Multi-column Chromatography Research Articles

Advancing biopharmaceutical manufacturing: economic and sustainability assessment of end-to-end continuous production of monoclonal antibodies

Monoclonal antibodies (mAbs) have become essential therapeutics for treating various diseases. The robust, cost-effective, and sustainable production of mAbs is crucial due to their growing clinical and commercial demand. Advances in bioprocessing, such as improved cell lines, perfusion bioreactors, multicolumn chromatography, and automation, can significantly increase productivity, making treatments more accessible. Streamlining the production process also aligns with environmental sustainability by reducing waste and energy consumption. This study quantifies the economic and environmental impacts of incorporating recent advances into end-to-end continuous bioprocessing of mAbs. The results demonstrate that, compared with an optimized best-in-class fed-batch process (with 15 g/l titer and multicolumn chromatography), continuous manufacturing can reduce the total annual production costs, facility footprint, plastic waste, and CO2 emissions by up to 23%, 51%, 57%, and 54%, respectively, in a producing clinical and commercial lots. Additionally, uncertainty analysis indicates that these gains are even more substantial under demand fluctuations.

Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid

Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTagTM affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTagTM for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99%) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTagTM as a universal capture platform for diverse classes of recombinant therapeutic proteins.

Comparison of batch and continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers

Protein A affinity membrane adsorbers are a promising alternative to resins to intensify the manufacturing of monoclonal antibodies. This study examined the process performance of convective diffusive membrane adsorbers operated in batch and continuous multi-column mode. Therefore, three different processes were compared regarding membrane utilization, productivity, and buffer consumption: the batch process, the rapid cycling parallel multi-column chromatography process, and the rapid cycling simulated moving bed process. The influence of the monoclonal antibody loading concentration (between 0.5 g L-1 and 5.2 g L-1) and the loading flow rate (between 1.25 MV min-1 and 10 MV min-1) on the monoclonal antibody binding behavior of the membrane adsorber were studied with breakthrough curve experiments. The determined breakthrough curves were used to calculate the monoclonal antibody dynamic binding capacity, the duration of the loading steps for each process, and the number of required membrane adsorbers for the continuous processes rapid cycling parallel multi-column chromatography and rapid cycling simulated moving bed. The highest productivity for the batch (176 g L-1 h-1) and rapid cycling parallel multi-column chromatography process (176 g L-1 h-1) was calculated for high monoclonal antibody loading concentrations and low loading flow rates. In contrast, the rapid cycling simulated moving bed process achieved the highest productivity (217 g L-1 h-1) for high monoclonal antibody loading concentrations and loading flow rates. Furthermore, due to the higher membrane utilization, the buffer consumption of the rapid cycling simulated moving bed process (1.1 L g-1) was up to 1.9 times lower than that of the batch or rapid cycling parallel multi-column chromatography operation (2.1 L g-1).

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Improved adeno-associated virus empty and full capsid separation using weak partitioning multi-column AEX chromatography.

Recombinant adeno-associated virus (rAAV) empty and full capsid separation has been a topic of interest in the rAAV gene therapy community for many years and the anion exchange chromatography (AEX) step has undergone various process optimizations to improve rAAV empty capsid separation, including AEX stationary phase, mobile phase, and process parameters. Here, we present a new AEX method that employs both weak partitioning chromatography (WPC) and multi-column chromatography (MCC) to achieve improved full rAAV percentage in the AEX pool. The WPC technology allows empty rAAV to be displaced by full rAAV during loading, while the MCC technology enables parallel column processing which further increases AEX step productivity. Our results show that, compared to baseline AEX batch chromatography, the AEX-WPC-MCC method demonstrated improvements in both AEX pool full rAAV percentage (∼ 20% increase) and rAAV genome recovery (∼ 20% increase). As a result, the productivity (full capsid generated per liter of AEX column per hour of processing time) of the AEX step increased by ∼34-fold from the baseline AEX batch run to the AEX-WPC-MCC run. It is foreseeable that this AEX-WPC-MCC method could find applications in large-scale rAAV manufacturing processes to improve AEX yield and reduce the cost of goods of rAAV manufacturing.

