Ultrafiltration/diafiltration Research Articles

Design and optimization of a continuous purification process using ion-exchange periodic counter-current chromatography for a low-titer enzyme

A continuous purification process can be beneficial to the purification of biologics due to its higher productivity and efficiency than a conventional batch purification process. However, regulatory issues and lack of established cases render deployment of the continuous process difficult in industrial settings. Here we report a case study for design and optimization of an advanced continuous process for purifying a low-titer enzyme as a model biologic. To convert a conventional batch process to an advanced continuous one in purification of biologics, conventional unit operations (UOs), including ultrafiltration/diafiltration (UF/DF) and batch chromatography, were replaced by advanced ones such as in-line dilution conditioning (IDC) and periodic counter-current chromatography (PCC). The UF/DF UO was changed to IDC UO to adjust pH and conductivity. The mixing ratio of the sample and the conditioning buffer in IDC was determined by experiments with three buffers. PCC was optimized with two variables, column height and sample loading residence time, as the delta pressure in the columns was less than 1.0 bar. A graph indicating the operating area was plotted to efficiently control the PCC. Although the sample volume increased in IDC, PCC had a complementary advantage in that purification was performed faster than batch chromatography. We observed at least 25% increase in economic advantage when the advanced continuous process was applied to purify a low-titer enzyme. We propose not only a continuous process with the substitution of UF/DF and batch chromatography with IDC and PCC but also a method to optimize PCC by plotting operating areas.

Ubiquitin: Characterization of a Host Cell Protein Covalently Attached to a Monoclonal Antibody Product by LC-MS/MS

Host cell protein (HCP) characterization is a crucial quality parameter for biotherapeutic drug safety and stability. With a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified ubiquitin in ultrafiltration/diafiltration (UF/DF) pools of one of our monoclonal antibody (mAb) products. Since ubiquitin occurs physiologically as a post-translational modification (PTM) involved in many cellular functions, we suspected the possibility that if identified as an HCP, it may occur as a covalent modification on the mAb. In fact, in this study we characterized and quantified the ubiquitin modification on the Fc domain of mAbX by data dependent acquisition (DDA) and data independent acquisition (DIA) – MS workflows. Covalent binding and site localization were confirmed by identifying a characteristic diglycine motif on the modified peptide. Initially observed reduced detectability of ubiquitin in samples prepared with native digestion was attributed to impaired digestion and subsequent removal along with the mAb in the precipitation step. Our work has contributed to a better understanding of ubiquitin as an HCP considering its specific features such as occurrence in different topologies and provided insight into how covalent binding to a drug product can affect its identification by MS when native digestion conditions are used.

A Review on Ultra- and Nanofiltration/Diafiltration Processes of the Food-Oriented Agro-industrial

This review was conducted to implement the potential use of ultra- and nanofiltration as a separation technique accompanied by a diafiltration (DF) technique. We start by introducing the principle of pressure-driven membrane-based separation process and DF process in general, and selected applications of ultra- and nanofiltration/diafiltration techniques on the food-oriented agro-industrial products in particular, such as nanofiltration/diafiltration (NF/DF) technique of mung beans autolysate, ultrafiltration/diafiltration (UF/DF) technique of forest honey products, and ultrafiltration/diafiltration (UF/DF) technique of fish gelatin. We also describe the potential benefits as green and sustainable and the drawbacks of membrane-based separation process. Both relevant information and data related to this review were obtained from various sources originating from international organizations (FAO and UNEP, United States Standards for Grades of Extracted Honey, USA and National Honey Board (NHB), USA), review of various scientific articles and scientific literature, handbook, and the selected result of experiment activity. We hope this review manuscript is useful for further research into membrane technology implementations in food-oriented agro-industrial products and in a wider range of applications.

Assessing the impact of sanitization methods for regenerated cellulose ultrafiltration/diafiltration membrane on membrane integrity and protein quality.

Ultrafiltration/diafiltration (UF/DF) is typically the final step in downstream processing of recombinant monoclonal antibody (mAb) products, which serves for protein concentration and buffer exchange. For UF/DF membranes composed of regenerated cellulose (RC), sanitization with 0.1 M sodium hydroxide is generally recommended by the supplier, but it may not be sufficient for reducing bioburden during large scale manufacturing. Therefore, more stringent sanitization methods for RC membranes are required. However, chemicals used in such sanitization step may disrupt membrane integrity, while the corresponding residuals may reduce product quality. Previous work has shown that high concentration of sodium hydroxide or addition of peracetic acid (PAA) can effectively reduce bioburden, but their effects on the RC membranes remain unknown. In this work, we assessed the impact of two sanitization methods, 0.5 M sodium hydroxide and 30 mM PAA in combination with 0.5 M sodium hydroxide, on membrane integrity and protein quality of Millipore and pall corporation (PALL) membranes. Both methods showed a similar impact as the control after performing 15 cycles. However, the addition of PAA may cause residual chemical concerns, therefore, 0.5 M sodium hydroxide was recommended as an effective and safe sanitization method for RC UF/DF membranes.

