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.     

Efficiency of ultrafiltration/diafiltration in removing organic and elemental process equipment related leachables from biological therapeutics

In the production of biological therapeutics such as monoclonal antibodies (mAbs), ultrafiltration and diafiltration (UF/DF) are widely regarded as effective downstream processing steps capable of removing process equipment related leachables (PERLs) introduced upstream of the UF/DF step. However, clearance data available in the literature are limited to species with low partition coefficients (log P) such as buffer ions, hydrophilic organic compounds, and some metal ions. Additional data for a wide range of PERLs including hydrophobic compounds and elemental impurities are needed to establish meaningful, comprehensive safety risk assessments. Herein, we report the results from studies investigating the clearance of seven different organic PERLs representing a wide range of characteristics (i.e., log P (−0.3 to 18)), and four model elements with different chemical properties spiked into a mAb formulation at 10 ppm and analyzed during clearance using gas chromatography–mass spectrometry (GC–MS), liquid chromatography-photodiode-array-mass spectrometry (LC-PDA-MS), and inductively coupled plasma mass spectrometry (ICP-MS). The clearance data showed ideal clearance and sieving of spiked organic PERLs with log P < 4, partial clearance of PERLs with 4 < log P < 9, and poor clearance of highly hydrophobic PERLs (log P > 9) after nine diafiltration volumes (DVs). Supplemental clearance studies on seven additional PERLs present at much lower concentration levels (0.1–1.5 ppm) in the mAb formulation upstream of UF/DF and three PERLs associated with the tangential flow filtration (TFF) equipment also demonstrated the similar correlations between log P and % clearance. For model elements, the findings suggest that UF/DF in general provides ideal clearance for elements. Evidence showed that the UF/DF process does not only help mitigate leachables risk from PERLs introduced upstream of UF/DF, but also from the TFF operation itself as all three TFF-related PERLs were effectively cleared. Overall, the UF/DF clearance presented in this work demonstrated whereas highly hydrophobic PERLs and elements that exist as charged species, particularly transition metal ions, may not be as effectively cleared and thus warrant further risk assessment; hydrophilic and some hydrophobic PERLs (log P < 4) are indeed well-cleared and thus present a lower overall safety risk.     

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.     

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.     

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.     

Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process

Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.     

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.     

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor? using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed‐batch were compared. Cell densities higher than 10⁸ cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell‐specific productivity was comparable at cell densities up to 1.3 × 10⁸ cells/mL in perfusion and was comparable or lower in fed‐batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed‐batch and 28× more in a 1‐month perfusion at 10⁸ cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 10⁸ and 10⁸ cells/mL was performed using cells from a perfusion run at 10⁸ cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013     

Purification of hemoglobin by tangential flow filtration with diafiltration

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189–199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC‐grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch‐mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS‐PAGE, and LC‐MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC‐grade bHb. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

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).     

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.     

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.     

Evaluation of single-use tangential flow filtration technology for purification of activated polysaccharides used in conjugate vaccine manufacturing

Over the past decade, single-use tangential flow filtration (TFF) technologies have emerged to reduce system preparation time, promote fast and flexible product change over, and ultimately shorten process development and manufacturing time/cost. In this study, the performance of a recently developed Pellicon® single-use TFF capsule was compared against traditional Pellicon® cassettes by assessing TFF process performance (such as flux, residuals clearance, and yield) and post-purification product attributes (such as concentration and mass-weighted average molecular weight). Good scaling was shown by comparing process performance and product attributes across different scales and formats. Additionally, similar TFF process performance and post-purification product attributes were observed for the single-use capsule compared to the reusable TFF cassettes. The capsule requires a smaller flush than the cassette, and it is easier to use since it does not require a compression holder or pre-sanitization. The results provide insight into the application of the single-use TFF capsule and scalability of TFF processes for the purification of conjugate vaccines.     

One-pot synthesis, purification, and formulation of bionanoparticle-CpG oligodeoxynucleotide hepatitis B surface antigen conjugate vaccine via tangential flow filtration

The synthesis and characterization of a Hepatitis B virus vaccine (HBsIC-ISS) candidate composed of Hepatitis B surface antigen (HBsAg) bionanoparticles conjugated to multiple copies of immunostimulatory sequence oligodeoxynucleotides is presented. An efficient tangential flow filtration (TFF) method has been developed to purify the conjugated bionanoparticles from the excess conjugation reagents. The TFF technique presented can serve as a rapid and convenient alternative to current methods like ultracentrifugation for the separation of excess small molecule/polymeric conjugation reagents from chemically modified viruses and other viruslike particles.     

Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.     

Considerations for operational space definition and optimization of a no-salt flowthrough hydrophobic interaction chromatography purification step

No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin.     

Enumeration and isolation of viral particles from oligotrophic marine environments by tangential flow filtration.

A method for concentrating, enumerating and isolating viral particles from marine water samples was developed and evaluated. The method consists of a concentration step by a tangential flow filtration (TFF) system, ultrafiltration by centrifugal concentrator, and visualization by transmission electron microscopy (TEM). This procedure allows to reduce volumes of ca. 21 of seawater to 10-20 microliters, which can be dispensed on electron microscopy grids to count total viral particles. This method allows the recovery of small numbers of viral particles from oligotrophic seawater samples, in which viral numbers ranged from 10(5) to 10(6) viral particles/ml. The tangential flow filtration system was evaluated as quantitative technique using suspensions of two different bacteriophages (T6 and phi X174) in autoclaved seawater. Recovery rates varied depending on both the viral morphology and flow rate; recovery percentages reached 117.4% for T6 and 60.6% for phi X174 using low flow rate.     

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow‐rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log₁₀ of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750–757, 2015