification process. Several mathematical models have been put forward to describe its operation. However, none of the models developed specifically for EBA allows simultaneous prediction of bed hydrodynamics, mass transfer/adsorption and (unwanted) interactions and fouling. This currently limits the development and early optimization of EBA‐based separation processes. In multiphase reactor engineering, the use of multiphase computational fluid dynamics has been shown to improve fundamental understanding of fluidized beds. To advance EBA technology, a combination of particle, equipment and process scale models should be used. By employing a cascade of multiscale simulations, the various challenges EBA currently faces can be addressed. This allows for optimal design and selection of equipment, materials and process conditions, and reduces risks and development times of downstream processes involving EBA. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: The use of antibodies, such as immunoglobulin G (IgG), has seen a significant growth in recent decades for biomedical and research purposes. However, antibodies are high cost biopharmaceuticals, for which the development of alternative and cost‐effective purification strategies is still in high demand. RESULTS: Aqueous biphasic systems (ABS) composed of poly(propylene glycol) (PPG) and cholinium‐based ionic liquids (ILs) were investigated for the separation of IgG. The ABS phase diagrams were determined whenever required. Initial studies with commercial IgG were carried out, followed by IgG separation from rabbit serum. In all ABS, IgG preferentially partitions to the IL‐rich phase, unveiling preferential interactions between IgG and ILs. Extraction efficiencies ranging between 93% and 100%, and recovery yields ranging between 20% and 100%, were obtained for commercial IgG. Two of the best and two of the worst identified ABS were then evaluated for their performance to separate IgG from rabbit serum, where extraction efficiencies of 100% and recovery yields >80% were obtained. Under the best conditions studied, IgG with a purity level of 49% was obtained in a single‐step. After an ultrafiltration step applied to the best ABS, allowing the IgG recovery from the IL‐rich phase, the IgG purity level increased to 66%. This purity level of IgG is higher than those previously reported using other IL–polymer ABS. CONCLUSION: IgG preferentially migrates to the IL‐rich phase in ABS formed by ILs and polymers, allowing the design of effective separation systems for its recovery from serum samples. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Integrated continuous production technology is of great interest in biopharmaceutical industry. Efficient, flexible and cost effective methods for continuous cell removal have to be developed, before a fully continuous and integrated product train can be realized. The paper describes the development and testing of such an integrated continuous and disposable set‐up for cell separation by flocculation combined with depth filtration. RESULTS: Screening of multiple flocculation agents, depth filters, and conditions demonstrated that the best performance was obtained with 0.0375% polydiallyldimethylammonium chloride (pDADMAC; a polycationic flocculation agent) in combination with Clarisolve® depth filters. Using this set‐up, a 4‐fold decrease of filtration area was achieved relative to standard filtration without flocculation, with yields of ≥97% and DNA depletion of up to 99%. Continuous operation was accomplished using a simple tubular reactor design with parallelization of the filtration. The reactor length was selected to allow a 13.2‐min residence time, which was sufficient to complete flocculation in batch experiments. Continuous flocculation performance was monitored on‐line using focused beam reflectance measurement. Filter switch cycles based on upstream pressure were controlled by in‐line pressure sensors, and were stable from one filter to the next. CONCLUSION: It was demonstrated that stable and efficient continuous flocculation associated with depth filtration can be easily accomplished using tubular reactors and parallelization. Continuous cell separation is essential for the development of fully continuous integrated process trains. This cost‐efficient disposable design run in continuous mode significantly reduces facility foot print, process costs and enables great flexbility. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Monoclonal antibodies are becoming a leading class of biopharmaceuticals but to increase their accessibility by the general population, new production processes must be developed in particular for the downstream processing. RESULTS: In this work, an alternative and innovative affinity chromatographic method using quaternary amine matrices is proposed. Separation is driven by the dual affinity ligand LYTAG‐Z, composed of a choline binding polypeptide (LYTAG) and the synthetic antibody binding Z domain. A two‐elution method was developed for the purification of mAbs and the performance of different anion exchangers containing quaternary amines that act as choline analogues – CIMmultus Q, Q Sepharose and gPore Q – were tested and compared, with both CIMmultus Q and Q Sepharose allowing a recovery of more than 94% of mAbs from a CHO cell supernatant with a purity greater than 95%. An integrated platform combining an initial affinity extraction step for the clarification and capture of mAbs and a subsequent chromatographic separation using Q‐matrices for the polishing of mAbs is also proposed. LYTAG‐Z triggers the extraction of 94.7 ± 1.7% mAbs to the PEG‐rich phase, as opposed to 26.9 ± 0.6% in the absence of the ligand, using 7% PEG 3350 and 6% dextran 500 k. Further purification using Q Sepharose allowed a mAb recovery of 95.3 ± 1.4% with a purity level of 91.4 ± 13.0%. CONCLUSION: An integrated platform based on two purification steps – affinity extraction and affinity chromatography – results in an overall process yield of 90%, allowing the processing of mAbs directly from a non‐clarified CHO cell culture. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Non‐mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof‐of‐concept for developing affinity peptide‐based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:987–998, 2018 [ABSTRACT FROM AUTHOR]