Model-based optimization of multistage ultrafiltration/diafiltration for recovery of canola protein

The ultrafiltration/diafiltration (UF/DF) process is a crucial step in the canola protein isolation process from rapeseed meal. The process involves using a multi-stage membrane system to separate components of the mixture. As diafiltration dilutes the feed stream in the ultrafiltration system, a large amount of diafiltration water is required. Reducing the diafiltrate for sustainability reasons can be done by carefully selecting process variables or using recycle streams. However, finding the optimum process variables can be a meticulous process if performed experimentally or via trial and error. In this study, we performed an optimization using a mechanistic model integrated with a genetic algorithm to aid in finding an optimum combination of process variables. The mechanistic ultrafiltration model was derived by taking into account transport phenomena within the filtration system. Parameters were characterized experimentally in term of viscosity coefficient, membrane resistance, cake porosity, aggregate diameter equivalent, and material compressibility factors. Using the mechanistic model-based optimization in combination with actual experimental values, the performance of a four-stage UF/DF system could already be improved despite fixing the configuration, albeit at the cost of a reduction in purity. Further improvement could be achieved by using recycle streams. The optimized system achieved a diafiltrate reduction of up to 79%, an increase of purity of up to 31%, and an increase of dry matter content of up to 18%, while maintaining the product purity of the reference set-up.

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode.

Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps and size-based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7->3.4 and 2.5-3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.

Use of ultrafiltration/diafiltration for the processing of antisense oligonucleotides.

Ultrafiltration/diafiltration (UF/DF) has been the hallmark for concentrating and buffer exchange of protein and peptide-based therapeutics for years. Here we examine the capabilities and limitations of UF/DF membranes to process oligonucleotides using antisense oligonucleotides (ASOs) as a model. Using a 3 kDa UF/DF membrane, oligonucleotides as small as 6 kDa are shown to have low sieving coefficients (<0.008) and thus can be concentrated to high concentrations (≤200 mg/mL) with high yield (≥95%) and low viscosity (<15 centipoise), provided the oligonucleotide is designed not to undergo self-hybridization. In general, the oligonucleotide should be at least twice the reported membrane molecular weight cutoff for robust retention. Regarding diafiltration, results show that a small amount of salt is necessary to maintain adequate flux at concentrations exceeding about 40 mg/mL. Removal of salts along with residual solvents and small molecule process-related impurities can be robust provided they are not positively charged as the interaction with the oligonucleotide can prevent passage through the membrane, even for common divalent cations such as calcium or magnesium. Overall, UF/DF is a valuable tool to utilize in oligonucleotide processing, especially as a final drug substance formulation step that enables a liquid active pharmaceutical ingredient.

Monitoring of ultra- and diafiltration processes by Kalman-filtered Raman measurements

Monitoring the protein concentration and buffer composition during the Ultrafiltration/Diafiltration (UF/DF) step enables the further automation of biopharmaceutical production and supports Real-time Release Testing (RTRT). Previously, in-line Ultraviolet (UV) and Infrared (IR) measurements have been used to successfully monitor the protein concentration over a large range. The progress of the diafiltration step has been monitored with density measurements and Infrared Spectroscopy (IR). Raman spectroscopy is capable of measuring both the protein and excipient concentration while being more robust and suitable for production measurements in comparison to Infrared Spectroscopy (IR). Regardless of the spectroscopic sensor used, the low concentration of excipients poses a challenge for the sensors. By combining sensor measurements with a semi-mechanistic model through an Extended Kalman Filter (EKF), the sensitivity to determine the progress of the diafiltration can be improved. In this study, Raman measurements are combined with an EKF for three case studies. The advantages of Kalman-filtered Raman measurements for excipient monitoring are shown in comparison to density measurements. Furthermore, Raman measurements showed a higher measurement speed in comparison to Variable Pathlength (VP) UV measurement at the trade-off of a slightly worse prediction accuracy for the protein concentration. However, the Raman-based protein concentration measurements relied mostly on an increase in the background signal during the process and not on proteinaceous features, which could pose a challenge due to the potential influence of batch variability on the background signal. Overall, the combination of Raman spectroscopy and EKF is a promising tool for monitoring the UF/DF step and enables process automation by using adaptive process control.

