Protein loss during membrane processes in biopharmaceutical manufacturing.

Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.

Effect of protein fouling on filtrate flux and virus breakthrough behaviors during virus filtration process.

Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log10 ). However, it is still constrained by protein fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.

Comparative Evaluation of the Performance of Sterile Filters for Bioburden Protection and Final Fill in Biopharmaceutical Processes.

Sterile filtration processes are widely used in the production of biotherapeutics for microorganism removal and product sterility. Sterile filtration processes can be applied to buffer preparation and cell culture media preparation in biotherapeutics processes, and to final sterilization or final filling in downstream processes. Owing to their broad range of applications in bioprocessing, various 0.2/0.22 µm sterile filters with different polymer materials (i.e., hydrophilic PVDF and PES) and nominal pore sizes are commercially available. The objective of this study was to evaluate two different commercial sterile filters in terms of filtration performance in various sterile filtration processes of biopharmaceutical production. The results demonstrate the importance of choosing the appropriate filter considering the process type and target removal/transport product to ensure efficient sterile filtration in the production of biotherapeutics.

Effects of Impurities from Sugar Excipient on Filtrate Flux during Ultrafiltration and Diafiltration Process.

Sugar excipients such as sucrose and maltose are widely used for biopharmaceutical formulation to improve protein stability and to ensure isotonicity for administration. However, according to recent literature, pharmaceutical-grade sucrose contained nanoparticulate impurities (NPIs) that result in protein aggregation and degradation. The objective of this study was to evaluate the filtrate flux behavior of sugar solution during ultrafiltration (UF) and diafiltration (DF). Filtrate flux data were obtained using either a tangential flow filtration (TFF) system for DF experiments or a normal flow filtration system for UF experiments. In diafiltration experiments, which were performed using 7 g/L of human immunoglobulin G in a 20 mM histidine buffer with the 100 mM sucrose or maltose, the filtrate flux with sucrose solution decreased significantly. In contrast, the one with maltose solution was in good correspondence with the calculated filtrate flux accounting for the effects of solution viscosity. This large decline in the flux was also observed during UF experiments, in which the presence of NPIs was identified by dynamic light scattering analysis and by capturing an SEM image of the membrane surface after filtration. In addition, highly purified sucrose resulted in a much lower flux decline in TFF in the absence of NPIs. These results provide important insights into the factors governing the optimization of the UF/DF process using appropriate excipients for biopharmaceutical formulation.

Beneficial effects of Vigna angularis extract in osteoporosis and osteoarthritis.

In Asia, Vigna angularis (azuki bean) has been used as a traditional medicine to treat various diseases because of its biological properties. Osteoarthritis (OA) and osteoporosis (OP) are common regenerative bone diseases that are characterized by deterioration of joint and bone structure. In this study, we evaluated the effects of Vigna angularis extract (VAE) on monosodium iodoacetate (MIA)-induced OA and ovariectomy (OVX)-induced OP models. In the MIA-induced OA results, severe OA was alleviated by the administration of VAE. Extensive local damage in the cartilage and hemorrhagic and edematous of surrounding tissues were decreased by VAE treatment. Articular cartilage was almost intact except for a focal mild abrasion, and the surface was glistening, similar to that of the normal joint. In the OVX-induced OP results, bone mineral content (BMC) and bone mineral density (BMD) were recovered by VAE treatment, and it improved the microstructures of bone. These results show that VAE could inhibit OA and OP symptoms.

Relationship between surface hydrophobicity and flux for membrane separation.

Surface hydrophobicity of anodic aluminum oxide (AAO) membranes was controlled via carbon coating using the CVD method or O2 plasma treatment with insignificant changes of pore diameter. This study first demonstrated that a larger hydrophobic pore surface and hydrophilic membrane surface are favorable for developing high performance membranes.

Stereospecific interactions between histidine and monoclonal antibodies.

Histidine is a frequently used buffer in the final formulation of many commercialized monoclonal antibodies (mAbs), with histidine helping to stabilize the antibody during storage in addition to its buffering function. The objective of this study was to examine the stereospecificity of any histidine-antibody interactions using a combination of experimental studies and molecular dynamics simulations. Isothermal titration calorimetry provided evidence of weak stereospecific interactions, with the antibody showing approximately two to four additional interaction sites for d- versus l-histidine. The greater interactions with d-histidine were confirmed by measurements of the net protein charge using electrophoretic light scattering. The reduction in the net negative charge of the antibody in d-histidine led to significantly different behavior during diafiltration due to Donnan exclusion effects. Molecular dynamics simulations corroborated the presence of additional d-histidine interaction sites. These results provide the first demonstration of weak stereospecific interactions between l- and d-histidine and a mAb and the implications of these interactions for antibody formulation.

