Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant.

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

Constructing Ag@SiO2-TiO2 Nanofiber Interlayers with a Three-Dimensional Lithiophilic Gradient Framework for an Ultrastable Lithium Metal Anode.

Lithium metal batteries have become one of the most promising rechargeable batteries due to the ultrahigh theoretical specific capacity of the Li metal anode. However, the Li dendrite growth and volume change of the Li metal anode during repeated Li plating-stripping cycles restrict the practical viability. Herein, a unique lithiophilic gradient structure of uniformly incorporating Ag nanoparticles into a three-dimensional (3D) nanofiber framework with amorphous SiO2 and TiO2 hybrids was prepared by an electrospinning process and used as a multifunctional interlayer between the pristine separator and Li metal foil. The 3D framework not only possesses excellent flexibility but also alleviates volume changes, which can withstand massive Li loading and promote uniform Li+ distribution. In addition, the 3D lithiophilic gradient structure allows for regulable Li+ flux and suppresses Li dendrite growth. Impressively, the Li||Li symmetric batteries with Ag@SiO2-TiO2 interlayers exhibit a prolonged lifespan of 1500 h at 0.5 mA cm-2 for 0.5 mAh cm-2. The full cells coupled with the Ag@SiO2-TiO2 interlayer show a capacity retention rate of 94.6% after 1000 cycles and a high rate capability. This work provides promising guidance for the design of a gradient-distributed lithiophilic structure toward an ultrastable Li metal anode.

Advances in high-throughput, high-capacity nonwoven membranes for chromatography in downstream processing: A review.

Nonwoven membranes are highly engineered fibrous materials that can be manufactured on a large scale from a wide range of different polymers, and their surfaces can be modified using a large variety of different chemistries and ligands. The fiber diameters, surface areas, pore sizes, total porosities, and thicknesses of the nonwoven mats can be carefully controlled, providing many opportunities for creative approaches for the development of novel membranes with unique properties to meet the needs of the future of downstream processing. Fibrous membranes are already finding use in ultrafiltration, microfiltration, depth filtration, and, more recently, in membrane chromatography for product capture and impurity removal. This article summarizes the various methods of manufacturing nonwoven fabrics, and the many methods available for the modification of the fiber surfaces. It also reviews recent studies focused on the use of nonwoven fabric devices in membrane chromatography and provides some perspectives on the challenges that need to be overcome to increase binding capacities, decrease residence times, and reduce pressure drops so that eventually they can replace resin column chromatography in downstream process operations.

Purification of Adeno-Associated Virus (AAV) Serotype 2 from Spodoptera frugiperda (Sf9) Lysate by Chromatographic Nonwoven Membranes.

The success of adeno-associated virus (AAV)-based therapeutics in gene therapy poses the need for rapid and efficient processes that can support the growing clinical demand. Nonwoven membranes represent an ideal tool for the future of virus purification: owing to their small fiber diameters and high porosity, they can operate at high flowrates while allowing full access to target viral particles without diffusional limitations. This study describes the development of nonwoven ion-exchange membrane adsorbents for the purification of AAV2 from an Sf9 cell lysate. A strong anion-exchange (AEX) membrane was developed by UV grafting glycidyl methacrylate on a polybutylene terephthalate nonwoven followed by functionalization with triethylamine (TEA), resulting in a quaternary amine ligand (AEX-TEA membrane). When operated in bind-and-elute mode at a pH higher than the pI of the capsids, this membrane exhibited a high AAV2 binding capacity (9.6 × 1013 vp·mL-1) at the residence time of 1 min, and outperformed commercial cast membranes by isolating AAV2 from an Sf9 lysate with high productivity (2.4 × 1013 capsids·mL-1·min-1) and logarithmic reduction value of host cell proteins (HCP LRV ~ 1.8). An iminodiacetic acid cation-exchange nonwoven (CEX-IDA membrane) was also prepared and utilized at a pH lower than the pI of capsids to purify AAV2 in a bind-and-elute mode, affording high capsid recovery and impurity removal by eluting with a salt gradient. To further increase purity, the CEX-IDA and AEX-TEA membranes were utilized in series to purify the AAV2 from the Sf9 cell lysate. This membrane-based chromatography process also achieved excellent DNA clearance and a recovery of infectivity higher that that reported using ion-exchange resin chromatography.

Application of 3D mullite fiber matrix as a lithiophilic interlayer in lithium metal anodes.