In-line monitoring of protein concentration with MIR spectroscopy during UFDF.

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.

Modification of fermented whey protein concentrates: Impact of sequential ultrasound and TGase cross-linking

This study aimed to examine the impact of fermentation process on whey protein and improve the general properties of fermented whey protein concentrate (FWPC) recovered by a combined ultrafiltration-diafiltration (UF-DF) operation. Impacts of sequential ultrasound (US) pretreatment and transglutaminase (TGase) crosslinking on structural, functional, and physicochemical properties of FWPCs were investigated. Partially denatured and hydrolyzed fermented whey protein could replace heat denaturation prior to the TGase addition to a whey protein system. Sequential treatment increased the molecular weight of FWPCs as exhibited by both SEM and SDS-PAGE, which demonstrates that modification can lead to the polymers and oligomers production. The zeta potential value increased significantly after US treatment and enzyme catalysis, and all the modified FWPCs were strongly negatively charged. Compared with the secondary structure of untreated FWPCs, the percentage of α-helix and random coil in modified FWPCs significantly increased, while the percentage of β-sheet and β-turns reduced. Solubility, free sulfhydryl groups, and surface hydrophobicity of all FWPCs were significantly improved compared to non-fermented WPC (P < 0.05). Sequential treatment induced a substantial impact on the emulsifying activity and stability of modified samples in comparison with untreated FWPCs. Scanning electron microscope pictures confirmed the positive effects of sequential treatments on texture and void size reduction. Therefore, the application of recovering modified FWPCs is fully recommended as a commercially viable approach for enhanced protein production at the industrial scale.

Predicting Formulation Conditions During Ultrafiltration and Dilution to Drug Substance Using a Donnan Model with Homology-Model Based Protein Charge

In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments.

Digital Twin for HIV-Gag VLP Production in HEK293 Cells

The development and adoption of digital twins (DT) for Quality-by-Design (QbD)-based processes with flexible operating points within a proven acceptable range (PAR) and automation through Advanced Process Control (APC) with Process Analytical Technology (PAT) instead of conventional process execution based on offline analytics and inflexible process set points is one of the great challenges in modern biotechnology. Virus-like particles (VLPs) are part of a line of innovative drug substances (DS). VLPs, especially those based on human immunodeficiency virus (HIV), HIV-1 Gag VLPs, have very high potential as a versatile vaccination platform, allowing for pseudotyping with heterologous envelope proteins, e.g., the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As enveloped VLPs, optimal process control with minimal hold times is essential. This study demonstrates, for the first time, the use of a digital twin for the overall production process of HIV-1 Gag VLPs from cultivation, clarification, and purification to lyophilization. The accuracy of the digital twins is in the range of 0.8 to 1.4% in depth filtration (DF) and 4.6 to 5.2% in ultrafiltration/diafiltration (UFDF). The uncertainty due to variability in the model parameter determination is less than 4.5% (DF) and less than 3.8% (UFDF). In the DF, a prediction of the final filter capacity was demonstrated from as low as 5.8% (9mbar) of the final transmembrane pressure (TMP). The scale-up based on DT in chromatography shows optimization potential in productivity up to a factor of 2. The schedule based on DT and PAT for APC has been compared to conventional process control, and hold-time and process duration reductions by a factor of 2 have been achieved. This work lays the foundation for the short-term validation of the DT and PAT for APC in an automated S7 process environment and the conversion from batch to continuous production.

Simulation-based evaluation of single pass continuous diafiltration with alternating permeate flow direction

Abstract In the framework of modern bioprocessing continuous ultrafiltration/diafiltration (UF/DF) is getting increasingly popular. However, while continuous UF can be easily implemented using a so-called single pass tangential flow filtration (SPTFF) module, continuous DF requires a more complicated setup including several SPTFF modules and intermittent dilution steps. Recently, we introduced a novel module design for continuous DF allowing simultaneous delivery of fresh buffer while withdrawing the permeate, thus achieving high degrees of buffer exchange within a single unit. In addition, the system allows to cyclically switch the flow direction of DF buffer through the membranes. Those uncommon features, however, also make it more difficult to determine an operation optimum experimentally by means of trial and error. Therefore, here a detailed finite element model of the physical processes within the module is presented, predicting key figures such as the obtained diafiltration efficiency and the resulting pressures. Because within the module all flow channels are filled by a 3D-printed porous grid supporting the membranes from both sides, the modified Brinkman equation was used to simulate the hydrodynamics, while common mass balance differential equations including accumulation, convection, and an anisotropic dispersion term were used for the simulation of concentration profiles of dissolved species. The predicted key figures are in good agreement with experimental results, obtained for feed solutions including up to 50 g/L of protein and being operated with and without switching the flow direction of the diafiltration buffer. A thorough parameter study reveals that the module shows the best performance for unidirectional flow of the diafiltration buffer, reaching diafiltration efficiencies independence to the applied diavolumes which are comparable to the ones of a conventional multi-stage setup using three SPTFF modules. Therefore, the simulation-based evaluation of optimum operation conditions reveals that the new module design has the potential to realize truly continuous diafiltration setups with high efficiency, requiring only one unit and no extra external piping for returning diafiltration in counterflow. Such simplified setups should be especially useful in small, flexible processing plants as they are increasingly demanded in the biopharmaceutical industry.

