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.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment

Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.     

Genotoxicity risk assessment: a proposed classification strategy

Recent advances in genetic toxicity (mutagenicity) testing methods and in approaches to performing risk assessment are prompting a renewed effort to harmonize genotoxicity risk assessment across the world. The US Environmental Protection Agency (EPA) first published Guidelines for Mutagenicity Risk Assessment in 1986 that focused mainly on transmissible germ cell genetic risk. Somatic cell genetic risk has also been a risk consideration, usually in support of carcinogenicity assessments. EPA and other international regulatory bodies have published mutagenicity testing requirements for agents (pesticides, pharmaceuticals, etc.) to generate data for use in genotoxicity risk assessments. The scheme that follows provides a proposed harmonization approach in which genotoxicity assessments are fully developed within the risk assessment paradigm used by EPA, and sets out a process that integrates newer thinking in testing battery design with the risk assessment process. A classification strategy for agents based on inherent genotoxicity, dose-responses observed in the data, and an exposure analysis is proposed. The classification leads to an initial level of concern for genotoxic risk to humans. A total risk characterization is performed using all relevant toxicity data and a comprehensive exposure evaluation in association with the genotoxicity data. The result of this characterization is ultimately used to generate a final level of concern for genotoxic risk to humans. The final level of concern and characterized genotoxicity risk assessment are communicated to decision makers for possible regulatory action(s) and to the public.     

Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.     

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up- and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.     

Separation of protein charge variants by ultrafiltration

The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine epsilon-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.     

State-of-the-art in downstream processing of monoclonal antibodies: Process trends in design and validation

Monoclonal antibodies (mAbs) are the most important family of biopharmaceutical compounds in terms of market share. At present, 30 mAbs have been approved and are now commercialized for therapeutic purposes. mAbs are typically produced by mammalian cell culture in bioreactors that range in scale of 1–20 m³. Regardless of scale, from laboratory to commercial settings, the recovery and purification of mAbs present important challenges. Depending on the scale, the particular product, and the type of production process (bioreactor operation, process time, complexity of the culture media, cell density, etc.), many possible downstream configurations are possible and have been used. In this contribution, we review each type of unit operation that forms a downstream train for mAb production. We provide information regarding typical operation settings and critical variables for centrifugation, ultrafiltration, affinity chromatography, ion exchange chromatography, and viral removal operations. In addition, we discuss some important considerations required for the formulation of drugs based on mAbs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 899–916, 2012     

Small molecule clearance in ultrafiltration/diafiltration in relation to protein interactions: Study of citrate binding to a Fab

Ultrafiltration/diafiltration (UFDF) is commonly utilized in the purification of recombinant proteins to concentrate and buffer exchange the product. It is often the final step in the purification process, placing the protein in its final formulation and clearing small molecules introduced in upstream purification steps. This article presents a case study of reduced small molecule clearance in ultrafiltration/diafiltration of an antigen‐binding fragment of a monoclonal antibody. Citrate, a commonly utilized small molecule in downstream processes, is shown to have reduced clearance due to specific interactions with the protein product. The study presents process solutions and utilizes a simple model to characterize clearance of small molecules which exhibit interactions with product protein. Biotechnol. Bioeng. 2009;102: 1718–1722. © 2008 Wiley Periodicals, Inc.     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

Extraction of rIL-2 inclusion bodies from Escherichia coli using cross-flow filtration

A cross-flow membrane filtration process was developed for the recovery of rIL-2 inclusion bodies from homogenized Escherichia coli. The membrane extraction process was comprised of a two-step diafiltration followed by an extraction with 7 M GuHCl and a 40-fold dilution of the solubilized inclusion bodies into 0.01 M Tris-HCl, 0.035 M NaCl, pH 7.9. The first diafiltration was with a 0.03 M Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8, followed by a diafiltration with 1.75 M GuHCl. All of the insoluble rIL-2 was retained behind the membrane, whereas a GuHCl wash solubilized approximately 15% of the rIL-2. The membrane process increased the yield of rIL-2 in the diluted extract by threefold as compared to a similar centrifuge process with a significant increase in purity as determined by reverse-phase high-performance liquid chromatography (HPLC). (c) 1994 John Wiley & Sons, Inc.     

