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%.

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.

Enrichment and recovery of oligonucleotide impurities by N-Rich twin-column continuous chromatography.

N-Rich is a twin-column continuous chromatography technology well suited for small-scale isolation and the enrichment of product related impurities. For the first time, N-Rich was used for impurity isolation from a double-stranded RNA (dsRNA) therapeutic synthetic oligonucleotide (ON), produced by solid-phase synthesis. By employing the N-Rich process, where the desired impurities are recycled and selectively enriched, and interfering substances are depleted, it was possible to obtain substantial amounts of high purity marginal impurities with a reproducible, automatized, and productive method. The productivity-purity tradeoff inherent to traditional impurity isolation methods, i.e., analytical chromatography, was effectively alleviated. Using N-Rich, satisfactory purity values and mass recoveries of several low-concentrated impurities could be obtained simultaneously. A performance comparison demonstrated an up to 15-fold increase for purity values and up to 20-fold mass impurity isolation and concentration with the N-Rich technology in comparison to conventional isolation procedures, drastically reducing processing times, manual handling, and waste production.

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.

Enrichment and purification of peptide impurities using twin-column continuous chromatography.

N-Rich is a twin-column chromatography process that enriches target compounds relative to other components in a mixture, thereby facilitating their isolation and characterization. This study demonstrates the performance of N-Rich for isolation of Angiotensin II peptide impurities compared with standard analytical and preparative chromatography approaches. Peptides have diverse chemical properties and are produced using a wide range of methods, resulting in products with complex impurity profiles. The characterization of impurities for clinical development is essential but obtaining high purity samples in sufficient quantities is often a difficult task when using standard chromatographic techniques. In contrast, by using cyclic continuous chromatography with UV-based process control, N-Rich enables automatic on-column accumulation of target impurities while other compounds in the mixture are depleted. This has multiple advantages compared to standard techniques. Firstly, at the end of the cyclic accumulation phase the highly enriched target is eluted in one step with high purity and concentration. This means fewer fractions for analysis are generated and up-concentration steps are reduced. Secondly, the purification of target impurities using semi-preparative scale chromatography becomes viable, even if initial resolution is poor compared to analytical HPLC. This allows for very significant increases in productivity for purification of difficult to isolate impurities. This study demonstrates two N-Rich strategies: Example 1: Purification of µg quantities of multiple Angiotensin II impurities with a >9-fold increase in productivity compared to analytical HPLC. Example 2: Specific isolation of 1 mg of a critical impurity at 88% purity. 79-fold increase in productivity and a 69-fold reduction in solvent consumption compared to analytical HPLC.

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.

Continued insights into virus clearance validation across continuous capture chromatography.

Multi-column capture chromatography (MCC) has gained increased attention lately due to the significant economic and process-related advantages it offers compared to traditional batch mode chromatography. However, for wide adoption of this technology in the clinical and commercial space, it requires scalable models for viral validation. In this study, additional viral validation studies were conducted under cGLP guidelines to assess retro-(X-MuLV) and parvo-virus (minute virus of mice) clearance across twin-column continuous capture chromatography (CaptureSMB) to supplement work previously performed. A surrogate model was validated using standard batch mode chromatography equipment based on flow path modifications to mimic the loading strategy employed in CaptureSMB. In addition, aged resin was used in this surrogate format to assess the impact of resin lifetime on viral clearance during continuous capture operation. The impact of column loading was also explored to shed light on the viral clearance mechanisms of protein A chromatography in overloading conditions. The proposed approach greatly simplifies MCC virus validation studies, and provides a robust strategy for regulatory filing of continuous biomanufacturing processes.

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification.

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.

Design space and robustness analysis of batch and counter-current frontal chromatography processes for the removal of antibody aggregates.