Membrane filtration is a key separations technique in downstream bioprocessing applications: for a commercial bioprocess operation, 10–20 membrane-related steps are typically required. At the laboratory scale, the evaluation of membrane separation performance often involves a ‘stirred cell device’; however, this simple tool is poorly suited for conducting high-throughput studies of separation performance. Here, we designed a high-throughput stirred cell (HTSC) device which is ideally suited for conducting optimization studies, especially at the early stages of bioprocess development when small volumes of feed material are available. The HTSC allows for up to six filtration experiments to be run simultaneously with continuous mixing above each membrane facilitated by suspended magnetic stir elements. Using fluorescently-labeled dextrans as model biomolecules and commercial ultrafiltration membranes, it was shown that the HTSC device gave the same separation performance as a traditional stirred cell, but with the added benefits of operating at a faster pace (due to its parallel nature) and requiring nearly an order of magnitude lower sample volumes. Furthermore, the utility of the HTSC was demonstrated by concurrently evaluating the performance of four different ultrafiltration membranes for the same model biomolecule and the membrane selectivity for a mixture of two model bio-macromolecules. [ABSTRACT FROM AUTHOR]

At present, polyhydroxyalkanoates (PHAs) have been considered as a promising alternative to conventional plastics due to their diverse variability in structure and rapid biodegradation. To ensure cost competitiveness in the market, thermoseparating aqueous two-phase extraction (ATPE) with the advantages of being mild and environmental-friendly was suggested as the primary isolation and purification tool for PHAs. Utilizing two-level full factorial design, this work studied the influence and interaction between four independent variables on the partitioning behavior of PHAs. Based on the experimental results, feed forward neural network (FFNN) was used to develop an empirical model of PHAs based on the ATPE thermoseparating input-output parameter. In this case, bootstrap resampling technique was used to generate more data. At the conditions of 15 wt % phosphate salt, 18 wt % ethylene oxide-propylene oxide (EOPO), and pH 10 without the addition of NaCl, the purification and recovery of PHAs achieved a highest yield of 93.9%. Overall, the statistical analysis demonstrated that the phosphate concentration and thermoseparating polymer concentration were the most significant parameters due to their individual influence and synergistic interaction between them on all the response variables. The final results of the FFNN model showed the ability of the model to seamlessly generalize the relationship between the input-output of the process. [ABSTRACT FROM AUTHOR]

The isolated Block V repeats-in-toxin (RTX) peptide domain of adenylate cyclase (CyaA) from Bordetella pertussis reversibly folds into a β-roll secondary structure upon calcium binding. In this review, we discuss how the conformationally dynamic nature of the peptide is being engineered and employed as a switching mechanism to mediate different protein functions and protein-protein interactions. The peptide has been used as a scaffold for diverse applications including: a precipitation tag for bioseparations, a cross-linking domain for protein hydrogel formation and as an alternative scaffold for biomolecular recognition applications. Proteins and peptides such as the RTX domains that exhibit natural stimulus-responsive behavior are valuable building blocks for emerging synthetic biology applications. [ABSTRACT FROM AUTHOR]