Mass Balance Model with Donnan Equilibrium Accurately Describes Unusual pH and Excipient Profiles during Diafiltration of Monoclonal Antibodies.

There is extensive experimental data showing that the final pH and buffer composition after protein diafiltration (DF), particularly with monoclonal antibodies, can be considerably different than that in the DF buffer due to electrostatic interactions between the charged protein and the charged ions. Previous models for this behavior have focused on the final (equilibrium) partitioning and are unable to explain the complex pH and concentration profiles during the DF process. The objective of this study is to develop a new model for antibody DF based on solution of the transient mass balance equations, with the permeate concentrations of the charged species evaluated assuming Donnan equilibrium across the semipermeable membrane in combination with electroneutrality constraints. Model predictions are in excellent agreement with experimental data obtained during DF of both acidic and basic monoclonal antibodies, with the protein charge determined from independent electrophoretic mobility measurements. The model is able to predict the entire pH/histidine concentration profiles during DF, providing a framework for the development of DF processes that yield the desired antibody formulation.

Intermolecular interactions in highly concentrated formulations of recombinant therapeutic proteins.

The subcutaneous administration of recombinant therapeutic proteins requires the use of highly concentrated protein formulations to provide the desired dosage in a single injection. These highly concentrated formulations can have very high viscosities, creating challenges in processing (e.g. by ultrafiltration), storage (e.g. enhanced aggregation), and delivery (e.g. injection through small bore needles). Recent work has begun to identify the key intermolecular interactions governing the behavior of these highly concentrated formulations, including the effects of different excipients that have been shown to reduce viscosity and enhance the stability of these formulations. These intermolecular interactions also have a significant effect on the filtrate flux and maximum achievable protein concentration that can be obtained during ultrafiltration used for final concentration and formulation of these therapeutic proteins.

pH variations during diafiltration due to buffer nonidealities.

Diafiltration is used for final formulation of essentially all biotherapeutics. Several studies have demonstrated that buffer/excipient concentrations in the final diafiltered product can be different than that in the diafiltration buffer due to interactions between buffer species and the protein product. However, recent work in our lab has shown variations in solution pH that are largely independent of the protein concentration during the first few diavolumes. Our hypothesis is that these pH variations are due to nonidealities in the acid-base equilibrium coefficient. A model was developed for the diafiltration process accounting for the ionic strength dependence of the pKa . Experimental results obtained using phosphate and histidine buffers were in excellent agreement with model predictions. A decrease in ionic strength leads to an increase in the pKa for the phosphate buffer, causing a shift in the solution pH, even under conditions where the initial feed and the diafiltration buffer are at the same pH. This effect could be eliminated by matching the ionic strength of the feed and diafiltration buffer. The experimental data and model provide new insights into the factors controlling the pH profile during diafiltration processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1555-1560, 2017.

Ultrafiltration behavior of monoclonal antibodies and Fc-fusion proteins: Effects of physical properties.

Ultrafiltration (UF) is used for the final concentration and formulation of essentially all antibody-based therapeutics including both monoclonal antibodies (mAbs) and Fc-fusion proteins. The objective of this study was to quantitatively compare the filtrate flux behavior for two highly purified mAbs and an Fc-fusion protein under identical flow and buffer conditions. Filtrate flux data were obtained using a Pellicon 3 tangential flow filtration cassette over a wide range of transmembrane pressures and bulk protein concentrations. Independent experimental measurements were performed to evaluate the protein osmotic pressure and solution viscosity. The maximum achievable protein concentration was directly correlated with the solution viscosity, which controls the pressure drop and extent of back-filtration in the cassette. The filtrate flux data were analyzed using a recently developed model that accounts for the effects of intermolecular interactions and transmembrane pressure gradients on the extent of concentration polarization. These results provide important insights into the factors controlling the filtrate flux during the UF of concentrated protein solutions and an effective framework for the design/analysis of UF processes for the formulation of antibody-based therapeutics. Biotechnol. Bioeng. 2017;114: 2057-2065. © 2017 Wiley Periodicals, Inc.