Functional properties of hemp protein concentrate obtained by alkaline extraction and successive ultrafiltration and spray‐drying

SummaryHemp protein concentrates were obtained from hemp seed cake by extraction at high alkaline pH (10 and 12) followed by an ultrafiltration/diafiltration (UF) purification step and spray‐drying. The composition, thermal, nutritional, and functional properties of the resulting protein samples were evaluated. The UF treatment increased protein purity of the protein extracts recovered at pH 10 and 12, from ˜61% and ˜70% to ˜85% and ˜88%, respectively. This protein enrichment did not change the polypeptide composition, as well as the protein solubility profile at each respective pH of extraction. However, the pH 12‐extracted proteins showed higher solubility (&gt;85%) notably at alkaline pH (7–9) compared with the pH 10‐extracted samples (˜40–80%). The highest in vitro protein digestibility values were obtained for the crude protein sample resulting from the extraction at pH 10 (˜89%) and the pH 12‐extracted protein sample after heat treatment (˜96%). The pH 12‐extracted hemp proteins presented excellent emulsifying properties, producing submicromic emulsions with high stability even after protein heating, whereas the emulsions prepared from the pH 10‐extracted proteins tended to be easily destabilised by flocculation and coalescence. The particular properties of the former protein sample could be explained by the denaturing polymerisation of protein during the extraction at pH 12 involving complexation with polyphenols.

Integration of Membrane Processes for By-Product Valorization to Improve the Eco-Efficiency of Small/Medium Size Cheese Dairy Plants.

Goat and second cheese whey from sheep’s milk are by-products of the manufacture of goat cheeses and whey cheeses from sheep. Due to their composition which, apart from water—about 92%—includes lactose, proteins, fat, and minerals, and the elevated volumes generated, these by-products constitute one of the main problems facing to cheese producers. Aiming to add value to those by-products, this study evaluates the efficiency of ultrafiltration/diafiltration (UF/DF) for the recovery of protein fraction, the most valuable component. For a daily production of 3500 and using the experimental results obtained in the UF/DF tests, a membrane installation was designed for valorization of protein fraction, which currently have no commercial value. A Cost–Benefit Analysis (CBA) and Sensitivity Analysis (SA) were performed to evaluate the profitability of installing that membrane unit to produce three new innovative products from the liquid whey protein concentrates (LWPC), namely food gels, protein concentrates in powder and whey cheeses with probiotics. It was possible to obtain LWPC of around 80% and 64% of crude protein, from second sheep cheese whey and goat cheese whey, respectively. From a survey of commercial values for the intended applications, the results of CBA and SA show that this system is economically viable in small/medium sized cheese dairies.

Streamlining process characterization efforts using the high throughput ambr® crossflow system for ultrafiltration and diafiltration processing of monoclonal antibodies.

Commercial process development for biopharmaceuticals often involves process characterization (PC) studies to gain process knowledge and understanding in preparation for process validation. One common approach to conduct PC activities is by using design-of-experiment, which can help determine the impact process parameter deviations may have on product quality attributes. Qualified scale-down systems are typically used to conduct these studies. For an ultrafiltration/diafiltration (UF/DF) application, however, a traditional scale-down still requires hundreds of milliliters of material per run and can only conduct one experiment at a time. This poses a challenge in resources as there could be 20+ experiments required for a typical UF/DF PC study. One solution to circumvent this is the use of high-throughput systems, which enable parallel experimentation by only using a fraction of the resources. Sartorius Stedim Biotech has recently commercialized the ambr® crossflow high-throughput system to meet this need. In this study, the performance of this system during a monoclonal antibody UF/DF step was first compared with a pilot- and a manufacturing-scale tangential flow filtration (TFF) system at a single operating condition. Due to material limitations, it was then compared to only the pilot-scale TFF system across wider ranges of transmembrane pressure; crossflow rate; and diafiltration concentration in a PC study. Permeate flux, aggregate content, process yield, pH/conductivity traces, retentate concentration, axial pressure drop, and turbidity values were measured at both scales. A good agreement was attained across scales, further supporting its potential use as a scale-down system.

