Diafiltration Process Research Articles

Modeling the Gibbs–Donnan effect during ultrafiltration and diafiltration processes using the Poisson–Boltzmann theory in combination with a basic Stern model

The Gibbs–Donnan effect is a well-known phenomenon causing ions to be distributed unevenly across semi-permeable membranes that are permeable to ions but not to larger macromolecules such as proteins. In protein ultrafiltration and diafiltration (UF/DF) processes, this effect often leads to discrepancies between the pH and excipient concentrations in the final drug substance and in the DF buffer. In this work, a model describing the retentate and permeate composition throughout combined UF/DF processes is introduced. The model accounts for volume exclusion effects and electrostatic interactions between ions and the protein based on the Poisson–Boltzmann theory in combination with a basic Stern model. Advantages and limitations of the proposed model were analyzed using UF/DF experiments with multiple diafiltration buffers and proteins. A comparison between simulated and experimental permeate data showed good agreement for low to moderate Donnan potentials but model limitations for high Donnan potentials at protein concentration larger 100 gL -1 . In contrast, simulated retentate data showed good agreement for both low and high Donnan potentials and for protein concentrations up to 190 gL -1 . It was demonstrated that in this high protein concentration regime, the applied basic Stern model provides more accurate predictions compared to previous theories based on the Poisson–Boltzmann theory alone. This makes the model a valuable tool to describe discrepancies between pH and excipient concentrations in the final drug substance and DF buffer for highly concentrated protein formulations. As model predictions are based solely on structural information on the protein and the composition of the DF buffer, the model is particularly beneficial at an early stage in process development to streamline process development and improve process understanding. • Introduction of a model to describe the Gibbs–Donnan effect in UF/DF processes. • Advanced implementation of the cell model approximation. • Calibration solely based on structural information on the protein. • Validation using various proteins (up to 190 g/L) and buffer systems.

Rapid removal of lactose for low-lactose milk by ceramic membranes

The dairy industry requires membranes with narrow-distributed pore size to separate lactose efficiently as the demand for low-lactose milk is increasing. In this study, ceramic membranes were used to remove lactose from skim milk. Ceramic microfiltration membranes were selected for filtration owing to their high permeation flux and high rejection performance for separating lactose and protein. The operating parameters of the microfiltration processes were systematically discussed and optimized, achieving 99% rejection of the proteins with 100% permeation of lactose, and the permeance was as high as 75 L m−2h−1 bar−1. A diafiltration model was proposed to optimize the diafiltration process; moreover, the optimum volume concentration ratio of the pre-concentration process was obtained to minimize the operation time. The diafiltration operation was then optimized to remove lactose from skim milk. The results showed that lactose removal and protein concentration were achieved simultaneously, and low-lactose milk with a lactose concentration of less than 5 g L−1 have been prepared. This study serves as a valuable guide for process design and practical operations in the dairy industry.

Energy Consumption of Nanofiltration Diafiltration Process: Identifying the Optimal Conditions of Continuous and Intermittent Feed Diafiltration

Energy Consumption of Nanofiltration Diafiltration Process: Identifying the Optimal Conditions of Continuous and Intermittent Feed Diafiltration

From sucrose to fructo-oligosaccharides: Production and purification of fructo-oligosaccharides by an integrated enzymatic catalysis and membrane separation process

Fructo-oligosaccharides (FOS) are a group of linear fructose oligomers with numerous health benefits, which can be synthesized by the transfructosylation of sucrose using fructosyltransferases (FTases). In this study, it was found that the selected FTases showed both hydrolytic and transfructosylating activities. For FOS production, the hydrolytic activities of FTases became significant when the reaction time was above 1 h. Thus, FTases needed to be inactivated or removed from the reactor after achieving the highest FOS conversion. We therefore developed an integrated ultrafiltration-diafiltration- concentration process for FTases reuse and FOS purification. Because of the 100% rejection of FTases activity and a higher average flux, a PES-10 ultrafiltration membrane was selected for FTases recovery, where the cake layer formation was the main membrane fouling mechanism. Then, a fed-batch reaction process was developed for FOS production, where the hydrolytic behavior of FTases was negligible, and the FTases dosage was reduced by 75% compared to single batch operation (four batches). Moreover, the FOS production was comparable, and 95.5% FTases was recovered by the PES10 membrane after the four fed-batches processes. Subsequently, a NF5 nanofiltration membrane was selected to further separate FOS from the FTases-free FOS solution by a constant volume diafiltration process under 45 °C, and the monosaccharides in the reaction mixture (glucose and fructose) were completely removed, increasing the FOS purity from 55.8% to 92.3%. Finally, the FOS and sucrose in the diafiltration permeate was recovered by a DL nanofiltration membrane under 60 °C. The outcome of this work offers a complete technical route for FOS production using sucrose as substrate.