Effects of Histidine and Sucrose on the Biophysical Properties of a Monoclonal Antibody.

PURPOSE: Histidine is a commonly used buffer in formulation of monoclonal antibodies (mAb), often with excipients like sucrose. The objective of this study was to examine the effects of both histidine and sucrose on the biophysical characteristics of a mAb. METHODS: The hydrodynamic radius of the mAb was determined by dynamic light scattering and confirmed by size exclusion chromatography. Data were also obtained for the osmotic virial coefficients (from osmotic pressure measurements), the solution viscosity, and the mAb thermal stability (using differential scanning calorimetry) at selected conditions. RESULTS: There were no significant changes in mAb conformation / stability as determined by DSC. The hydrodynamic radius initially increased with increasing histidine concentration, going through a maximum at a histidine concentration of about 20 mM. The addition of sucrose increased the mAb hydrodynamic radius at all histidine concentrations by about 0.5 nm. The observed effects of histidine and sucrose on the hydrodynamic radius were also reflected in changes in the osmotic pressure and solution viscosity. CONCLUSIONS: These results provide important insights into the effects of both histidine and sucrose on the behavior of concentrated mAb solutions, including the potential impact on ultrafiltration / diafiltration processes.

A Carbonaceous Membrane based on a Polymer of Intrinsic Microporosity (PIM-1) for Water Treatment.

As insufficient access to clean water is expected to become worse in the near future, water purification is becoming increasingly important. Membrane filtration is the most promising technologies to produce clean water from contaminated water. Although there have been many studies to prepare highly water-permeable carbon-based membranes by utilizing frictionless water flow inside the carbonaceous pores, the carbon-based membranes still suffer from several issues, such as high cost and complicated fabrication as well as relatively low salt rejection. Here, we report for the first time the use of microporous carbonaceous membranes via controlled carbonization of polymer membranes with uniform microporosity for high-flux nanofiltration. Further enhancement of membrane performance is observed by O2 plasma treatment. The optimized membrane exhibits high water flux (13.30 LMH Bar-1) and good MgSO4 rejection (77.38%) as well as antifouling properties. This study provides insight into the design of microporous carbonaceous membranes for water purification.

Autonomous Graphene Vessel for Suctioning and Storing Liquid Body of Spilled Oil.

Despite remarkable strides in science and technology, the strategy for spilled oil collection has remained almost the same since the 1969 Santa Barbara oil spill. The graphene vessel devised here can bring about an important yet basic change in the strategy for spilled oil collection. When it is placed on the oil-covered seawater, the graphene vessel selectively separates the oil, then collects and stores the collected oil in the vessel all by itself without any external power inputs. Capillarity and gravity work together to fill this proto-type graphene vessel with the spilled oil at a rate that is higher than 20,000 liters per square meter per hour (LMH) with oil purity better than 99.9%, and allow the vessel to withstand a water head of 0.5 m. The vessel also has a superb chemical stability and recyclability. An expanded oil contact area, considerably greater than the thickness of the oil layer, forms at the reduced graphene oxide (rGO) foam interface upon contact with the spilled oil. This expanded contact area does not change much even when the oil layer thins out. As a result, the high oil collection rate is maintained throughout the recovery of spilled oil.

Tunable Photonic Band Gap of PS-b-P2VP Lamellar Film Using Metal Ions and pH Gradation.

Optical properties of photonic crystal film were investigated by tuning photonic band gap (PBG). The lamellar-forming photonic films were prepared by nearly symmetric poly(styrene-b-2-vinyl pyridine) (PS-b-P2VP) block copolymers. Molecular weight of PS block and P2VP block is 52 kg/mol, and 57 kg/mol, respectively. When submerged in water, the lamellar films were swollen and show Bragg reflection in visible light region. We observed that the reflection color can be tuned by ion concentration (e.g., hydrogen or metal ion) in water. The higher concentration of hydrogen ion in solution, the longer reflectance wavelength shifted (from 537 nm to 743 nm). In addition, max-reflectance wavelength is dependent on both metal ion and the concentration. The max-reflectance wavelength is shifted from 653 nm (i.e., in water without ion) to 430 nm, 465 nm, and 505 nm for 120 mM of Ca2+, Fe2+, and Cu2+, respectively. Therefore, we can control the photonic band gap of photonic devices by changing the condition of swelling solution.

A carbon nanotube wall membrane for water treatment.

Various forms of carbon nanotubes have been utilized in water treatment applications. The unique characteristics of carbon nanotubes, however, have not been fully exploited for such applications. Here we exploit the characteristics and corresponding attributes of carbon nanotubes to develop a millimetre-thick ultrafiltration membrane that can provide a water permeability that approaches 30,000 l m(-2) h(-1) bar(-1), compared with the best water permeability of 2,400 l m(-2) h(-1) bar(-1) reported for carbon nanotube membranes. The developed membrane consists only of vertically aligned carbon nanotube walls that provide 6-nm-wide inner pores and 7-nm-wide outer pores that form between the walls of the carbon nanotubes when the carbon nanotube forest is densified. The experimental results reveal that the permeance increases as the pore size decreases. The carbon nanotube walls of the membrane are observed to impede bacterial adhesion and resist biofilm formation.

Carbon nanotube-bonded graphene hybrid aerogels and their application to water purification.

We present carbon nanotube (CNT)-bonded graphene hybrid aerogels that are prepared by growing CNTs on a graphene aerogel surface with nickel catalyst. The presence of bonded CNTs in the graphene aerogel results in vastly improved mechanical and electrical properties. A significant increase in specific surface area is also realized. The presence of the CNTs transforms the hybrid aerogels into a mesoporous material. The viscoelasticity of the hybrid aerogels is found to be invariant with respect to temperature over a range of between -150 °C and 450 °C. These characteristics along with the improved properties make the hybrid aerogels an entirely different class of material with applications in the fields of biotechnology and electrochemistry. The mesoporous nature of the material along with its high specific surface area also makes the hybrid aerogel attractive for application in water treatment. Both anionic and cationic dyes can be effectively removed from water by the hybrid aerogel. A number of organics and oils can be selectively separated from water by the hybrid aerogel. The hybrid aerogel is easy to handle and separate from water due to its magnetic nature, and can readily be recycled and reused.

High-performance reverse osmosis CNT/polyamide nanocomposite membrane by controlled interfacial interactions.

Polyamide reverse osmosis (RO) membranes with carbon nanotubes (CNTs) are prepared by interfacial polymerization using trimesoyl chloride (TMC) solutions in n-hexane and aqueous solutions of m-phenylenediamine (MPD) containing functionalized CNTs. The functionalized CNTs are prepared by the reactions of pristine CNTs with acid mixture (sulfuric acid and nitric acid of 3:1 volume ratio) by varying amounts of acid, reaction temperature, and reaction time. CNTs prepared by an optimized reaction condition are found to be well-dispersed in the polyamide layer, which is confirmed from atomic force microscopy, scanning electron microscopy, and Raman spectroscopy studies. The polyamide RO membranes containing well-dispersed CNTs exhibit larger water flux values than polyamide membrane prepared without any CNTs, although the salt rejection values of these membranes are close. Furthermore, the durability and chemical resistance against NaCl solutions of the membranes containing CNTs are found to be improved compared with those of the membrane without CNTs. The high membrane performance (high water flux and salt rejection) and the improved stability of the polyamide membranes containing CNTs are ascribed to the hydrophobic nanochannels of CNTs and well-dispersed states in the polyamide layers formed through the interactions between CNTs and polyamide in the active layers.

Feasibility of supercritical CO2 treatment for controlling biofouling in the reverse osmosis process.

Physical cleaning and/or chemical cleaning have been generally used to control biofouling in the reverse osmosis (RO) process. However, conventional membrane cleaning methods to control biofouling are limited due to the generation of by-products and the potential for damage to the RO membranes. In this study, supercritical carbon dioxide (SC CO(2)) treatment, an environmentally friendly technique, was introduced to control biofouling in the RO process. SC CO(2) (100 bar at 35°C) treatment was performed after biofouling was induced on a commercial RO membrane using Pseudomonas aeruginosa PA01 GFP as a model bacterial strain. P. aeruginosa PA01 GFP biofilm cells were reduced on the RO membrane by >8 log within 30 min, and the permeate flux was sufficiently recovered in a laboratory-scale RO membrane system without any significant damage to the RO membrane. These results suggest that SC CO(2) treatment is a promising alternative membrane cleaning technique for biofouling in the RO process.