Introduction and clearance of beta-glucan in the downstream processing of monoclonal antibodies.

β‐Glucan process‐related impurities can be introduced into biopharmaceutical products via upstream or downstream processing or via excipients. This study obtained a comprehensive process‐mapping dataset for five monoclonal antibodies to assess β‐glucan introduction and clearance during development and production runs at various scales. Overall, 198 data points were available for analysis. The greatest β‐glucan concentrations were found in the depth‐filtration filtrate (37–2,745 pg/ml). Load volume correlated with β‐glucan concentration in the filtrate, whereas flush volume was of secondary importance. Cation‐exchange chromatography significantly cleared β‐glucans. Furthermore, β‐glucan leaching from the Planova 20N virus removal filter was reduced by increasing the flush volume (1 vs. 10 L/m2). β‐glucan concentrations after filter flush with 10 L/m2 were consistently <10 pg/ml. No or only limited β‐glucan clearance was attained via ultrafiltration/diafiltration (UF/DF). However, during the first run with monoclonal antibody (mAb) 4, β‐glucan concentration in the UF/DF retentate was 10.8 pg/mg, potentially due to β‐glucan leaching from the first run with a regenerated cellulose membrane. Overall, β‐glucan levels in the final mAb drug substance were 1–12 pg/mg. Assuming high doses of 1,000–5,000 mg, a β‐glucan contamination at 20 pg/mg would translate to 20–100 ng/dose, which is below the previously suggested threshold for product safety (≤500 ng/dose).

A mechanistic model to account for the Donnan and volume exclusion effects in ultrafiltration/diafiltration process of protein formulations.

Ultrafiltration/diafiltration (UF/DF) is a typical step in protein drug manufacturing process to concentrate and exchange the protein solution into a desired formulation. However, significant offset of pH and composition from the target formulation have been frequently observed after UF/DF, posing challenges to the stability, performance, and consistency of the final drug product. Such shift can often be attributed to the Donnan and volume exclusion effects. In order to predict and compensate for those effects, a mechanistic model is developed based on the protein charge, mass and charge balances, as well as the equilibrium condition across the membrane. The integrated UF/DF model can be used to predict both the dynamic behavior and the final outcome of the process. Examples of the modeling results for the pH and composition variation during the UF/DF operations are presented for two monoclonal antibody proteins. The model predictions are in good agreement with a comprehensive experimental data set that covers different process steps, protein concentrations, solution matrices, and process scales. The results show that significant pH and excipient concentration shifts are more likely to occur for high protein concentration and low ionic strength matrices. As a special example, a self-buffering protein formulation shows unique pH behavior during DF, which could also be captured with the dynamic model. The capability of the model in predicting the performance of UF/DF process as a function of protein characteristics and formulation conditions makes it a useful tool to improve process understanding and facilitate process development.

Designing scalable ultrafiltration/diafiltration process of monoclonal antibodies via mathematical modeling by coupling mass balances and Poisson-Boltzmann equation.

Ultrafiltration/diafiltration (UF/DF) operations are employed for achieving the desired therapeutic monoclonal antibody (mAb) formulations. Due to electrostatic interactions between the charged proteins, solute ions, and uncharged excipients, the final pH and concentration values are not always equal to those in the DF buffer. At high protein concentrations, typical for industrial formulations, this effect becomes predominant. To account for challenges occurring in industrial environments, a robust mathematical framework enabling the prediction of pH and concentration profiles throughout the UF/DF process is provided. The proposed mechanistic model combines a macroscopic mass balance approach with a molecular approach based on a Poisson-Boltzmann equation dealing with electrostatic interactions and accounting for protein exclusion volume effect. The mathematical model was validated with experimental data of two commercially relevant mAbs obtained from an industrial UF/DF process using scalable laboratory equipment. The robustness and flexibility of the model were tested by using proteins with different isoelectric points and net charges. The latter was determined via a titration curve, enabling realistic protein charge-pH evaluation. In addition, the model was tested for different DF buffer types containing both monovalent and polyvalent ions, with various types of uncharged excipients. The model generality enables its implementation for the UF/DF processes of other protein varieties.

Strategies for high-concentration drug substance manufacturing to facilitate subcutaneous administration: A review.

To achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold-up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self-association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity-lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high-concentration drug substance manufacturing, such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high-concentration drug substance manufacturing.