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Downstream processing of monoclonal antibodies—Application of Platform Approaches
Abstract
This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.
... Low-pH viral inactivation (VI), operated in either batch or continuous mode, often follows the Protein A capture step in downstream purification due to the acidic condition of Protein A eluate (PAE; Parker et al., 2018). A typical low-pH VI operation includes an acidification (VIA) step, where PAE is adjusted to around pH 3.6 and held for up to several hours to achieve sufficient VI (Brorson et al., 2003;Mattila et al., 2016); and a neutralization (VIN) step, where the VIA pool is adjusted to a pH value close to neutral for further downstream processing (Shukla, Hubbard, Tressel, Guhan, & Low, 2007). ...
... The pH of PAE pool is typically adjusted to 3.5-3.8 by addition of an acid solution (Brorson et al., 2003;Shukla et al., 2007) and the VIA condition expectedly should not cause mAb aggregation under the sufficient mixing condition. It held true in bench scale experiments. ...
... This would ensure the designed protein loading at subsequent chromatographic polishing steps while achieving satisfactory step yield, final product quality, and overall robust downstream performance. A purification platform for mAbs typically includes two chromatographic polishing steps after VI operation (Fahrner et al., 2001;Shukla et al., 2007), for example, AEX/HIC, AEX/CEX, and so forth (Shukla, Leslie, Wolfe, Mostafa, & Norman, 2017). In the case here, a polishing step yield was positively correlated the protein loading but inversely correlated with HMW %, VIN (data not shown). ...
Significant amounts of soluble product aggregates were observed in the low‐pH viral inactivation (VI) opertion during an initial scale‐up run for an IgG4 monoclonal antibody (mAb IgG4‐N1). Being earlier in development, a scale‐down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)‐based high molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4‐N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN) (Jin et al. 2019). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low‐pH zone (≤ pH 3.3) profiles and PAE properties, the integrated low‐pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50‐200L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale‐up runs, resulted in 100% success rate of the consecutive 12 pilot‐scale runs. This work demonstrated that the CFD‐based HMW prediction model could be used as a tool to facilitate the scale up of the low‐pH VI process directly from bench to pilot/production scale.
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... This technique relies on the highly specific interaction between the Fc domain of antibodies with protein A ligand. A single step of protein A chromatography may result in >98% purity even with complex cell culture media as a starting material (Shukla et al., 2007). Protein A chromatography is effective in removing process-related contaminants such as HCP, DNA, and virus, relieving the burden on subsequent intermediate and polishing steps (Pinto et al., 2015). ...
... Generally, the protein binds to the column at neutral pH, and elutes at acidic pH (Ghose et al., 2006;Jazayeri and Carroll, 2008). Low pH values may negatively impact the integrity and biological activity of Fc-fusion proteins, or promote the formation of HMWA (Shukla et al., 2007). Product aggregation may be prevented by addition of stabilizers, such as arginine or glycine into the elution buffer (Arakawa et al., 2004). ...
... While protein A chromatography is a straightforward choice for the capture step of Fc-fusion proteins, the selection of the chromatographic techniques and sequence of polishing steps directly depends on the remaining product and impurities to be removed (Pinto et al., 2015). Polishing steps are used to remove misfolded proteins, remaining HCP, DNA, LMW, HMWA, viral particles, and leached Protein A (Shukla et al., 2007;. In addition, to achieve enough viral inactivation, an acid hold step is introduced post protein A chromatography (Shukla and Aranha, 2015). ...
Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.
... Nevertheless, several chromatography steps are required after the capture step in order to remove host cell proteins (HCPs), DNA, and product-related impurities to reach desired CQAs [39]. A further important reason for subsequent chromatographic steps is the leaching of affinity ligands due to harsh elution conditions [40,41]. The different chromatographic methods for capture and purification/polishing are listed in Table 1, with a focus on the methods used for mAbs and antibody fragments. ...
... Although affinity chromatography is widely used as an initial step, it is expensive and requires harsh elution conditions (pH~3), leading to decreased column stability, ligand leaching, and possible activity loss of the target product [52,55,56]. However, the acidic conditions are an advantage for the required viral inactivation in mammalian production processes [41]. Other chromatographic methods, such as ion exchange (IEX), are mainly used as additional purification steps to remove leached affinity ligands, HCPs, and DNA [41]. ...
... However, the acidic conditions are an advantage for the required viral inactivation in mammalian production processes [41]. Other chromatographic methods, such as ion exchange (IEX), are mainly used as additional purification steps to remove leached affinity ligands, HCPs, and DNA [41]. These purification and polishing steps can include up to three or four different chromatographic steps to achieve the required product quality ( Figure 3) [24]. ...
In the past 30 years, highly specific drugs, known as antibodies, have conquered the biopharmaceutical market. In addition to monoclonal antibodies (mAbs), antibody fragments are successfully applied. However, recombinant production faces challenges. Process analytical tools for monitoring and controlling production processes are scarce and time-intensive. In the downstream process (DSP), affinity ligands are established as the primary and most important step, while the application of other methods is challenging. The use of these affinity ligands as monitoring tools would enable a platform technology to monitor process steps in the USP and DSP. In this review, we highlight the current applications of affinity ligands (proteins A, G, and L) and discuss further applications as process analytical tools.
... cancer, inflammation and autoimmune disorders (Kaplon and Reichert 2019). Therapies benefit from their high target specificity and affinity in vivo which reduces the risk of side effects compared to their small molecule counterparts (Shukla et al. 2007;Gaughan 2016;Leader, Baca, and Golan 2008). ...
... undesired product variants, aggregates, fragments, and process derived impurities such as host cell proteins (HCP), DNA, endotoxins, leached Protein A as well as process buffer and media supplements to acceptable levels (Shukla et al. 2007). ...
... First and foremost, bioprocesses are developed to provide safe, stable and efficacious high-quality products that satisfy regulatory requirements. Whilst patient safety is the primary concern, the cost of manufacturing is becoming an increasingly important focus of process development activities, bringing other criteria into play to deliver processes that are commercially viable, such as speed of development and time needed before a candidate can enter clinical trials, productivity of the process, scalability and robustness (Łącki, Joseph, and Eriksson 2018;Shukla et al. 2007). There are many potential obstacles the product and process need to surmount along their way. ...
A key challenge for early stage bioprocess development is the need to deliver effective operating conditions against aggressive timelines and stringent material constraints. For antibodies, the largest class of biopharmaceuticals to date, chromatography constitutes a key tool for product purification. The development of these steps typically involves multi-variate and multi-objective optimisation problems with various constraints and often complex trade-offs. This research investigates data-driven experimental optimisation strategies based on the Nelder-Mead simplex algorithm, and other more traditional approaches using DoE and fundamental chromatography understanding. Through three case studies, the polishing chromatography process was developed for a series of industrially-relevant binary and ternary mAb feeds. Bind-and-elute cation-exchange chromatography operations were optimised with regards to the loading and elution conditions, employing step and linear gradients. Experiments were conducted using conventional laboratory-scale chromatography. In the third case study, these were compared head-to-head with micro-scale chromatography methods, on a Tecan Fluent liquid handling station. Process insight was generated, and trends visualised and interpreted for key performance outputs such as product mass, recovery and productivity against pre-defined requirements for product purity. These trends were shown to be reproducible at micro-scale with comparable chromatographic resolution, employing residence time scaling. The proposed experimental optimisation strategies were shown to effectively deal with the high variability typical of antibodies, revealing interesting trade-offs for high loading conditions and in the vicinity of product breakthrough, leading to high-performing counter-intuitive operating regions. In these practical scenarios, the simplex was demonstrated to be an easy-to-use and dynamic tool that guides development efforts towards feasible and promising operating regions whilst also being interactive by accepting information from different sources to maximise the value gained from the available experimental data. Hence, this work has demonstrated how state-of-the-art experimental optimisation methods can be deployed for rapid and rational process development, enabling the identification of high-performing operating conditions that may not necessarily align with conventional assumptions and traditional expectations.
... Low pH viral inactivation in mAb manufacturing process is typically performed after Protein A capture chromatography. Since, protein elutes at a lower pH from the Protein A chromatography, it is convenient to follow it up with viral inactivation [10]. This low pH incubation step is targeted at inactivation of enveloped C-type retroviruses [8] (like Murine Leukemia Virus and Lentivirus). ...
... This low pH incubation step is targeted at inactivation of enveloped C-type retroviruses [8] (like Murine Leukemia Virus and Lentivirus). Viral inactivation is typically performed at a pH < 4, by incubating the sample at this pH value for 30− 120 min [10][11][12]. Selection of the inactivation condition depends on the viral log reduction kinetics and the stability profile of the mAb molecule under consideration [8]. ...
