Periodic Counter-current Chromatography Research Articles

Design and optimization of a continuous purification process using ion-exchange periodic counter-current chromatography for a low-titer enzyme

AbstractA continuous purification process can be beneficial to the purification of biologics due to its higher productivity and efficiency than a conventional batch purification process. However, regulatory issues and lack of established cases render deployment of the continuous process difficult in industrial settings. Here we report a case study for design and optimization of an advanced continuous process for purifying a low-titer enzyme as a model biologic. To convert a conventional batch process to an advanced continuous one in purification of biologics, conventional unit operations (UOs), including ultrafiltration/diafiltration (UF/DF) and batch chromatography, were replaced by advanced ones such as in-line dilution conditioning (IDC) and periodic counter-current chromatography (PCC). The UF/DF UO was changed to IDC UO to adjust pH and conductivity. The mixing ratio of the sample and the conditioning buffer in IDC was determined by experiments with three buffers. PCC was optimized with two variables, column height and sample loading residence time, as the delta pressure in the columns was less than 1.0 bar. A graph indicating the operating area was plotted to efficiently control the PCC. Although the sample volume increased in IDC, PCC had a complementary advantage in that purification was performed faster than batch chromatography. We observed at least 25% increase in economic advantage when the advanced continuous process was applied to purify a low-titer enzyme. We propose not only a continuous process with the substitution of UF/DF and batch chromatography with IDC and PCC but also a method to optimize PCC by plotting operating areas.

A Novel 3D-Printed and Miniaturized Periodic Counter Current Chromatography System for Continuous Purification of Monoclonal Antibodies.

Continuous chromatography has emerged as one of the most attractive methods for protein purification. Establishing such systems involves installing several chromatographic units in series to enable continuous separation processes and reduce the cost of the production of expensive proteins and biopharmaceuticals (such as monoclonal antibodies). However, most of the established systems are bulky and plagued by high dead volume, which requires further optimization for improved separation procedures. In this article, we present a miniaturized periodic counter-current chromatography (PCCC) system, which is characterized by substantially reduced dead volume when compared to traditional chromatography setups. The PCCC device was fabricated by 3D printing, allowing for flexible design adjustments and rapid prototyping, and has great potential to be used for the screening of optimized chromatography conditions and protocols. The functionality of the 3D-printed device was demonstrated with respect to the capture and polishing steps during a monoclonal antibody purification process. Furthermore, this novel miniaturized system was successfully used for two different chromatography techniques (affinity and ion-exchange chromatography) and two different types of chromatographic units (columns and membrane adsorbers). This demonstrated versability underscores the flexibility of this kind of system and its potential for utilization in various chromatography applications, such as direct product capture from perfusion cell cultures.

Evaluation of dynamic control of continuous capture with periodic counter-current chromatography under feedstock variations

Multi-column periodic counter-current chromatography is a promising technology for continuous antibody capture. However, dynamic changes due to disturbances and drifts pose some potential risks for continuous processes during long-term operation. In this study, a model-based approach was used to describe the changes in breakthrough curves with feedstock variations in target proteins and impurities. The performances of continuous capture of three-column periodic counter-current chromatography under ΔUV dynamic control were systematically evaluated with modeling to assess the risks under different feedstock variations. As the concentration of target protein decreased rapidly, the protein might not breakthrough from the first column, resulting in the failure of ΔUV control. Small reductions in the concentrations of target proteins or impurities would cause protein losses, which could be predicted by the modeling. The combination of target protein and impurity variations showed complicated effects on the process performance of continuous capture. A contour map was proposed to describe the comprehensive impacts under different situations, and nonoperation areas could be identified due to control failure or protein loss. With the model-based approach, after the model parameters are estimated from the breakthrough curves, it can rapidly predict the process stability under dynamic control and assess the risks under feedstock variations or UV signal drifts. In conclusion, the model-based approach is a powerful tool for continuous process evaluation under dynamic changes and would be useful for establishing a new real-time dynamic control strategy.