Background The production of polymers constitutes the major mass flow in the chemical industry. The foreseeable future change from fossil to renewable resources leads to the demand for more production routes based on biotechnological conversions. These routes preferably require biocompatible components and possess certain limitations of temperature and pH value for the corresponding downstream processing, which can be met by liquid-liquid extraction. This study investigates the extraction of the monomers hexane-1,6-diamine and butane-1,4-diamine from fermentation media using bis(2-ethylhexyl) hydrogen phosphate and isostearic acid as reactive extractants and kerosene as well as oleyl alcohol as diluents. Results As expected, the degree of extraction strongly depends on the pH-value but only weakly on the diluents. A higher diluent polarity leads to a slightly higher degree of extraction at a given pH value. The fraction of reactive extractant can be used to adjust the minimal pH value to reach the desired degree of extraction. No significant influence of fermentation medium, buffer, presence of an E. coli microorganism, or temperature was detected. Conclusion Reactive extraction of bio-based diamines with organic acids is feasible. The degree of extraction can be adjusted by the fraction of reactive extractant. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Precipitation with polyelectrolytes is a promising alternative to conventional methods for purification of monoclonal antibodies (mAb). This study focuses on clarification of a monoclonal antibody by precipitation of the model impurity bovine serum albumin (BSA) with cationic polyelectrolytes. Different types of cationic polyelectrolytes with different functional amine groups and molecular weights are screened for their capability to precipitate BSA in mixture with mAb. Due to the high BSA depletion and mAb recovery achieved, two cationic polyelectrolytes, polyethyleneimine (PEI) and polyallylamine (PAA) with a molecular weight of 65 kDa were selected as most suitable precipitation agents. Additionally, influences of different parameters (polyelectrolyte, BSA and NaCl concentration and pH value) were analyzed by the use of design of experiments (DoE). Herewith, a fast identification of influencing factors and factor interaction was achieved. With DoE, two regression models for the analysis of BSA depletion in dependence of influencing factors were established. This enabled the analysis of the robustness of precipitation as initial clarification step of downstream process of mAb. [ABSTRACT FROM AUTHOR]

Obtaining adequate viral clearance in the manufacture of biopharmaceuticals is a major challenge. While virus filtration is routinely used for this purpose, there have been few attempts to model virus filter performance. Here, virus filter performance is modeled in consideration of a multilayered membrane structure with a membrane pore size distribution that is assumed to be the same log-normal distribution in each membrane layer. Comparison of experimental data both for constant pressure and constant flux filtration to the model calculations showed good agreement. This method relies on three fitted parameters that can easily be determined from experimental data: particle size that blocks the membrane pores, particle density and the maximum pore size that can capture a particle. In addition, high virus removability was confirmed by the model calculations. By applying the calculation method introduced here, it becomes possible to quantitatively reproduce and analyze the filtration characteristics of virus filters. Based on analysis using the numerical calculation method presented in this study, we demonstrated the equivalence of constant pressure filtration and constant flux filtration. This method also shows potential for predicting filtration behavior based on minimal experimental data. [Display omitted] • Multilayer membranes with pore size distribution are key features of virus filter. • Numerical model of filtration behavior based on filter properties was established. • Numerical model uses only physical parameters without any empirical parameters. • Calculation method applies to both constant pressure and constant filtration modes. • Theoretical prediction of filtration behavior is possible with optimized parameters. [ABSTRACT FROM AUTHOR]

ABSTRACT There is growing interest in the possibility of developing truly continuous processes for the large-scale production of high value biological products. Continuous processing has the potential to provide significant reductions in cost and facility size while improving product quality and facilitating the design of flexible multi-product manufacturing facilities. This paper reviews the current state-of-the-art in separations technology suitable for continuous downstream bioprocessing, focusing on unit operations that would be most appropriate for the production of secreted proteins like monoclonal antibodies. This includes cell separation/recycle from the perfusion bioreactor, initial product recovery (capture), product purification (polishing), and formulation. Of particular importance are the available options, and alternatives, for continuous chromatographic separations. Although there are still significant challenges in developing integrated continuous bioprocesses, recent technological advances have provided process developers with a number of attractive options for development of truly continuous bioprocessing operations. Biotechnol. Bioeng. 2016;113: 465-475. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

pH is a common mobile phase variable used to control protein separations due to the tunable nature of amino acid and adsorbent charge. Like other column variables such as column density and ligand loading density, pH is usually optimized empirically. Single-molecule spectroscopy extracts molecular-scale data to provide a framework for mechanistic optimization of pH. The adsorption and diffusion of a model globular protein, α-lactalbumin, was studied by single-molecule microscopy at a silica-aqueous interface analogous to aqueous normal phase and hydrophilic interaction chromatography and capillary electrophoresis interfaces at varied pH. Electrostatic repulsion resulting in free diffusion was observed at pH above the isoelectric point of the protein. In contrast, at low pH strong adsorption and surface diffusion with either no ( D ∼ 0.01 μm2/s) or translational ( D ∼ 0.3 μm2/s) motion was observed where the protein likely interacted with the surface through electrostatic, hydrophobic, and hydrogen bonding forces. The fraction of proteins immobilized could be increased by lowering the pH. These results show that retention of proteins at the silica interface cannot be viewed solely as an adsorption/desorption process and that the type of surface diffusion, which ultimately leads to ensemble chromatographic separations, can be controlled by tuning long-range electrostatic and short-range hydrophobic and hydrogen bonding forces with pH. [ABSTRACT FROM AUTHOR]

