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

The impact of two different quality feeds, derived using two different harvest clarification processes, on protein A periodic counter‐current chromatography (PCC) design and performance is investigated. Data from batch experiments were input into a model to design optimal PCC operating parameters specific to each feed material. The two clarification methods were: depth filtration using a wetlaid matrix which has Q‐functionality; and a combination of depth filtration and chromatographic clarification, using a Q‐functional nonwoven with a high anion exchange capacity (Emphaze™ AEX Hybrid Purifier) in which key impurities such as host cell DNA (HCDNA) and host cell proteins (HCP) are removed. The model predicted 34% better productivity for the chromatographically clarified cell culture fluid (CCCF) using a 4 column system, and productivity gains of 28% using only 3 columns enabling the option to simplify the protein A PCC strategy. Experimental validation of the predicted optimized PCC operating parameters using industrially relevant monoclonal antibody (mAb) CCCF feedstock over 100 cycles showed productivity gains of 49% for the chromatographically clarified material. HCP concentration was 11‐fold lower, and HCDNA concentration was reduced by 4.4 Log Reduction Value (LRV) in the protein A PCC eluates. This work, therefore, demonstrates that the removal of HCDNA and HCP during clarification is an effective strategy for improving protein A PCC performance. This was achieved using the Emphaze™ AEX Hybrid Purifier which can be easily incorporated into a batch or continuous process, in a scalable fashion, without adding additional separate unit operations. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1380–1392, 2018

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two‐stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two‐stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre‐existing high‐molecular‐weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter‐current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow‐through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

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.

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.

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%.

Definition and dynamic control of a continuous chromatography process independent of cell culture titer and impurities

Integrated and continuous processing of recombinant proteins offers several advantages over batch or semi-batch processing used traditionally in the biotechnology industry. This paper presents a theoretical and practical approach for designing a periodic counter-current chromatography (PCC) operation as a continuous capture purification step that is integrated with a perfusion cell culture process. The constraints for continuous and optimal PCC operation govern the selection of residence time and number of columns. The flexibility available in PCC design for selection of these parameters is dictated by the binding characteristics of the target protein on the capture resin. Using an empirical model for the protein breakthrough curve, analytical solutions to determine these conditions were derived and verified with experimental results for three different proteins: two relatively unstable proteins (recombinant enzymes) and a relatively stable protein (monoclonal antibody). The advantages of a continuous downstream capture step are highlighted for the three case studies in comparison with the existing batch chromatography processes. The use of PCC leads to improvements in process economics due to higher resin capacity utilization and correspondingly lower buffer consumption. Furthermore, integrated and continuous bioprocessing results in a smaller facility footprint by elimination of harvest hold vessels and clarification, as well as by reducing the capture column size by one to two orders of magnitude.

There is a growing interest in continuous biopharmaceutical processing due to the advantages of small footprint, increased productivity, consistent product quality, high process flexibility and robustness, facility cost-effectiveness, and reduced capital and operating cost. To support the decision making of biopharmaceutical manufacturing, comparisons between conventional batch and continuous processing are provided. Various process unit operations in different operating modes are summarized. Software implementation, as well as computational methods used, are analyzed pointing to the advantages and disadvantages that have been highlighted in the literature. Economic analysis methods and their applications in different parts of the processes are also discussed with examples from publications in the last decade. The results of the comparison between batch and continuous process operation alternatives are discussed. Possible improvements in process design and analysis are recommended. The methods used here do not reflect Lilly's cost structures or economic evaluation methods. This paper provides a review of the work that has been published in the literature on computational process design and economic analysis methods on continuous biopharmaceutical antibody production and its comparison with a conventional batch process.

Continuous bioprocessing with Protein A affinity chromatography has demonstrated great potential to increase productivity and reduce the cost of goods in monoclonal antibody (mAb) production. However, maintaining process stability and responding to dynamic changes remains significant challenges, particularly in the real-time optimization and control of multi-column periodic counter-current chromatography (PCC) for Protein A affinity chromatography, due to the computational complexity of rapidly solving mechanistic models. To address this challenge, this study developed distilled physics-informed neural networks (PINNs) based on the general rate model (GRM) to accelerate and enhance the breakthrough curve fitting and four-column PCC (4C-PCC) process optimization. The distilled PINNs achieved a balance between prediction accuracy and computational speed. The 157k-parameter distilled PINN enabled the breakthrough curve fitting and 4C-PCC process optimization approximately 10 times faster than numerical methods while improving accuracy by about 40%. A smaller 2k-parameter model achieved a 22-fold acceleration with an acceptable trade-off in accuracy, and the optimization time was reduced to 1.44 s. Explainability analyses confirmed the PINN's capability to capture nonlinear and interactive effects among key process parameters. The PINN-accelerated GRM was then integrated with real-time model predictive control (MPC) and applied to a lab-scale continuous manufacturing process. PINN-based MPC maintained robust control of binding capacity and yield, achieving a productivity of 35 g/L resin/h and resin capacity utilization of 90%, despite resin capacity decay and upstream variability. This work demonstrates that the PINNs can provide a computationally efficient and physically consistent framework for real-time optimization and control of continuous processes. Integrating a mechanistic model with neural networks can enhance process understanding and robustness, supporting the implementation of continuous biomanufacturing for therapeutic proteins.

Model-assisted process design for better evaluation and scaling up of continuous downstream bioprocessing

Introduction The pharmaceutical industry is gradually changing batch-wise manufacturing processes to continuous manufacturing processes, due to the advantages it has to offer. The final product quality and process efficiency of continuous manufacturing processes is among others impacted by the properties of the raw materials. Existing knowledge on the role of raw material properties in batch processing is however not directly transferable to continuous processes, due to the inherent differences between batch and continuous processes. Areas covered A review is performed to evaluate the role of excipient properties for different unit operations used in continuous manufacturing processes. Unit operations that will be discussed include feeding, blending, granulation, final blending, and compression. Expert opinion Although the potency of continuous manufacturing is widely recognized, full utilization still requires a number of challenges to be addressed effectively. An expert opinion will be provided that discusses those challenges and potential solutions to overcome those challenges. The provided overview can serve as a framework for the pharmaceutical industry to push ahead process optimization and formulation development for continuous manufacturing processes.

Batch chromatography has several disadvantages, such as insufficient utilization of the capacity of the resin, high buffer consumption and discontinuity. Considering the high costs for downstream processing, a continuously working chromatographic system with three membrane adsorber units was designed, tested and put into operation. The basic principle of the setup is periodic counter-current chromatography (PCCC). The PCCC system was used for capturing and purifying Candida antarctica lipase B (CalB) directly from cell lysate in one single unit operation. The best purification result was achieved by means of anion-exchange chromatography. The dynamic binding capacity with Sartobind® Q 75 amounted to 4.2mg (56g/cm2). After transferring the method to the 3MA-PCCC, 0.22g CalB (73U/mg) were obtained from 0.9 L E.coli lysate within 6 h and a recovery of 80%. Compared to the batch process, the productivity could be increased by 36% and the buffer consumption could be reduced by about 20%. Although the purification of CalB from lysate by means of anion-exchange chromatography was not selective and quantitative using the 3MA-PCCC device, it could be shown that the concept of the system was successfully implemented and led to a significant improvement of CalB purification.

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

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.