Increasing molecular diversity and market competition requires biopharmaceutical manufacturers to intensify their processes. In this respect, frontal chromatography on cation exchange resins has shown its potential to effectively remove aggregates. However, yield losses during the wash step need to be accepted in order to ensure robust product quality. In this work, we present a novel counter-current frontal chromatography process called Flow2, which uses inline dilution during an interconnected wash phase to allow high monomer recovery without contaminating the product pool with impurities. Its model-based design spaces under purity and yield constraints are compared with those corresponding to traditional batch processes in terms of size and process attributes yield and productivity. The Flow2 process shows the largest extent of feasible operating points independent of feed conditions. Thereby, it allows the implementation of higher ionic strength wash, thus widening the range of operating conditions resulting in yields above 95% compared to batch processes. Productivities of batch and counter-current processes are the same at short regeneration times and equal residence time. However, long regeneration times, while influencing the size of the Flow2 design space, are not detrimental for its productivity resulting in twice as high values as obtained for the batch process. Furthermore, process robustness is evaluated by the ability of the process to maintain the required product quality when subjected to process parameter perturbations. It is found that the Flow2 process is able to retain a larger design space associated also with higher yields showing its ability to improve process attributes without sacrificing robustness at the same time.

Virus clearance validation across continuous capture chromatography.

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover.

Model-assisted process characterization and validation for a continuous two-column protein A capture process.

In this study we introduce three process characterization approaches toward validation of continuous twin-column capture chromatography (CaptureSMB), referred to as "standard," "model assisted," and "hybrid." They are all based on a traditional risk-based approach, using process description, risk analysis, design-of-experiments (DoE), and statistical analysis as essential elements. The first approach, the "standard" approach uses a traditional experimental DoE to explore the design space of the high-ranked process parameters for the continuous process. Due to the larger number of process parameters in the continuous process, the DoE is extensive and includes a larger number of experiments than an equivalent DoE of a single column batch capture process. In the investigated case, many of the operating conditions were practically infeasible, indicating that the design space boundaries had been chosen inappropriately. To reduce experimental burden and at the same time enhance process understanding, an alternative "model assisted" approach was developed in parallel, employing a chromatographic process model to substitute experimental runs by computer simulations. Using the "model assisted" approach only experimental conditions that were feasible in terms of process yield constraints (>90%) were considered for statistical analysis. The "model assisted" approach included an optimization part that identified potential boundaries of the design space automatically. In summary, the "model assisted" approach contributed to increased process understanding compared to the "standard" approach. In this study, a "hybrid" approach was also used containing the general concepts of the "standard" approach but substituting a number of its experiments by computer simulations. The presented approaches contain essential elements of the Food and Drug Administration's process validation guideline.

Purification of Human Monoclonal Antibodies and Their Fragments.

This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion-exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.

Model assisted comparison of Protein A resins and multi-column chromatography for capture processes.

Effect of particle size (85µm vs. 50µm) on the performance of continuous capture chromatography using Protein A affinity was evaluated in combination with varied feed titers, loading flow rates and target breakthrough using a Design of Experiments (DoE) approach. In comparison to previous studies, higher cell culture titers on the order of 5-15 g/L, relevant to current high productivity industrial cell lines, were evaluated. Further, three modes of capture continuous chromatography were included in the DoE: single column batch, 2-column CaptureSMB and 4-column periodic counter-current chromatography (PCC). The breakthrough percentage at the outlet of the first column being loaded showed the most significant impact on process performance, confirming the advantage of multi-column over batch chromatography processes. Out of the two resins, the one with smaller particle size displayed significantly better performance. To verify and generalize these results, a shrinking core model for protein A chromatography has been developed and validated. The model was used to optimize the processes with respect to capacity utilization (load per cycle) and productivity (load per time). The smaller particle size resin (50µm) produced steeper breakthrough curves and allowed for better capacity utilization at any given productivity value. The improvement in loading was around 15% on average in comparison to the 85µm bead size in spite of the ligand density being same. The 50µm resin also allowed for higher maximum productivity values compared to the 85µm resin (improvements of 25-50%, depending on the process), despite lower maximum flow rate due to increased pressure drop. In addition, it is worth noting that recovery and regeneration rather than the maximum flow rate (pressure drop) became the limiting factor for process optimization in almost all considered scenarios.

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.

Model based adaptive control of a continuous capture process for monoclonal antibodies production.

A two-column capture process for continuous processing of cell-culture supernatant is presented. Similar to other multicolumn processes, this process uses sequential countercurrent loading of the target compound in order maximize resin utilization and productivity for a given product yield. The process was designed using a novel mechanistic model for affinity capture, which takes both specific adsorption as well as transport through the resin beads into account. Simulations as well as experimental results for the capture of an IgG antibody are discussed. The model was able to predict the process performance in terms of yield, productivity and capacity utilization. Compared to continuous capture with two columns operated batch wise in parallel, a 2.5-fold higher capacity utilization was obtained for the same productivity and yield. This results in an equal improvement in product concentration and reduction of buffer consumption. The developed model was used not only for the process design and optimization but also for its online control. In particular, the unit operating conditions are changed in order to maintain high product yield while optimizing the process performance in terms of capacity utilization and buffer consumption also in the presence of changing upstream conditions and resin aging.

