Multicolumn Countercurrent Solvent Gradient Purification Process Research Articles

Enhancing the purification of crocin-I from saffron through the combination of multicolumn countercurrent chromatography and green solvents

Crocin-I, a valuable natural compound found in saffron (Crocus sativus L.), is the most abundant among the various crocin structures. Developing a cost-effective and scalable purification process to produce high-purity crocin-I is of great interest for future investigations into its biological properties and its potential applications in the treatment of neurological disorders. However purifying crocin-I through single-column preparative chromatography (batch) poses a yield-purity trade-off due to structural similarities among crocins, meaning that the choice of the collection window sacrifices either yield in benefit of higher purity or vice versa. This study demonstrates how the continuous countercurrent operating mode resolves this dilemma. Herein, a twin-column MCSGP (multicolumn countercurrent solvent gradient purification) process was employed to purify crocin-I. This study involved an environmentally friendly ethanolic extraction of saffron stigma, followed by an investigation into the stability of the crocin-I within the feed under varying storage conditions to ensure a stable feed composition during the purification. Then, the batch purification process was initially designed, optimized, and subsequently followed by the scale-up to the MCSGP process. To ensure a fair comparison, both processes were evaluated under similar conditions (e.g., similar total column volume). The results showed that, at a purity grade of 99.7%, the MCSGP technique demonstrated significant results, namely + 334% increase in recovery + 307% increase in productivity, and − 92% reduction in solvent consumption. To make the purification process even greener, the only organic solvent employed was ethanol, without the addition of any additive. In conclusion, this study presents the MCSGP as a reliable, simple, and economical technique for purifying crocin-I from saffron extract, demonstrating for the first time that it can be effectively applied as a powerful approach for process intensification in the purification of natural products from complex matrices.Graphical

Improved process design for monoclonal antibody charge variants separation with multicolumn counter-current solvent gradient purification

The multicolumn counter-current solvent gradient purification (MCSGP) method has proven effective in addressing the issue of elution profile overlap for difficult-to-separate proteins, leading to improved purity and recovery. However, during the MCSGP process, the flow rate and proportion of loaded proteins undergo changes, causing a significant discrepancy between the elution profiles of batch process design and the actual MCSGP process. This mismatch negatively impacts the purity and recovery of the target protein. To address this challenge, an improved process design (reDesign) was proposed with the first-run MCSGP to mimic the actual continuous process and obtain elution profiles that closely resemble the real ones. The reDesign was demonstrated with both a model protein mixture and a sample of monoclonal antibody (mAb) with charge variants. For model protein mixture, the reDesign-based MCSGP process (reMCSGP) showed a remarkable improvement in recovery, increasing from 83.6% to 97.8% while maintaining a purity of more than 95%. For mAb sample, the recovery of reMCSGP improved significantly to 93.9%, surpassing the performance of normal MCSGP processes at a given purity level of more than 84%. In general, the new process design strategy developed in this work could generate a more representative elution profile that closely mirrors actual conditions in continuous processes, which enhances the separation performance of MCSGP.

Purification of a GalNAc-cluster-conjugated oligonucleotide by reversed-phase twin-column continuous chromatography

Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a continuous chromatography technique used to maximize purification yields compared to traditional batch purification methods. Here we apply MCSGP for the reversed phase purification of a N-acetylgalactosamine (GalNAc)-cluster-conjugated DNA-LNA gapmer oligonucleotide therapeutic using a twin-column chromatography system. Based on a batch process as a starting point, MCSGP was designed, optimized and compared with the batch process regarding process performance and scale-up requirements. Product yields increased from 52.7% using batch chromatography to 91.5% using MCSGP, with purity, productivity, and buffer consumption otherwise comparable. In a manufacturing scenario, use of MCSGP would allow the downscaling of oligonucleotide synthesis by 42.5%, which would result in a significant cost reduction and increased throughput. Moreover, the equipment, chemicals and methodology used in MCSGP are analogous to a standard reversed phase purification allowing for a “like for like” transition to the upgraded MCSGP process.

Experimental Design of the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) Unit for the Separation of PEGylated Proteins

The rapid growth and diffusion of biopharmaceuticals, including polyethylene glycol (PEG)–protein conjugates, required to set up stringent regulatory requirements, in terms of purity and clinical safety. In order to produce proteins that meet these requirements, the development of robust purification methods has been one of the main goals in the field of downstream processing in the last years. Most of these methods rely on single or sequential batch chromatographic separations, enabling one to reach the purity specification but often at the expense of low yield. An appealing alternative is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP). Thanks to the internal recycling of the fractions where product and impurities coelute, this process can provide a significant improvement in the process yield, preserving the purity specification. The drawback lies in the increased number of process parameters, compared to the batch, which complicates the design and optimization of this unit. In this work, we propose an ad hoc design procedure for the optimization of central-cut separations at a target purity. Using PEGylated lysozyme as a model system, we illustrate the use of this procedure to identify the load, the elution gradient as well as the collection intervals for optimal yield and productivity, at fixed purity specifications, for a batch (single) column system. The obtained optimal process parameters are subsequently transferred to the MCSGP unit. The so-designed MCSGP process exhibited superior performances compared to the corresponding optimal batch process by increasing the yield and productivity by 17.0% and 2.1%, respectively, at the purity specification of 80%.