Advanced control strategies for continuous capture of monoclonal antibodies based upon biolayer interferometry.

The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield. This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi-column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow-through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement.

Continuous multi-membrane chromatography of large viral particles

Continuous multi-column chromatography (CMCC) has been successfully implemented to address biopharmaceutical biomolecule instability, to improve process efficiency, and to reduce facility footprint and capital cost. This paper explores the implementation of a continuous multi-membrane chromatography (CMMC) process, using four membrane units, for a large viral particle in just few weeks. CMMC improves the efficiency of the chromatography step by enabling higher loads with smaller membranes for multiple cycles of column use and enables steady-state continuous bioprocessing. The separation performance of CMMC was compared to a conventional batch chromatographic capture step used at full manufacturing scale. The product step yield was 80% using CMMC versus 65% in batch mode while increasing slightly the relative purity. Furthermore, the total amount of membrane area required for the CMMC approach was approximately 10% of the area needed for batch operation, while realizing similar processing times. Since CMMC uses smaller membrane sizes, it can take advantage of the high flow rates achievable for membrane chromatography that are not typically possible at larger membrane scales due to skid flow rate limitations. As such, CMMC offers the potential for more efficient and cost-effective purification trains.

Rhamnogalacturonan I-Enriched Pectin, Flavonoids, and Alkaloids from Lotus Leaf Infusion in Regulating Glycolipid Absorption and Metabolism: Isolation, In Vitro Bioactivity Verification, and Structural Characterization.

Lotus leaf is effective in regulating glycolipid absorption and metabolism, but the roles of small-molecule compounds and polysaccharides are unknown. In this study, the small-molecule compounds including flavonoids, alkaloids, and polysaccharides were gradually isolated from lotus leaf infusion by multi-column chromatography and applied to in vitro activity verification and structural characterization. Although flavonoids and alkaloids were effective in inhibiting pancrelipase and α-glucosidase, polysaccharides more effectively bounded bile acids, inhibited cholesterol micelle solubility, and stimulated the growth of Bifidobacterium than lotus leaf infusion. Polysaccharides, presented as spherical conformation in water, were identified as rhamnogalacturonan I-enriched (93%) low-ester pectin with multiple branches mainly composed of arabinan, arabinogalactan-type II, and galactan formed by →3)-Galp-(1→, →5)-Araf-(1→ and →4)-Galp-(1→ residues. Polysaccharides, which were a key constituent of lotus leaf infusion in regulating glycolipid absorption and metabolism, should be paid more attention and developed as a functional food ingredient.

Purification and identification of globulin-1 S allele as a novel allergen with N-glycans in wheat (Triticum aestivum)

Wheat (Triticum aestivum) contains various allergenic proteins and induces diverse allergic reactions. Although many allergens are found to be glycoproteins, research on glycosylated allergens in wheat is rare. In this study, the main glycoprotein in wheat soluble protein was purified by sequential multi-column chromatography including affinity chromatography, gel filtration chromatography, and ion-exchange chromatography. The purified proteins were identified as globulin-1 S allele by liquid chromatography-tandem mass spectrometry and characterized by circular dichroism spectra. The strong IgE-binding capacity of the purified fractions was further demonstrated, suggesting globulin-1 S allele to be a novel allergen in wheat. Further de-N-glycosylated treatment by N-Glycosidase F showed that N-glycosylation enhanced the IgE-binding of globulin-1 S allele. These results identified globulin-1 S allele as a novel allergen in wheat, demonstrated the role of glycosylation in IgE-binding, and provided new insights into the prevention and treatment of wheat allergy.