Membrane diafiltration for enhanced purification of biologically active compounds from goji berries extracts

• The recovery of phenolic compounds from aqueous goji berries extract was investigated. • Three tight ultrafiltration membranes with different cut-off (1, 2.5 and 3.5 kDa) were studied. • Diafiltration was implemented to improve the separation of phenolic compounds from sugars. • Membrane performance was evaluated in terms of productivity and selectivity towards target compounds. • All selected membranes allowed high recovery of phenolic compounds into the retentate. Goji berries represent a valuable source of phenolic compounds with a strong antioxidant and free radical scavenging activity combined to huge quantities of small sugars. This study was aimed at evaluating the potential of three tight ultrafiltration (UF) membranes with molecular weight cut-off (MWCO) from 1.0 to 3.5 kDa in the fractionation of phenolic compounds from sugars in the aqueous extract of dried goji berries. The aqueous extract produced in optimized conditions of water-to-solid ratio and temperature (6.66 mL/g and 65 °C, respectively) was preliminarily clarified by UF in order to remove suspended solids and colloidal particles. A membrane diafiltration (DF) process was developed in order to fractionate phenolic compounds from sugars with selected UF membranes from the clarified extract. The membrane performance was evaluated in terms of productivity and selectivity towards target compounds. All selected membranes allowed high recovery (more than 80%) of phenolic compounds into the retentate. On the other hand, the yield of carbohydrates increased in the permeate by increasing the diafiltration volume. Among the selected membranes, the 2.5 kDa membrane allowed to reduce the concentration of total carbohydrates from 26.5 up to 2.2 g glucose/L (a reduction of 91.7%) in a diafiltration/batch concentration process producing a concentrated extract enriched in phenolic compounds of interest for pharmaceutical or nutraceutical applications.

A Combination of Aqueous Extraction and Ultrafiltration for the Purification of Phycocyanin from Arthrospira maxima.

The purification of phycocyanin (PC) from Spirulina generally involves a combination of different techniques. Here, we report the results on PC yields from a combined aqueous extraction-ultrafiltration (UF) process of a strain of Arthrospira maxima cultivated in a farm devoted to producing PC with food-grade purity. Samples optimized from different biomass/solvent ratios were purified by using a polyethersulphone (PES) membrane with a molecular weight cut-off (MWCO) of 20 kDa. The UF system was operated at 2.0 ± 0.1 bar and at 24 ± 2 °C up to a volume concentration factor (VCF) of 5. A diafiltration (DF) process was conducted after UF in order to increase the PC recovery in the retentate. Samples were collected during both UF and DF processes in order to evaluate membrane productivity and PC purity. The average permeate fluxes of about 14.4 L/m2h were measured in the selected operating conditions and more than 96% of PC was rejected by the UF membrane independently ofthe extraction yields and times. The concentration of PC in the final retentate was 1.17 mg/mL; this confirmed the observed rejection and the final VCF of the process (about 5-fold when compared to the concentration of PC in the crude extract). In addition, the combination of UF and diafiltration allowed the removal of about 91.7% of the DNA from the crude extract, thereby improving the purity of the phycocyanin in the retentate fraction.

A comparison of zinc interactions with succinylated milk protein concentrate and sodium caseinate

Milk proteins with substantial reactive groups enable new protein chemistries and succinylation being a potential post-translational modification plays a significant role in regulating milk protein configuration properties. The presence of additional carboxylic group due to succinylation also improves the divalent metal binding efficiency of milk proteins. Therefore, in the present research work, the interaction of zinc (Zn) with native and succinylated milk protein concentrate (MPC) and sodium caseinate (NaCN) was assessed. For the optimization of milk protein-Zn complexes, a diafiltration process was adopted and this process efficiently removed the free form of succinic acid which was confirmed by scanning electron microscopy. Maximum Zn binding ability of MPC and succinylated MPC (SMPC) was observed at 2.0 and 7.0 mmol/L Zn concentration, whereas NaCN and SNaCN showed maximum Zn binding ability at 2.4 and 7.6 mmol/L Zn concentrations, respectively. Processing variables such as pH and thermal treatment did not alter the Zn holding capacity of SMPC and SNaCN, however, ionic concentration ≥0.3 mol/L NaCl significantly decreased (P < 0.05) it. Therefore, based on the key findings, the milk protein-Zn complex can be utilized for the fortification of various food products to eradicate Zn deficiency.

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.

Recovery of Cr(III) from Tannery Effluents by Diafiltration Using Chitosan Modified Membranes

The selective recovery of chromium remaining in tannery effluents after the leather tanning process is highly desirable to potentiate its reuse, simultaneously minimizing the ecotoxicity of these effluents. To the best of our knowledge, this work evaluates for the first time the ability of a chitosan-based membrane for selective recovery of chromium from a tannery wastewater by subsequent diafiltration and selective chromium desorption, envisaging their integration after tannery wastewater treatment by reverse osmosis (RO). A polyethersulfone (PES) microfiltration membrane top-coated with a chitosan layer (cs-PES MF022) was used for selective recovery of Cr(III), from concentrate streams obtained by treatment of synthetic and real tannery effluents through reverse osmosis (RO), through a diafiltration process. The diafiltration of the RO concentrates was conducted by an intermittent addition of water acidified to pH 3.6. The prepared cs-PES MF022 membranes were able to retain 97% of the total mass of Cr(III) present in the RO concentrates, from a real tannery effluent, with a selectivity of 4.2 and 5 in reference to NH4+ and Cl−, respectively, 12.9 and 14.6 in reference to K and Na, and &gt;45 in reference to Mg, Ca, and S. Such a high selectivity is explained by the preferential adsorption of Cr(III) onto chitosan, and by the relatively high permeability of cs-PES MF022 membranes to the other ionic species. Proof of concept studies were performed to investigate the desorption of Cr(III) at pH 2 and 5.8. A higher Cr(III) desorption degree was obtained at pH 2, leading to a final solution enriched in Cr(III), which may be re-used in tannery operations, thus improving the process economy and reducing the hazardous impact of the effluents discharged by this industry.

Dual-gating pH-responsive membranes with the heterogeneous structure for whey protein fractionation

Whey protein is a mixture of globular proteins with high nutritional value and excellent functional properties availability. Purified individual whey proteins exhibit better functionality than in their original mixture, thus there is an increasing interest in developing more efficient methods to recover individual whey protein fractions, especially membrane technology. However, whey protein fractionation from the protein mixture is still a huge challenge for membrane separation. Herein, a dual-gating pHresponsive membrane with the heterogeneous structure was developed for whey protein fractionation. The heterogeneous structure of the membranes was obtained by grafting the dimethylaminoethyl methacrylate (DMAEMA) and 4-vinylpyridine (4VP) near the topside and inside the pores of the ethylene vinyl alcohol copolymer (EVAL) membranes based on selective modification of grafting sites. The PDMAEMA and P4VP gates responded to pH individually and provided a multi-stage regulation of the surface charge and sieving property of the membranes. The major whey protein fractionation from the mixture could be achieved by single dual-gating pH-responsive membrane via the sequential stepwise pH gradient from 3.0 to 8.0. The α-Lactalbumin (α-La), β-Lactoglobulin (β-Lg), lactoferrin (LF) and bovine serum albumin (BSA) were separated and enriched in the permeate at pH 3.0, 4.2, 6.4 and 8.0 by the diafiltration process, respectively. This work not only proposed a novel concept of pHresponsive membrane, but also offered a sustainable solution to fractionate the whey protein from the complicated mixture.

Effect of different diafiltration process on the protein fractionation of skim milk by cross flow microfiltration

Diafiltration had been widely used in milk protein fractionation for almost thirty years to produce casein-enriched retentate. Among them, intermittent feed diafiltration was the only way ever used. But we noted some other diafiltration types had been used in other fields such as chemical engineering. In this study. The separation performance, chemical properties, component resistance and mass balance analysis were compared under different diafiltration types, aiming to find suitable diafiltration processes for milk protein fractionation. Research suggested that different diafiltration process may have different advantages. From the separation performance point of view, the highest removal rate of native whey proteins was observed under variable volume diafiltration (VVD) mode. But in terms of average flux, constant volume diafiltration (CVD) mode was utmost time and energy saving with an average flux of 81.25 kg m−2h−1. In addition, the percentage loss of protein under CVD and constant volume concentration ratio diafiltration (CVCRD) mode were significantly lower than that of other diafiltration modes (P < 0.05), indicating fewer protein were absorbed or compacted by the membrane. Therefore, diafiltration type should be selected according to the purpose of production.

Process analytical technology for on-line monitoring of quality attributes during single-use ultrafiltration/diafiltration.

Process analytical technology (PAT) is a fast-growing field within bioprocessing that enables innovation in biological drug manufacturing. This study demonstrates novel PAT methods for monitoring multiple quality attributes simultaneously during the ultrafiltration and diafiltration (UF/DF) process operation, the final step of monoclonal antibody (mAb) purification. Size exclusion chromatography (SEC) methods were developed to measure excipients arginine, histidine, and high molecular weight (HMW) species using a liquid chromatography (LC) system with autosampler for both on-line and at-line PAT modes. The methods were applied in UF/DF studies for the comparison of single-use tangential flow filtration (TFF) cassettes to standard reusable cassettes to achieve very high concentration mAb drug substance (DS) in the order of 100-200 g/L. These case studies demonstrated that single-use TFF cassettes are a functionally equivalent, low-cost alternative to standard reusable cassettes, and that the on-line PAT measurement of purity and excipient concentration was comparable to orthogonal offline methods. These PAT applications using an on-line LC system equipped with onboard sample dilution can become a platform system for monitoring of multiple attributes over a wide dynamic range, a potentially valuable tool for biological drug development and manufacturing.

Safety risk management for low molecular weight process-related impurities in monoclonal antibody therapeutics: Categorization, risk assessment, testing strategy, and process development with leveraging clearance potential.

Process‐related impurities (PRIs) derived from manufacturing process should be minimized in final drug product. ICH Q3A provides a regulatory road map for PRIs but excludes biologic drugs like monoclonal antibodies (mAbs) that contain biological PRIs (e.g. host cell proteins and DNA) and low molecular weight (LMW) PRIs (e.g., fermentation media components and downstream chemical reagents). Risks from the former PRIs are typically addressed by routine tests to meet regulatory expectations, while a similar routine‐testing strategy is unrealistic and unnecessary for LMW PRIs, and thus a risk‐assessment‐guided testing strategy is often utilized. In this report, we discuss a safety risk management strategy including categorization, risk assessment, testing strategy, and its integrations with other CMC development activities, as well as downstream clearance potentials. The clearance data from 28 mAbs successfully addressed safety concerns but did not fully reveal the process clearance potentials. Therefore, we carried out studies with 13 commonly seen LMW PRIs in a typical downstream process for mAbs. Generally, Protein A chromatography and cation exchange chromatography operating in bind‐and‐elute mode showed excellent clearances with greater than 1,000‐ and 100‐fold clearance, respectively. The diafiltration step had better clearance (greater than 100‐fold) for the positively and neutrally charged LMW PRIs than for the negatively charged or hydrophobic PRIs. We propose that a typical mAb downstream process provides an overall clearance of 5,000‐fold. Additionally, the determined sieving coefficients will facilitate diafiltration process development. This report helps establish effective safety risk management and downstream process design with robust clearance for LMW PRIs.

Ultrafiltration and Diafiltration Processes for Concentration and Formulation of Antibody-based Therapeutics

Ultrafiltration and Diafiltration Processes for Concentration and Formulation of Antibody-based Therapeutics

Purification of Cas9-RNA complexes by ultrafiltration.

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.

Production and characterization of cow milk based low-protein milk protein concentrate (MPC) powders.

Ultrafiltration and Diafiltration processes are used to concentrate proteins present in defatted milk in order to manufacture milk protein concentrate (MPC) powders. Selective passage of the water-soluble components causes retention as well as concentration of colloidal milk components in these processes. Increase in calcium and casein contents decreases the stability of milk proteins present in ultrafiltered retentates and negatively influence properties of manufactured MPC powders. Homogenization, diafiltration and disodium phosphate induced changes in properties of low-protein MPC powders were targeted in this study. Applied treatments significantly (P < 0.05) improved foaming and emulsification, solubility, viscosity, heat stability, dispersibility, specific surface area and buffer index of resultant MPC powders over control. Fresh, treated low-protein MPC powders showed significantly higher (< 0.05) solubility values over control sample, which remains higher even after 60days of storage at 25 ± 1°C. The rheological behaviour of reconstituted low-protein MPC solutions was also studied. It was best explained as Herschel-Bulkley rheological behaviour. Low-protein MPC powders with improved functional properties may find better use as a protein ingredient in different dairy and food applications.