A novel coiled flow inversion reactor (CFIR) has recently been utilized for continuously carrying out various downstream unit operations that require reaction and mixing like refolding, PEGylation and precipitation. In this study, we demonstrate utility of the CFIR for continuous virus inactivation. Low pH virus inactivation for a mAb therapeutic was performed in continuous mode using this reactor and its performance was compared to a parallel batch setup. It was found that both batch and continuous processes yield about equal level of virus inactivation with comparable logarithmic reduction value (LRV) for XMuLV of 4.32 ± 0.26 in batch versus 4.12 ± 0.08 in continuous over a period of 55 minutes. The data clearly demonstrates that the reactor configuration can be used in a continuous train for virus inactivation without any reduction in inactivation efficiency or inactivation kinetics. Integration of the reactor with the continuous downstream train has also been evaluated by highlighting different possible configurations. Two different case studies depicting the integration of the reactor to a continuous Protein-A capture chromatography under different set of operational conditions have been discussed. In all cases, consistent process performance and product quality attributes were obtained. The proposed reactor offers a suitable configuration for continuous viral inactivation for mAb continuous processing platforms.
... The resins can be costly (up to $20,000 per liter), they require special techniques for packing, they require cleaning and validation after use, and the pressure drop in the column limits the residence time and productivity of the process. Due to this, the column chromatography step, on its own, contributes a significant portion of overall manufacturing costs [10,12]. ...
... For these reasons, membrane chromatography has become a promising alternative to columns due to the very low membrane production costs, and their potential to serve as single-use devices [12,13]. Membrane ion-exchange chromatography promises several advantages over column chromatography, including low operating pressure and fast flow rates, ease-of-use, and elimination of cleaning and validation costs in single use applications [14][15][16]. ...
This study presents the preparation and characterization of UV-grafted polybutylene terepthalate (PBT) ion exchange nonwoven membranes for chromatographic purification of biomolecules. The PBT nonwoven was functionalized with sulfonate and secondary amine for cation and anion exchange (CEX and AEX), respectively. The anion exchange membrane showed an equilibrium static binding capacity of 1300 mg BSA/g of membrane, while the cationic membranes achieved a maximum equilibrium binding capacity of over 700 mg hIgG/g of membrane. The CEX and AEX membranes resulted in dynamic binding capacities under flow conditions, with a residence time of 0.1 min, of 200 mg hIgG/mL of membrane and 55 mg BSA/mL of membrane, respectively. The selectivity of the PBT-CEX membranes was demonstrated by purifying antibodies and antibody fragments (hIgG and scFv) from CHO cell culture supernatants in a bind-an-elute mode. The purity of the eluted samples exceeded 97%, with good log removal values (LRV) for both host cell proteins (HCPs) and DNA. The PBT-AEX nonwoven membranes exhibited a DNA LRV of 2.6 from hIgG solutions in a flow-through mode with little loss of product. These results indicate that these membranes have significant potential for use in downstream purification of biologics.
... Of the overall cost of mAb downstream processing, chromatographic separations account for 64.1% of overall processing time and 55.8% of the overall cost (Farid, 2007). Fig. 1 shows the classical downstream processing outline for the mAb therapeutics (Shukla et al., 2007). Protein A chromatography is ubiquitously used as the capture step, followed by other chromatographic steps like ion-exchange and hydrophobic interaction for removal of the various product-related impurities (Ng et al., 2012). ...
... Dissociation of IgG from the SPA-IgG complex is achieved by lowering the pH to an acidic environment (pH < 4.00). The acidic elution pH is responsible for the formation of soluble aggregates in the mAb product, which exhibit reduced biological activity and at times invoke an immunogenic response in patients (Shukla et al., 2005(Shukla et al., , 2007Wu et al., 2014). Acidic elution pH in Protein A chromatography can also lead to the formation of insoluble aggregates due to precipitation of the various host cell proteins. ...
Protein A chromatography is one of the most widely used purification steps in the manufacturing of the various classes of recombinant and non-recombinant antibodies. Due to the higher cost, lower binding capacity, and limited life cycle of Protein A ligand, this affinity-based purification step is often one of the most significant contributors to the cost of manufacturing of monoclonal antibody (mAb) products. In the last decade, there has been significant progress in improving the Protein A chromatography throughput by designing new engineered Staphylococcal Protein A (SPA) variants with higher dynamic binding capacity, considerable alkaline tolerance, and mild acidic elution pH. This review aims at summarizing the various protein engineering approaches used for improving the throughput of the Protein A-based affinity purification of various immunoglobulins. With biopharmaceutical producers operating under ever-increasing pressure towards reducing the cost of manufacturing, these advances in engineered protein A variants will help in processing larger cell culture volumes with high throughput and thereby significantly lower the cost of raw materials.
... 8 A key unit operation for the purification of antibodies is staphylococcal Protein A affinity. 9 Rendering batch chromatography into a continuous result is a semicontinuous process where the product is eluted in a cyclic fashion. Countercurrent loading and twincolumn simulated moving beds (SMB) are the most popular ways of achieving continuous protein A chromatography. ...
BACKGROUND
A major improvement in biomanufacturing will arise with the transition from batch processing to continuous processing. Two important challenges to address in this change are batch definition and the ability to trace raw material through the process.
RESULTS
We used an established simulation of a process train to compare the conventional batch definition based on a fixed time to a new batch definition method based on the greatest common divisor (GCD) of the time period of the unit operations. We successfully demonstrated that, by using the new concept based on GCD, we will have a constant periodic concentration of product. With this basis, we can define batches in a continuous process, which will lead to higher control over the process, and we will be able to trace the material through the process.
CONCLUSION
We achieved better control over the process using the batch definition based on the GCD method. In comparison to collecting the outlet products over arbitrary hours or days, collecting the product based on a section using the GCD method meets the criteria for knowing the residence-time distribution of the process, as advised by regulatory authorities. This method can be used in a continuous process or a hybrid process in which there are only a few continuous unit operations along with batch process operations.
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... While it is difficult to adapt a truly pre-defined process for different individual mAb purifications, the platform development approaches based on common unit operations have been proposed by many biopharmaceutical companies (Kelley et al., 2009;Shukla et al., 2007). Since disulfide bond reduction mainly happens at the beginning of the purification process, the cell culture harvest and Protein A chromatography could be the main unit operations to align the strategy for disulfide bond reduction elimination and recovery approaches. ...
Antibody disulfide bond reduction has been a challenging issue in monoclonal antibody manufacturing. It could lead to decrease of product purity and failure to meet targeted product profile and/or specifications. More importantly, disulfide bond reduction could also impact drug safety and efficacy. Scientists across industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategies and developing novel approaches to maintain high product quality. Additionally, in recent years as more complex molecules (such as bispecific and trispecific antibodies) emerge, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative challenging issue. Given the disulfide reduction challenges that biotech industry is facing, in this review, we provide a comprehensive scientific summary of the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and its potential remediated recovery based on published papers. First, this paper intends to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discusses disulfide bond reduction impact on product stability, associated analytical methods for disulfide bond reduction detection and characterization, process control strategies as well as their manufacturing implementation. In addition, brief perspectives on the development of future mitigation strategies are also reviewed, including platform alignment, mitigation strategy application for the emerging new modalities such as bispecific and trispecific antibodies as well as using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from the published papers. This article is protected by copyright. All rights reserved.
... While it is difficult to adapt a truly pre-defined process for different individual mAb purification, the platform development approaches based on common unit operations have been proposed by many biopharmaceutical companies (Kelley, Blank, & Lee, 2009;Shukla, Hubbard, Tressel, Guhan, & Low, 2007). Since disulfide bond reduction mainly happens at the beginning of the purification process, the cell culture harvest and Protein A chromatography could be the main unit operations to align the strategy for disulfide bond reduction elimination and recovery approaches. ...
Disulfide bond reduction has been a challenging issue in antibody manufacturing, as it leads to reduced product purity, failed product specifications and more importantly, impacting drug safety and efficacy. Scientists across industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high-titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategy and development of novel approaches to achieve high product quality. Additionally, in recent years as more complex molecules emerge such as bispecific and trispecific antibodies, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative issue. Given the disulfide reduction challenges that our industry are facing, in this review, we provide a comprehensive contemporary scientific insight into the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and recovery based on our expertise in commercial and clinical manufacturing of biologics. First, this paper intended to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discussed disulfide bond reduction impact to product stability and process performance, analytical methods for detection and characterization, process control strategies and their manufacturing implementation. In addition, brief perspectives on development of future mitigation strategies will also be reviewed, including platform alignment, mitigation strategy application for bi- and tri-specific antibodies and using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from both the published papers and our internal development work.
... Due to the structural similarities between mAb therapeutics, this platform process has been widely adopted and currently considered industry standard. 36,37 The same platform process for mAb therapeutics was adopted in the study so as to make the findings applicable and relatable to global mAb manufacturing practices. The process for mAb manufacturing for the study included the following steps-cell culture, centrifugation, depth filtration, chromatography, and final ultrafiltration-diafiltration. ...
Continuous processing offers a promising approach to revolutionize biotherapeutics manufacturing as reflected in recent years. The current study offers a comparative economic assessment of batch and continuous processing for the production of biotherapeutic products. Granulocyte-colony stimulating factor (GCSF), a protein expressed in E. coli, and an IgG1 monoclonal antibody, were chosen as representatives of microbial and mammalian derived products for this assessment. Economic indicators-cost of goods (COGs), net present value (NPV), and payback time have been estimated for the assessment. For the case of GCSF, conversion from batch to integrated continuous manufacturing induced a $COGs/g reduction of 83% and 73% at clinical and commercial scales, respectively. For the case of mAb therapeutic, a 68% and 35% reduction in $COGs/g on translation from batch to continuous process was projected for clinical and commercial scales, respectively. Upstream mAb titer was also found to have a significant impact on the process economics. With increasing mAb titer, the $COG/g decreases in both operating modes. With titer increasing from 2 to 8 g/L, the $COG/g of batch process was reduced by 53%, and that of the continuous process was reduced by 63%. Cost savings in both the cases were attributed to increased productivity, efficient equipment and facility utilization, smaller facility footprint, and reduction in utilization of consumables like resin media and buffers actualized by the continuous processing platform. The current study quantifies the economic benefits associated with continuous processing and highlights its potential in reducing the manufacturing cost of biotherapeutics.
... DoEs are widely applied in mAb downstream process development. [33][34][35] Generally, their designs take into account the factors number and type, existing information, and reliability of the results to design the experiments. Thus, several factors can be changed in one set of the experiments, and the influences of these factors could be evaluated by a small number of experiments. ...
Disulfide bonds play a crucial role in folding and structural stabilization of monoclonal antibodies (mAbs). Disulfide bond reduction may happen during the mAb manufacturing process, resulting in low molecular weight species and possible failure to meet product specifications. Although many mitigation strategies have been developed to prevent disulfide reduction, to the best of our knowledge, reforming disulfide bonds from the reduced antibody in manufacturing has not previously been reported. Here, we explored
a novel rescue strategy in the downstream process to repair the broken disulfide bonds via in-vitro redox reactions on Protein A resin. Redox conditions including redox pair (cysteine/cystine ratio), pH, temperature, and reaction time were examined to achieve high antibody purity and a high reaction rate. Under the optimal redox condition, >90% reduced antibody could be reoxidized to form an intact antibody on Protein A resin in an hour. In addition, this study showed high flexibility on the range of the intact mAb fraction in the initial reduced mAb sample (the lower limit of intact mAb faction could be 14% based on the data reported in this study). Furthermore, a kinetic model based on elementary oxidative reactions was constructed to help optimize the reoxidation conditions and to predict product purity. Together, the deep understanding of interchain disulfide bond reoxidation, combined with the predictive kinetic model, provided a good foundation to implement a rescue strategy to generate high-purity antibodies with
substantial cost savings in manufacturing processes.
... Thus, one set of the experiments can change several factors to evaluate their influences efficiently. [32][33][34] As illustrated in Table 2, antibody purities of 69% -97% were achieved in the studied ranges of Protein A loading amount 10-50 g/L resin, LMW level of mAb load 10-90%, and mAb/redox contact time 15-60 minutes. Statistical analysis ( Figure 3) showed that the resultant mAb purity was primarily affected by load LMW level (p = .004) ...
Disulfide bond reduction, which commonly occurs during monoclonal antibody (mAb) manufacturing processes, can result in a drug substance with high levels of low molecular weight (LMW) species that may fail release specifications because the drug's safety and the efficiency may be affected by the presence of this material. We previously studied disulfide reoxidation of mAbs and demonstrated that disulfide bonds could be reformed from the reduced antibody via redox reactions under an optimal redox condition on Protein A resin. The study here implements a redox system in a manufacturing setting to rescue the reduced mAb product and to further eliminate LMW issues in downstream processing. As such, we incorporate the optimized redox system as one of the wash buffers in Protein A chromatography to enable an on-column disulfide reoxidation to form intact antibody in vitro. Studies at laboratory scale (1 cm (ID) x 20 cm (Height), MabSelect SuRe LX) and pilot scale (30 cm (ID) x 20 cm (Height), MabSelect SuRe LX) were performed to demonstrate the effectiveness and robustness of disulfide formation with multiple mAbs using redox wash on Protein A columns. By applying this rescue strategy using ≤50 g/L-resin loading, the intact mAb purity was improved from <5% in the Protein A column load to >90% in the Protein A column elution with a product yield of >90%. Studies were also done to confirm that adding the redox wash has no negative impact on process yield or impurity removal or product quality. The rescued mAbs were confirmed to form complete interchain disulfide bonds, exhibiting comparable biophysical properties to the reference material. Furthermore, since the redox wash is followed by a bridging buffer wash before the final elution, no additional burden is involved in removing the redox components during the downstream steps. Due to its ease of implementation, significant product purity improvement, and minimal impact on other product quality attributes, we demonstrate that the on-column reoxidation using a redox system is a powerful, simple, and safe tool to recover reduced mAb during manufacturing. Moreover, the apparent benefits of using a high-pH redox wash may further drive the evolution of Protein A platform processes.
... The subsequent chromatographic steps in downstream processing, also referred as the polishing steps, entails anion exchange chromatography (AEX), cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC). The aim of these polishing steps is the reduction of process and product related impurities such as: protein A leachates, clips, high molecular weight aggregates and host cell proteases [36]. The use of inexpensive ion exchange resins such as CEX or AEX has demonstrated a protein purity of !99.5% and aggregate levels 5% [37]. ...
Monoclonal antibodies are proteinaceous in nature and are subject to instability issues. Stability testing of monoclonal antibodies is a critical regulatory requirement in their development and commercialization as therapeutic biological molecules. This article reviews the numerous drug manufacturing processes such as: upstream processing, downstream purification and aseptic filling along with physical and chemical factors such as protein concentration, structure, pH, temperature, light, agitation, deamidation, oxidation, glycation leading to instabilities in monoclonal antibodies and it spotlights the variety of analytical techniques employed to investigate and generate information on stability studies and henceforth, helps in developing the stability-indicating methods. In addition, this paper aims to discuss the ICH regulatory guideline (s) for the stability assessment of biological products (Drug Substance and Drug Product).
... been employed for purifying mAb [5][6][7]. In this protocol, protein A chromatography serves as core platform techniques for antibody purification and has become the golden standard in mAb production pipeline [8]. ...
The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high‐capacity polymer‐grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD‐grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD‐grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high‐capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300–350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD‐grafted protein A gels than non‐grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.
... Liu, Nguyen, Andya, & Shire, 2005;Luo et al., 2014;Vázquez-Rey & Lang, 2011) grow across cell culture, recovery, purification, and storage, their chromatographic separation from monomers is difficult due to their similar isoelectric points and hydrophobicities. Since mAb aggregates also contain the Fc domain and cannot be separated by protein A chromatography (Marichal-Gallardo & Álvarez, 2012;Shukla, Hubbard, Tressel, Guhan, & Low, 2007), they are often separated using cation-exchange (CEX) chromatography in bind-and-elute mode (Borg et al., 2014). Although more effectively achieved by protein A chromatography, reductions in the residual HCP after a CEX step are also routinely observed (Levy, Valente, Lee, & Lenhoff, 2016;Liu & Tang, 2008). ...
An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host-cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind-and-elute cation-exchange (CEX) chromatography polishing step. A variant of the bind-and-elute mode is the displacement mode, which allows strongly-binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this work we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002-1.4%) using different multi-column designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low-prevalence HCPs resulted in an effectively bimodal-like distribution of HCPs along the length of a multi-column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of low-prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography. This article is protected by copyright. All rights reserved.
... While the understanding of small protein binding in multimodal chromatographic systems is valuable, it is important to extend these studies to more complex and industrially relevant biomolecules. One such protein is the F C domain, which is highly conserved across a given class of mAbs and which plays an important role in a number of biotherapeutics such as bispecific antibodies and fusion proteins (Shukla, Hubbard, Tressel, Guhan, & Low, 2007). Robinson et al. has examined the domain contributions of mAb binding using a strategic set of chromatographic experiments and shown a shift in domain dominance with pH and resin type (Robinson, Roush, & Cramer, 2018, 2020. ...
... Furthermore, antibodies with undesired folding are not able to effectively engage their target antigen or to mediate effector functions, have unfavourable pharmacokinetics, and tend to aggregate. Besides these biological limitations, purification of antibody products contaminated with aggregated or misfolded mAbs is a major hindrance in the downstream processing of therapeutic molecules and is currently the topic of numerous studies [9][10][11][12]. ...
Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner.
... Preparative modes of chromatography involving Protein A affinity, anion and cation exchange chromatography are commonly used. 7,8 Substantial efforts, however, may be required to separate the product from product-related impurities, such as aggregates and fragments, which are monitored as high molecular weight (HMW) and low molecular weight (LMW) species during analytical size-exclusion chromatography (SEC) experiments. ...
The discovery of therapeutic monoclonal antibodies (mAbs) primarily focuses on their biological activity favoring the selection of highly potent drug candidates. These candidates, however, may have physical or chemical attributes that lead to unfavorable chemistry, manufacturing, and control (CMC) properties, such as low product titers, conformational and colloidal instabilities, or poor solubility, which can hamper or even prevent development and manufacturing. Hence, there is an urgent need to consider the developability of mAb candidates during lead identification and optimization. This work provides a comprehensive proof of concept study for the significantly improved developability of a mAb variant that was optimized with the help of sophisticated in silico tools relative to its difficult-to-develop parental counterpart. Interestingly, a single amino acid substitution in the variable domain of the light chain resulted in a three-fold increased product titer after stable expression in Chinese hamster ovary cells. Microscopic investigations revealed that wild type mAb-producing cells displayed potential antibody inclusions, while the in silico optimized variant-producing cells showed a rescued phenotype. Notably, the drug substance of the in silico optimized variant contained substantially reduced levels of aggregates and fragments after downstream process purification. Finally, formulation studies unraveled a significantly enhanced colloidal stability of the in silico optimized variant while its folding stability and potency were maintained. This study emphasizes that implementation of bioinformatics early in lead generation and optimization of biotherapeutics reduces failures during subsequent development activities and supports the reduction of project timelines and resources.
... Detecting mAb aggregation at varying pHs is important because a low pH can be required, such as during downstream processing of mAbs. 43,44 These tests were first performed with trastuzumab, mAb1, and mAb11, and the lowest level of aggregation was observed near neutral pH for all mAbs. Unexpectedly, mAb1 and mAb11 (both IgG1 subclass) were observed to have better stability at pH 4 compared to pH 5. ...
Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of protein-based drugs can endanger patient safety due, for example, to increased immunogenicity. Aggregation can also inactivate protein drugs and prevent target engagement, and thus regulatory requirements are strict regarding drug stability monitoring during manufacturing and storage. Many of the current technologies for aggregation monitoring are time- and material-consuming and require specific instruments and expertise. These types of assays are not only expensive, but also unsuitable for larger sample panels. Here we report a label-free time-resolved luminescence-based method using an external Eu³⁺-conjugated probe for the simple and fast detection of protein stability and aggregation. We focused on monitoring the properties of IgG, which is a common format for biological drugs. The Protein-Probe assay enables IgG aggregation detection with a simple single-well mix-and-measure assay performed at room temperature. Further information can be obtained in a thermal ramping, where IgG thermal stability is monitored. We showed that with the Protein-Probe, trastuzumab aggregation was detected already after 18 hours of storage at 60°C, 4 to 8 days earlier compared to SYPRO Orange- and UV250-based assays, respectively. The ultra-high sensitivity of less than 0.1% IgG aggregates enables the Protein-Probe to reduce assay time and material consumption compared to existing techniques.
In this paper, we determined the optimal flow rate trajectory during the loading phase of a mAb capture column. For this purpose, a multi-objective function was used, consisting of productivity and resin utilization. Several general types of trajectories were considered, and the optimal Pareto points were obtained for all of them. In particular, the presented trajectories include a constant-flow loading process as a nominal approach, a stepwise trajectory, and a linear trajectory. Selected trajectories were then applied in experiments with the state-of-the-art protein A resin mAb Select PrismATM, running in batch mode on a standard single-column chromatography setup, and using both a purified mAb solution as well as a clarified supernatant. The results show that this simple approach, programming the volumetric flow rate according to either of the explored strategies, can improve the process economics by increasing productivity by up to 12% and resin utilization by up to 9% compared to a constant-flow process, while obtaining a yield higher than 99%. The productivity values were similar to the ones obtained in a multi-column continuous process, and ranged from 0.23 to 0.35 mg/min/mL resin. Additionally, it is shown that a model calibration carried out at constant flow can be applied in the simulation and optimization of flow trajectories. The selected processes were scaled up to pilot scale and simulated to prove that even higher productivity and resin utilization can be achieved at larger scales, and therefore confirm that the trajectories are generalizable across process scales for this resin.
Monoclonal antibodies are key molecules in medicine and pharmaceuticals. A potentially crucial drawback for faster advances in research here is their high price due to the extremely expensive antibody purification process, particularly the affinity capture step. Affinity chromatography materials have to demonstrate high binding capacity and recovery efficiency as well as superior chemical and mechanical stability. Low-cost materials and robust, faster processes would reduce costs and enhance industrial immunoglobulin purification. Therefore, exploring the use of alternative materials is necessary. In this context, we conduct the first comparison of the performance of magnetic nanoparticles with commercially available chromatography resins and magnetic microparticles with regard to immobilizing Protein G ligands and to recovering Immunoglobulin G (IgG). Simultaneously, we demonstrate the suitability of bare as well as silica-coated and epoxy-functionalized magnetite nanoparticles for that purpose. All materials applied have a similar specific surface area but differ in the nature of their matrix and surface accessibility. The nanoparticles are present as micrometer agglomerates in solution. The highest Protein G density can be observed on the nanoparticles. IgG adsorbs as a multilayer on all materials investigated. However, the recovery of IgG after washing indicates a remaining monolayer which points to the specificity of the IgG binding to the immobilized Protein G. One important finding is the impact of the ligand binding stoichiometry (Protein G surface coverage) on IgG recovery, reusability and the ability to withstand long-term sanitization. Differences in the materials’ performances are attributed to mass transfer limitations and steric hindrance. These results demonstrate that nanoparticles represent a promising material for the economical and efficient immobilization of proteins and the affinity purification of antibodies, promoting innovation in downstream processing.
In this study, the binding of multimodal chromatographic ligands to the IgG1 FC domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15 N-labeled FC domain indicated that while single-mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the FC . The multimodal ligand-binding sites on the FC were concentrated in the hinge region and near the interface of the CH 2 and CH 3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on FC corroborated molecular-level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand-FC binding in these preferred regions was shown to be electrostatic interactions and π-π stacking of surface-exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular-level understanding of multimodal ligand-FC interactions and sets the stage for future analyses of even more complex biotherapeutics.
Recent studies have shown that by combining orthogonal, non-affinity chromatography steps, it is possible to rapidly develop efficient purification processes for molecules of interest. Here, we build upon previous work to develop a flexible framework for identifying resins that remove optimally orthogonal sets of impurities for a wide variety of products. Our approach involves screening a library of proteins with diverse properties (pI ranging from 5.0-11.4 and varying hydrophobicity measured by retention in a HIC gradient) on a library of resins and quantifying each resin’s ability to separate every set of protein pairs in the library. Orthogonality is then defined as the degree to which two resins separate mutually exclusive sets of protein pairs. We applied this approach to a library of model proteins and a series of strong, salt tolerant, and multimodal ion exchangers and evaluated which resin combinations performed well and which performed poorly. In particular, we found that strong cation and strong anion exchangers were orthogonal, while strong and salt tolerant anion exchangers were not orthogonal. Interestingly, salt tolerant and multimodal cation exchangers were found to be orthogonal and the best resin combination included a multimodal cation exchange resin and a tentacular anion exchange resin. This approach for quantifying orthogonality is valuable in that it can be used both as a criteria for resin design as well as process design. We envision that, using this framework, it will be possible to design a set of next generation chromatography ligands that are explicitly engineered to optimize separability and orthogonality.
Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a non‐homogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in‐line spiking. This is challenging to be implemented at the CRO performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow through anion exchange chromatography step in a monoclonal antibody manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAb were introduced in concentrated peaks, or as a homogeneous batch, the clearance of MMV was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of AEX unit operating in continuous processing.
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The potential of continuous bioprocessing is hindered by the bottlenecks of chromatography processing, which continues to be executed in batch mode. Highlighting the critical drawbacks of batch chromatography, this review underscores the transition that the industry has made by implementing continuous upstream process without devising a working model for downstream chromatography operations. Even though multitude of process development initiatives have commenced, the review emphasizes the first principle models of chromatography on which these initiatives are built. Various models of continuous chromatography, which are essential, but not limited to multi-column systems, employed to congeal a unified process are reviewed. Advancements made by several mechanistic models and simulations to maximize productivity and performance are described, in an attempt to provide the integral tools. The modeling tools can be used for development of a strong model based control strategy and can be embedded into the continuous chromatography framework. The review addresses the limitations and challenges of the current modeling methods for development of robust mechanistic modeling and efficient unit operation platform in continuous chromatography.
An alternative downstream process for the purification of a monoclonal antibody (mAb) based on aqueous two-phase extraction as clarification, capture and primary purification step was developed. For further purification unit operations which are commonly used for mAb platform processes were utilized. A diafiltration approach was used to combine virus inactivation and removal of phase forming components as well as low molecular weight impurities in one step followed by cation and anion exchange chromatography. Starting from cell containing cultivation broth, an overall mAb yield of 74 % was achieved within an application study. The process was optimized regarding mAb yield and the clearance of process related impurities like deoxyribonucleic acid as well as host cell proteins, which were removed to approximately 60 and 6000 ppm respectively. Critical product quality attributes, regarding glycosylation patterns, were also examined and remained unimpaired after the aqueous two-phase extraction. The alternative downstream process presented in this study offers great potential to improve mAb manufacturing.
A mechanistic model for describing unfolding of a monoclonal antibody (mAb) in ion exchange chromatography has been developed. The model reproduced retention behavior characteristic for conformational changes of antibodies upon adsorption, including: multi-peak elution, aggregate formation, and recovery reduction. Two competitive paths in the adsorption mechanism of the unfolded protein were assumed: refolding in the adsorbed phase to the native form followed by its desorption, or direct desorption followed by instantaneous aggregation in the liquid phase. The reduction in recovery of the eluted protein was attributed to spreading of the unfolded protein on the adsorbent surface, which enhanced the binding affinity.
The model was formulated based on the analysis of retention behavior of a model mAb that was eluted in pH gradients on a strong cation exchange resin.
The pH profile was found to be distorted in the presence of the protein, which was ascribed to dissociation of ionizable groups of the protein in the adsorbed phase. Since the protein retention was strongly pH dependent, that phenomenon was also accounted for in mathematical modeling. A series of independent experiments was designed to evaluate the model parameters that quantified the process thermodynamics and kinetics: the Henry constants of the native, unfolded, spread and aggregated forms of the protein along with underlying kinetic coefficients. The model was efficient in reproducing the retention pattern of the protein and the aggregate content in eluting band profiles. After proper calibration, the model can potentially be used to quantify protein unfolding and elution in other ion exchange systems.
Intensified high cell density (HCD) processes for the production of therapeutic proteins have already reached cell concentrations up to 100 million cells/mL (> 300 g/L wet cell weight, WCW). The clarification of such HCD processes using technologies established for conventional fed-batch processes (< 100 g/L WCW) is faced with low biomass loading capacities and relatively low product recoveries, which limit their applicability. This and the growing demand for single-use (SU) technologies increases the pressure to develop scalable, robust and cost-effective SU based HCD clarification solutions.
Therefore, in this study a SU fluidized bed centrifuge (FBC) system was investigated to clarify HCD broths with 100 million cells/mL from a monoclonal antibody production process. FBC parameters including process volumes and flow rates during the FBC operating steps were optimized so that an almost complete removal of biomass and product recoveries of 95 % were achieved. In addition, the mild centrifugation conditions were demonstrated by the superior cell viability for the separated cells while minimizing release of host cell impurities. Furthermore, a post-centrifugal filter screening showed acceptable turbidities below 5 NTU of the sterile filtrate. This concept of FBC with subsequent filtration offers excellent potential to achieve a robust HCD clarification process solution.
A fast method for assessing the stability of monoclonal antibodies (mAbs) adsorbed on ion exchange resins has been developed. The method exploited a real time polymerase chain reaction equipment to determine the temperature of protein phase transition, i.e., the so called melting temperature, based on differential scanning fluorimetry.
Changes to the melting temperature were screened under various adsorption conditions and correlated with the protein stability upon adsorption.
The method was tested for two different mAbs bound to various types of strong cation exchangers at different pH and loading concentrations. The mAbs destabilized upon adsorption due to strong binding, which manifested itself in aggregate formation and recovery reduction. The phenomenon depended on the resin type and binding conditions. However, regardless of the process conditions and resins used, drop in the melting temperatures to a critical value of about 30° could serve as an indicator of destructive changes in the protein structure in the adsorbed phase. The measurements were simultaneously accomplished for a number of samples with very small material consumption. Therefore, the method may be applied for screening resins and operating variables for a given mAb to exclude conditions that induce structure destabilization and aggregation.
The application of recombinant DNA technology to the production of protein therapeutics has undergone tremendous progress over the past decades. Considerable scientific effort has been devoted to developing robust processes that produce large amounts of complex proteins with the desired product quality attributes. An overview of the contributions from the four major disciplines working in concert to deliver these processes and methods will be covered. This article will discuss some of the tools, methods, and approaches used to produce, purify, formulate, and analyze the drugs of modern biotechnology. Future trends in each of the disciplines will also be presented.
Over the past 30 years, a multitude of separations science–based methods for analysis of proteins, and particularly antibodies, have been developed and standardized within the biotechnology industry. In addition to the profound improvements in instrumentation and associated consumables such as columns and chemical reagents that have supported these developments, a framework for characterizing and defining the criticality of the species separated and quantified by these methods has been developed as part of the concept of quality by design. This chapter provides an overview of the chief methods used for separations-based testing of monoclonal antibodies and the quality and control system frameworks within which these technologies and methodologies are utilized.
Cation exchange chromatography (CEX) is a widely used technique for the removal of monoclonal antibody (mAb) aggregates. At present, resins are mainly used for this purpose, as convective types of adsorbents such as membrane adsorbers (MAs) have often not demonstrated overall comparable performance for this particular application. Fiber-based adsorbents can overcome the current limitations of MAs with respect to permeability, binding capacity, and adsorbent cost, and could therefore be a viable alternative to resins for the removal of mAb aggregates. It has not been evaluated, however, whether and under which conditions the use of such adsorbents is feasible for this purpose.
In the present study, the use of fiber-based CEX adsorbents for mAb aggregate removal was examined. Two types of fiber-based adsorbents, an uncontrolled grafted and a controlled grafted fiber-based adsorbent, were evaluated with respect to permeability, dynamic mAb binding capacity (DBC), resolution capabilities, and the performance in bind and elute (B/E) and frontal chromatography mode with respect to typical performance indicators. The permeabilities of the fiber-based adsorbents ranged from 200-1700 mD, making it possible to use the fiber-based adsorbents at larger bed heights than membrane adsorbers with fast mobile phase velocities. Antibody DBCs ranged from 20-41 g/L at 150 cm/h, and at higher mobile phase velocities exceeded the DBC of an existing resin material, Poros 50 HS, which has frequently been used for aggregate removal. Both fiber types showed good resolution capabilities of monomer and aggregates, and provided better resolution per column length than Poros 50 HS. Typical purity and yield constraints were fulfilled for both fiber types in both B/E and frontal chromatography mode for mobile phase velocities ranging up to 480 cm/h and 1060 cm/h. The overall performance of the controlled grafted fibers was found to be superior to the performance of uncontrolled grafted fiber-based adsorbents due to higher productivity and lower buffer consumption. The overall performance of the fiber-based adsorbents was found to be comparable to the performance of Poros 50 HS at typical operating conditions.
The results in this study indicate that the use of fiber-based adsorbents for mAb aggregate removal is feasible with a performance that is comparable to the performance of an existing resin material. Depending on the cost of the adsorbents and the use scenario, the usage of such adsorbents could be beneficial.
High throughput screening (HTS) approaches are commonly used to accelerate downstream process development. While most HTS approaches use batch isothermal data (KP screen) or bind and elute mode as screening procedure, different or new process designs are rarely investigated. In this paper a mechanistic model case study for the separation of two different two‐component solutions was conducted and confirmed prior evidence. With these outcomes, a novel HTS screening procedure was developed including the determination of competitive adsorption‐based displacement effects and key parameter identification. The screening procedure employing an overload bind and elute mode (OBE) is presented in a case study dealing with IgG aggregate removal in a typical monoclonal antibody purification step, applying a Sartobind® S membrane adsorber (MA). Based on a MA scale down device, the OBE mode allows the determination of classical process parameters and dynamic effects, such as displacement effects. Competitive adsorption‐based displacement effects are visualized by introducing a displacement identifier (DI) leading to a displacement process map. Based on this map, the approach is transferred to and confirmed by the OBE recycle experiments with 4.6 mL and 8.2 mL benchtop scale devices resulting in 45 % reduced IgG monomer and 88 % increased HMWS binding capacities. This article is protected by copyright. All rights reserved
The purification of monoclonal antibodies and Fc fusion proteins consist of several unit operations operated commonly as a platform approach, starting with Protein A chromatography. The first capture step, the following low pH virus inactivation, and subsequent ion exchange chromatography steps are mostly able to remove any impurities, like host cell proteins, aggregates, and viruses. The changes in pH and conductivity during these steps can lead to additional unwanted product species like aggregates. In this study, excipients with stabilizing abilities, like polyols, were used as buffer system additives to study their impact on several aspects during Protein A chromatography, low pH virus inactivation, and cation exchange chromatography. The results show that excipients, like PEG4000, influence antibody elution behavior, as well as host-cell protein elution behavior in a pH-gradient setup. Sugar excipients, like Sucrose, stabilize the antibody during low pH virus inactivation. All excipients tested show no negative impact on virus inactivation and dynamic binding capacity in a subsequent cation exchange chromatography step. This study indicates that excipients and, possibly excipient combinations, can have a beneficial effect on purification without harming subsequent downstream processing steps.
Batch low-pH hold is a common processing step to inactivate enveloped viruses for biologics derived from mammalian sources. Increased interest in the transition of biopharmaceutical manufacturing from batch to continuous operation resulted in numerous attempts to adapt batch low-pH hold to continuous processing. However, control challenges with operating this system have not been directly addressed. This article describes a low-cost, column-based continuous viral inactivation system constructed with off-the-shelf components. Model-based, reaction-invariant pH controller is implemented to account for the nonlinearities with Bayesian estimation addressing variations in the operation. The residence time distribution is modeled as a plug flow reactor with axial dispersion in series with a continuously stirred tank reactor, and is periodically estimated during operation through inverse tracer experiments. The estimated residence time distribution quantifies the minimum residence time, which is used to adjust feed flow rates. Controller validation experiments demonstrate that pH and minimum residence time setpoint tracking and disturbance rejection are achieved with fast and accurate response and no instability. Viral inactivation testing demonstrates tight control of logarithmic reduction values over extended operation. This work provides tools for the design and operation of continuous viral inactivation systems in service of increasing productivity, improving product quality, and enhancing patient safety.
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Although the field of antibody drugs has grown larger, the antibody production still faces several challenges. Effective antibody purification is required, but the conventional purification method for antibodies is cost intensive and often causes aggregation problems, indicating the need for new alternative antibody purification methods. In the present study, a constant temperature antibody purification system for use with a thermo-responsive polymer column was developed based on switching of anion species in eluents. By adjusting the temperature for each antibody, the developed column enabled separation of the therapeutic monoclonal antibodies, rituximab and trastuzumab, from contaminants without changing salt concentration or pH of the eluents. The thermo-responsive hydrogel-modified column packing material was synthesized by introducing n-butyl methacrylate, acrylic acid, N,N’-methylenebisacrylamide and N-isopropylacrylamide to the surface of silica beads with an initiator by a graft-from approach. Elution behavior of antibodies with three types of anions, such as citrate, phosphate, and chloride were tested under three different temperature conditions. It was demonstrated that the thermo-responsive hydrogel grafted column showed a switchable antibody retention behavior at constant temperature and salt concentration, with antibody adsorption by NaCl eluent and desorption by citric acid buffer eluent.
Process validation is a key component of the licensing process for a recombinant protein. It represents the collection and evaluation of process knowledge that was obtained during process development and scale-up. This process knowledge is accumulated throughout the product life cycle and must be appropriately documented and summarized. It uses a risk- and science-based approach to evaluate parameters. The culmination of process validation activities occurs during execution of qualification lots that represent the integration of the production process with other validated systems and processes at the commercial facility. Process validation provides documented evidence that the process can be run consistently to deliver the desired product quality attributes. It should not be thought of as a single event that occurs late in the development cycle but as a series of activities that will continue throughout the product life cycle. Process verification after approval serves as a vital link to demonstrate that the validated process remains in a state of control.
As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical parameters that has become clear in the field is the importance of mAb 'developability'. Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes.
In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale‐dependent fashion using a standard platform purification process is described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesised to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to germline of these residues was carried out. The resulting mutants have improved expression titres and recovered stability within a forced precipitation assay, without any change to biological activity.
Given the time pressures of drug development in industry, process optimisation of the lead molecule was carried out in parallel to the 'retrospective' mutagenesis approach. Bespoke process optimisation for large‐scale manufacture was successful. However, we propose that such context‐dependent sequence liabilities should be included in the arsenal of in‐silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.
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Protein A chromatography is a key technology in the industrial production of antibodies, and a variety of commercial protein A adsorbents are available in shelf. High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography. Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols (e.g. washing with sodium hydroxide solution), and suffered from low binding capacity, harsh elution, ligand leakage and other problems involved in industrial applications. During the last three decades, various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications. This mini-review focuses on the technical explorations in protein A-based affinity adsorbents, especially protein A-based ligands, including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization. Moreover, the efforts to develop short peptide ligands based on the structure of protein A, including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed. These peptide and peptide-based hybrid ligands exhibit high affinity and selectivity to antibodies, but noteworthy differences in the binding mechanism of antibody from protein A. As a result, bound antibody to the ligands could be effectively eluted under mild conditions. Perspectives for the development of the protein A-based peptide ligands have been extensively discussed, suggesting that the ligands represent a direction for technological development of antibody purification.
A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early‐phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time‐to‐market pressure and an abundance of mAbs in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform.
This study investigates the influence of amino acid substitutions in the complementarity‐determining region (CDR) of a full‐length IgG1 mAb on the elution behavior in preparative cation exchange (CEX) chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared to the wild type antibody. Further, the substitution of tryptophan with lysine in the H‐CDR3 increased charge heterogeneity of the product. A multi‐scale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing.
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An increasing number of non‐mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log10 reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.
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 (37–2745 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 20 N 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 1000–5000 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).
Few monoclonal antibodies are currently approved for treating infectious diseases, but multiple products are in development against a broad range of infectious diseases, including Ebola, influenza, hepatitis B, HIV, dengue, and COVID‐19. The maturity of mAb technologies now allow us to identify and advance neutralizing mAb products to the clinic at “pandemic pace”, as the pipeline of mAbs targeting SARS‐CoV‐2 has demonstrated. Ensuring global access to these products for passive immunization, however, will require both low manufacturing cost and multi‐ton production capacity – particularly for those infectious diseases where the geographic burden falls mostly in low‐ and middle‐income countries or those with pandemic potential. Analysis of process economics and manufacturing technologies for antibody and other parenteral protein therapeutics demonstrates the importance of economies of scale to reducing the cost of goods for drug substance manufacturing. There are major benefits to convergence on a standardized platform process for antibody production that is portable to most existing very large‐scale facilities, carries low risk for complications during process transfer and scale‐up, and has a predictable timeline and probability of technical and regulatory success. In the case of an infectious disease with pandemic potential which could be treated with an antibody, such as COVID‐19 or influenza, these advantages are paramount. This article is protected by copyright. All rights reserved.
The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.
Protein A affinity chromatography has been widely used for both laboratory scale purification and commercial manufacturing of monoclonal antibodies and Fc-fusion proteins. Protein A purification is specific and efficient. However, there still remain several issues to be addressed, such as incomplete clearance of impurities including host cell proteins, DNA, aggregates, etc. In addition, the effects of wash buffers in protein A purification on the physicochemical characteristics of antibodies have yet to be fully understood. Here we found a new purification protocol for monoclonal antibodies that can improve physicochemical properties of monoclonal antibodies simply by inserting an additional wash step with a basic buffer after the capture step to the conventional protein A purification. The effects of the alkaline wash on monoclonal antibodies were investigated in terms of physicochemical characteristics, yields, and impurity clearance. The simple insertion of an alkaline wash step resulted in protection of antibodies from irreversible aggregation, reduction in free thiols and impurities, an improvement in colloidal and storage stability, and enhanced yields. This new procedure is widely applicable to protein A affinity chromatography of monoclonal antibodies.
Protein A affinity chromatography is widely used for capturing therapeutic antibodies. It offers high binding capacity, selectivity, and resin reusability while delivering high yield and product purity. The Protein A step is ubiquitous in its presence in purification platforms for production of antibody products due to the effective clearance it offers of impurities, high and low molecular weight species (HMW and LMW), host cell proteins (HCP), and DNA. In this chapter, we describe a highly selective Protein A affinity chromatography protocol for purification of monoclonal antibodies.
Multimodal chromatography uses multiple modes of interaction such as charge, hydrophobic, or hydrogen bonding to separate proteins. Recently, we used molecular dynamics (MD) simulations to show that ligands immobilized on surfaces can interact and associate with neighboring ligands to form hydrophobic and charge patches, which may have important implications for the nature of protein-surface interactions. Here, we study interfacial systems of increasing complexity-from a single immobilized multimodal ligand to high density surfaces-to better understand how ligand behavior is affected by the presence of a surface and the presence of other ligands in the vicinity, and how this behavior scales to larger systems. We find that tethering a ligand to a surface restricts its conformations to a subset of those observed in free solution, yet the ligand maintains flexibility in the plane of the surface and can form contacts with neighboring ligands. We find that although the formation of a contact between two neighboring ligands is slightly unfavorable, three neighboring ligands exhibit a preference for the formation of a fully connected cluster. To explore how these trends in ligand association extend to a larger surface with high density of ligands, we performed coarse-grained Monte Carlo (MC) simulations of a 132-ligand surface using ligand interactions parametrized based on free energies obtained from the three-ligand MD simulations. Despite their simplicity, the coarse-grained simulations qualitatively capture the cluster size distribution of ligands observed in detailed MD simulations. Quantitative differences between the two suggest opportunities for improvements in the coarse-grained energy function for efficient predictions of cluster and pattern formations. Our approach presents a promising route to the engineering of multimodal patterns for future chromatographic resin design.
Low-molecular-weight synthetic molecules that mimic the activity of native biological macromolecules have therapeutic potential, utility in large-scale production of biopharmaceuticals, and the capacity to act as probes to study molecular recognition events. We have developed a nonpeptidyl mimic for Staphylococcus aureus Protein A (SpA). The specific recognition and complexation elements between the B domain (Fb) of SpA and the Fc fragment of IgG were identified from the x-ray crystallographic structure. Computer-aided molecular modeling was used to design a series of biomimetic molecules around the Phe132-Tyr133 dipeptide involved in its binding to IgG. One of the ligands binds IgG competitively with SpA in solution and when immobilized on agarose beads, with an affinity constant of 10(5)-10(6) M-1. The immobilized artificial Protein A was used to purify IgG from human plasma and murine IgG from ascites fluid, and to remove bovine IgG from fetal calf serum.
Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s. This increase in volumetric productivity has resulted mainly from improvements in media composition and process control. Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering.
Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.
There are currently 26 modern antibody-based therapeutic agents approved by the US and EU, with 500 more products in the global development pipeline. Total market value of these therapeutics could reach more than $17 billion by the close of 2010. This market overview includes all 26 products (plus two that were withdrawn) and their originators, their therapeutic indications, and their approval records.
Small, non-enveloped viruses can be difficult to inactivate or remove from source plasma. Virus filtration is a useful clearance method for these viruses. High-flux filters promise to be economical.
With West Nile virus in the news, viral inactivation is under public scrutiny. Yet, the culture media, the therapeutic product, and the potential viral contaminants can all affect the inactivation method chosen during production. Viral inactivation techniques - those tried or used in manufacturing or in final biopharmaceuticals, vaccines, and media products - have been gathered from recent scientific literature and organized in this article series as an addition to your process development toolbox.
Thorough process characterization can improve success rates in manufacturing, reduce the number of process related incidents, and significantly improve process yields.
Methodology for the purification of monoclonal antibodies from large-scale mammalian cell culture systems has been investigated. The concentration of monoclonal antibodies in conditioned cell culture media was generally found to be in the milligram per liter range, requiring 100–1,000 liters for production of gram quantities. To reduce the volumes, several ultrafiltration systems, including hollow fiber, plate, and frame, and spiral cartridges were investigated and found to be effective for large-scale work. Once a concentrated product was obtained, several methods including ammonium sulfate precipitation, ion exchange, protein. A agarose, and size exclusion chromatograhy were utilized and compared. The best results were obtained when concentrated conditioned medium was either diluted or diafiltered (to reduce the high ionic strength of conditioned medium) and fractionated by cation exchange chromatography. Variations in isoelectric points for different monoclonal antibodies require that specific pH and ionic strength parameters be determined to optimize binding and elution. Additional purification was achieved by further ion exchange steps or the use of ammonium sulfate precipitation. For particularly difficult purifications, protein A affinity chromatography was used. Once a purity of 90–95% was achieved, size exclusion chromatography was used as a final step to remove aggregates, process chemicals, contaminant proteins, and to exchange the antibody into the formulation buffer.
Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M–1. A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M–1, and ΔH = –7.1 ± 0.4 kcal mol–1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E-domain indicating that the E-domain binding site on Fab resides within VH. E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M–1, and ΔH = –24.6 ± 0.6 kcal mol–1. Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.
Production rate is an important parameter in the design of efficient protein A affinity chromatography processes for purifying recombinant monoclonal antibodies. A simple equation was derived that expresses production rate in terms of flow rate and column length. Changes in flow rate and column length will not affect the antibody and are therefore easily varied for bioprocess applications. In the equation, production rate depends on dynamic capacity, which can be expressed as a function of the load flow rate and column length. The only empirical data needed for production rate optimization is the relationship of dynamic capacity to load flow rate and column length, which was quickly determined by using an on-line assay. The optimal production rate was found at a high flow rate, a low column length, and a low dynamic capacity, which has several implications for using high production rate protein A affinity chromatography for antibody manufacturing.
Tangential flow filtration has traditionally been scaled up by maintaining constant the filtrate volume to membrane surface area ratio, membrane material and pore size, channel height, flow path geometry and retentate and filtrate pressures. Channel width and the number of channels have been increased to provide increased membrane area. Several other parameters, however, have not been maintained constant. A new comprehensive methodology for implementation of linear scale up and scale down of tangential flow filtration processes has been developed. Predictable scale up can only be achieved by maintaining fluid dynamic parameters which are independent of scale. Fluid dynamics are controlled by operating parameters (feed flow rate, retentate pressure, fed batch ratio and temperature), geometry (channel length, height, turbulence promoter and entrance/exit design), materials (membrane, turbulence promoter, and encapsulant compression), and system geometry (flow distribution). Cassette manufacturing procedures and tolerances also play a significant role in achieving scale independent performance. Extensive development work in the aforementioned areas has resulted in the successful implementation of linear scale up of ultrafiltration processes for recovery of human recombinant DNA derived pharmaceuticals. A 400-fold linear scale up has been achieved without intermediate pilot scale tests. Scale independent performance has a direct impact on process yield, protein quality and product economics and is therefore particularly important in the biotechnology industry. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 737-746, 1997.
We describe the performance characteristics of five Protein A affinity-chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host-cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.
A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.
Efficient harvest and recovery of high-purity monoclonal antibodies was achieved using hydrophobic charge induction chromatography (HCIC). Both simple and complex feedstocks were studied, including protein-free cell culture supernatant and the clarified/concentrated milk of transgenic goats. Viral clearance studies demonstrated a 4-log reduction of MVM virus (minute virus of mice), along with substantial reduction of DNA content. Sorbent characterization studies confirmed that HCIC is based on the pH-dependent behavior of a dual-mode, ionizable ligand. Binding, based on hydrophobic interaction, was achieved under near-physiological conditions, and in the absence of lyotropic salt. Desorption was accomplished under mild conditions--pH 4.0. At this pH, both ligand and antibody carry a net positive charge, and desorption occurs on the basis of electrostatic charge repulsion. pH-based control of chromatographic function was demonstrated. Chromatography on this antibody-selective HCIC sorbent was evaluated as a cost-effective, process-compatible alternative to affinity chromatography protein A sorbents.
With the technological advances made during the past decade, antibodies now represent an important and growing class of biotherapeutics. With the potential new targets resulting from genomics and with methods now in place to make fully human antibodies, the potential of antibodies as valuable therapeutics in oncology, inflammation and cardiovascular disease can be fully realised. Systems to produce these antibodies as full-length molecules and as fragments include expression in both mammalian and bacterial cells grown in bioreactors and in transgenic organisms. Factors including molecular fidelity and the cost of goods are critical in evaluating expression systems. Mammalian cell culture and transgenic organisms show the greatest promise for the expression of full-length, recombinant human antibodies, and bacterial fermentation seems most favorable for the expression of antibody fragments.
Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications.
Antibodies and antibody derivatives constitute twenty five percent of therapeutics currently in development, and a number of therapeutic monoclonal antibodies have recently reached the market. All antibodies approved by the US Food and Drug Administration, however, contain mouse protein sequences. These partially murine antibodies, therefore, have the potential to elicit allergic or other complications when used in human patients. Recent developments aim to reduce or eliminate murine components, and fully human antibodies are rapidly becoming the norm. A number of technologies exist which enable the development of 100% human antibodies.
Will we replace oil with wheat or corn as a feedstock for producing natural plastic? The success of biotechnology for bulk product manufacturing will heavily depend on engineering solutions in the downstream processes in which separation and purification have a crucial role with respect to commercial development. Development of efficient bioseparation methods is important for a broad range of business areas including pharmaceuticals, nutrition and health products, bio-based materials and crop protection chemicals. Depending on the value of the end product and the scale of production, the processing required varies significantly. Key factors that have an impact on the choice of separation strategy include process throughput, particle size of the product and impurities and the desired end-product concentration. The development of efficient, economical and selective separation methods will be required for successful commercialization of bioprocesses. Despite this well-recognized need, there are relatively few available methods for commercial implementations. Development of novel mechanical systems for selective separation of solid and liquid mixtures must become a top priority for current research investment to reduce the reliance on expensive chromatographic and thermal separation methods.
Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.
Protein A is a popular generic ligand for purification of monoclonal and recombinant antibodies. The performance of 15 commercially available protein A media was studied. Equilibrium and dynamic binding capacity for human IgG was determined and the capture of IgG from a crude feed-stock was investigated. For initial screening the dynamic binding capacity was determined at small scale. Media with good performance were further tested with increased column height. Comparing the data from the two different column heights it could be shown that the dynamic capacity strongly depends on the residence time. Agarose based media exhibited higher binding capacity at residence times longer than 3 min whereas polymeric media or media based on porous glass showed a lesser dependence on the flow velocity and the residence time. A quantitative description of this behavior was derived by determination of the adsorption isotherms and fitting the breakthrough profiles with the Thomas solution. Agarose based media exhibited higher maximum equilibrium binding capacities and the dissociation constants derived from adsorption isotherms were smaller. The other media exhibited higher apparent rate constants, indicating a faster mass transfer. This can be explained by the smaller particle diameter of these media and it can be assumed that constant pattern conditions are thereby obtained more quickly. Selectivity was tested by performing antibody purification under standardized conditions. Polyclonal human IgG in cell culture supernatant containing 2.5% fetal calf serum was used as a representative feed-stock. Under the applied conditions several sorbents showed very tight binding of IgG and in some cases most of the sample remained on the sorbent. The study can be useful as a guide for optimization of large-scale purification processes.
The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.
Protein A affinity chromatography is often employed as a capture step to meet the purity, yield, and throughput requirements for pharmaceutical antibody purification. However, a trade-off exists between step performance and price. Protein A resin removes 99.9% of feed stream impurities; however, its price is significantly greater than those of non-affinity media. With many therapeutic indications for antibodies requiring high doses and/or chronic administration, the consideration of process economics is critical. We have systematically evaluated the purification performance of cation-exchange, anion-exchange, hydroxyapatite, hydrophobic interaction, hydrophobic charge induction, and small-molecule ligand resins in each step of a three-step chromatographic purification process for a CHO-derived monoclonal antibody. Host cell proteins were removed to less-than-detectable for three processes (cation-exchange-anion-exchange-hydrophobic interaction chromatography, cation-exchange-anion-exchange-mixed cation-exchange chromatography, and cation-exchange-mixed cation-exchange-anion-exchange chromatography). The order of the process steps affected purification performance significantly.
Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.
The past 5 years have seen the commercialization of two recombinant protein products from transgenic plants, and many recombinant therapeutic proteins produced in plants are currently undergoing development. The emergence of plants as an alternative production host has brought new challenges and opportunities to downstream processing efforts. Plant hosts contain a unique set of matrix contaminants (proteins, oils, phenolic compounds, etc.) that must be removed during purification of the target protein. Furthermore, plant solids, which require early removal after extraction, are generally in higher concentration, wider in size range, and denser than traditional bacterial and mammalian cell culture debris. At the same time, there remains the desire to incorporate highly selective and integrative separation technologies (those capable of performing multiple tasks) during the purification process from plant material. The general plant processing and purification scheme consists of isolation of the plant tissue containing the recombinant protein, fractionation of the tissue along with particle size reduction, extraction of the target protein into an aqueous medium, clarification of the crude extract, and finally purification of the product. Each of these areas will be discussed here, focusing on what has been learned and where potential concerns remain. We also present details of how the choice of plant host, along with location within the plant for targeting the recombinant protein, can play an important role in the ultimate ease of recovery and the emergence of regulations governing plant hosts. Major emphasis is placed on three crops, canola, corn, and soy, with brief discussions of tobacco and rice.
A quantitative understanding of how proteins interact with hydrophobic charge induction chromatographic resins is provided. Selectivity on this mode of chromatography for monoclonal antibodies as compared to other model proteins is probed by means of a linear retention vs pH plot. The pH-dependent adsorption behavior on this mode of chromatography for a hydrophobic, charged solute is described by taking into account the equilibrium between a hydrophobic, charged solute and an ionizable, heterocyclic ligand. By analogy, an equation that is seen to adequately describe macromolecular retention under linear conditions over a range of pH is developed. A preparative, nonlinear isotherm that can capture both pH and salt concentration dependency for proteins is proposed by using an exponentially modified Langmuir isotherm model. This model is seen to successfully simulate adsorption isotherms for a variety of proteins over a range of pHs and mobile phase salt concentrations. Finally, the widely differing retention characteristics of two monoclonal antibodies are used to derive two different strategies for improving separations on this mode of chromatography. A better understanding of protein binding to this class of resins is seen as an important step to future exploitation of this mode of chromatography for industrial scale purification of proteins.
In this paper, a wide range of antibodies from various subclasses and subfamilies are employed to evaluate the creation of generic separation processes using Protein A chromatography. The reasons for elution pH differences amongst several IgG1s, IgG2s, antibody fragments, and Fc-fusion proteins during Protein A chromatography are investigated using several complimentary techniques. The results indicate that variable region interactions play a major role in determining elution pH for VH3 subfamily antibodies while using traditional protein A chromatographic materials. On the other hand, experiments with a resin which employs a ligand consisting solely of B domain of Protein A indicate that variable region interactions can be mitigated, enabling the use of a single elution pH for a range of antibodies. Finally, the moderation of elution conditions associated with this engineered ligand are shown to minimize problems associated with low pH induced aggregation. It is expected that the findings reported in this paper will facilitate faster process development cycle times for this important class of human therapeutics.
Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.
Protein recovery from a bacterial lysate was accomplished using microfiltration membranes in a flat crossflow filter and in a cylindrical rotary filter. Severe membrane fouling yielded relatively low long-term permeate flux values of 10(-4)-10(-3) cm/s (where I cm/s = 3.6 x 10(4) L/m(2) - h). The permeate flux was found to be nearly independent of transmembrane pressure and to increase with increasing shear rate and decreasing solids concentration. The flux increased with shear to approximately the one-third power or greater for the flat filter and the one-half power or greater for the rotary filter; the stronger dependence for the rotary filter is thought to result from Taylor vortices enhancing the back transport of debris carried to the membrane surface by the permeate flow. The average protein transmission or sieving coefficient was measured at approximately 0.6, but considerable scatter in the transmission data was observed. The largest sieving coefficients were obtained for dilute suspensions at high shear rate. The rotary filter provided higher fluxes than did the flat filter for dilute suspensions, but not for concentrated suspensions. (c) 1995 John Wiley & Sons, Inc.
The Use of NaOH for CIP of rProtein A Media: a 300 Cycle Study Waterside Conference
- H Johansson
- A Bergenstable
- G Rodrigo
- K Oberg