The lithium (Li) metal anode is an intriguing choice for Li metal batteries because of its considerably high theoretical capacity. However, the commercialization of unimproved lithium metal batteries is impeded due to the severe security issues related to uncontrollable Li dendrite growth. Herein, we fabricated three-dimensional (3D) mullite sheets via a high temperature annealing process in the presence of silica sol and used such sheets as interlayers between Li anodes and separators for assembling cells. As both Al2O3 and SiO2, the two main components of mullite fibers, are lithiophilic, the deposition of Li during charging processes could be regulated and the growth of lithium dendrites and accumulation of dead Li could be efficiently suppressed. In a Li||Cu half-cell, the mullite fiber/Li composite electrode delivers a coulombic efficiency of above 98% under a current density of 1.0 mA cm-2. For a Li||LiFePO4 full cell, the same composite electrode exhibited improved capacity retention (>89%, after 800 cycles) and rate capability at 5.0C.

Iminodiacetic Acid (IDA) Cation-Exchange Nonwoven Membranes for Efficient Capture of Antibodies and Antibody Fragments.

There is strong need to reduce the manufacturing costs and increase the downstream purification efficiency of high-value therapeutic monoclonal antibodies (mAbs). This paper explores the performance of a weak cation-exchange membrane based on the coupling of IDA to poly(butylene terephthalate) (PBT) nonwoven fabrics. Uniform and conformal layers of poly(glycidyl methacrylate) (GMA) were first grafted to the surface of the nonwovens. Then IDA was coupled to the polyGMA layers under optimized conditions, resulting in membranes with very high permeability and binding capacity. This resulted in IgG dynamic binding capacities at very short residence times (0.1-2.0 min) that are much higher than those achieved by the best cation-exchange resins. Similar results were obtained in the purification of a single-chain (scFv) antibody fragment. As is customary with membrane systems, the dynamic binding capacities did not change significantly over a wide range of residence times. Finally, the excellent separation efficiency and potential reusability of the membrane were confirmed by five consecutive cycles of mAb capture from its cell culture harvest. The present work provides significant evidence that this weak cation-exchange nonwoven fabric platform might be a suitable alternative to packed resin chromatography for low-cost, higher productivity manufacturing of therapeutic mAbs and antibody fragments.

Protection of lithium anodes by fibrous silica nanospheres.

Lithium metal (anode) has attracted significant attention for use in lithium-metal batteries due to its high energy density, but its practical application is still hindered by the dendrite growth during the battery charging process. Here, fibrous silica nanospheres were prepared via a direct hydrothermal reaction and coated on a separator to form a composite electrode with lithium sheets. Upon using this composite electrode in a symmetrical cell, the charge and discharge curves became more stable and the overpotential was alleviated compared with that of the bare lithium metal electrode. Meanwhile, the coulombic efficiency obtained from the Li‖Cu cell remained above 95.9% after 200 cycles at 0.5 mA h cm-2. The validity of using this composite electrode in the Li‖LFP (LiFePO4, lithium iron phosphate) cells was also evaluated. The results show that the composite electrode can help restrict the growth of lithium dendrites and the accumulation of dead lithium.

The efficacy of ropivacaine and bupivacaine in the caesarean section and the effect on the vital signs and the hemodynamics of the lying-in women.

OBJECTIVE: To investigate the efficacy of ropivacaine and bupivacaine in caesarean section and vital signs and the hemodynamics of the lying-in women. METHODS: A total of 480 lying-in women who were admitted to this hospital for treatment between December 2017 and June 2018 were enrolled into this study as the subjects, which were divided into the experiment group and the control group, with 240 subjects in each group. In the experiment group, subjects received the local anesthesia by infusion of 1.5 mL ropivacaine (0.75%), while those in the control group also took the local anesthesia by infusion of 1.5 mL bupivacaine (0.75%). Thereafter, we observed the differences in the anesthetic efficiency, vital signs and hemodynamics of the lying-in women between two groups. RESULTS: The excellent and good rates of the anesthesia in two groups were 92.1% and 87.9%, showing no obvious difference; in the experiment group, the average arterial pressures and systolic pressures at 5 min and 10 min after combined spinal and epidural analgesia (CSEA) were all elevated when comparing to the control group (all P < 0.05); in the experiment group, the onset time was obviously extended, while duration of sensory and motor block and the duration of motor block were all shorter than those in the control group (all P < 0.05). During anesthesia, the incidence rate of the adverse reactions in the control group was 2.50%, significantly higher than 0.83% in the experiment group (P < 0.05). CONCLUSION: Despite that ropivacaine and bupivacaine are efficient in anesthesia in the CSEA in the caesarean section, ropivacaine is more recommended for little influence on the hemodynamics, shorter duration of sensory block and motor block and low incidence rate of adverse reactions, which are conducive to the recovery and also safe to the patients.