Bioseparations conducted in aqueous two-phase systems offer a great number of advantages over the conventional separation techniques. Among them the most relevant are rapid mass transfer due to low interfacial tension, rapid and selective separation, easiness of operation mode, reliability in scale-up biocompatibility and environment-friendly features, and possibility of process integration when applied in biomolecule production. Upon overcoming the major problem - mostly empirical establishment of operating conditions bioseparations in aqueous two-phase systems will become a necessary step in both existing and newly developed bioprocesses for the primary recovery of products.

The isolated Block V repeats-in-toxin (RTX) peptide domain of adenylate cyclase (CyaA) from Bordetella pertussis reversibly folds into a β-roll secondary structure upon calcium binding. In this review, we discuss how the conformationally dynamic nature of the peptide is being engineered and employed as a switching mechanism to mediate different protein functions and protein-protein interactions. The peptide has been used as a scaffold for diverse applications including: a precipitation tag for bioseparations, a cross-linking domain for protein hydrogel formation and as an alternative scaffold for biomolecular recognition applications. Proteins and peptides such as the RTX domains that exhibit natural stimulus-responsive behavior are valuable building blocks for emerging synthetic biology applications.

Wall modified photonic crystal fibre capillary columns for in-capillary micro-extraction and liquid chromatographic separations is presented. Columns contained 126 internal parallel 4 μm channels, each containing a wall bonded porous monolithic type polystyrene-divinylbenzene layer in open tubular column format (PLOT). Modification longitudinal homogeneity was monitored using scanning contactless conductivity detection and scanning electron microscopy. The multichannel open tubular capillary column showed channel diameter and polymer layer consistency of 4.2 ± 0.1 μm and 0.26 ± 0.02 μm respectively, and modification of 100% of the parallel channels with the monolithic polymer. The modified multi-channel capillaries were applied to the in-capillary micro-extraction of water samples. 500 μL of water samples containing single μg L −1 levels of polyaromatic hydrocarbons were extracted at a flow rate of 10 μL min −1 , and eluted in 50 μL of acetonitrile for analysis using HPLC with fluorescence detection. HPLC LODs were 0.08, 0.02 and 0.05 μg L −1 for acenaphthene, anthracene and pyrene, respectively, with extraction recoveries of between 77 and 103%. The modified capillaries were also investigated briefly for direct application to liquid chromatographic separations, with the retention and elution of a standard protein (cytochrome c) under isocratic conditions demonstrated, proving chromatographic potential of the new column format, with run-to-run retention time reproducibility of below 1%. [ABSTRACT FROM AUTHOR]

Industrial production of menaquione-7 by Bacillus subtilis natto is associated with major drawbacks. To address the current challenges in menaquione-7 fermentation, studying the effect of magnetic nanoparticles on the bacterial cells can open up a new domain for intensified menqainone-7 process. This article introduces the new concept of production and application of L-lysine coated iron oxide nanoparticles (L-Lys@IONs) as a novel tool for menaquinone-7 biosynthesis. L-Lys@IONs with the average size of 7 nm were successfully fabricated and were examined in a fermentation process of L-Lys@IONs decorated Bacillus subtilis natto. Based on the results, higher menaquinone-7 specific yield was observed for L-Lys@IONs decorated bacterial cells as compared to untreated bacteria. In addition, more than 92% removal efficacy was achieved by using integrated magnetic separation process. The present study demonstrates that L-Lys@IONs can be successfully applied during a fermentation of menaquinone-7 without any negative consequences on the culture conditions. This study provides a novel biotechnological application for IONs and their future role in bioprocess intensification. [ABSTRACT FROM AUTHOR]

Surfactin, one of the most powerful biosurfactants, is a lipopeptide-type biosurfactant that is generated by the gram-positive, endospore-producing, microorganism, Bacillus subtilis . The chemical structure of surfactin is composed of seven amino acids that are bonded to the carboxyl and hydroxy groups on long chain fatty acids (C 13 –C 15 ). The potential applications of surfactin are therapeutic applications, and environmental applications. However, the high cost of surfactin production and its low yields limit its range of commercial applications. This work therefore develops the natural production a surface active agent by microorganisms and evaluates the microbial production system, the purification process, the identification process and the potential applications of surfactin. [ABSTRACT FROM AUTHOR]

