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Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation
Abstract
Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra‐/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography‐based antibody standard purification; (ii) the continuous precipitation‐based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation‐based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small‐scale production in parallel fashion, which is of importance for antibody production in non‐privileged countries and for personalized medicine.
See accompanying commentary by Ajoy Velayudhan DOI: 10.1002/biot.201300098
... The rise of biosimilars means the production of mAb-based therapeutics will increase in scale to provide more affordable mAb-based therapeutics for patients. In order to benefit from the economy of scale, pharmaceutical companies will require more cost-effective manufacturing technologies of mAb, especially for replacing the affinity chromatography step that typically accounts for more than half of the downstream purification cost due to the high cost of protein-A resin [6,10,11]. "Anything but conventional chromatography" is a concerted effort in the academia and industry to develop alternative biopurification technologies including crystallisation [12][13][14], precipitation [15], filtration [16][17][18] and extraction [19][20][21][22][23] to achieve the same high product purity as protein-A chromatography, while overcoming the throughput limited nature of chromatography and avoiding the use of expensive consumables (i.e. resin) [7,24,25]. ...
... As purification cost usually accounts for >50% of the entire production cost and affinity chromatography (i.e. with protein-A resin) accounts for ~70% of the overall purification cost [6,10,11], the successful replacement of affinity chromatography with crystallisation can achieve a substantial reduction (i.e. ~20 -30%) in the production cost of mAb-based therapeutics (note: this estimate is agreed by industrial producers of mAb that we have collaborated with over the past few years), which will subsequently translate into more affordable therapeutics and less heavy financial burdens for patients. ...
... In terms of scale, crystallisation can be conducted in industrial bioreactors where the synthesis of mAb takes place. These bioreactors have volume normally between a few hundred to approximately ten thousand litres and the typical titre is ~10 g/L [6,10,11]. This means crystallisation can process up to 100 kg mAb per batch in a 10,000 L bioreactor. ...
Therapeutics based on monoclonal antibody (mAb) represent one of the most advanced biopharmaceuticals, being able to treat a wide range of challenging diseases such as cancers and arthritis. As the scale of mAb production steadily increases with the demand for mAb-based therapeutics, the downstream biopurification continues to experience significant bottleneck due to the throughput limited nature of the current purification technology. Over the last decades, significant advances have been made in protein (and especially mAb) crystallisation as an alternative biopurification technology that offers high product stability and purity as well as scalability. This review starts with the introduction of general physicochemical properties of mAb before moving on to the in-depth discussion of distinct phase behaviour of mAb in comparison with conventional globular proteins such as lysozyme. The final part of this review presents a summary of successful demonstrations of crystallisation scale-ups of mAb and discusses the critical factors (i.e. mixing and temperature control) to be considered.
... Biopharmaceutical processing is typically divided into two parts, upstream and downstream, and is mainly run in a batch mode. The scale of production ranges from the experimental bench scale [16] to the commercial manufacturing scale [17][18][19]. Biopharmaceutical processing transitions through three phases: preclinical, clinical, and commercial production. In the early phases of development, small quantities are produced (grams), but as of the protein therapeutics development progresses, kilograms of products are produced to support different operations including clinical trials. ...
... However, this process has not been used in large-scale manufacturing of mAbs [38,39]. In recent years, several continuous or semi-continuous methods have been developed for primary capture including periodic counter-current chromatography (PCC) [40], multi-column countercurrent solvent gradient purification (MCSGP) [9,28], continuous countercurrent tangential chromatography (CCTC) [9,28,[41][42][43][44], and continuous precipitation process [18]. ATPS can also be modified for continuous operation with packed columns [45,46]. ...
... The software contains yearly updated built-in cost data library, including equipment, consumables, and solution components. The software allows users to customize cost data based on models [18,35]. However, this software is not able to capture the dynamic nature of the process. ...
Purpose
There is a growing interest in continuous biopharmaceutical processing due to the advantages of small footprint, increased productivity, consistent product quality, high process flexibility and robustness, facility cost-effectiveness, and reduced capital and operating cost. To support the decision making of biopharmaceutical manufacturing, comparisons between conventional batch and continuous processing are provided.
Methods
Various process unit operations in different operating modes are summarized. Software implementation as well as computational methods used are analyzed pointing to the advantages and disadvantages that have been highlighted in the literature. Economic analysis methods and their applications in different parts of the processes are also discussed with examples from publications in the last decade.
Results
The results of the comparison between batch and continuous process operation alternatives are discussed. Possible improvements in process design and analysis are recommended. The methods used here do not reflect Lilly’s cost structures or economic evaluation methods.
Conclusion
This paper provides a review of the work that has been published in the literature on computational process design and economic analysis methods on continuous biopharmaceutical antibody production and its comparison with a conventional batch process.
... This increase in product titer has created new opportunities for the development of alternative, lower cost, downstream operations. Several studies have demonstrated that precipitation can provide an attractive alternative to Protein A chromatography for initial antibody purification [5][6][7][8][9][10][11], with the use of tubular flow precipitation reactors followed by either centrifugation [5] or tangential flow filtration (TFF) [7,8,11] for dewatering and washing the precipitated protein. ...
... However, a hollow fiber module with partially blocked fibers will show a non-zero pressure intercept due to the finite pressure drop associated with the feed flow through the lumen of the open fibers (ΔP) since the flow through the blocked fibers occurs from the back end of the module as shown in Fig. 5. The value of the intercept provides a direct measure of the fraction of the fibers that have been blocked, i.e., f = 2*TMP int ΔP (6) where TMP int is the x-intercept of the linear regression fit to the water flux. The data in Fig. 4 for the fouled hollow fiber module give f = 0.35 ± 0.03 (with TMP int = 0.15 and ΔP = 0.85 ± 0.15 kPa, with the latter evaluated from the measured pressure drop through the fouled module). ...
Recent increases in monoclonal antibody titer from Chinese Hamster Ovary cell culture has led to renewed interest in precipitation for the initial capture/purification of these high-value proteins. In this work, we examined the effect of the membrane module geometry on the sustainable flux and fouling mechanisms using human serum Immunoglobulin G (IgG) precipitated with 10 mM zinc chloride and 7 w/v % polyethylene glycol as a model system. The sustainable flux was evaluated using flux stepping experiments for both open channel cassettes and hollow fiber membrane modules. The hollow fiber modules had relatively low sustainable flux due to clogging of the fibers at the module inlet, with this behavior confirmed using both SEM imaging and hydraulic permeability data for the fouled modules. In contrast, the open channel cassettes showed no evidence of channel clogging, enabling continuous operation for at least 24 h at a filtrate flux below the experimentally determined sustainable flux. These results provide important insights into the origin of the sustainable flux during tangential flow filtration of precipitated proteins, greatly facilitating the design of precipitation-filtration processes for continuous purification of monoclonal antibody products.
... Precipitation offers an interesting approach for capturing of biopharmaceuticals, as it usually has a high yield of over 90% and is able to reduce process volume significantly in a single step. In contrast to crystallization, the influence of reaction time on the process outcome is insignificant (Hammerschmidt et al., 2014;Neugebauer and Khinast, 2015;van Alstine et al., 2018;Burgstaller et al., 2019). Hammerschmidt et al. (2014) examined the possibilities of precipitation in a continuous manner for the purification of antibodies and developed different processes. ...
... In contrast to crystallization, the influence of reaction time on the process outcome is insignificant (Hammerschmidt et al., 2014;Neugebauer and Khinast, 2015;van Alstine et al., 2018;Burgstaller et al., 2019). Hammerschmidt et al. (2014) examined the possibilities of precipitation in a continuous manner for the purification of antibodies and developed different processes. They compared a continuous process for mAbs using four precipitation steps (Caprylic acid, PEG, CaCl2 and cold ethanol) combined with a flow-through anion exchange (AEX) step and a standard chromatographic purification, and showed that the continuous process provides cost reduction at all scales. ...
Continuous production delivers higher productivity and better quality than traditional batch-wise approaches. It intensifies production lowering capital cost and enables better control. Despite obvious advantages, continuous processing has not yet guided biomanufacturing to 21st Century Advanced Manufacturing status. Production relies primarily on batch-wise methods that have served the industry through its infancy. While great improvements have been achieved on the upstream side, downstream processing lacks development in continuous processing and is now the handbrake on modernisation. Nevertheless, there are hopeful advances. Research on continuous chromatographic purification for antibodies is maturing, and work has commenced on other unit operations and on process system integration. This exciting field of process intensification research is at a turning point, though considerably more research is needed. This review aims to summarize the latest developments and capabilities of continuous downstream processing applied in biopharmaceutical research and gives an overview of recent developments.
... Ovary (CHO) cell cultivation were employed: The fed-batch process (nutrients for the CHO cells are supplied for a complete manufacturing process, followed by harvest of the entire batch), the hybrid process (cultivation is performed in a fed-batch bioreactor, followed by continuous or semicontinuous purification of the produced antibodies), and the continuous perfusion process (cultivated cells are retained in the bioreactor, while the growth medium containing the antibodies is continuously substituted with fresh medium in a perfusion bioreactor; the used medium undergoes a continuous or semi-continuous purification process in order to isolate the antibodies; Figure 1; Hammerschmidt et al., 2014;Walsh, 2014). Each manufacturing method was then combined with a downstream process based on either chromatographic or caprylic acid purification. ...
... with five independent experts from five different companies in Germany, Denmark, and the United Kingdom (Farid, 2007;Rasmussen et al., 2012;Hammerschmidt et al., 2014;Walsh, 2014;Klutz et al., 2015;Laustsen et al., 2017). The assumed volume was based on a previous assessment for what the need for a sub-Saharan antivenom would be to establish the approximate scale of manufacture. ...
Snakebite envenoming is a neglected tropical disease that affects millions of people across the globe. It has been suggested that recombinant antivenoms based on mixtures of human monoclonal antibodies, which target key toxins of medically important snake venom, could present a promising avenue toward the reduction of morbidity and mortality of envenomated patients. However, since snakebite envenoming is a disease of poverty, it is pivotal that next-generation therapies are affordable to those most in need; this warrants analysis of the cost dynamics of recombinant antivenom manufacture. Therefore, we present, for the first time, a bottom-up analysis of the cost dynamics surrounding the production of future recombinant antivenoms based on available industry data. We unravel the potential impact that venom volume, abundance of medically relevant toxins in a venom, and the molecular weight of these toxins may have on the final product cost. Furthermore, we assess the roles that antibody molar mass, manufacturing and purification strategies, formulation, antibody efficacy, and potential cross-reactivity play in the complex cost dynamics of recombinant antivenom manufacture. Notably, according to our calculations, it appears that such next-generation antivenoms based on cocktails of monoclonal immunoglobulin Gs (IgGs) could be manufacturable at a comparable or lower cost to current plasma-derived antivenoms, which are priced at USD 13-1120 per treatment. We found that monovalent recombinant antivenoms based on IgGs could be manufactured for USD 20-225 per treatment, while more complex polyvalent recombinant antivenoms based on IgGs could be manufactured for USD 48-1354 per treatment. Finally, we investigated the prospective cost of manufacturing for recombinant antivenoms based on alternative protein scaffolds, such as DARPins and nanobodies, and highlight the potential utility of such scaffolds in the context of low-cost manufacturing. In conclusion, the development of recombinant antivenoms not only holds a promise for improving therapeutic parameters, such as safety and efficacy, but could possibly also lead to a more competetive cost of manufacture of antivenom products for patients worldwide.
... Furthermore, it is able to deal with high process volumes and even more important with rising titers. In addition to that, it is easy scalable and various working groups have already proven the feasibility of continuous operation [10][11][12][13]. ...
... [9] [4,8] [ 10] 2. Precipitation kinetics: solubility, mass balances [11] Figure 15. Model development and validation concept for the implementation of physico-chemical process models in biologics manufacturing. ...
The demand on biologics has been constantly rising over the past decades and has become crucial in modern medicine. Promising approaches to cope with widespread diseases like cancer and diabetes are gene therapy, plasmid DNA, virus-like particles, and exosomes. Due to progress that has been made in upstream processing (USP), difficulties arise in downstream processing and demand for innovative solutions. This work focuses on the integration of precipitation using a quality by design (QbD) approach for process development. Selective precipitation is achieved with PEG 4000 resulting in an HCP depletion of ≥80% respectively to IgG. Dissolution was executed with a sodium phosphate buffer (pH = 5/50 mM) reaching an IgG recovery of ≥95%. However, the central challenge in process development is still an optimal process design, which is transferable for a broad molecular variety of new products. This is where rigorous modeling becomes vital in order to generate digital twins to support early-stage process development and reduce the experimental overhead. Therefore, a model development and validation concept for construction of a process model for precipitation is also presented.
... The comparison between batch and continuous processing can be evaluated by using economic analysis. Hammerschmidt et al. 24 compared fed-batch (with fed-batch upstream and batch downstream), hybrid and fully continuous process by economic analysis. Instead of using continuous chromatography, the continuous process used precipitation for protein purification. ...
... Thus, the operating cost of the continuous process can be further reduced. Hammerschmidt et al. 24 considered the fed-batch process plus the chromatography system and perfusion plus the precipitation system and compared the COG values for different upstream titers. The results showed that, for the fedbatch system, as the titer increased, the ratio between upstream and downstream decreased which means the cost shifts from upstream to downstream. ...
This work aims to evaluate the benefits of the continuous process in biopharmaceutical manufacturing of monoclonal antibodies (mAbs). An integrated continuous process is designed and built using a process simulator and compared with a fed-batch process production line. The comparisons are based on cost of goods (COG/g) calculation and sensitivity analysis. The fed-batch process results in a total of COG/g of $99/g in mAbs production whereas continuous process accounts for $51/g. Due to the smaller footprint and fewer storage tanks required in the continuous process, the facility cost reduced by 66% compared to the fed-batch. In continuous production, there is a better utilization of the primary capture resin, which leads to a reduction in consumables cost by 68%. Sensitivity analysis is used to evaluate the process in different manufacturing scales (50-1200 kg/yr), upstream titers (1.5-5.5 g/L) and downstream yield (70%-80%). Cost savings can be observed through the whole range.
... However, this dramatic productivity increase in upstream processing has led to a bottleneck in downstream processing of the created product (Gottschalk, 2008;Milne, 2017). Downstream processing does not benefit from economies of scale, and additional costs such as increased amounts of protein A resin for antibody purification may in some cases surpass the additional revenue gained from upscaling (Hammerschmidt et al., 2014). The reason for this is that the producer cell is a highly complex system that synthesizes and secretes not only the recombinant protein of choice, but also a huge number of other proteins, which have to be separated from the desired product (Chollangi et al., 2015). ...
... This results in greater purity of the produced protein therapeutic, and should generally lead to easier downstream purification processing. For example, in the case of IgGs, the increased purity may even allow elimination of protein A columns, which make up a substantial proportion of the total downstream processing costs, from the purification process (Hammerschmidt et al., 2014). The very reason why IRES SVV+55nt is resistant to PKR-induced translational downregulation, namely its independence from translational initiation factors, also precluded us from reaching industrial productivity standards which can make use of the full ribosomal machinery. ...
Capitalizing on the ability of mammalian cells to conduct complex post-translational modifications, most protein therapeutics are currently produced in cell culture systems. Addition of a signal peptide to the product protein enables its accumulation in the cell culture supernatant, but separation of the product from endogenously secreted proteins remains costly and labor-intensive. We considered that global downregulation of translation of non-product proteins would be an efficient strategy to minimize downstream processing requirements. Therefore, taking advantage of the ability of mammalian protein kinase R (PKR) to switch off most cellular translation processes in response to infection by viruses, we fused a caffeine-inducible dimerization domain to the catalytic domain of PKR. Addition of caffeine to this construct results in homodimerization and activation of PKR, effectively rewiring rapid global translational downregulation to the addition of the stimulus in a dose-dependent manner. Then, to protect translation of the target therapeutic, we screened viral and cellular internal ribosomal entry sites (IRESes) known or suspected to be resistant to PKR-induced translational stress. After choosing the best-in-class Seneca valley virus (SVV) IRES, we additionally screened for IRES transactivation factors (ITAFs) as well as for supplementary small molecules to further boost the production titer of the product protein under conditions of global translational downregulation. Importantly, the residual global translation activity of roughly 10% under maximal downregulation is sufficient to maintain cellular viability during a production timeframe of at least five days. Standard industrially used adherent as well as suspension-adapted cell lines transfected with this synthetic biology-inspired Protein Kinase R-Enhanced Protein Production (PREPP) system could produce several medicinally relevant protein therapeutics, such as the blockbuster drug rituximab, in substantial quantities and with significantly higher purity than previous culture technologies. We believe incorporation of such purity-by-design technology in the production process will alleviate downstream processing bottlenecks in future biopharmaceutical manufacturing.
... Economic analysis showed that precipitation-based processes reduce costs at all stages compared to protein A based systems [13,14]. In these Costs of Goods (CoGs) studies, the main cost contribution of materials in precipitation was the use of large amounts of concentration stock solutions of precipitating agents, washing and redissolution buffers [15,16]. ...
The economic benefits of continuous PEG precipitation of antibodies compared to continuous capture by protein A affinity chromatography have been always lowered by the excess amount of material needed for the process. PEG is added as a concentrated stock solution and therefore liquid handling is increased during this step. To fully exploit the benefits of PEG precipitation, the precipitant must be added in solid form. We used an in-line feeding device with a screw conveyor delivery system for the continuous addition of PEG6000 in a powder form. The powder feeding device was connected to a tubular reactor where the precipitation occurs. Protein precipitation was continuously performed for 4 h. A yield of 76 % and 79 % with a purity of 98 % was achieved. The total cost of goods and the environmental footprint were compared with typical chromatography-based purification methods; batch and continuous periodic countercurrent protein A affinity chromatography with four columns. Solid PEG precipitation showed a remarkable reduction in water consumption and equipment size, reducing production costs by 45 % compared to liquid PEG and 53 % cheaper than Protein A periodic counter-current chromatography. Process mass intensity was reduced by 55 % and carbon emissions by 60 %. The reduction of water by the direct addition of PEG also impacted the environmental footprint and process costs. This is an attractive approach for a continuous capture step yielding an uninterrupted mass flow of the product and will pave the way for PEG precipitation as a capture step.
... PEG-aided precipitation has received also interest in the field of monoclonal antibody (mAb) purification (Giese et al., 2013;Hammerschmidt et al., 2014Hammerschmidt et al., , 2015Knevelman et al., 2010;Kuczewski et al., 2011;Sommer et al., 2015). Kuczewski and Giese (Kuczewski et al., 2011) reported that product selectivity can be improved by tuning the precipitant conditions and selection of the PEG type in single and two-step precipitation. ...
Microheterogeneity of mAbs can impact their activity and stability. Formation of charge variants is considered as the most important source the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variant in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was as a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs. This article is protected by copyright. All rights reserved.
... Nonetheless, economic analyses for the CM of biopharmaceuticals have shown conflicting results. On one hand, studies conducted by Pollock et al. [75] and Klutz et al. [84] on continuous antibody production confirm the economical advantages of the hybrid approach compared with end-to-end CM, Hammerschmidt et al. [85] concluded that fully continuous processes lead to the most significant cost savings in long term. Moreover, the gap between the equipment and knowledge that have been developed mainly in academic institutions and their adaptability to the vaccine manufacturing environment is another hurdle on the way of implementing CM in vaccine manufacturing. ...
Pandemics and epidemics are continually challenging human beings’ health and imposing major stresses on the societies particularly over the last few decades, when their frequency has increased significantly. Protecting humans from multiple diseases is best achieved through vaccination. However, vaccines thermal instability has always been a hurdle in their widespread application, especially in less developed countries. Furthermore, insufficient vaccine processing capacity is also a major challenge for global vaccination programs. Continuous drying of vaccine formulations is one of the potential solutions to these challenges. This review highlights the challenges on implementing the continuous drying techniques for drying vaccines. The conventional drying methods, emerging technologies and their adaptation by biopharmaceutical industry are investigated considering the patented technologies for drying of vaccines. Moreover, the current progress in applying Quality by Design (QbD) in each of the drying techniques considering the critical quality attributes (CQAs), critical process parameters (CPPs) are comprehensively reviewed. An expert advice is presented on the required actions to be taken within the biopharmaceutical industry to move towards continuous stabilization of vaccines in the realm of QbD.
... PEG-aided precipitation has received also interest in the field of monoclonal antibody (mAb) purification (Giese et al., 2013;Hammerschmidt et al., 2014Hammerschmidt et al., , 2015Knevelman et al., 2010;Kuczewski et al., 2011;Sommer et al., 2015). Kuczewski and Giese (Kuczewski et al., 2011) reported that product selectivity can be improved by tuning the precipitant conditions and selection of the PEG type in single and two-step precipitation. ...
... This is then washed in a two-step process in hollow fiber TFF, resulting in an intermediate concentrated product with a purity and yield comparable with protein A chromatography [38]. Combining four different precipitation and dissolution techniques based on caprylate, PEG, CaCl 2 , and cold ethanol followed by a flow through ion exchange chromatography enables full continuous processing when connected to a perfusion bioreactor [39]. ...
Process intensification is being used primarily in the chemical industry since almost 50 years and was only recently adopted by biotechnology. The different or combined process intensification strategies should lead to simpler, more robust, and more efficient processes. Based on the depicted standard process, several entry points for the introduction of disposable solutions for process intensification can be identified. When working with a new recombinant protein, the first step certainly is process development. A wide range of different single-use bioreactors is nowadays available for perfusion and continuous processes. The chapter describes the current range of stationary phases that are available as disposable devices. Process intensification is certainly heavily influenced by the practical operations on the shop floor. However, it should be noted that process analytical technology can contribute a great deal in increasing the productivity. Process intensification can be implemented in most processes but should be evaluated already during the development.
... 2,6,7 A single example of the application of each of these three techniques to continuous protein purification has been published. [10][11][12] In addition to reducing facility footprints, precipitation, ATPE, and crystallization have the potential to dramatically increase equipment utilization, allowing the biopharmaceutical sector to realize higher productivities and improved operational flexibility. 2,3,7 These methods could also support the robust control of short product residence times, allowing for the rapid recovery of labile proteins and standardization of critical quality attributes across each lot. ...
Accompanied with the growth of the biopharmaceuticals market has been an interest in developing processes with increased control of product quality attributes at low manufacturing cost, with one of the...
... At larger scale, the costs would not be expected to increase significantly. Also, the use of disposables can be considered, making cleaning-in-place and sanitisation-in-place procedures redundant, further reducing the costs (Hammerschmidt et al., 2014). ...
The scale-up of protein precipitation processes proves to be a challenging task due to the complexity of the reactions and transport processes involved. A good understanding of the molecular processes underpinning precipitate formation and the reaction kinetics are therefore required in order to devise a scale-up strategy. The doctoral project was first set out to establish micro-mixing as an engineering tool for the scale-up of antibody precipitation from cell culture, and secondly to design a downstream process with the goal of purifying a therapeutic mAb to clinical grade levels. Studies were first conducted in batch and transferred to a continuous process, with the scale-up approach focusing on the latter. Interactions between precipitation conditions and centrifugal recovery were then examined by employing an ultra scale-down (USD) methodology to mimic large-scale centrifugation. The downstream process design was on the basis of integrating precipitation with non-affinity chromatography steps to avoid the cost of affinity chromatography. Precipitate formation in batch and continuous settings was governed by the mixing at the molecular scale, which determined the final particle properties. Based on this, the mean energy dissipation rate for a continuous precipitation process proved an effective scale-up criterion, enabling high process throughputs relative to batch operation. The strength of protein precipitates, as evaluated by exposing particles to turbulent shear in a rotating disc device, was shown to correlate with particle fractal dimensions. Despite excellent precipitate solids removal from the USD methodology, these could not be predicted by disc-stack centrifugation. Differences in hindered settling between the systems were proposed to explain this observation which suggests routes to resolve this scale-up challenge. To provide an integrated DSP solution for therapeutic mAbs processes anion exchange and mixed-mode chromatography steps subsequent to precipitation were designed. Parameter ranges were studied to identify the optimal conditions in maximising antibody yield and HCP removal. Using optimal conditions, precipitation and anion exchange demonstrated an 18-fold removal in HCPs, whilst precipitation and mixed-mode provided a 40-fold removal. For a three step process comprising the sequence precipitation, anion exchange and mixed mode, an overall HCP removal of 260-fold was seen; however such levels remain at least 38-fold higher than the typical specification of a clinical grade product. This therefore necessitates further optimisation in one or more steps.
... Shifting the mode of manufacturing for biopharmaceuticals from batch to continuous has emerged as a potential solution to this predicament. It has been shown that continuous processing yields a significant reduction in the manufacturing costs [2,3] by increasing productivity, decreasing working volume, and increasing utilization of equipment's and column [4][5][6]. ...
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.
... Continuous reactors, such as tubular reactors, offer a promising approach to precipitation unit operations as these can achieve higher throughputs. [20][21][22][23] Moreover, these systems can achieve superior mixing times based on the specific design elements, including mixer selection and bore size. This is particularly important for fast precipitation reactions where the nucleation and growth processes occur spontaneously. ...
BACKGROUND
Precipitation has seen application for the processing of important therapeutics including monoclonal antibodies (mAbs). The scale‐up proves to be a challenging task due to the complexity of the reactions and transport processes involved. This requires a good understanding of the molecular processes underpinning precipitate formation.
The aim of the study was to build a micro‐mixing model for the precipitation of a mAb in continuous tubular reactors using ammonium sulphate. The effect of micro‐mixing on precipitate formation with respect to size, strength and nature was evaluated. An ultra scale‐down (USD) centrifugation methodology was applied to determine the ease of precipitate clarification.
RESULTS
The results demonstrated that the final mean particle size decreased with increased micro‐mixing, and was obtained with short residence times. Antibody yields in the tubular reactors were consistently above 90% and shown to be independent of the mixing. Similar particle sizes between a lab and pilot‐scale reactor were correlated with the average energy dissipation rate. The smaller particles obtained from improved micro‐mixing had higher fractal dimensions which correlated with minimal breakage upon exposure to turbulent shear. Precipitates were easily clarified at the USD scale (> 95% clarification) but less so at pilot‐scale (< 80 % clarification).
CONCLUSION
The precipitation is a rapid process with final precipitate properties controlled by the flow conditions. Therefore, the process can be controlled to acquire a certain particle size range. A high‐throughput precipitation process is also possible. Further investigation into large‐scale precipitate recovery is required.
This article is protected by copyright. All rights reserved.
... Additionally, initial aptamer generation costs are high. However, once a suitable aptamer has been generated, the production costs of the aptamer are likely to be lower than antibody-generation costs (e.g., <USD 50/g for aptamers supplied by Aptagen LLC [93] compared with~USD 300/g for recombinant monoclonal antibodies produced using mammalian cell lines [94]). ...
Aptamers are a novel technology enabling the continuous measurement of analytes in blood and other body compartments, without the need for repeated sampling and the associated reagent costs of traditional antibody-based methodologies. Aptamers are short single-stranded synthetic RNA or DNA that recognise and bind to specific targets. The conformational changes that can occur upon aptamer-ligand binding are transformed into chemical, fluorescent, colour changes and other readouts. Aptamers have been developed to detect and measure a variety of targets in vitro and in vivo. Gonadotropin-releasing hormone (GnRH) is a pulsatile hypothalamic hormone that is essential for normal fertility but difficult to measure in the peripheral circulation. However, pulsatile GnRH release results in pulsatile luteinizing hormone (LH) release from the pituitary gland. As such, LH pulsatility is the clinical gold standard method to determine GnRH pulsatility in humans. Aptamers have recently been shown to successfully bind to and measure GnRH and LH, and this review will focus on this specific area. However, due to the adaptability of aptamers, and their suitability for incorporation into portable devices, aptamer-based technology is likely to be used more widely in the future.
... The increasing need to develop new biopharmaceuticals and reduce costs have led to higher demands on processes being more versatile and efficient (Zydney, 2015). The opportunities and rationale for integrated continuous processes have been discussed in several studies (Hammerschmidt, Tscheliessnig, Sommer, Helk, & Jungbauer, 2014;Konstantinov & Cooney, 2015;Papathanasiou & Kontoravdi, 2020), and integrated continuous processes is also encouraged by the U.S. Food and Drug Administration (FDA) (Woodcock, 2014). There is also a trend towards more stratified therapies, which will lead to an increase in the demand for flexible multi-purpose facilities for smaller-scale production (Gronemeyer, Ditz, & Strube, 2014). ...
... Regarding downstream, they found that continuous processing is slightly beneficial as cost of goods decrease from 12 €/g to 6-9€/g when employing continuous protein A chromatography (Klutz, et al., 2015). Similar findings were obtained by Hammerschmidt et al., pointing out that a hybrid process results in the lowest cost of goods for annual production of a protein (Hammerschmidt, et al., 2014). ...
For the first time to our knowledge the implementation of a continuous protein A capture process for antibody applications (CoPACaPAnA) embedded in an end-to-end single-use 500 L GMP manufacturing downstream process of a multispecific monoclonal antibody (mAb) using a single-use SMB system was conducted. Throughout the last years, a change concerning the pipelines in pharmaceutical industry could be observed, moving to a more heterogeneous portfolio of antibodies, fusion proteins and nanobodies. Trying to adjust purification processes to these new modalities, a higher degree of flexibility and lower operational and capital expenditure is desired. The implementation of single-use equipment is a favored solution for increasing manufacturing agility and it has been demonstrated that continuous processing can be beneficial concerning processing cost and time. Reducing protein A resin resulted in 59% cost reduction for the protein A step, with additional cost reduction also for the intermediate and polishing step due to usage of disposable technology. The downstream process applied here consisted of three chromatography steps that were all conducted on a single-use SMB system, with the capture step being run in continuous mode while intermediate and polishing was conducted in batch mode. Further, two steps dedicated to virus inactivation/ removal and three filtration steps were performed, yielding around 100 g of drug substance going into clinical phase I testing. Therefore, in this study it has been demonstrated that employing a continuous capture within a GMP single-use downstream processing chain is feasible and worthy of consideration among the biotech industry for future application to modality-diverse pipelines.
... A recent survey of 222 biopharmaceutical decision-makers identified a number of key trends in biomanufacturing including the "adoption of single-use systems" and the "use of continuous processing" [1]. Several studies have discussed the rationale and economic potential of continuous bioprocessing systems [2][3][4]. The membrane industry has been active in responding to both of these trends, including the use of ultrafiltration for debottlenecking the downstream process, i.e., reducing process volumes to meet tankage or pumping constraints [5][6][7][8][9]. ...
Increasing pressures on biomanufacturing costs have led to renewed interest in the development of single-use technologies that can be readily adapted to continuous processing. The objective of this study was to use commercially available hollow fiber membranes, originally designed for high-flux hemodialysis, for single pass tangential flow filtration with high conversions. Experiments were performed with solutions of Immunoglobulin G (IgG) using polysulfone hollow fiber membrane cartridges with a surface area of 1.9 m². The hollow fiber modules were able to provide more than 10-fold concentration of IgG in a single-pass with exit concentrations greater than 200 g/L. Stable operation was achieved during a continuous run for over a 120-h period with feed side pressure drops less than 1 kPa and transmembrane pressure drops less than 60 kPa. A single dialyzer was able to process 1.4 kg of IgG per day with a total cost of less than $0.004 per g of IgG. Flux-pressure profiles were in good qualitative agreement with a simple model accounting for concentration polarization effects and the variation of flux and pressure with axial position in the module. These results clearly demonstrate the potential of using these low-cost single-use hollow fiber modules for single-pass tangential flow filtration in continuous biomanufacturing.
... The fields of neglected tropical diseases, other infectious diseases, and intoxications may even represent a scientific Eldorado for areas such as understanding and designing cross-reactivity for monoclonal antibodies [107], use of oligoclonal antibody cocktails for multi-target neutralization [1,17,108], novel immunization approaches [109], targeting of evasive pathogens [9,10], and exploitation of novel therapeutic mechanisms of action [41]. Such potential developments will likely be further catalyzed by advances in antibody production technologies that may enable low-cost antibody manufacture [17,110] and, in turn, better affordability of antibody-based therapies on a global scale. Despite their currently overlooked status, neglected tropical diseases, other infectious diseases, and intoxications may one day find themselves in the limelight of antibody developers, who may recognize the potential for improving therapies for victims worldwide, while simultaneously experimenting with innovative antibody-based approaches and elucidating fundamentally novel antibody properties. ...
Introduction: Monoclonal antibody-based therapies now represent the single-largest class of molecules undergoing clinical investigation. Although a handful of different monoclonal antibodies have been clinically approved for bacterial and viral indications, including rabies, therapies based on monoclonal antibodies are yet to fully enter the fields of neglected tropical diseases and other infectious diseases.
Areas covered: This review presents the current state-of-the-art in the development and use of monoclonal antibodies against neglected tropical diseases and other infectious diseases, including viral, bacterial, and parasitic infections, as well as envenomings by animal bites and stings. Additionally, a short section on mushroom poisonings is included. Key challenges for developing antibody-based therapeutics are discussed for each of these fields.
Expert opinion: Neglected tropical diseases and other infectious diseases represent a golden opportunity for academics and technology developers for advancing our scientific capabilities within the understanding and design of antibody cross-reactivity, use of oligoclonal antibody mixtures for multi-target neutralization, novel immunization methodologies, targeting of evasive pathogens, and development of fundamentally novel therapeutic mechanisms of action. Furthermore, a huge humanitarian and societal impact is to gain by exploiting antibody technologies for the development of biotherapies against diseases, for which current treatment options are suboptimal or non-existent.
... By comparison, antibodies expressed in cell cultures can be purified using caprylic acid precipitation and/or protein A affinity chromatography (Liu et al., 2010;Morais et al., 2014). This is routinely performed within industrial antibody manufacturing (Hammerschmidt et al., 2014;Liu et al., 2010), with the consequence that these purification methods have been extensively optimized and are highly standardized. Therefore, when choosing a production strategy, it is highly relevant to take the downstream processing, that follows each method of (bio) synthesis, into account. ...
Snakebite envenoming is a devastating Neglected Tropical Disease, the treatment of which has seen relatively little innovation since the invention of antivenom serotherapy in 1894. Current antivenoms have been and continue to be invaluable in saving thousands of lives. However, these medicines are associated with a number of drawbacks pertaining to availability, safety, and efficacy. Fortunately, with the advent of novel methodologies, such as antibody discovery technologies, high-throughput drug discovery approaches, and improved methods for protein engineering, we are starting to see scientific advances in the field. This review presents relevant engineering and design considerations for exploiting these methodologies to develop next-generation antivenoms with improved safety, efficacy, and affordability. The pros and cons of different treatment modalities are discussed with regards to immunogenicity, the suitability of preclinical efficacy assays, availability of discovery methods, economic viability of production schemes, and possible regulatory approval paths.
... Most of the antibodies need to be purified prior to use. The purification step both increases the production costs and inversely affects the activity of the antibody [26,27], so the availability of a feasible purification scheme is another important criteria. The antibody should either show sufficient performance when it is not fully purified, or a cost-effective purification scheme that does not affect antibody activity should be established. ...
Antibodies are the basic components of immunoanalytical systems used for detection of a wide range of analytes. Although there are some ground rules for antibody selection, analyte- and assay-specific criteria are the ones that determine the ultimate success of the immunoassays. In this study, we introduced an effective antibody selection procedure for the development of immunoaffinity columns for aflatoxins. The designed scheme puts emphasis on solvent- and matrix-related characterization steps and was used to comparatively evaluate eight monoclonal antibodies. The selected antibody was tolerant to 40% methanol, 20% acetonitrile, 30% acetone and 40% ethanol and did not interact with corn, red pepper or hazelnut extracts. Immunoaffinity columns developed with the selected antibody were validated by 15 independent aflatoxin analysis laboratories.
... Especially the major biopharmaceutical product group of monoclonal antibodies and their main production host Chinese hamster ovary (CHO) cells are in the focus of research (Ecker et al., 2015). As a result, product titers have increased substantially over the past years (Kunert and Reinhart, 2016) and process intensification toward continuous processes (Hammerschmidt et al., 2014) as well as an increased use of singleuse bioreactors (Löffelholz et al., 2013) has taken place. But since fed-batch cultivation remains the predominant large-scale process in industry, a large number of studies focusing on fed-batch processes have been published over the past decades. ...
Biopharmaceutical production processes strive for the optimization of economic efficiency. Among others, the maximization of volumetric productivity is a key criterion. Typical parameters such as partial pressure of CO2 (pCO2) and pH are known to influence the performance although reasons are not yet fully elucidated. In this study the effects of pCO2 and pH shifts on the phenotypic performance were linked to metabolic and energetic changes. Short peak performance of qmAb (23 pg/cell/day) was achieved by early pCO2 shifts up to 200 mbar but followed by declining intracellular ATP levels to 2.5 fmol/cell and 80% increase of qLac. On the contrary, steadily rising qmAb could be installed by slight pH down-shifts ensuring constant cell specific ATP production (qATP) of 27 pmol/cell/day and high intracellular ATP levels of about 4 fmol/cell. As a result, maximum productivity was achieved combining highest qmAb (20 pg/cell/day) with maximum cell density and no lactate formation. Our results indicate that the energy availability in form of intracellular ATP is crucial for maintaining antibody synthesis and reacts sensitive to pCO2 and pH-process parameters typically responsible for inhomogeneities after scaling up.
... This observation is particularly important as it mirrors the problem that viable cell densities can be improved by smart process engineering, whereas improvements of cell-specific productivities typically require detailed knowledge of intracellular metabolic control mechanisms. The need of understanding may even increase with the envisaged development of continuous production processes (Hammerschmidt et al., 2014) that demand for optimized operating set points. It has been shown that ATP availabilities positively correlate with cell specific production rates of monoclonal antibodies . ...
To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. Various production-like growth conditions ranging from ample glucose and amino acid supply via moderate to severe nitrogen limitation were investigated in batch cultures. The combined application of quantitative metabolite pool analysis, 13C tracer studies and non-stationary flux calculations revealed that Pyr/H+ symport (MPC1/2) bore the bulk of the mitochondrial transport under ample nutrient supply. Glutamine limitation induced the concerted adaptation of the bidirectional Mal/aKG (OGC) and the Mal/HPO42− antiporter (DIC), even installing completely reversed shuttle fluxes. As a result, NADPH and ATP formation were adjusted to cellular needs unraveling the key role of cytosolic malic enzyme for NADPH production. Highest cell specific IgG1 productivities were closely correlated to a strong mitochondrial malate export according to the anabolic demands. The requirement to install proper NADPH supply for optimizing the production of monoclonal antibodies is clearly outlined. Interestingly, it was observed that mitochondrial citric acid cycle activity was always maintained enabling constant cytosolic adenylate energy charges at physiological levels, even under autophagy conditions.
... Continuous and batch precipitation methods were described by our group and others as an economically viable option for the continuous capture of antibodies from clarified culture supernatants (Grosshans, Wang, Fischer, & Hubbuch, 2018;N. Hammerschmidt, Hintersteiner, Lingg, & Jungbauer, 2015;Nikolaus Hammerschmidt, Hobiger, & Jungbauer, 2016;N. Hammerschmidt, Tscheliessnig, Sommer, Helk, & Jungbauer, 2014;Kateja, Agarwal, Saraswat, Bhat, & Rathore, 2016;Rathore, Kateja, Agarwal, & Sharma, 2016;Tscheliessnig et al., 2014). This method is quite robust; it readily accommodates varying feed concentrations; and it can be combined with flocculation. Converting the process into a continuous operation is relatively simple with tubular reactors (B ...
Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG 6000 and Zn⁺⁺ in a tubular reactor integrated with a two‐stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor re‐solubilization. We developed a new two‐stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a five‐fold reduction in pre‐existing high‐molecular‐weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter‐current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow‐through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.
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... In the research and development stage, there are many methods and tools for removing impurities, and there are many resins of different qualities to choose from. However, many times, a process solves the problem of impurities in an early laboratory, but it could not be industrialized because the resin used does not meet the GMP production standards [43][44][45][46]. If conditions permit, the life cycle of a resin should also be verified as soon as possible. ...
Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.
The significant increase in product titers, coupled with the growing focus on continuous bioprocessing, has renewed interest in using precipitation as a low‐cost alternative to Protein A chromatography for the primary capture of monoclonal antibody (mAb) products. In this work, a commercially relevant mAb was purified from clarified cell culture fluid using a tubular flow precipitation reactor with dewatering and washing provided by tangential flow microfiltration. The particle morphology was evaluated using an inline high‐resolution optical probe, providing quantitative data on the particle size distribution throughout the precipitation process. Data were obtained in both a lab‐built 2‐stage countercurrent washing system and a commercial countercurrent contacting skid that provided 4 stages of continuous washing. The processes were operated continuously for 2 h with overall mAb yield of 92 ± 3% and DNA removal of nearly 3 logs in the 4‐stage system. The high DNA clearance was achieved by selective redissolution of the mAb using a low pH acetate buffer. Host cell protein clearance was 0.59 ± 0.08 logs, comparable to that based on model predictions. The process mass intensity was slightly better than typical Protein A processes and could be significantly improved by preconcentration of the antibody feed material.
As biomanufacturers consider the transition from batch to continuous processing, it will be necessary to re-examine the design and operating conditions for many downstream processes. For example, the integration of virus removal filtration in continuous biomanufacturing will likely require operation at low and constant filtrate flux instead of the high (constant) transmembrane pressures (TMPs) currently employed in traditional batch processing. The objective of this study was to examine the effect of low operating filtrate flux (5-100 L/m2 /h) on protein fouling during normal flow filtration of human serum Immunoglobulin G (hIgG) through the Viresolve® Pro membrane, including a direct comparison of the fouling behavior during constant-flux and constant-pressure operation. The filter capacity, defined as the volumetric throughput of hIgG solution at which the TMP increased to 30 psi, showed a distinct minimum at intermediate filtrate flux (around 20-30 L/m2 /h). The fouling data were well-described using a previously-developed mechanistic model based on sequential pore blockage and cake filtration, suitably modified for operation at constant flux. Simple analytical expressions for the pressure profiles were developed in the limits of very low and high filtrate flux, enabling rapid estimation of the filter performance and capacity. The model calculations highlight the importance of both the pressure-dependent rate of pore blockage and the compressibility of the protein cake to the fouling behavior. These results provide important insights into the overall impact of constant-flux operation on the protein fouling behavior and filter capacity during virus removal filtration using the Viresolve® Pro membrane.
The WHO estimates that 8–10% of couples are facing fertility problems, often due to inaccuracy in predicting the female's ovulation period controlled by four key hormones. The quantification and monitoring of such key hormones are crucial for the early identification of infertility, but also in improving therapeutic management associated with hormonal imbalance. In this review, we extensively summarize and discuss: i) drawbacks of laboratory methods for fertility testing (costly, invasive, complex) and commercially available point-of-care tests (measuring only one/two of the four key hormones), ii) the understanding of different biosensors for fertility monitoring, and iii) an in-depth classification and overview of aptamer-based sensing of the hormones of interest. This review provides insights on hormone detection strategies for fertility, with a focus on the classification of the current ‘aptasensing’ strategies, aiming to assist as a basic guide for the development of accurate fertility window monitoring tools based on aptamers.
Monoclonal antibodies were already one of the fastest growing sectors of biopharmaceutical industry [1]. Recent research on the significant benefits of various antibodies in reducing the risks of fatality or reducing the symptoms of COVID-19, e.g., tocilizumab and sarilumab (Cortegiani et al. 2021), inevitably increases the importance of the rapid discovery of mAbs and the development of efficient manufacturing processes. Research reports frequently concentrated on the rapid discovery of new mAbs, but the developability and the manufacturability of mAbs were less explored until recently [1–4]. This chapter adopts the framework of quality by design (QbD), concentrating particularly on the model-based risk assessment of mAb developability. A case study highlights specific areas where advanced modelling approaches can contribute to speeding up the manufacturability and developability of mAbs and demonstrates the benefits and the challenges of this approach.
Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2ʹ and EGFP-RBDʹ, exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBDʹ to SV2C-LD was determined to be 5.45 μM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2ʹ, respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBDʹ to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2ʹ, respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBDʹ. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.
Therapeutic monoclonal antibodies (mAbs) are the fastest-growing class of biotherapeutics. They are mainly used to treat cancer, inflammatory, metabolic and autoimmune diseases. Their commercial production processes are mainly based on Chinese hamster ovary (CHO) suspension cells, which are currently cultivated in fed-batch mode at cubic meter scale. The annually growing market for therapeutic mAbs and the pressure on producers to reduce their manufacturing costs have led to the increasing development of intensified and continuous production processes in recent years. Single-use systems are used in both upstream and downstream processing. This book chapter describes the main intensification approaches and operational architectures of continuous processes realized today, based on the developmental status of single-use technologies used for the up- and downstream processing of mAbs. In this context, the terms “process intensification” and “continuous process” are defined, while the preferential application of single-use systems is described using literature, and further by own studies. Based on the findings, the main challenges for the implementation of intensified and continuous mAb production using single-use systems are identified.KeywordsCHO cellsConcentrated fed-batchFlow-through modeHigh-seed fed-batchMulti-column-chromatographyN-1 perfusionSingle-use capture and polishingSingle-use formulationSingle-use virus inactivation and removal
Biopharmaceuticals market has been constantly increasing during the last years, what is transforming the manufacturing industry of biomolecules. The efforts have been put into better understanding how bioprocesses are regulated in order to firstly, build the quality of the biopharmaceuticals into the bioprocess, and secondly, to be able to develop suitable culture strategies for the implementation of intensified bioprocess while preserving the product quality attributes (product quality). Both targets strongly depend on the development of reliable monitoring tools able to measure the key bioprocess variables and parameters. Among all the available monitoring tools, spectroscopic techniques are called to be the predominant as they can offer multicomponent mixtures composition. However, such techniques have not been widely incorporated due to its complexity which hampers their adoption by the industry. Alternatively, other “soft sensors” like oxygen uptake rate (OUR), have been successfully applied for the monitoring of cell activity, being very sensible to changes in metabolic behaviour or other biochemical changes suffered by the cells. Therefore, OUR has been applied to determine the key time points (Time of Action, TOA), for example the proper time for nutrients feeding in intensified cultures, or also the Time of Harvest in virus-host cells systems. TOAs detection would allow to automate and control the bioprocesses achieving higher productivities and product quality.KeywordsPATCell culture monitoringOURHCDCFed-batchPerfusionBioprocess automation
Since the application of recombinant DNA technology was first successfully exploited to produce human insulin in a heterologous expression system, there has been an explosion in biotherapeutic proteins produced to treat chronic diseases. The biotechnology behind many biological drugs, cell therapies and vaccines, such as those recently developed against COVID-19, relies on the industrial nature of heterologous protein expression. Whether it is the production of blockbuster monoclonal antibody treatments with Chinese Hamster Ovary cells, or using Human Embryonic Kidney cells to produce vaccines, culturing at high density in scales from litres to thousands of litres in bioreactors across the globe ensures global supplies of these sophisticated biologics and therapies. This global scale of production generates vast volumes of waste in the form of single use culture media. Equally, as society considers the impact of its carbon footprint, the bioprocessing community also has a responsibility to contribute to the global push towards a sustainable bioeconomy. The scope of this chapter is to highlight how biotechnology and systems biology has made significant progress toward the goal of valorising waste streams, and how we might learn from these to use the waste generated from animal cell culture as a source of new value.KeywordsBioeconomyValorization of wasteBioprocessingChemically defined mediaMicrobial fermentation
The biopharmaceutical industry has been growing and evolving at a pace that's hard to match, especially in terms of manufacturing. Besides in-line analytics methods included in the process analytical technology (PAT) concepts, the key technology has been the generation of decisive validated digital twins based on process models. For process development, a sophisticated PAT concept has to be developed parallel to upstream processing and downstream process modeling to generate digital twins, later on supporting model validation, piloting, and production. In liquid–liquid extraction, the most important kinetic parameter is the mass transfer coefficient. The continuous single-pass tangential flow filtration version is analogous, but due to the complexity of additional setup variations. The biologic pharmaceutical boom is not only apparent regarding the ever-growing market size but steadily increases molecular variety. Process integration is essential to provide a comprehensive solution, not leaving weak spots, which may be causing approval troubles.
Recombinant peptides were designed using the C -terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli . A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC5328 BoNT/A1, referred to EGFP-A2ʹ and EGFP-RBDʹ, exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant ( K D ) of EGFP-RBDʹ to SV2C-LD was determined to be 5.45 mM, which is 33.87- and 15.67-times smaller than the K D values for EGFP and EGFP-A2ʹ, respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBDʹ to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2ʹ, respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBDʹ. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.
Continuous cultivation of mammalian cells originated in chemostats in the 1950s and, through a series of technological advancements, has evolved into modern day high-density perfusion processes with volumetric productivities >5 g/L-d over extended cultivation periods. With appropriate process development, these productivities can be achieved with no adverse impact to cell line stability and product quality. Developments in downstream processing and automation have enabled the integration of drug substance manufacturing processes suitable for next-generation plants that require lower capital deployment with the potential for rapid construction and validation for clinical and commercial production. Perfusion has also been successfully used to generate high-density cell banks (> 50 x 10⁶ cells/mL) and for seed-train optimization. They have resulted in significant productivity increases (10-fold increase in volumetric productivity) from fed-batch bioreactors by providing a high-density seeding culture that elevates the inoculation density in a production bioreactor (> 5 x 10⁶ cells/mL). Related advancements in cell culture medium formulations and cell line performance typically are independent of bioreactor format and have broad applicability for biotherapeutic manufacturing. The last decade has seen renewed interest in high-density perfusion cultures and technological advances spanning the entire space of drug substance manufacturing – from cell line development to the final purification step. Continually evolving, these new technologies are now being introduced into clinical and commercial manufacturing. We anticipate the general principles and learnings from process intensification will extend well beyond classical perfusion cultivation and into all aspects of mammalian cell culture.
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Bio-pharmaceutics is one of the most science-intensive industries. Annually a lot of money is spent on applied research aimed at development and commercialization of new medications. Many pharmaceutical companies try to have in their product line or pipeline drugs on the basis of monoclonal antibodies, i.e. a class of biotechnological preparations that are used to combat oncologic and autoimmune diseases and are based on target therapy principle. Because of the high interest in bio-pharmaceutical industry on the part of businessmen, state and science any advanced data dealing with the situation inside the market can be useful for shaping the adequate picture of the present day condition and for making managerial decisions on state and private level. The article provides information about global sales of preparations based on monoclonal antibodies. Apart from sales in terms of money the author calculates the natural volume of products being sold based on price analysis of products. The article gives a list of preparations registered on EU and US markets rated by their sales. By analyzing preparation prices corrected to dosage it was possible to find the most expensive and the cheapest medications in their class. Information concerning the natural volume of drug being sold can help understand the scale of preparation production.
Continuous capture with affinity chromatography is one of the most important units for continuous manufacturing of monoclonal antibody (mAb). Due to the complexity of three‐column periodic counter‐current chromatography (3C‐PCC), three approaches (experimental, model‐based and simplified approaches) were studied for process development and optimization. The effects of residence time for interconnected load (RTC), breakthrough percentage of the first column for interconnected load (s) and feed protein concentration (c0) on productivity and capacity utilization were focused. The model‐based approach was found superior to the experimental approach in process optimization and evaluation. Two phases of productivity were observed and the optimal RTC for the maximum productivity was located at the boundary of the two phases. The comprehensive effects of the operating parameters (RTC, s and c0) were evaluated by the model‐based approach, and the operation space was predicted. The best performance of 34.5 g/L/h productivity and 97.6% capacity utilization were attained for MabSelect SuRe LX resin under 5 g/L concentration at RTC = 2.8 min and s = 87.5%. Moreover, a simplified approach was suggested to obtain the optimal RTC for the maximum productivity. The results demonstrated that model‐assisted tools are useful to determine the optimum conditions for 3C‐PCC continuous capture with high productivity and capacity utilization.
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Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two‐system setup that can be used as a general downstream setup offering multi‐product and multi‐purpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step. A real‐time controller was implemented to ensure stable operation by adapting the downstream process to external changes. A concentration disturbance was introduced to test the controller. After the disturbance was applied, the product output recovered within 6 hours, showing the effectiveness of the controller. In a comparison of the process with and without the controller, the product output per cycle increased by 27%, the resin utilization increased from 71.4% to 87.9%, and the specific buffer consumption was decreased by 21% with the controller, while maintaining a similar yield and purity as in the process without the disturbance. In addition, the integrated continuous process outperformed the batch process, increasing the productivity by 95% and the yield by 28%.
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In recent years process modelling has become an established method which generates digital twins of manufacturing plant operation with the aid of numerically solved process models. This article discusses the benefits of establishing process modelling, in-house or by cooperation, in order to support the workflow from process development, piloting and engineering up to manufacturing. The examples are chosen from the variety of botanicals and biologics manufacturing thus proving the broad applicability from variable feedstock of natural plant extracts of secondary metabolites to fermentation of complex molecules like mAbs, fragments, proteins and peptides.Consistent models and methods to simulate whole processes are available. To determine the physical properties used as model parameters, efficient laboratory-scale experiments are implemented. These parameters are case specific since there is no database for complex molecules of biologics and botanicals in pharmaceutical industry, yet.Moreover, Quality-by-Design approaches, demanded by regulatory authorities, are integrated within those predictive modelling procedures. The models could be proven to be valid and predictive under regulatory aspects. Process modelling does earn its money from the first day of application. Process modelling is a key-enabling tool towards cost-efficient digitalization in chemical-pharmaceutical industries.
The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.
Membrane systems play an essential role in the production of all biopharmaceuticals. This includes initial clarification of cell culture fluid by both depth filtration and tangential flow filtration, biomolecule purification by membrane adsorbers, sterilizing grade filtration and bioburden reduction by normal flow filtration, and virus removal by specially designed virus filtration membranes. Recent developments in biomanufacturing are driving new innovations in membrane technology to address the needs associated with continuous bioprocessing, single-use flexible manufacturing, and the production of more complex biotherapeutics for gene and cell-based therapies. This Review examines how the membrane industry has responded to these challenges, with a focus on recent developments in membranes, modules, and processes specifically targeted to biomanufacturing. A brief discussion is also provided of future bioprocessing challenges that will provide unique opportunities for membrane technologies.
Continuous crystallization has been proposed as the downstream processing step for the purification of pharmaceutical proteins aimed at alleviating the manufacturing bottleneck caused by the limitations of chromatography-based operations at high upstream production titers. Herein we reviewed the current state of research in continuous protein crystallization from which future research directions were identified. While the benefits of batch-to-continuous manufacturing transformation have been long established, progress in continuous protein crystallization lags behind its small-molecule counterpart. The reasons are because the challenging nature of protein crystallization, even when performed in the batch platform, and the lack of well-understood proteins available for thorough study. Nevertheless, successful batch-to-continuous transformations in both mixed-suspension-mixed-product-removal crystallizer and tubular crystallizers (i.e. slug flow, oscillatory baffled flow) have been demonstrated using lysozyme or monoclonal antibody as the model protein. Compared to the batch platform, the continuous platform produces comparable crystallization yield but with higher production capacity (g/h). Strategies to optimize the crystallizer's performance based on modelling and simulation results are also available. Future research should (1) study a wider range of proteins with impurities incorporated in the raw material streams, and (2) adopt advancements in continuous crystallization of small-molecule pharmaceuticals to improve the crystal quality and yield.
Non-protein A-based purification technology is increasingly needed in that protein A chromatography possesses mang inherent problems, such as the high material and operational costs, limited productivity, and leaching of the protein A ligand. In this study, antibody purification was first achieved by precipitation of the antibody from the cell culture supernatant (CCS) in a tubular reactor with a static mixer. The protein precipitates were efficiently intercepted by incorporating a depth filtration membrane directly after the mixer. Chromatin-directed cell culture clarification was shown to enable the full usage of the load of the filter membrane and also significantly reduced the burden of the subsequent purification step. The addition of NaCl to the CCS significantly increased the permeabilization of the filter membrane by increasing the loading capacity by up to 20%. The re-dissolved and eluted antibodies were further polished using single multimodal chromatography, which supported direct sample loading. The content of non-histone host cell protein (n-h-HCP) in the final product was less than 20 ppm, DNA was less than 1 ppb, and the aggregates was less than 0.05%. The overall antibody recovery was ˜82%. This method is a feasible alternative to the current protein A-dominant antibody purification platform and has high value and potential for widespread implementation.
Continuous virus inactivation remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting virus inactivation (VI) to continuous operation are two‐fold: (1) achieving fluid homogeneity and (2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, we implemented a dynamic active in‐line mixer and a packed‐bed continuous virus inactivation reactor (CVIR), which acts as a narrow RTD incubation chamber. The developed concept was applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in‐line mixer was characterized and enabled mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor’s RTD was characterized and additional control experiments confirmed that the VI was due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before 2 reactor volumes and the logarithmic reduction values (LRV) of the continuous inactivation process were identical to those obtained by the traditional batch operation. The packed‐bed reactor for continuous virus inactivation unites fully continuous processing with very low pressure drop and scalability.
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Integrated and continuous processing of recombinant proteins offers several advantages over batch or semi-batch processing used traditionally in the biotechnology industry. This paper presents a theoretical and practical approach for designing a periodic counter-current chromatography (PCC) operation as a continuous capture purification step that is integrated with a perfusion cell culture process. The constraints for continuous and optimal PCC operation govern the selection of residence time and number of columns. The flexibility available in PCC design for selection of these parameters is dictated by the binding characteristics of the target protein on the capture resin. Using an empirical model for the protein breakthrough curve, analytical solutions to determine these conditions were derived and verified with experimental results for three different proteins: two relatively unstable proteins - recombinant enzymes and a relatively stable protein - monoclonal antibody. The advantages of a continuous downstream capture step are highlighted for the three case studies in comparison with the existing batch chromatography processes. The use of PCC leads to improvements in process economics due to higher resin capacity utilization and correspondingly lower buffer consumption. Furthermore, integrated and continuous bioprocessing results in a smaller facility footprint by elimination of harvest hold vessels and clarification, as well as by reducing the capture column size by 1-2 orders of magnitude.
In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
Manufacturing processes for therapeutic monoclonal antibodies (mAbs) have evolved tremendously since the first licensed mAb product in 1986. The rapid growth in product demand for mAbs triggered parallel efforts to increase production capacity through construction of large bulk manufacturing plants as well as improvements in cell culture processes to raise product titers. This combination has led to an excess of manufacturing capacity, and together with improvements in conventional purification technologies, promises nearly unlimited production capacity in the foreseeable future. The increase in titers has also led to a marked reduction in production costs, which could then become a relatively small fraction of sales price for future products which are sold at prices at or near current levels. The reduction of capacity and cost pressures for current state-of-the-art bulk production processes may shift the focus of process development efforts and have important implications for both plant design and product development strategies for both biopharmaceutical and contract manufacturing companies.
Monoclonal antibodies (mAbs) comprise the majority of protein candidates currently in clinical development because of their versatility as therapeutic agents. While traditionally associated with the biotechnology industry, mAb therapeutics are now being developed and marketed by most major pharmaceutical firms. A total of 21 products are approved in the US, with additional products marketed outside the US, and over 200 mAb candidates are currently undergoing clinical study. Benchmark data for mAb therapeutics, such as clinical development and US Food and Drug Administration approval times, approval success rates, and clinical phase transition probabilities, are critical for strategic planning purposes. Trends in these benchmarks for various types of mAbs, with an emphasis on those studied as anticancer and immunological therapeutics, are discussed.
Staphylococcal protein A can selectively interact with immunoglobulins. This protein is widely used as a ligand for affinity chromatography to purify therapeutic antibodies on an industrial scale. This type of affinity chromatography constitutes a generic step in processing antibodies. Questions of scale-up, design of chromatographic conditions, clearance of adventitious agents and operational modes, such as continuous operation or purification of antibodies in expanded-bed mode, will be addressed in this review.
Progress in downstream processes have not kept pace with increases in upstream yields. It is time for protein purification to make a comeback.
Improved feedstream titers are driving a demand for increased downstream processing productivity as manufacturers seek to lower the costs of monoclonal technologies for MAb purification. Platform technologies and chromatography resin improvements can help manufacturers achieve rapid and economical process development aria scale-up. A multiprong approach can enable downstream process scientists to enhance productivity.
The analysis of host cell proteins (HCPs) is one of the most important analytical requirements during bioprocess development of therapeutic moieties. In this review, we focus on the comparison of different methods for the analysis of HCPs and how cell lines, fermentation conditions, and unit operations influence HCP distribution during the process chain. Current guidelines typically require reduction of HCPs to the ppm level, depending on the intended use, the route of administration of the product, and the production system. A range of immunospecific and non-specific methods are available that have been globally accepted by regulatory bodies. Immunospecific methods, such as ELISA, are simple to use in routine analysis and can quantify low levels of HCPs when specific antibodies are available. Non-specific methods are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Different methods for the comparison of bioprocessing strategies during scale-up and purification development are compared herein. The methods include immunospecific methods, such as ELISA, western blot, and threshold, and non-specific methods, such as 2D-DIGE and 2D-HPLC combined with MS.
Monoclonal antibodies are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is separation efficiency - a way to increase this separation efficiency is by using magnetic stimuli-responsive polymer nanoparticles. In this work thermo-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we have evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a dialyzed CHO cell culture supernatant by cation exchange. The adsorption step was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure fractions with host cell protein removals greater than 98%. By exploring the stimuli-responsive proprieties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity.
An aqueous two-phase extraction (ATPE) process based on a polyethylene glycol /phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80 and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100% and 95% host cell proteins purity were attained, representing a 22-fold reduction in the host cell proteins/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation and economical feasibility.
IntroductionChallenges When Striving for the Cost-Effective Manufacture of mAbsCost Definitions and Benchmark ValuesEconomies of ScaleOverall Process Economic DriversDSP Drivers at High TitersProcess Economic Trade-Offs for DSP BottlenecksSummary and OutlookReferences
In manufacturing of biological drugs, product quality is defined by the process (e.g. equipment, sequence of unit operations, operation parameters) because no complete analysis of these complex molecules is possible. Therefore, the process is unavoidably fixed after the first clinical lot production in a pilot plant. There is no further process optimization option parallel to production, which, in the case of small molecule productions, allows further process optimization.Process development times will not increase in future due to increasing pressure on time to market. In addition to that, no change in paradigm seems possible, as complete analysis of complex biomolecules comparable to small synthetic drugs is not seen in near future.As a consequences the challenge is to establish generic processes for different drug classes and to find consistent process development methods, which allow a reliable prediction of large-scale production.Generic in this sense is not understood as a fixed sequence of unit operations with a certain set of generic process parameters. Here, generic means that a typical arrangement of unit operations is set up in an efficient sequence to fulfil the separation task.
Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time.
Recombinant Chinese hamster ovary cells (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Recent advances in cell culture technology for rCHO cells have achieved significant improvement in protein production leading to titer of more than 10 g/L to meet the huge demand from market needs. This achievement is associated with progression in the establishment of high and stable producer and the optimization of culture process including media development. In this review article, we focus on current strategies and achievements in cell line development, mainly in vector engineering and cell engineering, for high and stable protein production in rCHO cells. The approaches that manipulate various DNA elements for gene targeting by site-specific integration and cis-acting elements to augment and stabilize gene expression are reviewed here. The genetic modulation strategy by "direct" cell engineering with growth-promoting and/or productivity-enhancing factors and omics-based approaches involved in transcriptomics, proteomics, and metabolomics to pursue cell engineering are also presented.
The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion.
PEGs for protein precipitation are usually classified by molecular weight. The higher molecular weight precipitants are more efficient but result in higher viscosity. Following empirical evidence that the precipitation efficiency is more comprehensively characterized by PEG hydrodynamic radius (r(h,PEG)) than molecular weight, this paper proposes a model to explicate the significance of r(h,PEG). A general expression was formulated to characterize the PEG effect exclusively by r(h,PEG). The coefficients of a linearized form were then fitted using empirical solubility data. The result is a simple numerical relation that models the efficiency of general-shaped PEG precipitants as a function of r(h,PEG) and protein hydrodynamic radius (r(h,prot)). This equation also explains the effects of environmental conditions and PEG branching. While predictions by the proposed correlation agree reasonably well with independent solubility data, its simplicity gives rise to potential quantitative deviations when involving small proteins, large proteins and protein mixtures. Nonetheless, the model offers a new insight into the precipitation mechanism by clarifying the significance of r(h,PEG). This in turn helps to refine the selection criterion for PEG precipitants.
Sales of monoclonal antibody (mAbs) therapies exceeded $ 40 billion in 2010 and are expected to reach $ 70 billion by 2015. The majority of the approved antibodies are targeting cancer and autoimmune diseases with the top 5 grossing antibodies populating these two areas. In addition over 100 monoclonal antibodies are in Phase II and III of clinical development and numerous others are in various pre-clinical and safety studies. Commercial production of monoclonal antibodies is one of the few biotechnology manufacturing areas that has undergone significant improvements and standardization over the last ten years. Platform technologies have been established based on the structural similarities of these molecules and the regulatory requirements. These improvements include better cell lines, advent of high-performing media free of animal-derived components, and advances in bioreactor and purification processes. In this chapter we will examine the progress made in antibody production as well as discuss the future of manufacturing for these molecules, including the emergence of single use technologies.
Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG(1)) in a high-density suspension culture of PER.C6 cells. The cell culture harvest was clarified by enhanced cell settling (ECS) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train.
A highly productive chemically defined fed-batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell-based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc-fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale.
Over the past four years, several new types of experimental biologic treatment have received commercial registration, but the emergence of biosimilars represents the biggest shift in the biologic approval landscape.
Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.
The rapid development of high-yielding and robust manufacturing processes for monoclonal antibodies is an area of significant focus in the biopharmaceutical landscape. Advances in mammalian cell culture have taken titers to beyond the 5 g/l mark. Platform approaches to downstream process development have become widely established. Continuous evolution of these platforms is occurring as experience with a wider range of products is accrued. The increased cell culture productivity has shifted the attention of bioprocess development to operations downstream of the production bioreactor. This has rejuvenated interest in the use of non-chromatographic separation processes. Here, we review the current state-of-the-art industrial production processes, focusing on downstream technologies, for antibodies and antibody-related products and discuss future avenues for evolution.
The number of biotechnology-based pharmaceuticals in the late-stage pipeline has been increasing more than ever. As a result, there is an enhanced demand for more efficient and cost-effective processes. During the last years, the upstream technology for the production of biopharmaceuticals has been considerably improved. Continuous discoveries in molecular biology and genetics, combined with new advances in media and feed development, have significantly increased the production titres. In order to keep up this gain, it is now essential to design new, as well as to improve the existing downstream processes that remain an unresolved bottleneck. This review evaluates several alternatives to the currently established platforms for the downstream processing biopharmaceuticals, with main focus on aqueous two-phase extraction.
The large-scale production of recombinant monoclonal antibodies demands economical purification processes with high throughputs. The potential for ion-exchange membrane adsorbers to replace traditional ion-exchange columns was evaluated. Breakthrough capacities of commercially available cation-exchange membranes were determined as a function of flow-rate and layer number. Due to economic and process restrictions, cation-exchange membranes may not currently be advantageous for process-scale antibody purification in a bind and elute mode. However, anion-exchange membranes in a flow-through mode may provide a reasonable alternative to columns for the removal of low levels of impurities such as DNA, host cell protein, and virus.
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.
Sustained approvals of new biopharmaceuticals supported by a sparkling pipeline are the drivers for the above-market growth of biopharmaceuticals. Due to usually high therapeutic dose of monoclonal antibodies, they are demanding for high capacity needs. This requires significant capital investment and stimulates innovation for process improvement to decrease cost of goods and to save capital investments. Such process improvements are either ongoing along the learning curve or result from significant process changes through regulatory authorities impact. Both approaches require extensive protein analytical guidance to maintain product quality, safety and equivalency. In addition to second generation processes, second generation products have the feature of optimizing the physiological principle of biopharmaceuticals to the therapeutic need and to decrease the therapeutic dose, which goes along with investment savings and lower cost of goods.
Staphylococcal protein A can selectively interact with immunoglobulins. This protein is widely used as a ligand for affinity chromatography to purify therapeutic antibodies on an industrial scale. This type of affinity chromatography constitutes a generic step in processing antibodies. Questions of scale-up, design of chromatographic conditions, clearance of adventitious agents and operational modes, such as continuous operation or purification of antibodies in expanded-bed mode, will be addressed in this review.
The clinical and commercial success of monoclonal antibodies has led to the need for very large-scale production in mammalian cell culture. This has resulted in rapid expansion of global manufacturing capacity [1], an increase in size of reactors (up to 20,000 L) and a greatly increased effort to improve process efficiency with concomitant manufacturing cost reduction. This has been particularly successful in the upstream part of the process where productivity of cell cultures has improved 100 fold in the last 15 years. This success has resulted from improvements in expression technology and from process optimisation, especially the development of fed-batch cultures. In addition to improving process/cost efficiencies, a second key area has been reducing the time taken to develop processes and produce the first material required for clinical testing and proof-of-principle. Cell line creation is often the slowest step in this stage of process development. This article will review the technologies currently used to make monoclonal antibodies with particular emphasis on mammalian cell culture. Likely future trends are also discussed.
Pressures for cost-effective manufacture of antibodies are growing given their high doses and increasing market potential that have resulted in significant increases in total site capacities of up to 200,000 L. This paper focuses on the process economic issues associated with manufacturing antibodies and reviews the cost studies published in the literature; many of the issues highlighted are not only specific to antibodies but also apply to recombinant proteins. Data collated at UCL suggest current benchmark investment costs of $660-$1580/ft2 ($7130-$17,000/m2) and $1765-$4220/L for antibody manufacturing facilities with total site capacities in the range of 20,000-200,000 L; the limitations of the data are highlighted. The complications with deriving benchmark cost of goods per gram (COG/g) values are discussed, stressing the importance of stating the annual production rate and either titre or fermentation capacity with the cost so as to allow comparisons. The uses and limitations of the methods for cost analysis and the available software tools for process economics are presented. Specific examples found in the literature of process economic studies related to antibody manufacture for different expression systems are reviewed. The key economic drivers are identified; factors such as fermentation titre and overall yield are critical determinants of economic success. Future trends in antibody manufacture that are driven by economic pressures are discussed, such as the use of alternative expression systems (e.g. transgenics, E. coli and yeast), disposables, and improvements to downstream technology. The hidden costs and the challenges in each case are highlighted.
Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.
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.
Up to now, the productivity of mammalian cell culture has been perceived as limiting the productivity of the industrial manufacture of therapeutic monoclonal antibodies. Dramatic improvements in cell culture performance have changed this picture, and the throughput of antibody purification processes is gaining increasing attention. Although chromatographic separations currently are the centerpiece of antibody purification, mostly due to their high resolving power, it becomes more and more apparent that there may be limitations at the very large scale. This review will discuss a number of alternatives to chromatographic antibody purification, with a particular emphasis on the ability to increase throughput and overcome traditional drawbacks of column chromatography. Specifically, precipitation, membrane chromatography, high-resolution ultrafiltration, crystallization, and high-pressure refolding will be evaluated as potential large scale unit operations for industrial antibody production.
The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.
Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.
Improvements in upstream production have boosted productivity in the biomanufacturing industry, but this is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. Although chromatography remains an indispensible component of downstream processing due to its simplicity and high resolving power (The Good), there is virtually no economy of scale effect so more product translates almost linearly into greater production costs. Bind-and-elute processes (such as the initial capture step in antibody manufacturing) are volume-driven and therefore have knock-on effects that impact on the entire production facility since the space required for preparation, storage, and cleaning steps has to be similarly adapted (The Bad). During long-term operations with multiple cycles, thorough cleaning is necessary to prevent progressive fouling and microbial contamination (The Ugly). Innovative solutions are required, which may include revisiting simpler and less expensive separation technologies, the use of disposable modules, and the integration of improved processes that are scalable to cope with increased demands. Among the alternatives that have been put forward, membrane adsorbers are beginning to make a real impact on the industry, particularly for flow-through applications such as polishing and viral clearance.
Introduction: The Renaissance of Protein Purifica-tion. BioPharm Internat
- U Gottschalk
Gottschalk, U., Introduction: The Renaissance of Protein Purifica-tion. BioPharm Internat. 2006, 19, 8.
201400012 Research Article In vivo and in vitro activity of an immunoglobulin Fc fragment (Fcab) with engineered Her-2/neu binding sites Max Woisetschläger
- Kamila Napora-Wijata
- Gernot A Strohmeier
- Margit Winkler
Biotechnology
Journal
Kamila Napora-Wijata, Gernot A. Strohmeier
and Margit Winkler
http://dx.doi.org/10.1002/biot.201400012
Research Article
In vivo and in vitro activity of an immunoglobulin Fc fragment
(Fcab) with engineered Her-2/neu binding sites
Max Woisetschläger, Bernhard Antes, Radha Borrowdale,
Susanne Wiederkum, Manuela Kainer, Herta Steinkellner
Gordana Wozniak-Knopp, Kevin Moulder, Florian Rüker
and Geert C. Mudde
http://dx.doi.org/10.1002/biot.201300387
Research Article
Microscopic monitoring provides information on structure
and properties during biocatalyst immobilization
Sarka Bidmanova, Eva Hrdlickova, Josef Jaros, Ladislav
Ilkovics, Ales Hampl, Jiri Damborsky and Zbynek Prokop
http://dx.doi.org/10.1002/biot.201300049
Introduction: The Renaissance of Protein Purification.
- Gottschalk U.
Downstream Processing: Improving Productivity in Downstream Processing.
- Gail Sofer L. C. C.