Facile and green fabrication of cation exchange membrane adsorber with unprecedented adsorption capacity for protein purification.

Fabricating membrane adsorbers with high adsorption capacity and appreciable throughput for the separation and purification of protein products is challenging in biomedical and pharmaceutical industries. Herein, we report the synthesis of a novel membrane adsorber by functionalizing a nylon microfiltration membrane with alginate dialdehyde (ADA) followed by sulphonic addition, without any solvent usage, and its successful application in the purification of lysozyme. Taking advantage of abundant dual cation exchange (CEX) groups on sulphonic-ADA (S-ADA) ligands, this novel S-ADA-nylon membrane adsorber showed an unprecedented static binding capicity of 286mg/mL for lysozyme adsorption. Meanwhile, the prepared membrane adsorber could be easily regenerated (complete protein elution) under mild conditions and be reused at least for five times. Featured with a unique selectivity, the S-ADA-nylon membrane also captured lysozyme from chicken egg white solution with a high purity (100%) and a high recovery of 98%. The purified lysozyme showed similar specific activity as commercial product. The present work provides a facile, green and low-cost approach for the preparation of high-performance membrane adsorbers, which has a great potential in protein production.

Facile preparation of salt-tolerant anion-exchange membrane adsorber using hydrophobic membrane as substrate.

In this study, a polyvinylidene fluoride (PVDF) hydrophobic membrane with high mechanical property was used as substrate to prepare salt-tolerant anion-exchange (STAE) membrane adsorber. Effective hydrophilization and functionalization of PVDF membrane was realized via polydopamine (PDA) deposition, thus overcoming the drawbacks of hydrophobic substrates including poor water permeability, inert property as well as severe non-specific adsorption. The following polyallylamine (PAH) coupling was carried out at pH 10.0, where unprotonated amine groups on PAH chains were more prone to couple with PDA. This membrane adsorber could remain 75% of protein binding capacity when NaCl concentration increased from 0 to 150mM, while its protein binding capacity was independent of flow rate from 10 to 100 membrane volume (MV)/min due to its high mechanical strength (tensile strength: 43.58±2.30MPa). With 200mM NaCl addition at pH 7.5, high purity (above 99%) and high recovery (almost 100%) of Immunoglobulin G (IgG) were obtained when using the STAE membrane adsorber to separate IgG/human serum albumin (HSA) mixture, being similar to that without NaCl at pH 6.0 (both under the flow rate of 10-100MV/min). Finally, the reliable reusability was confirmed by five reuse cycles of protein binding and elution operations. In comparison with commercial membrane adsorber, the new membrane adsorber exhibited a better mechanical property, higher IgG polishing efficiency and reusability, while the protein binding capacity was lower due to less NH2 density on the membrane. The outcome of this work not only offers a facile and effective approach to prepare membrane adsorbers based on hydrophobic membranes, but also demonstrates great potential of this new designed STAE membrane adsorbers for efficient monoclonal antibody (mAb) polishing.

Polydopamine meets porous membrane: A versatile platform for facile preparation of membrane adsorbers.

Polydopamine, as an intermediate layer coated on PES membrane, was applied to fabricate various membrane adsorbers. Anion-exchange, hydrophobic interaction and affinity membrane adsorbers prepared by this facile method exhibited a high selectivity in fractionation of IgG (immunoglobulin)/HSA (human serum albumin) mixture. The anion-exchange membrane adsorber containing polyethylenimine (PEI) improved the HSA purity from 17.7% to 96.7%; The hydrophobic interaction membrane adsorber with Dodecyl mercaptan (DDM) as ligand obtained an IgG purity of 94.6%; Histidine attached affinity membrane chromatography achieved nearly a 100% purity of IgG. The present work indicated that the polydopamine layer not only activated membrane surface to attach various adsorptive ligands under the mild condition, but also reduced non-specific adsorption. Due to the versatile conjunction function, this facile mussel-inspired coating is also promising for the preparation of diverse membrane adsorbers.

Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV.

The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography.