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.

Continuous countercurrent chromatographic twin-column purification of oligonucleotides: The role of the displacement effect.

Oligonucleotides (ONs) are breaking through in the biopharmaceutical industry as a promising class of biotherapeutics. The main success of these molecules is due to their peculiar way of acting in the cellular process, regulating the gene expression and hence influencing the protein synthesis at a pretranslational level. Although the Food and Drug Administration (FDA) already approved a few ON-based therapeutics, their production cost strongly limits large-scale manufacturing: a situation that can be alleviated through process intensification. In this study, we address this problem by developing an efficient and continuous chromatographic purification process for ONs. In particular, we considered the chromatographic purification of an ON crude prepared by chemical synthesis using anion exchange resins. We demonstrate that in this system the competitive adsorption of the various species on the same sites of the resin leads to the displacement of the more weakly adsorbing species by the more strongly adsorbing ones. This phenomenon affects the behavior of the chromatographic units and it has been investigated in detail. Then, we developed a continuous countercurrent solvent gradient purification (MCSGP) process, which can significantly improve the productivity and buffer consumption compared to a classical single-column, batch chromatographic 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.

Multifraction separation in countercurrent chromatography (MCSGP).

The multicolumn countercurrent solvent gradient purification (MCSGP) process is a continuous countercurrent multicolumn chromatography process capable of performing three fraction separations while applying a linear gradient of some modifier. This process can then be used either for the purification of a single species from a multicomponent mixture or to separate a three component mixture in one single operation. In this work, this process is extended to the separation of multifractions, in principle with no limitation. To achieve this goal the MCSGP standard process is extended by introducing one extra separation section per extra fraction to be isolated. Such an extra separation section is realized in this work through a single additional column, so that a n fraction MCSGP process can be realized using a minimum of n columns. Two separation processes were considered to experimentally demonstrate the possibility of realizing a four-fraction MCSGP unit able to purify two intermediate products in a given multicomponent mixture. The first one was a model mixture containing four different proteins. The two proteins eluting in the center of the chromatogram were purified with yields equal to 95% for the early eluting and 92% for the later eluting one. The corresponding purities were 94% and 97%, respectively. Such performance was well superior to that of the batch operation with the same modifier gradient which for the same purity values could not achieve yields larger than 67% and 81%, respectively. Similar performance improvements were found for the second separation where two out of seven charge variants which constitute the mAb Cetuximab currently available on the market have been purified in one single operation using a four-fraction MCSGP unit. In this case, yields of 81% and 65% were obtained with purities of 90% and 89%, respectively. These data compare well with the corresponding data from batch chromatography where with the same gradient and for the same purities, yield values not larger than 49% and 34%, respectively, could be achieved.

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.

Continuous Countercurrent Chromatography in Protein Purification.

Continuous countercurrent chromatography can be applied for both capture and polishing steps in the downstream processing of biopharmaceuticals. This chapter explains the concept of countercurrent operation, focusing on twin-column processes and how it can be used to alleviate the trade-offs of traditional batch chromatography with respect to resin utilization/productivity and yield/purity. CaptureSMB and MCSGP, the main twin-column continuous countercurrent chromatography processes, are explained, and the metrics by which they are compared to single-column chromatography are identified. Practical hints for process design and application examples are provided. Finally, regulatory aspects, scale-up, and UV-based process control are covered.

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.

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.

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.

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.

Continuous and Integrated Expression and Purification of Recombinant Antibodies.

This chapter introduces the necessary concepts to design continuous expression and purification processes for monoclonal antibodies. The operation of a perfusion bioreactor is discussed containing the preparation procedures, the seeding train and the preparation and control of a long-term production run. The downstream processes exploit the benefits of countercurrent chromatography. Their design from batch experiments is presented. The CaptureSMB process is introduced for continuous capturing while for polishing applications the design of the two-column MCSGP process is described. The chapter also puts these processes together in the context of their integration to an end-to-end production process.

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.

Continuous counter-current chromatography for capture and polishing steps in biopharmaceutical production.

The economic advantages of continuous processing of biopharmaceuticals, which include smaller equipment and faster, efficient processes, have increased interest in this technology over the past decade. Continuous processes can also improve quality assurance and enable greater controllability, consistent with the quality initiatives of the FDA. Here, we discuss different continuous multi-column chromatography processes. Differences in the capture and polishing steps result in two different types of continuous processes that employ counter-current column movement. Continuous-capture processes are associated with increased productivity per cycle and decreased buffer consumption, whereas the typical purity-yield trade-off of classical batch chromatography can be surmounted by continuous processes for polishing applications. In the context of continuous manufacturing, different but complementary chromatographic columns or devices are typically combined to improve overall process performance and avoid unnecessary product storage. In the following, these various processes, their performances compared with batch processing and resulting product quality are discussed based on a review of the literature. Based on various examples of applications, primarily monoclonal antibody production processes, conclusions are drawn about the future of these continuous-manufacturing technologies.