Integrated continuous downstream process of monoclonal antibody developed by converting the batch platform process based on the process characterization.

A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic-counter current chromatography (PCCC) with two protein A columns was used for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow-through chromatography (FTC) with two connected columns of different separation modes (anion-mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected-column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5-10 L/day) and feed concentrations (1-3.2 g/L) were performed with protein A columns of 1-5 mL and FTC columns of 3-10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank.

Automated quality analysis in continuous downstream processes for small-scale applications

Development of integrated, continuous biomanufacturing (ICB) processes brings along the challenge of streamlining the acquisition of data that can be used for process monitoring, product quality testing and process control. Manually performing sample acquisition, preparation, and analysis during process and product development on ICB platforms requires time and labor that diverts attention from the development itself. It also introduces variability in terms of human error in the handling of samples. To address this, a platform for automatic sampling, sample preparation and analysis for use in small-scale biopharmaceutical downstream processes was developed. The automatic quality analysis system (QAS) consisted of an ÄKTA Explorer chromatography system for sample retrieval, storage, and preparation, as well as an Agilent 1260 Infinity II analytical HPLC system for analysis. The ÄKTA Explorer system was fitted with a superloop in which samples could be stored, conditioned, and diluted before being sent to the injection loop of the Agilent system. The Python-based software Orbit, developed at the department of chemical engineering at Lund university, was used to control and create a communication framework for the systems.To demonstrate the QAS in action, a continuous capture chromatography process utilizing periodic counter-current chromatography was set up on an ÄKTA Pure chromatography system to purify the clarified harvest from a bioreactor for monoclonal antibody production. The QAS was connected to the process to collect two types of samples: 1) the bioreactor supernatant and 2) the product pool from the capture chromatography. Once collected, the samples were conditioned and diluted in the superloop before being sent to the Agilent system, where both aggregate content and charge variant composition were determined using size-exclusion and ion-exchange chromatography, respectively. The QAS was successfully implemented during a continuous run of the capture process, enabling the acquisition of process data with consistent quality and without human intervention, clearing the path for automated process monitoring and data-based control.

Continuous protein refolding and purification by two-stage periodic counter-current chromatography

Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size exclusion chromatography (SEC) has been extensively used for MAR because of its ability to load and refold proteins at high concentrations. However, the SEC-based batch MAR processes have the disadvantages of requiring longer columns for better separation and product dilution due to a high column-to-sample volume ratio. In this work, a modified operational scheme is developed for continuous MAR of L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC). The volumetric productivity of the modified SEC-PCC process is 6.8-fold higher than the batch SEC process. In addition, the specific buffer consumption decreased by 5-fold compared to the batch process. However, the specific activity of the refolded protein (110–130 IU/mg) was less due to the presence of impurities and additives in the refolding buffer. To address this challenge, a 2-stage process was developed for continuous refolding and purification of IBs using different matrices in sequential PCCs. The performance of the 2-stage process is compared with literature reports on single-stage IMAC-PCC and conventional pulse dilution processes for refolding L-asparaginase IBs. The 2-stage process resulted in a refolded protein with enhanced specific activity (175–190 IU/mg) and a high recovery of 84%. The specific buffer consumption (6.2 mL/mg) was lower than the pulse dilution process and comparable to the single-stage IMAC-PCC. A seamless integration of the two stages would considerably increase the throughput without compromising other parameters. High recovery, throughput, and increased operational flexibility make the 2-stage process an attractive option for protein refolding.

Continuous precipitation of antibodies by feeding of solid polyethylene glycol

The economic benefits of continuous PEG precipitation of antibodies compared to continuous capture by protein A affinity chromatography have been always lowered by the excess amount of material needed for the process. PEG is added as a concentrated stock solution and therefore liquid handling is increased during this step. To fully exploit the benefits of PEG precipitation, the precipitant must be added in solid form. We used an in-line feeding device with a screw conveyor delivery system for the continuous addition of PEG6000 in a powder form. The powder feeding device was connected to a tubular reactor where the precipitation occurs. Protein precipitation was continuously performed for 4 h. A yield of 76 % and 79 % with a purity of 98 % was achieved. The total cost of goods and the environmental footprint were compared with typical chromatography-based purification methods; batch and continuous periodic countercurrent protein A affinity chromatography with four columns. Solid PEG precipitation showed a remarkable reduction in water consumption and equipment size, reducing production costs by 45 % compared to liquid PEG and 53 % cheaper than Protein A periodic counter-current chromatography. Process mass intensity was reduced by 55 % and carbon emissions by 60 %. The reduction of water by the direct addition of PEG also impacted the environmental footprint and process costs. This is an attractive approach for a continuous capture step yielding an uninterrupted mass flow of the product and will pave the way for PEG precipitation as a capture step.

Residence time distribution in counter-current protein A affinity chromatography using an inert tracer

The trend in the biopharmaceutical industry is changing from batch process to continuous process. For continuous biomanufacturing, traceability of the material is required by regulatory authorities. The recent ICH draft guideline Q13 on continuous manufacturing of drug substances and drug products requests an “understanding of process dynamics as a function of input material attributes (e.g., potency, material flow properties), process conditions (e.g., mass flow rates) … One common approach is characterization of residence time distribution (RTD) for the individual unit operations and integrated system.” Thus, it is necessary to trace material through individual continuous unit operations and the integrated process. The RTD of a process is obtained experimentally by injecting a pulse of an inert tracer into the inlet and measuring the broadening of the injected pulse in the outlet. We investigated the RTD of three-column periodic counter-current chromatography (PCC) using staphylococcal protein A affinity chromatography, with a focus on how the material distributes over subsequent cycles. A fluorescent-labeled antibody was used as the inert tracer under high salt concentration. The tracer was injected once in each run but at different points of the loading phase. We then analyzed the outlet of the column. In the elution phase, regardless of the point of injection, we observed an even distribution of the tracer. In the loading phase, a constant exchange between the antibody in the solid phase and the liquid phase was observed, meaning that sending the outlet of one chromatography column into another column to improve resin utilization causes higher residence time in the system for some portion of the material.

Model-based evaluation and model-free strategy for process development of three-column periodic counter-current chromatography

Multi-column counter-current chromatography is an advanced technology used for continuous capture processes to improve process productivity, resin capacity utilization and product consistency. However, process development is difficult due to process complexity. In this work, some general and convenient guidances for three-column periodic counter-current chromatography (3C-PCC) were developed. Boundaries and distributions of operating windows of 3C-PCC processes were clarified by model-based predictions. Interactive effects of feed concentration (c0), resin properties (qmax and De), recovery and regeneration times (tRR) were evaluated over a wide range for maximum productivity (Pmax). Furthermore, variation of Pmax was analyzed considering the constraint factors (capacity utilization target and flow rate limitation). The plateau value of Pmax was determined by qmax and tRR. The operating conditions for Pmax were controlled by qmax, tRR and c0 interactively, and a critical concentration existed to judge whether the operating conditions of Pmax under constraints. Based on the comprehensive understanding on 3C-PCC processes, a model-free strategy was proposed for process development. The optimal operating conditions could be determined based on a set of breakthrough curves, which was used to optimize process performance and screen resins. The approach proposed was validated using monoclonal antibody (mAb) capture with a 3C-PCC system under various mAb and feed concentrations. The results demonstrated that a thorough model-based process understanding on multi-column counter-current chromatography is important and could improve process development and establish a model-free strategy for more convenient applications.

Continuous Affinity Purification of Adeno-Associated Virus Using Periodic Counter-Current Chromatography.

Replacing batch unit operations of biopharmaceuticals by continuous manufacturing is a maturing concept, with periodic counter-current chromatography (PCC) favoured to replace batch chromatography. Continuous affinity capture of adeno-associated virus (AAV) using PCC has the potential to cope with the high doses required for AAV therapies thanks to its inherent high throughput. The implementation of continuous AAV affinity capture using a four-column PCC process is described herein. First, elution buffer screening was used to optimize virus recovery. Second, breakthrough curves were generated and described using a mechanistic model, which was later used to characterize the loading zone of the PCC. The experimental runs achieved a stable cyclic steady state yielding virus recoveries in line with the optimized batch process (>82%), with almost a three-fold improvement in productivity. The PCC affinity capture process developed here can bolster further improvements to process economics and manufacturing footprint, thereby contributing to the integrated continuous manufacturing concept.