Productivity for free: Residence time gradients during loading increase dynamic binding capacity and productivity

• An algorithm to improve productivity and dynamic binding capacity in protein chromatography. • A controller to optimize the flow rate gradient during loading. • The theoretical concept has been validated by chromatography experiments. • The algorithm allows a productivity increase of 68% in Protein A chromatography without additional expense. Traditional chromatographic purification steps consist of long loading steps at a constant residence time, especially during early stages of the downstream process. The breakthrough behavior of the product is usually well defined. This information can be used to optimize the loading phase by starting the loading phase at a low residence time and gradually increasing the residence time to boost productivity and resin utilization. Process modeling of the loading of lysozyme on a Toyopearl SP-650C stationary phase, a strong cation exchanger, revealed optimal residence time gradients. These gradients result in a faster breakthrough in the time domain, while having the same breakthrough as constant residence time loading, in the volume domain. Modeling also revealed that a delayed start of the gradient further increases productivity. Eventually, several mathematical optimizations were employed to ensure the optimality of the idea. We confirmed the process model results in lab experiments using lysozyme breakthrough in ion-exchange chromatography and monoclonal antibody breakthrough in protein A affinity chromatography. Productivity of the loading step could be increased by 68 % from 40.8 to 68.7 g L -1 h −1 resin for protein A chromatography while retaining the same breakthrough behavior and better manufacturability of unstable proteins. Consequently, the bound fraction of protein is maintained above 99 %. This affords similar productivity gains as multicolumn chromatography while employing an easy-to-implement process model that can be used on conventional systems.

Evaluation of the Efficiency of Continuous Capture Chromatography

Multi-column chromatography (periodic counter-current chromatography, PCCC) with protein A affinity resins is used as an efficient monoclonal antibody (mAb) continuous capture process. However, the evaluation of the efficiency of PCCC process compared with repeated batch (or cyclic) operation (RBO) is still not standardized. In this study, we proposed two criteria for evaluating the capture process efficiency, productivity P and buffer consumption per load amount, BC. These criteria were calculated for 2 column, 3 column and 4 column-PCCC and for RBO. For PCCC, P was proportional to mAb concentration C0 and inversely proportional to the number of columns, nC since the loaded amount of mAb ML was fixed for the calculation. Bc did not depend on C0 or nC as ML was the same. For RBO, BC was larger by 20% at residence time (RT) = 4 minutes, and by 50% at RT = 2 minutes whereas P was similar to or slightly lower than that for 2C-PCCC at the same RT (4 minutes).

A regressive approach to the design of continuous capture process with multi-column chromatography for monoclonal antibodies

Although empirical methods have been introduced in the process development of continuous chromatography, the common approach to optimize a multi-column continuous capture chromatography (periodic counter-current chromatography, PCCC) process heavily relies on numerical model simulations and the number of experiments. In addition, different multi-column settings in PCCC add more design variables in process development. In this study, we have developed a rational method for designing PCCC processes based on iterative calculations by mechanistic model-based simulations. Breakthrough curves of a monoclonal antibody were measured at different residence times for three protein A resins of different particle sizes and capacities to obtain the parameters needed for the simulation. Numerical calculations were performed for the protein sample concentration in the range of 1.5 to 4 g/L. Regression curves were developed to describe the relative process performances compared with batch operation, including the resin capacity utilization and the buffer consumption. Another linear correlation was established between breakthrough cut-off (BT%) and a modified group composed of residence time, mass transfer coefficient, and particle size. By normalizing BT% with binding capacity and switching time, the linear regression curves were established for the three protein A resins, which are useful for the design and optimization of PCCC to reduce the process development time.

Current status and future challenges in transitioning to continuous bioprocessing of virus‐like particles

AbstractVirus‐like particles (VLPs) are bioengineered protein complexes that can serve as prophylactic vaccines due to favorable immunological characteristics like size, repetitive surface geometry and ability to induce both innate and adaptive immune responses, without being infectious. Large‐scale manufacturing of VLPs presents some interesting and unique challenges associated with removal of refractory contaminants to attain high product purity, improvement in structural homogeneity and colloidal stability. Recent advancements in production and purification of VLP‐based vaccines, such as application of monolithic multicolumn chromatography and inline concentration and diafiltration, have set a stage for implementation of continuous bioprocessing for commercial manufacturing of VLPs. The benefits of continuous bioprocessing have been well demonstrated for biotherapeutics, particularly monoclonal antibodies and small peptides. With the growing demand for affordable vaccines, the biopharmaceutical industry has shown increasing interest in establishing end‐to‐end continuous platforms. This article reviews major developments that have occurred in continuous processing for the manufacturing of VLPs. The major problems inherent in transitions from batch to continuous processing are also discussed, along with potential strategies to overcome the challenges. © 2021 Society of Chemical Industry (SCI).