From batch to continuous chromatographic purification of a therapeutic peptide through multicolumn countercurrent solvent gradient purification

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated. Moreover, the purification process can be completely automatized.Aim of this work is to present a simple procedure for the development of the MCSGP process based on a single batch experiment, in the case of a therapeutic peptide of industrial relevance. This allowed to recover roughly 90% of the injected glucagon in a purified pool with a purity of about 90%.A comparison between the performance of the MCSGP process and the classical single column batch process indicates that percentage increase in the recovery of target product is +23% when transferring the method from batch conditions to MCSGP, with an unchanged purity of around 89%. This improvement comes at the expenses of a reduction of about 38% in productivity.

Process for Continuous Fab Production by Digestion of IgG

Intensified processing and end-to-end integrated continuous manufacturing are increasingly being considered in bioprocessing as an alternative to the current batch-based technologies. Similar approaches can also be used at later stages of the production chain, such as in the post-translational modifications that are often considered for therapeutic proteins. In this work, a process to intensify the enzymatic digestion of immunoglobulin G (IgG) and the purification of the resulting Fab fragment is developed. The process consists of the integration of a continuous packed-bed reactor into a multicolumn chromatographic process. The integration is realized through the development of a novel multicolumn countercurrent solvent gradient purification (MCSGP) process, which, by adding a third column to the classical two-column MCSGP process, allows for continuous loading and then straight-through processing of the mixture leaving the reactor.

Experimental Evaluation of the Impact of Intrinsic Process Parameters on the Performance of a Continuous Chromatographic Polishing Unit (MCSGP).

The semicontinuous twin-column multicolumn countercurrent solvent gradient purification (MCSGP) process improves the trade-off between purity and yield encountered in traditional batch chromatography, while its complexity, in terms of hardware requirements and process design, is reduced in comparison to process variants using more columns. In this study, the MCSGP process is experimentally characterized, specifically with respect to its unique degrees of freedom, i.e., the four switching times, which alternate the columns between interconnected and batch states. By means of isolation of the main charge isoform of an antibody, it is shown that purity is determined by the selection of the product collection window with negligible influence from the recycle phases. In addition, the amount of weak and strong impurities can be specifically attributed to the start and end of the collection, respectively. Due to higher abundance of weakly adsorbing impurities, the start of product collection influences productivity and yield more than the other switching times. Furthermore, most of the encountered tendencies scale between different loadings. The found trends can be rationalized from the corresponding batch chromatogram and therefore used during process design to obtain desirable process performances without extensive trial-and-error experimentation or complete model development and calibration.

Equilibrium Theory Based Design Space for the Multicolumn Countercurrent Solvent Gradient Purification Process

A procedure for designing the operation parameter space for the twin-column multicolumn countercurrent solvent gradient purification (MCSGP) process for the purification of therapeutic proteins is derived. This is based on the equilibrium theory, which assumes instantaneous equilibrium conditions. As the MCSGP process allows protein separation with a linear modifier gradient, all equations are derived in terms of the covered distance as a function of the modifier concentration in ion-exchange chromatography. All constraints, which need to be fulfilled in order to obtain a stable process with maximum yield and purity, are described as a function of the different process parameters. For operation parameters within the parameter space where all constraints are fulfilled, a stable process is predicted. Additionally, on the boundary of this region, the optimal operation point in terms of buffer consumption and productivity can be found. Besides, the presented design space can help to analyze the impact of diff...

Experimental design of a twin-column countercurrent gradient purification process

As typical for separation processes, single unit batch chromatography exhibits a trade-off between purity and yield. The twin-column MCSGP (multi-column countercurrent solvent gradient purification) process allows alleviating such trade-offs, particularly in the case of difficult separations. In this work an efficient and reliable procedure for the design of the twin-column MCSGP process is developed. This is based on a single batch chromatogram, which is selected as the design chromatogram. The derived MCSGP operation is not intended to provide optimal performance, but it provides the target product in the selected fraction of the batch chromatogram, but with higher yield. The design procedure is illustrated for the isolation of the main charge isoform of a monoclonal antibody from Protein A eluate with ion-exchange chromatography. The main charge isoform was obtained at a purity and yield larger than 90%. At the same time process related impurities such as HCP and leached Protein A as well as aggregates were at least equally well removed. Additionally, the impact of several design parameters on the process performance in terms of purity, yield, productivity and buffer consumption is discussed. The obtained results can be used for further fine-tuning of the process parameters so as to improve its performance.

Intelligent, model-based control towards the intensification of downstream processes

Process Intensification (PI) has been gaining increasing interest as industrial trends urge a shift towards more eco-efficient processes of significantly decreased operation and capital costs. In this direction we focus on the development of advanced control strategies of the Multicolumn Countercurrent Solvent Gradient Purification Process (MCSGP), an industrial, semi-continuous, chromatographic process, used for the purification of several biomolecules. We present a novel control approach that manages to drive the process towards continuous, sustainable operation. The presented controllers are designed within the PARametric Optimization and Control (PAROC) framework/software platform that enables the development of intelligent, model-based controllers through a step-by-step approach. The controllers are successfully tested against various disturbance profiles and they manage to track the predefined setpoints without significant offset.

