Multicolumn Countercurrent Solvent Gradient Purification Research Articles

Model-based optimization strategy for intensification in the chromatographic purification of oligonucleotides

Oligonucleotides (ONs) are acquiring clinical relevance and their demand is expected to grow. However, the ON production capacity is currently limited by high manufacturing costs. Since the purification of the target ON sequence from molecularly similar variants represents a major bottleneck, this work presents a resource-effective strategy for the optimization of their preparative reversed-phase chromatographic purification. First, a model based on the equilibrium-dispersive theory was introduced to describe the chromatographic operation. Considering a deoxyribose nucleic acid with 20 nucleobases as case study, a genetic algorithm was developed to efficiently determine the adsorption isotherm and mass transfer parameters for the target ON and impurities. After the estimation of these parameters, a strategy for the in-silico optimization of the operation was established. The product collection window, gradient duration, and resin loading were considered as process variables and their influence on yield and productivity was investigated after setting a purity specification of 99.0%. The optimal process parameters identified through this analysis were experimentally verified, confirming the reliability of the model, calibrated with only 5 experimental runs. In addition, this optimal setpoint was exploited to design the multicolumn countercurrent solvent gradient purification (MCSGP) of this ON mixture, which allowed to boost the yield of the process and to work at cyclic steady state, while respecting the purity constraint. This study confirmed the potential of this in-silico optimization strategy in both improving the performance of the traditional single-column operations and in the rapid development of multicolumn processes.

UV-based dynamic control improves the robustness of multicolumn countercurrent solvent gradient purification of oligonucleotides.

Therapeutic oligonucleotides (ONs) have great potential to treat many diseases due to their ability to regulate gene expression. However, the inefficiency of standard purification techniques to separate the target sequence from molecularly similar variants is hindering development of large scale ON manufacturing at a reasonable cost. Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a valuable process able to bypass the purity-yield tradeoff typical of single-column operations, and hence to make the ON production more sustainable from both an economic and environmental point of view. However, operating close to the optimum of MCSGP can be challenging, resulting in unstable process performance and in a drift in product quality, especially when running a continuous process for extended periods where process parameters such as temperature are prone to variation. In this work, we demonstrate how greater process robustness is introduced in the design and execution of MCSGP for the purification of a 20mer single-stranded DNA sequence through the implementation of UV-based dynamic control. With this novel approach, the cyclic steady state was reached already in the third cycle and disturbances coming from fluctuations in the feed quality, loading amount and temperature were effectively compensated allowing a stable operation close to the optimum. In response to the perturbations, the controlled process kept the standard deviation on product recovery below 3.4%, while for the non-controlled process it increased up to 27.5%.

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.

Role of the gradient slope during the product internal recycling for the multicolumn countercurrent solvent gradient purification of PEGylated proteins

Protein PEGylation, i.e. functionalization with poly(ethylene glycol) chains, has been demonstrated an efficient way to improve the therapeutic index of these biopharmaceuticals. We demonstrated that Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is an efficient process for the separation of PEGylated proteins (Kim et al., Ind. and Eng. Chem. Res. 2021, 60, 29, 10764–10776), thanks to the internal recycling of product-containing side fractions. This recycling phase plays a critical role in the economy of MCSGP as it avoids wasting valuable product, but at the same time impacts its productivity extending the overall process duration. In this study, our aim is to elucidate the role of the gradient slope within this recycling stage on the yield and productivity of MCSGP for two case-studies: PEGylated lysozyme and an industrially relevant PEGylated protein. While all the examples of MCSGP in the literature refer to a single gradient slope in the elution phase, for the first time we systematically investigate three different gradient configurations: i) a single gradient slope throughout the entire elution, ii) recycling with an increased gradient slope, to shed light on the competition between volume of the recycled fraction and required inline dilution and iii) an isocratic elution during the recycling phase. The dual gradient elution proved to be a valuable solution for boosting the recovery of high-value products, with the potential for alleviating the pressure on the upstream processing.

Design and economic investigation of a Multicolumn Countercurrent Solvent Gradient Purification unit for the separation of an industrially relevant PEGylated protein

Conjugation of biopharmaceuticals to polyethylene glycol chains, known as PEGylation, is nowadays an efficient and widely exploited strategy to improve critical properties of the active molecule, including stability, biodistribution profile, and reduced clearance. A crucial step in the manufacturing of PEGylated drugs is the purification. The reference process in industrial settings is single-column chromatography, which can meet the stringent purity requisites only at the expenses of poor product recoveries. A valuable solution to this trade-off is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which allows the internal and automated recycling of product-containing side fractions that are typically discarded in the batch processes. In this study, an ad hoc design procedure was applied to the single-column batch purification of an industrially relevant PEGylated protein, with the aim of defining optimal collection window, elution duration and elution buffer ionic strength to be then transferred to the MCSGP. This significantly alleviates the design of the continuous operation, subjected to manifold process parameters. The MCSGP designed by directly transferring the optimal parameters allowed to improve the yield and productivity by 8.2% and 17.8%, respectively, when compared to the corresponding optimized batch process, ensuring a purity specification of 98.0%. Once the efficacy of MCSGP was demonstrated, a detailed analysis of its cost of goods was performed and compared to the case of single-column purification. To the best of our knowledge, this is the first example of a detailed economic investigation of the MCSGP across different manufacturing scenarios and process cadences of industrial relevance, which demonstrated not only the viability of this continuous technology but also its flexibility.