Development and optimization of multicolumn chromatography for mAb perfusion processes

AbstractThe maturation of the bioprocessing industry is driving the desire to reduce manufacturing costs through process intensification. Here we discuss the motivation for more productive upstream perfusion processes and the implications for downstream purification. The focus of the article is how to optimize the downstream process chromatography steps based on perfusion upstream. A range of different chromatography solutions are explored and their possible impact on the purification process discussed. © 2021 Society of Chemical Industry

Enhancing protein A productivity and resin utilization within integrated or intensified processes.

Recent interest in continuous manufacturing of biologics has driven the development and evaluation of multicolumn chromatography systems to drive down resin costs by increasing productivity and maximizing resin utilization, especially for the expensive protein A capture step. Single-pass tangential flow filtration can be used to reduce the volume of perfusion harvest, enabling a further increase in the productivity of the capture step by up to fivefold. However, there are expected to be practical limits for the productivity of the capture step, which must be determined based on the manufacturing batch size, duration, and frequency, especially as it relates to efficient utilization of the column lifetime. For short fed-batch manufacturing campaigns, intensified capture processes may result in up to 82% lower resin consumption, while avoiding the long-term storage of used resin. For perfusion processes and longer fed-batch campaigns, it may be more efficient to operate at a lower productivity that enables the column lifetime to be routinely achieved and achieves the desired resin and buffer savings without introducing unnecessary process risk or complexity. An intensified batch capture process, "super-batch," will be compared as an alternative to multicolumn chromatography processes to achieve high productivity and resin utilization with a potentially simpler process.

基于集成化多柱二维液相色谱系统分析血清中的氨磺必利

快速准确的治疗药物监测对于临床上确保患者用药有效性及安全性至关重要,同时也能够确定患者用药依从性,制定个性化给药方案。该文以两支疏水性略有差异的反相分离柱Supersil ODS2和SinoChrom ODS-BP,及强阳离子交换捕集柱Supersil SCX构建了基于集成化的多柱二维液相色谱系统。通过二维色谱接口,以pH 3.0的磷酸缓冲液调整第一维分离后的洗脱液组成,降低有机相含量并维持pH,改善了中心切割模式下样品转移和捕集的效率。利用该多柱二维液相色谱系统发展了血清中氨磺必利的二维液相色谱检测方法,血清样品经过高氯酸和甲醇混合液沉淀蛋白质并离心后直接300 μL大体积进样,以乙腈/磷酸缓冲液(25 mmol/L, pH 3.0)(20/80, v/v)作为第一维分离流动相,磷酸缓冲液(25 mmol/L, pH 3.0)作为捕集过程的稀释流动相,乙腈/磷酸缓冲液(25 mmol/L, pH 7.0)(25/75, v/v)作为第二维分离流动相,12 min内即可完成分析。方法在10~200 ng/mL的范围内线性相关性良好(r=0.9998)。样品在50 ng/mL和100 ng/mL两个加标浓度下的回收率稳定,在73.7%~76.8%之间。方法的检出限为7.28 ng/mL,定量限为24.27 ng/mL,能够满足《神经精神药理学治疗药物检测共识指南》中推荐的药物监控范围要求。由于该系统日常使用及维护成本较低,且能够实现自动化分析,故该方法适合在临床上用于治疗药物监测研究。

Gamma irradiating chromatography columns enables bioburden-free integrated continuous biomanufacturing.

