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.     

Theoretical analysis of excipient concentrations during the final ultrafiltration/diafiltration step of therapeutic antibody

Diafiltration of a protein solution into a new buffer is a common final step in biopharmaceutical manufacturing. However, the excipient concentrations in the retentate are not always equal to their corresponding concentrations in the new buffer (diafiltration buffer). This phenomenon was observed repeatedly during diafiltration of different therapeutic monoclonal antibodies in which the concentrations of histidine and either sorbitol or sucrose (depending on which was chosen for the diafiltration buffer) in the retentate were lower than in the diafiltration buffer. Experimental studies and theoretical analyses of the ultrafiltration/diafiltration (UF/DF) step were carried out to determine the primary causes of the phenomenon and to develop a mathematical model capable of predicting retentate excipient concentrations. The analyses showed that retentate histidine concentration was low primarily because of repulsive charge interactions between positively‐charged histidine molecules and positively‐charged protein molecules, and that volume exclusion effects were secondary for like‐charged molecules. The positively‐charged protein molecules generate an electrical potential that cause an uneven distribution of charged histidine molecules. This interaction was used to construct a mathematical model based on the Poisson‐Boltzmann equation. The model successfully predicted the final histidine concentration in the diafiltered product (retentate) from the UF/DF development and production runs, with good agreement across a wide range of protein and histidine concentrations for four therapeutic monoclonal antibodies. The concentrations of uncharged excipients (sorbitol or sucrose) were also successfully predicted using previously established models, with volume exclusion identified as the primary cause of differences in uncharged excipient concentrations in the retentate and diafiltration buffer. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Multi‐criteria manufacturability indices for ranking high‐concentration monoclonal antibody formulations

The need for high‐concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high‐concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high‐concentration mAb formulation conditions in terms of their ease of manufacture. This is illustrated by analyzing published high‐throughput biophysical screening data that explores the influence of different formulation conditions (pH, ions, and excipients) on the solution viscosity and product thermostability. A decision tree classification method, CART (Classification and Regression Tree) is used to identify the critical formulation conditions that influence the viscosity and thermostability. In this work, three different multi‐criteria data analysis frameworks were investigated to derive manufacturability indices from analysis of the stress maps and the process conditions experienced in the final UF/DF step. Polynomial regression techniques were used to transform the experimental data into a set of stress maps that show viscosity and thermostability as functions of the formulation conditions. A mathematical filtrate flux model was used to capture the time profiles of protein concentration and flux decay behavior during UF/DF. Multi‐criteria decision‐making analysis was used to identify the optimal formulation conditions that minimize the potential for both viscosity and aggregation issues during UF/DF. Biotechnol. Bioeng. 2017;114: 2043–2056. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Perodicals, Inc.     

Clearance of solvents and small molecule impurities in antibody drug conjugates via ultrafiltration and diafiltration operation

Ultrafiltration and diafiltration (UF/DF) processes by tangential flow filtration (TFF) are frequently used for removal of solvents and small molecule impurities and for buffer exchange for biopharmaceutical products. Antibody-drug conjugates (ADCs) as an important class of biological therapeutics, carry unique solvents and small molecule impurities into the final UF/DF step as compared to standard antibody preparation. The production process of ADCs involves multiple chemical steps, for example, reduction and conjugation. The clearance of these solvents and small molecules by UF/DF, specifically the DF step, has been assessed and described herein. The rates of clearance for all the impurities in this study are close to the ideal clearance with no apparent interaction with either the protein or the TFF membrane and system. The effect of process variables during DF, such as pH, temperature, membrane loading, transmembrane pressure, and cross flow rate, has also been evaluated and found to have minimal impact on the clearance rate. These results demonstrate efficient clearance of solvents and small molecule impurities related to the ADC process by the DF process and provide a general data package to facilitate risk assessments based on the sieving factors and program specific needs.     

Predicting diafiltration solution compositions for final ultrafiltration/diafiltration steps of monoclonal antibodies

Many liquid formulations for monoclonal antibodies (MAbs) require the final ultrafiltration/diafiltration step to operate at high protein concentrations, often at or above 100 g/L. When operating under these conditions, the excipient concentrations and pH of the final diafiltered retentate are frequently not equal to the corresponding excipient concentrations and pH of the diafiltration buffer. A model based on the Poisson-Boltzmann equation combined with volume exclusion was extended to predict both pH and excipient concentrations in the retentate for a given diafiltration buffer. This model was successfully applied to identify the diafiltration buffer composition required to achieve the desired pre-formulated bulk drug substance (retentate) conditions. Predictions were in good agreement with the experimental results, and reduced the number of experimental iterations needed to define the diafiltration buffer composition. Additionally, the predictive model was applied in a sensitivity analysis across ranges of protein charge, protein concentration, and diafiltration buffer pH and excipient concentration. This sensitivity analysis can facilitate the design of experiments for robustness testing, and allow for generalized predictions across classes of molecules such as MAbs.     

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.     

Real-time monitoring of quality attributes by in-line Fourier transform infrared spectroscopic sensors at ultrafiltration and diafiltration of bioprocess

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.     

Continuous ultrafiltration/diafiltration using a 3D-printed two membrane single pass module

A 3D printed ultrafiltration/diafiltration (UF/DF) module is presented allowing the continuous, simultaneous concentration of retained (bio-)molecules and reduction or exchange of the salt buffer. Differing from the single-pass UF concepts known from the literature, DF operation does not require the application of several steps or units with intermediating dilution. In contrast, the developed module uses two membranes confining the section in which the molecules are concentrated while the sample is passing. Simultaneously to this concentration process, the two membranes allow a perpendicular in and outflow of DF buffer reducing the salt content in this section. The module showed the continuous concentration of a dissolved protein up to a factor of 4.6 while reducing the salt concentration down to 47% of the initial concentration along a flow path length of only 5 cm. Due to single-pass operation the module shows concentration polarization effects reducing the effective permeability of the applied membrane in case of higher concentration factors. However, because of its simple design and the capability to simultaneously run UF and DF processes in a single module, the development could be economically beneficial for small scale UF/DF applications.     