Demonstrating β-glucan and yeast peptide clearance in biopharmaceutical downstream processes

The use of yeast- and plant-derived hydrolysates in cell culture production processes has sparked concerns over the potential immunogenicity risk posed by β-glucans and yeast peptides contained in these raw materials. This article utilizes a combination of in-process testing from large-scale manufacturing and scale-down spiking studies to demonstrate the clearance of β-glucans and yeast peptides through chromatographic steps in the downstream purification process for a monoclonal antibody. β-Glucans were found to flow through most all three modes of chromatography (Protein A, cation and anion exchange) without binding to the resins or the product. Protein A affinity chromatography was found to provide the best clearance factor. The efficacy of the resin sanitization and storage procedures to prevent carryover from one run to the next was also demonstrated. Yeast peptides were found to be metabolized during the cell culture process and were undetectable after the Protein A purification step. The data presented here serve to allay concerns about the use of hydrolysates in cell culture production. The methodology presented here provides a template to demonstrate clearance of β-glucans and yeast peptides through chromatographic steps in downstream processing.     

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products

The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.     

Genotoxicity risk assessment: a proposed classification strategy

Recent advances in genetic toxicity (mutagenicity) testing methods and in approaches to performing risk assessment are prompting a renewed effort to harmonize genotoxicity risk assessment across the world. The US Environmental Protection Agency (EPA) first published Guidelines for Mutagenicity Risk Assessment in 1986 that focused mainly on transmissible germ cell genetic risk. Somatic cell genetic risk has also been a risk consideration, usually in support of carcinogenicity assessments. EPA and other international regulatory bodies have published mutagenicity testing requirements for agents (pesticides, pharmaceuticals, etc.) to generate data for use in genotoxicity risk assessments. The scheme that follows provides a proposed harmonization approach in which genotoxicity assessments are fully developed within the risk assessment paradigm used by EPA, and sets out a process that integrates newer thinking in testing battery design with the risk assessment process. A classification strategy for agents based on inherent genotoxicity, dose-responses observed in the data, and an exposure analysis is proposed. The classification leads to an initial level of concern for genotoxic risk to humans. A total risk characterization is performed using all relevant toxicity data and a comprehensive exposure evaluation in association with the genotoxicity data. The result of this characterization is ultimately used to generate a final level of concern for genotoxic risk to humans. The final level of concern and characterized genotoxicity risk assessment are communicated to decision makers for possible regulatory action(s) and to the public.     

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.     

Viral clearance by flow‐through mode ion exchange columns and membrane adsorbers

Anion exchange (AEX) is a common downstream purification operation for biotechnology products manufactured in cell culture such as therapeutic monoclonal antibodies (mAbs) and Fc‐fusion proteins. We present a head‐to‐head comparison of the viral clearance efficiency of AEX adsorbers and column chromatography using the same process fluids and comparable run conditions. We also present overall trends from the CDER viral clearance database. In our comparison of multiple brands of resins and adsorbers, clearance of three model viruses (PPV, X‐MuLV, and PR772) was largely comparable, with some exceptions which may reflect run conditions that had not been optimized on a resin/membrane specific basis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:124–131, 2014     

Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human,cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach

The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of ‘typical’ popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.     

Assuring the quality, safety, and efficacy of DNA vaccines

Scientists in academia whose research is aimed at the development of a novel vaccine or approach to vaccination may not always be fully aware of the regulatory process by which a candidate vaccine becomes a licensed product. It is useful for such scientists to be aware of these processes as the development of a novel vaccine could be problematic owing to the starting material often being developed in a research laboratory under ill-defined conditions. This paper examines the regulatory process with respect to the development of a DNA vaccine. DNA vaccines present unusual safety considerations that must be addressed during preclinical safety studies, including adverse immunopathology, genotoxicity through integration into a vaccinees chromosomes, and the potential for the formation of anti-DNA antibodies.     

Validation and optimization of viral clearance in a downstream continuous chromatography setting

Continuous bioprocessing holds the potential to improve product consistency, accelerate productivity, and lower cost of production. However, switching a bioprocess from traditional batch to continuous mode requires surmounting business and regulatory challenges. A key regulatory requirement for all biopharmaceuticals is virus safety, which is assured through a combination of testing and virus clearance through purification unit operations. For continuous processing, unit operations such as capture chromatography have aspects that could be impacted by a change to continuous multicolumn operation, for example, do they clear viruses as well as a traditional batch single column. In this study we evaluate how modifying chromatographic parameters including the linear velocity and resin capacity utilization could impact virus clearance in the context of moving from a single column to multicolumn operation. A Design of Experiment (DoE) approach was taken with two model monoclonal antibodies (mAbs) and two bacteriophages used as mammalian virus surrogates. The DoE enabled the identification of best and worst-case scenario for virus clearance overall. Using these best and worst-case conditions, virus clearance was tested in single column and multicolumn modes and found to be similar as measured by Log Reduction Values (LRV). The parameters identified as impactful for viral clearance in single column mode were predictive of multicolumn modes. Thus, these results support the hypothesis that the viral clearance capabilities of a multicolumn continuous Protein A system may be evaluated using an appropriately scaled-down single mode column and equipment.     

Preferential precipitation of acidic variants from monoclonal antibody pools

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.