An important consideration for integrated continuous biomanufacturing is that the downstream chromatography steps integrated with the bioreactor should maintain a low bioburden state throughout the entire duration of the operation. One potential strategy to achieve this is to start bioburden-free and functionally close the chromatography system. While chromatography skids themselves can be rendered bioburden-free, limitations exist in applying these methods to chromatography columns. The small column sizes used in continuous multicolumn chromatography enable gamma irradiation of disposable columns to render them bioburden-free. However, this approach has not been widely implemented, likely because gamma irradiation can negatively impact resin performance. Here, several protective mobile-phase modifiers were screened and shown to help chromatography resins retain naïve-like performance. Gamma irradiated columns were then integrated into perfusion bioreactors for continuous capture. Successful integrated continuous capture downstream of perfusion bioreactors for greater than 40 days using protein A, custom affinity, and non-affinity capture resins for multiple biologic modalities is demonstrated in development and commercial settings. No indications of time-based performance decline or bioburden growth have been observed. This strategy enables bioburden-free integrated continuous biomanufacturing operations and could allow full process closure and decreased environmental control requirements for facilities; thus, permitting simultaneous multi-product operations in a ballroom arrangement.

Model-assisted approaches for continuous chromatography: Current situation and challenges

Continuous bioprocessing is a promising trend in biopharmaceutical production, and multi-column continuous chromatography shows advantages of high productivity, high resin capacity utilization, small footprint, low buffer consumption and less waste. Due to the complexity and dynamic nature of continuous processing, traditional experiment-based approaches are often time-consuming and inefficient. In this review, model-assisted approaches were focused and their applications in continuous chromatography process development, validation and control were discussed. Chromatographic models are useful in describing particular process performances of continuous capture and polishing with multi-column chromatography. Model-assisted tools showed powerful ability in evaluating multiple operating parameters and identifying optimal points over the entire design space. The residence time distribution models, model-assisted process analytical technologies and model-predictive control strategies were also developed to reveal the propagation of disturbances, enhance real time monitor and achieve adaptive control to match the transient disturbances and deviations of continuous processes. Moreover, artificial neural networks and machine learning concepts were integrated into modeling approaches to improve data treatment. In general, further development in research and applications of model-assisted approaches for continuous chromatography are needed urgently to support the continuous manufacturing.

Simulations of unsteady flows with adsorption equilibrium in dynamic axial compression column

Simulated moving bed chromatography process, which is a multicolumn chromatography process, has been used in various industrial applications. Dynamic axial compression columns are key elements in simulated moving beds, and their flow characteristics are worth exploring using state-of-the-art numerical methodologies. In this study, new fluid distributors for the dynamic axial compression column were designed and fabricated based on mass conservation in fluid mechanics and the computer-aided design in the preliminary stage. Computational fluid dynamics was employed to resolve the flow field, and the numerical chromatograms were validated by laboratory experiments. For the computational fluid dynamics–based simulation of flow in the dynamic axial compression, the transient laminar flow fields were described by the momentum and species transport equations with Darcy’s law to model the porous zone in the packed bed. In addition, reverse engineering processes were applied to obtain the unknown physical parameters, such as viscous resistance and adsorption equilibrium coefficients. Moreover, including the adsorption equilibrium equation in the fundamental governing equations made the simulated results agree with the experimental data in chromatograms, providing a more feasible result for practical applications.

Batch chromatography with recycle lag. I—Concept and design

This is the first of a two-part study in which we explore the concept of batch chromatography with recycle lag, concluding with the design, construction, and experimental validation of a prototype that embodies the physical realization of this concept. Moreover, the apparatus is simple to set up in particular in view of large-scale applications. Here the theory behind batch chromatography with recycle lag is revisited and extended, with emphasis on the mathematical formulation and procedure for deriving the single-column batch analogue of any variant of multicolumn simulated countercurrent chromatography. By resorting to selected examples, namely GE Healthcare Bio-science’s three-column periodic countercurrent chromatography, Novasep’s sequential multicolumn chromatography, and a few hypothetical multicolumn processes, we discuss how the theory can be operationalized. Finally, we conclude by describing the design of a device or apparatus—an eluate recycling device (ERD)—to physically realize the proposed concept. The ERD implements an approximate “first in, first out” method for organizing and manipulating the to-be-recycled fractions of eluate collected from the chromatography column, where the oldest (first) amount fluid, or ‘head’ of the fraction, is the first to exit and be recycled to the column.