Advanced control strategies for the multicolumn countercurrent solvent gradient purification process

The multicolumn countercurrent solvent gradient purification process (MCSGP) is a semicontinuous, chromatographic separation process used in the production of monoclonal antibodies) . The process is characterized by high model complexity and periodicity that challenge the development of control strategies, necessary for feasible and efficient operation and essential toward continuous production. A novel approach for the development of control policies for the MCSGP process, which enables efficient continuous process control is presented. Based on a high fidelity model, the recently presented PAROC framework and software platform that allows seamless design and in‐silico validation of advanced controllers for complex systems are followed. The controller presented in this work is successfully tested against disturbances and is shown to efficiently capture the process periodic nature. © 2016 American Institute of Chemical Engineers AIChE J, 62: 2341–2357, 2016

Model-Based Comparison of Batch and Continuous Preparative Chromatography in the Separation of Rare Earth Elements

The demand for rare earth elements (REEs) is growing, while the future supply is uncertain. Their unique electronic characteristics make them irreplaceable, and the commercial value of pure fractions is high. A model-based simulation study is presented that compares batch chromatography with the twin-column MCSGP (multicolumn countercurrent solvent gradient purification) process for ion-exchange chromatography of the four-component system neodymium, samarium, europium, and gadolinium. The last three components are considered products with individual purity requirements of 99%. The twin-column process has been shown to be a good alternative to the batch process regarding modifier consumption and productivity since it enables internal recycling to achieve high purities. A new cut strategy for MCSGP is applied where subfractions are taken from each outlet. Two multiobjective optimizations with yield, solvent productivity, and productivity objectives show that the MCSGP process is a better alternative than batch chromatography.

Closed loop control of the multi-column solvent gradient purification process

A PID controller able to support the operator in the operation of the Multi-column Countercurrent Solvent Gradient Purification (MCSGP) process which is a continuous, countercurrent chromatographic process has been developed. As measurement, only the online UV signals at each column outlet are used. This guarantees a simple and cheap control implementation and a fast control action. Accordingly, the controller does not guarantee any purity or yield value, but simply that the withdrawn window of the product is centered in a specific region of the UV chromatogram where the purity specifications are expected to be satisfied. This can be determined by the operator based on the batch chromatogram selected for designing the MCSGP operating conditions. Thus the controller provides a reliable and efficient tool for the operator to run properly a MCSGP unit in combination with suitable offline analytics for the quantification of purity and yield. The applications are discussed involving the purification of a model protein and a peptide. It is shown that the developed controller is effective in driving the unit to steady state during start up and in keeping a stable steady state while rejecting external disturbances.

Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP)

A two-step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP-captured material in batch mode. Using multi-modal anion exchange in bind-elute mode, mAb was produced consistently within the purity specification. The benchmark was a state-of-the-art 3-step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2-step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase.

Role of Cleaning-in-Place in the Purification of mAb Supernatants Using Continuous Cation Exchange Chromatography

Monoclonal antibodies (mAbs) are among the most important therapeutic proteins for the treatment of cancer. They also find wide application in the field of diagnostics. The market for mAbs was US$ 5.4 billion in 2002 and is expected to triple by 2010 (1). MAbs are mainly produced in cell culture and purified by chromatography. Apart from the mAb, the downstream processing is confronted with a multitude of impurities from the upstream process. Some of these impurities bind strongly to the chromatographic stationary phase and can only be removed by separate cleaning solutions. Depending on the type of media used, e.g. affinity or ion exchange, differing cleaning regimes may have to be applied. In addition, sanitization is required to prevent microbial contamination. Both steps are closely tied to the mAb production step and need to be repeated in regular intervals. In this work, the impact of irreversible impurity adsorption on the separation performance is investigated experimentally; first for a batch column and then for a continuous chromatographic process. At first, the retention time of a model substance is established as a suitable measure to describe the degree of irreversible adsorption. The impact of the presence and the absence of cleaning-in-place (CIP) on the retention time of the model substance is demonstrated. Since the issue of column cleaning is particularly important if processes are operated in continuous mode, in the second part of this work the introduction of a CIP step in the continuous multicolumn countercurrent solvent gradient purification (MCSGP) process is investigated. The process purifies a mAb from a cell culture supernatant that also contains irreversibly adsorbing impurities—or impurities that desorb in a later cycle of the process as a contamination. Following the same approach as for the batch experiments, the MCSGP process is operated in the presence and the absence of CIP in order to track prospective changes of the process performance caused by changes in the product retention time. Instead of the model substance, a cell culture supernatant, providing a real case impurity profile in an industrial application is used for the process. Although an impact of the product retention time change on the process yield is not observed it is shown that CIP is required for a stable long-term operation in terms of purity and system pressure drop. With the introduction of a CIP step the process was successfully operated for 9000 min (6 days) without interruption.

Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP)

Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.