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.

Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography.

The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification problem. Different modes of chromatography that can cover almost every separation case are reviewed in this article. Additionally, an outlook to a very recent continuous chromatographic process (namely Multicolumn Countercurrent Solvent Gradient Purification, MCSGP) and future perspectives regarding purification strategies will be considered at the end of this review.

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%.

Process Intensification for the Purification of Peptidomimetics: The Case of Icatibant through Multicolumn Countercurrent Solvent Gradient Purification (MCSGP)

Biopharmaceuticals are subjected to very strict purity requirements to be marketed. At the same time, peptides and other biomolecules are industrially synthesized through techniques (e.g., solid-phase synthesis) often leading to the formation of many impurities with molecular characteristics very similar to the target product. Therefore, the purification of these mixtures via preparative chromatography can be very challenging. This typically involves ternary or central-cut separations, characterized by chromatograms where the central peak, corresponding to the target product, exhibits significant overlapping on both sides with impurities slightly more or less adsorbable. In single-column (batch) preparative chromatography, this leads to a typical yield-purity tradeoff, meaning that high purity can be obtained at the cost of low yield and vice versa, with obvious consequences on the overall production costs. This study demonstrates how this limitation can be alleviated using the continuous countercurrent operating mode, conducted on a multicolumn system, as a tool for process intensification. In particular, the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been applied to the purification of an industrial crude mixture of icatibant, which is a peptidomimetic antagonist of bradykinin B2-receptor that has been recently also considered for the treatment of patients affected by COVID-19 disease. It is shown that MCSGP allows conjugating process simplicity (using only two columns) with a significant improvement in process performance, compared to the corresponding batch process. This includes all process performance parameters: yield, productivity, and buffer consumption for a given purity specification of icatibant.

Using continuous chromatography methodology to achieve high-productivity and high-purity enrichment of charge variants for analytical characterization

Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants.

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...

Advanced model‐based control strategies for the intensification of upstream and downstream processing in mAb production

Current industrial trends encourage the development of sustainable, environmentally friendly processes with minimal energy and material consumption. In particular, the increasing market demand in biopharmaceutical industry and the tight regulations in product quality necessitate efficient operating procedures that guarantee products of high purity. In this direction, process intensification via continuous operation paves the way for the development of novel, eco-friendly processes, characterized by higher productivity and lower production costs. This work focuses on the development of advanced control strategies for (i) a cell culture system in a bioreactor and (ii) a semicontinuous purification process. More specifically, we consider a fed-batch culture of GS-NS0 cells and the semicontinuous Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) for the purification process. The controllers are designed following the PAROC framework/software platform and their capabilities are assessed in silico, against the process models. It is demonstrated that the proposed controllers efficiently manage to increase the system productivity, returning strategies that can lead to continuous, stable process operation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:966-988, 2017.

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.

Open-loop optimal control of batch chromatographic separation processes using direct collocation

This contribution presents a novel model-based methodology for open-loop optimal control of batch high-pressure liquid chromatographic (HPLC) separation processes. The framework allows for simultaneous optimization of target component recovery yield and production rate with respect to a parameterization of the input elution trajectory and fractionating interval endpoints. The proposed methodology implies formulating and solving a large-scale dynamic optimization problem (DOP) constrained by partial differential equations (PDEs) governing the multi-component system dynamics. It is based on a simultaneous method where both the control and state variables are fully discretized in the temporal domain, using direct local collocation on finite elements, and the state variables are discretized in the spatial domain, using an adaptive finite volume weighted essentially non-oscillatory (WENO) scheme. The direct transcription of the DOP described by Modelica, and its extension Optimica, code into a sparse nonlinear programming problem (NLP) is thoroughly presented. The NLP was subsequently solved using CasADi's (Computer algebra system with Automatic Differentiation) interface to the primal-dual interior point method IPOPT. The advantages of the open-loop optimal control strategy are highlighted through the solution of a challenging ternary complex mixture separation problem of human insulin analogs, with the intermediately eluting component as the target, for a hydrophobic interaction chromatography system. Moreover, the high intercorrelation between the shape of the optimal elution trajectories and the fractionation interval endpoints is thoroughly investigated. It is also demonstrated that the direct transcription methodology enabled accurate and efficient computation of optimal cyclic-steady-state solutions, which govern that state and control variables conform to periodicity constraints imposed on column re-generation and re-equilibration. By these means, the generic methods and tools developed here are applicable to continuous chromatographic separation technologies, including the continuous simulated moving bed (SMB) and the multicolumn counter-current solvent gradient purification (MCSGP) process.

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