Process challenges relating to hematopoietic stem cell cultivation in bioreactors

Hematopoietic stem cells (HSCs) are extremely useful in treating a wide range of diseases and have a variety of useful research applications. However, the routinely generated low in vitro concentrations of HSCs from current bioreactor manufacturing systems has been a hindrance to the full-scale application of these essential cellular materials. This has made the search for novel bioreactor systems for high-concentration HSC production a major research endeavour. This review addresses process challenges in relation to bioreactor development and optimisation for high-density HSC production under effective monitoring of essential culture parameters, such as pH, dissolved oxygen and nutrient uptake. It discusses different process strategies and bioreactor configurations for HSCs production from a commercial viability perspective, and also discusses recent advances in the field.     

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.     

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).     

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.     

Effect of protein and solution properties on the Donnan effect during the ultrafiltration of proteins

Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, improve the target product profile, and allow greater flexibility in dosing methods. The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc-fusion proteins. In this study, new equations for the Donnan effect during the diafiltration and concentration of proteins in solutions containing monovalent and divalent ions were derived. The new Donnan models obey mass conservation laws, account for the buffering capacity of proteins, and account for protein-ion binding. Data for the pH offsets of an Fc-fusion protein and a monoclonal antibody were predicted in both monovalent and divalent buffers using these equations. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used. The Donnan offset observed during the concentration of an acidic Fc-fusion protein was mitigated by operating at low temperature. It is important to account for the Donnan effect during preformulation studies. The excipients levels in an ultrafiltration pool may differ from the levels in a protein solution obtained by adding buffers into concentrated protein solutions due to the Donnan effect.     

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.     

Cationic polymer precipitation for enhanced impurity removal in downstream processing

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation–depth filtration and the current platform process for the purification of biologics.     

Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration

We designed small-scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small-scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.     

High concentration biotherapeutic formulation and ultrafiltration: Part 1 pressure limits

High therapeutic dosage requirements and the desire for ease of administration drive the trend to subcutaneous administration using delivery systems such as subcutaneous pumps and prefilled syringes. Because of dosage volume limits, prefilled syringe administration requires higher concentration liquid formulations, limited to about 30 cP or roughly 100–300 g L^?1 for mAb's. Ultrafiltration (UF) processes are routinely used to formulate biological therapeutics. This article considers pressure constraints on the UF process that may limit its ability to achieve high final product concentrations. A system hardware analysis shows that the ultrafiltration cassette pressure drop is the major factor limiting UF systems. Additional system design recommendations are also provided. The design and performance of a new cassette with a lower feed channel flow resistance is described along with 3D modeling of feed channel pressure drop. The implications of variations in cassette flow channel resistance for scaling up and setting specifications are considered. A recommendation for a maximum pressure specification is provided. A review of viscosity data and theory shows that molecular engineering, temperature, and the use of viscosity modifying excipients including pH adjustment can be used to achieve higher concentrations. The combined use of a low pressure drop cassette with excipients further increased final concentrations by 35%. Guidance is provided on system operation to control hydraulics during final concentration. These recommendations should allow one to design and operate systems to routinely achieve the 30 cP target final viscosity capable of delivery using a pre‐filled syringe. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:113–124, 2017     

A two-stage ultrafiltration and nanofiltration process for recycling dairy wastewater

A two-stage ultrafiltration and nanofiltration (UF/NF) process for the treatment of model dairy wastewater was investigated to recycle nutrients and water from the wastewater. Ultracel PLGC and NF270 membranes were found to be the most suitable for this purpose. In the first stage, protein and lipid were concentrated by the Ultracel PLGC UF membrane and could be used for algae cultivation to produce biodiesel and biofuel, and the permeate from UF was concentrated by the NF270 membrane in the second stage to obtain lactose in retentate and reusable water in permeate, while the NF retentate could be recycled for anaerobic digestion to produce biogas. With this approach, most of dairy wastewater could be recycled to produce reusable water and substrates for bioenergy production. Compared with the single NF process, this two-stage UF/NF process had a higher efficiency and less membrane fouling.     

Membrane‐Based Approach for the Downstream Processing of Influenza Virus‐Like Particles

Currently, marketed influenza vaccines are only efficient against homologous viruses, thus requiring a seasonal update based on circulating subtypes. This constant reformulation adds several challenges to manufacturing, particularly in purification due to the variation of the physicochemical properties of the vaccine product. A universal platform approach capable of handling such variation is therefore of utmost importance. In this work, a filtration‐based approach is explored to purify influenza virus‐like particles. Switching from adsorptive separation to size‐based purification allows overcoming the differences in retention observed for different influenza strains. The proposed process employs a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step. Different process parameters are assessed in terms of product recovery and impurities’ removal. Membrane chemistry, pore size, operation modes, critical flux, transmembrane pressure, and permeate control strategies are evaluated. After membrane selection and parameter optimization, concentration factors and diafiltration volumes are also defined. By optimizing the filtration mode of operation, it is possible to achieve product recoveries of approximately 80%. Overall, the process time is decreased by 30%, its scalability is improved, and the costs are reduced due to the removal of chromatography and associated buffer consumptions, cleaning, and its validation steps.