Fractionation of Arthrospira platensis (Spirulina) water soluble proteins by membrane diafiltration

Membrane diafiltration process was developed in order to fractionate the water soluble proteins of the supernatant obtained after Spirulina bead milling disruption and centrifugation. The obtained crude extract was filtered with 50 kDa, 150 kDa, 300 kDa or 0.2 µm membranes. Phycocyanin purified extracts were produced after filtration on the 300 kDa or 0.2 µm membranes. The filtration on 0.2 µm membrane allowed high recovery (more than 89%) of Phycocyanin A and C into the retentate and permeate as well as separation of both molecules (depletion of C-PC into the retentate relatively to the A-PC; enrichment into the permeate). Blue colour permeates, poor in chlorophyll, were successfully produced. Concentrated solutions with more than 80% (dry weight basis) of proteins were obtained. The most severe fouling occurred with the 300 kDa membrane leading to low permeate fluxes (65L.h−1.m−2). A fractionation of proteins by the size was possible for the 50, 150 and 300 kDa diafiltrations.

Coupling Ultrafiltration-Based Processes to Concentrate Phenolic Compounds from Aqueous Goji Berry Extracts.

In this work, a membrane-based process for the purification and concentration of antioxidant compounds from aqueous Goji (Lycium barbarum L.) berry extracts was investigated. The aqueous extract was previously clarified with hollow fiber ultrafiltration (UF) membranes in order to remove suspended solids and β-carotene and to produce a clarified extract enriched in phenolic compounds. Then, three UF flat sheet polyamide membranes with a molecular weight cut-off (MWCO) in the range 1000–3500 Da were tested to purify and concentrate phenolic compounds from the clarified extract. The effect of MWCO and transmembrane pressure (TMP) on the performance of selected membranes in terms of productivity and selectivity towards total dissolved solids (TDS), total phenolic compounds (TPC), total carbohydrates (TC) and total antioxidant activity (TAA) was evaluated. Experimental results indicated that the 2500 Da membrane exhibited a lower fouling index, higher cleaning efficiency, lower rejection towards carbohydrates (lower than 30%) and higher rejection towards phenolic compounds (higher than 50%) in comparison to the other investigated membranes. The inclusion of a diafiltration process in the treatment of the clarified extract with this membrane in a spiral-wound configuration improved the concentration of sugar compounds in the permeate stream and increased the purification of phenolic compounds in the retentate fraction.

Modeling and optimal operation of intermittent feed diafiltration for refining oligodextran using nanoporous ceramic membranes

Developing a facile and efficient model for the prediction and optimization of diafiltration processes is desired in the separation and purification of functional oligosaccharides. Herein, a data-driven model was proposed to describe the intermittent feed diafiltration process. To evaluate this model, oligodextran was applied as a representative oligosaccharide, and a nanoporous ZrO2 ceramic membrane was employed to narrow the molecular weight distribution of oligodextran. The role of concentration polarization effect in affecting the flux and solute rejection was well considered in the modeling of the diafiltration process. The permeation flux and yields predicted by this model agreed well with the experimental data. More importantly, the maximum productivity could be determined by this model, which takes the diluent consumption and the operation time into account, simultaneously, in optimizing of the diafiltration process. The proposed diafiltration model performs a good potential to be a candidate in the auxiliary design for the separation and purification processes of functional oligosaccharides.

PH and excipient profiles during formulation of highly concentrated biotherapeutics using bufferless media.

Several models have been developed to describe the shifts in pH and excipient concentrations seen during diafiltration of monoclonal antibody (mAb) products accounting for both Donnan equilibrium and electroneutrality constraints. However, these models have assumed that the mAb charge is either constant or only a function of pH, assumptions that will not be valid when formulating highly concentrated mAbs using bufferless or low-buffered media due to the change in local H+ concentration at the protein surface. The objective of this study was to incorporate the effects of both pH and ionic strength on the mAb charge, through the use of a charge regulation model based on the amino acid sequence of the mAb, into an appropriate mass balance model to describe the pH and excipient profiles during diafiltration. The model involves no adjustable parameters, with the protein charge evaluated directly from the protonation/deprotonation of the ionizable amino acids accounting for the electrostatic interactions between the charged mAb and the H+ ions. Model predictions are in excellent agreement with experimental data for the pH and ion concentrations during diafiltration of a mAb and fusion protein with different isoelectric points and different formulation conditions. Model simulations are then used to obtain fundamental insights into the factors controlling the diafiltration behavior as well as guidelines for development of diafiltration processes to achieve target bufferless formulation conditions.