An integrated and continuous downstream process for microbial virus-like particle vaccine biomanufacture.

In this study, we present the first integrated and continuous downstream process for the production of microbial virus‐like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus‐like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow‐through chromatography step followed by periodic counter‐current chromatography (PCC) (bind‐elute) using salt‐tolerant mixed‐mode resin and subsequent in‐line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml−1 with only minimal adjustments needed, and produced continuously high‐quality virus‐like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h−1 mlresin −1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.

Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand

Monoclonal antibodies (mAb) are used as therapeutics and for diagnostics of a variety of diseases, and novel antibodies are continuously being developed to find treatments for new diseases. Therefore, the manufacturing process must accommodate a range of mAb characteristics. Acid-sensitive mAbs can severely compromise product purity and yield in the purification process due to the potential formation of aggregates. To address this problem, we have developed an integrated downstream process for the purification of pH-sensitive mAbs at mild conditions. A calcium-dependent Protein A-based ligand, called ZCa, was used in the capture step in a 3-column periodic counter-current chromatography operation. The binding of ZCa to antibodies is regulated by calcium, meaning that acidic conditions are not needed to break the interaction and elute the antibodies. Further, the virus inactivation was achieved by a solvent/detergent method, where the pH could remain unchanged. The polishing steps included a cation and an anion exchange chromatography step, and screening of the capture and polishing steps was performed to allow for a seamless integration of the process steps. The process was implemented at laboratory scale for 9 days obtaining a high yield, and a consistently pure drug substance, including high reduction values of the host cell protein and DNA concentrations, as well as aggregate levels below the detection limit, which is attributed to the mild conditions used in the process.

Evaluation of the Efficiency of Continuous Capture Chromatography

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

Continuous refolding of L-asparaginase inclusion bodies using periodic counter-current chromatography

Chromatography-based refolding is emerging as a promising alternative to dilution-refolding of solubilized inclusion bodies (IBs). The advantages of this matrix-assisted refolding (MAR) lie in its ability to reduce aggregate formation, leading to better recovery of active protein, and enabling refolding at higher protein concentration. However, batch chromatography has the disadvantage of ineffective solvent utilization, under-utilization of resin, and low throughput. In this work, we overcome these challenges by using a 3-column Periodic Counter-current Chromatographic (PCC) system for continuous refolding of IBs, formed during the production of L-asparaginase by recombinant E. coli cultures. Initial experiments were conducted in batch processes using single-column immobilized metal-affinity chromatography. Different gradient operations were designed to improve the protein loading for the single-column, batch-MAR processes. Optimized conditions, based on the batch-MAR experiments, were used for designing the continuous-MAR processes using the PCC system. The continuous-MAR experiments were carried out over 3 cycles (∼ 30 h) in the PCC system. A detailed quantitative comparison based on recovery, throughput, buffer consumption, and resin utilization was made for the three modes of operation: pulse-dilution, single-column batch-MAR, and 3-Column PCC-based continuous-MAR processes. While recovery (73%) and throughput (11 mg/h) were the highest in PCC, specific buffer consumption (6.9 ml/mg) was the least. Also, during PCC operation, resin utilization improved by 92% in comparison to the single-column batch-MAR process. These quantitative comparisons clearly establish the advantages of the continuous-MAR process over the batch-MAR and other conventional refolding techniques.