BACKGROUND The emergence of monoclonal antibodies ( mAbs) as new biopharmaceutical products requires the development of new purification methods that are not only effective but are able to reduce production costs. To address the problematic recovery of mAbs, gum arabic ( GA) coated magnetic particles ( MPs) were used for the purification of human antibodies from animal cells supernatants. RESULTS MPs were synthesized via co-precipitation and exhibited a spherical-like physical aspect, with an average hydrodynamic diameter of 473 nm and a zeta potential of -26 mV. The adsorption and elution of IgG on these adsorbents was thoroughly studied. Adsorption of human IgG was enhanced at pH 6, for which a qmax of 244 mg IgG g−1 MPs and Kd of 25 mg L−1 were obtained. Increasing salt concentrations at a basic pH (1 mol L−1 NaCl at pH 11) were found to improve the elution of bound IgG. The MPs were challenged with an artificial protein mixture containing human IgG, albumin, insulin and apo-transferrin. An overall yield of 84% was achieved, retrieving 92% of bound IgG. CONCLUSIONS MPs were successfully used for the capture of monoclonal antibodies from two distinct mammalian cell cultures, a Chinese hamster ovary ( CHO) and a hybridoma cell culture supernatants. The elution yields were high, ranging between 84% and 94%, with overall yields ranging from 72% to 88%. Final purities of 85% were reached for hybridoma cell supernatants. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Bacterial membrane vesicles (BMVs) are bi-layered nanostructures derived from Gram-negative and Gram-positive bacteria. Among other pathophysiological roles, BMVs are critical messengers in intercellular communication. As a result, BMVs are emerging as a promising technology for the development of numerous therapeutic applications. Despite the remarkable progress in unveiling BMV biology and functions in recent years, their successful isolation and purification have been limited. Several challenges related to vesicle purity, yield, and scalability severely hamper the further development of BMVs for biotechnology and clinical applications. This review focuses on the current technologies and methodologies used in BMV production and purification, such as ultracentrifugation, density-gradient centrifugation, size-exclusion chromatography, ultrafiltration, and precipitation. We also discuss the current challenges related to BMV isolation, large-scale production, storage, and stability that limit their application. More importantly, the present work explains the most recent strategies proposed for overcoming those challenges. Finally, we summarize the ongoing applications of BMVs in the biotechnological field., (© 2022 Wiley-VCH GmbH.)

Galacto-oligosaccharides are typically produced by an enzymatic reaction when the post-reaction mixture contains considerable amounts of lactose and glucose and a smaller amount of galactose. In order to develop a process of chromatographic removal of saccharide impurities, adsorption equilibria and kinetics of these di- and monosaccharides were investigated for Diaion UBK 530, an industrialgrade strong cation-exchanger in the Na form. Frontal chromatographic experiments were carried out in the temperature range of 30-70 °C and a broad interval of saccharide concentrations up to 350 g L. Breakthrough curves were described using the equilibrium-dispersive model with the linear adsorption isotherm. Both the distribution and the axial dispersion coefficient values depended on the saccharide molecule type and size. No significant effect of temperature or concentration on the distribution coefficient was observed. The apparent dispersion coefficients of all saccharides exhibited some decrease with the temperature, which was caused by the decrease of the intraparticle mass transfer resistance. An analysis showed that both the intraparticle mass transfer and the axial dispersion had a significant influence on the front dispersion. [ABSTRACT FROM AUTHOR]

Microfluidics and lab-on-a-chip technology offers unique advantages for the next generation devices for diagnostic therapeutic applications. For chemical, biological and biomedical analysis in microfluidic systems, there are some fundamental operations such as separation, focusing, filtering, concentration, trapping, detection, sorting, counting, washing, lysis of bio-particles, and PCR-like reactions. The combination of these operations led to the complete analysis systems for specific applications. Manipulation of the bio-particles is the key ingredient for these applications. Therefore, microfluidic bio-particle manipulation has attracted a significant attention from the academic community. Considering the size of the bio-particles and the throughput of the practical applications, manipulation of the bio-particles is a challenging problem. Different techniques are available for the manipulation of bio-particles in microfluidic systems. In this review, some of the techniques for the manipulation of bio-particles; namely hydrodynamic based, electrokinetic-based, acoustic-based, magnetic-based and optical-based methods have been discussed. The comparison of different techniques and the recent applications regarding the microfluidic bio-particle manipulation for different biotechnology applications are presented. Finally, challenges and the future research directions for microfluidic bio-particle manipulation are addressed. [ABSTRACT FROM AUTHOR]