Comparison of batch and continuous multi-column protein A capture processes by optimal design.

Multi-column capture processes show several advantages compared to batch capture. It is however not evident how many columns one should use exactly. To investigate this issue, twin-column CaptureSMB, 3- and 4-column periodic counter-current chromatography (PCC) and single column batch capture are numerically optimized and compared in terms of process performance for capturing a monoclonal antibody using protein A chromatography. Optimization is carried out with respect to productivity and capacity utilization (amount of product loaded per cycle compared to the maximum amount possible), while keeping yield and purity constant. For a wide range of process parameters, all three multi-column processes show similar maximum capacity utilization and performed significantly better than batch. When maximizing productivity, the CaptureSMB process shows optimal performance, except at high feed titers, where batch chromatography can reach higher productivity values than the multi-column processes due to the complete decoupling of the loading and elution steps, albeit at a large cost in terms of capacity utilization. In terms of trade-off, i.e. how much the capacity utilization decreases with increasing productivity, CaptureSMB is optimal for low and high feed titers, whereas the 3-column process is optimal in an intermediate region. Using these findings, the most suitable process can be chosen for different production scenarios.

Optimal model-based design of the twin-column CaptureSMB process improves capacity utilization and productivity in protein A affinity capture.

Multi-column chromatographic processes have recently been developed for protein A affinity chromatography to efficiently capture monoclonal antibodies from cell culture supernatant. In this work, the novel twin-column CaptureSMB process was compared to a batch capture process with dual loading flow rate to identify performance gains. As a case study, the isolation of a monoclonal antibody with the Amsphere JWT-203 protein A resin was investigated. Using model based optimization, both processes were optimized and compared over a wide range of operating conditions. A trade-off between productivity and capacity utilization was found, and the resulting pareto-curves showed that CaptureSMB dominates batch, except at very low productivity values. With a feed titer of 1.2 mg mL(-1) , CaptureSMB could reach a productivity of up to 19.5 mg mL(-1) h(-1) experimentally, while maintaining relatively high capacity utilization of 63.8%. On the other hand, at maximum capacity utilization of 95.5%, a productivity of 10.2 mg mL(-1) h(-1) could be reached. This corresponds to a performance improvement with respect batch operation of about 25% in capacity utilization and 40% in productivity, for given yield and purity. CaptureSMB therefore offers a greatly increased performance over batch capture.

Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

Role of urea on recombinant Apo A-I stability and its utilization in anion exchange chromatography.

Apolipoprotein A-I (Apo A-I) is an important lipid-binding protein involved in the transport and metabolism of cholesterol. High protein purity, in particular with respect to endotoxins is required for therapeutic applications. The use of urea during the purification process of recombinant Apo A-I produced in Escherichia coli has been suggested so as to provide high endotoxin clearance. In this work, we show that urea can be used as a sole modifier during the ion exchange chromatographic purification of Apo A-I and we investigate the molecular mechanism of elution by correlating the effect of urea on self-association, conformation and adsorption equilibrium properties of a modified model Apo A-I. In the absence of urea the protein was found to be present as a population of oligomers represented mainly by trimers, hexamers and nonamers. The addition of urea induced oligomer dissociation and protein structure unfolding. We correlated the changes in protein association and conformation with variations of the adsorption equilibrium of the protein on a strong anion exchanger. It was confirmed that the adsorption isotherms, described by a Langmuir model, were dependent on both protein and urea concentrations. Monomers, observed at low urea concentration (0.5M), were characterized by larger binding affinity and adsorption capacity compared to both protein oligomers (0M) and unfolded monomers (2-8M). The reduction of both the binding strength and maximum adsorption capacity at urea concentrations larger than 0.5M explains the ability of urea of inducing elution of the protein from the ion exchange resin. The dissociation of the protein complexes occurring during the elution could likely be the origin of the effective clearance of endotoxins originally trapped inside the oligomers.

Purification of human monoclonal antibodies and their fragments.

This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have been proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.