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

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

A novel framework of surrogate-based feasibility analysis for establishing design space of twin-column continuous chromatography

Multi-column periodic counter-current chromatography (PCC) has attracted wide attention for the primary capture for the purpose of achieving continuous biomanufacturing. Consequently, determining the design space of the continuous capture process is very important to facilitate process understanding and improving product quality. In this work, we proposed a novel approach to identify the design space of continuous chromatography to balance the computational complexity and model predictions. Specifically, surrogate-based feasibility analysis with adaptive sampling is applied to establish the design space of twin-column CaptureSMB process. The surrogate model is constructed based on the developed mechanistic model for the identification of the design space. The effects of process variables (including interconnected loading time, interconnected flowrate, and batch flowrate) on the design space are comprehensively examined based on an active set strategy. Besides, essential factors like recovery-regeneration time and constraints of column performance parameters (yield, productivity, and capacity utilization) are thoroughly investigated. The impact of design variables such as column length is also studied.

Oncolytic virus purification with periodic counter-current chromatography.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and(iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

An integrated continuous downstream process with real-time control: A case study with periodic countercurrent chromatography and continuous virus inactivation.

Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two-system setup that can be used as a general downstream setup offering multiproduct and multipurpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step. A real-time controller was implemented to ensure stable operation by adapting the downstream process to external changes. A concentration disturbance was introduced to test the controller. After the disturbance was applied, the product output recovered within 6 h, showing the effectiveness of the controller. In a comparison of the process with and without the controller, the product output per cycle increased by 27%, the resin utilization increased from 71.4% to 87.9%, and the specific buffer consumption was decreased by 21% with the controller, while maintaining a similar yield and purity as in the process without the disturbance. In addition, the integrated continuous process outperformed the batch process, increasing the productivity by 95% and the yield by 28%.

Process development and optimization of continuous capture with three-column periodic counter-current chromatography.

Continuous capture with affinity chromatography is one of the most important units for continuous manufacturing of monoclonal antibody (mAb). Due to the complexity of three-column periodic counter-current chromatography (3C-PCC), three approaches (experimental, model-based, and simplified approaches) were studied for process development and optimization. The effects of residence time for interconnected load (RT C ), breakthrough percentage of the first column for interconnected load (s) and feed protein concentration (c 0 ) on productivity and capacity utilization were focused. The model-based approach was found superior to the experimental approach in process optimization and evaluation. Two phases of productivity were observed and the optimal RT C for the maximum productivity was located at the boundary of the two phases. The comprehensive effects of the operating parameters (RT C , s, and c 0 ) were evaluated by the model-based approach, and the operation space was predicted. The best performance of 34.5 g/L/h productivity and 97.6% capacity utilization were attained for MabSelect SuRe LX resin under 5 g/L concentration at RT C = 2.8 min and s = 87.5%. Moreover, a simplified approach was suggested to obtain the optimal RT C for the maximum productivity. The results demonstrated that model-assisted tools are useful to determine the optimum conditions for 3C-PCC continuous capture with high productivity and capacity utilization.

Antibody capture with twin-column continuous chromatography: Effects of residence time, protein concentration and resin

The rapid development of the biopharmaceutical industry has gradually shifted the focus of R&D to integrated continuous manufacturing. In this study, the effects of residence time and protein concentration on the performance of twin-column periodic counter-current chromatography for continuous antibody capture with new affinity resin Praesto Jetted A50 were evaluated. The results showed that the continuous process exhibited better performance with higher productivity, higher capacity utilization and lower buffer consumption when compared with the batch process. Meanwhile, the increase of protein concentration could increase productivity, while it showed minor impact on capacity utilization and buffer consumption. Furthermore, monoclonal antibody (mAb) continuous capture from clarified cell culture supernatant was investigated. The results showed that the qualities of the products obtained from the continuous and batch processes were similar. The performance of the continuous capture process at 1 min residence time was significantly better than the batch process with the improvements in productivity (37.5 g/L/h) and capacity utilization (77.8%) as well as reducing buffer consumption by 60%. Finally, effects of Protein A resins on the continuous process were discussed, and the results indicated that the Protein A resins with higher dynamic binding capacity at low residence time would be more suitable for continuous processes. In general, continuous capture under the suitable operation conditions shows good performance and has great potential for mAb production.