A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process

Biomolecules are often purified via solvent gradient batch chromatography. Typically suitable smooth linear solvent gradients are applied to obtain the separation between the desired component and hundreds of impurities. The desired product is usually intermediate between weakly and strongly adsorbing impurities, and therefore a central cut is required to get the desired pure product. The stationary phases used for preparative and industrial separations have a low efficiency due to strong axial dispersion and strong mass transfer resistances. Therefore a satisfactory purification often cannot be achieved in a single chromatographic step. For large scale productions and for very valuable molecules, countercurrent operation such as the well known SMB process, is needed in order to increase separation efficiency, yield and productivity. In this work a novel multicolumn solvent gradient purification process (MCSGP-process) is introduced, which combines two chromatographic separation techniques, which are solvent gradient batch and continuous countercurrent SMB. The process consists of several chromatographic columns, which are switched in position opposite to the flow direction. Most of the columns are equipped with a gradient pump to adjust the modifier concentration at the column inlet. Some columns are interconnected, so that non pure product streams are internally, countercurrently recycled. Other columns are short circuited and operate in batch mode. As a working example the purification of an industrial stream containing 46% of the hormone Calcitonin is considered. It is found that for the required purity the MCSGP unit achieves a yield close to 100% compared to a maximum value of a single column batch chromatography of 66%.     

Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP)

Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.     

Parametric study of a 6-column countercurrent solvent gradient purification (MCSGP) unit

The novel "multicolumn countercurrent solvent gradient purification" (MCSGP) process has been modeled for the purification of a polypeptide mixture characterized by a strong non-linear competitive adsorption isotherm. As a model system, the purification of an industrial polypeptide mixture containing 46% of the hormone calcitonin has been selected. The many impurities contained in the mixture have been lumped into three key impurities, which are selected as the ones eluting closer to the main component. The simulation model allows for a better understanding of the complex operating behavior of the multicolumn system, which has been experimentally investigated in a previous work. Through a systematic parametric analyses of the model behavior, the main operating parameters controlling the process performance in terms of purity and yield are investigated. The study of internal liquid and adsorbed phase concentration profiles along the unit for the different operating conditions allow elucidating the working principle of the new separation process. It is found that the MCSGP unit achieves much higher yields for a given product purity than the corresponding single-column batch units.     

Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)

The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion‐exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652–662. © 2010 Wiley Periodicals, Inc.     

A semicontinuous 3-column countercurrent solvent gradient purification (MCSGP) process

The recently developed continuous Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) Process has been reduced to a fully equivalent semicontinuous setup with only three chromatographic columns and three gradient pump modules. Actually the 3-column MCSGP unit can even achieve better performance than the original 6-column process due to an additional degree of freedom, that is a different switching time for the "batch lane" and the "interconnected lane." Experimental results for the 3-column MCSGP unit of the purification of an industrial multicomponent peptide mixture containing 46% of Calcitonin on a reversed phase resin are compared with model simulations. It is concluded, that the model is well suited to predict the system behavior and therefore to design its optimal operating conditions.     

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.     

Performance optimization of continuous countercurrent tangential chromatography for antibody capture

Recent studies have demonstrated that continuous countercurrent tangential chromatography (CCTC) can effectively purify monoclonal antibodies from clarified cell culture fluid. CCTC has the potential to overcome many of the limitations of conventional packed bed protein A chromatography. This paper explores the optimization of CCTC in terms of product yield, impurity removal, overall productivity, and buffer usage. Modeling was based on data from bench‐scale process development and CCTC experiments for protein A capture of two clarified Chinese Hamster Ovary cell culture feedstocks containing monoclonal antibodies provided by industrial partners. The impact of resin binding capacity and kinetics, as well as staging strategy and buffer recycling, was assessed. It was found that optimal staging in the binding step provides better yield and increases overall system productivity by 8–16%. Utilization of higher number of stages in the wash and elution steps can lead to significant decreases in buffer usage (～40% reduction) as well as increased removal of impurities (～2 log greater removal). Further reductions in buffer usage can be obtained by recycling of buffer in the wash and regeneration steps (～35%). Preliminary results with smaller particle size resins show that the productivity of the CCTC system can be increased by 2.5‐fold up to 190 g of mAb/L of resin/hr due to the reduction in mass transfer limitations in the binding step. These results provide a solid framework for designing and optimizing CCTC technology for capture applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:430–439, 2016     

Evaluation of several protein a resins for application to multicolumn chromatography for the rapid purification of fed‐batch bioreactors

Most of the existing production capacity is based on fed‐batch bioreactors. Thanks to the development of more efficient cell lines and the development of high‐performance culture media, cell productivity dramatically increased. In a manufacturing perspective, it is necessary to clear as quickly as possible the protein A capture step to respect the manufacturing agenda. This article describes the methodology applied for the design of a multicolumn chromatography process with the objective of purifying as quickly as possible 1,000 and 15,000 L fed‐batch bioreactors. Several recent and reference protein A resins are compared based on characteristic values obtained from breakthrough curves. The importance and relevance of resin parameters are explained, and purposely simple indicators are proposed to quickly evaluate the potential of each candidate. Based on simulation data, the optimum BioSC systems associated with each resin are then compared. The quality of the elution delivered by each resin is also compared to complete the assessment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:941–953, 2017     

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.     

Continuous precipitation for monoclonal antibody capture using countercurrent washing by microfiltration

There is renewed interest in the possibility of using precipitation for initial capture of high-value therapeutic proteins as part of an integrated continuous downstream process. Precipitation is greatly facilitated by the high product titers now achieved in most cell culture processes, in sharp contrast to chromatographic processes whose performance is reduced at high titers. The current study used a combination of reversible cross-linking (zinc chloride, ZnCl₂) and volume exclusion (polyethylene glycol) agents to precipitate a monoclonal antibody product directly from harvested cell culture fluid using a continuous tubular precipitation reactor. The precipitates were then dewatered and continuously washed using tangential flow filtration, with a countercurrent-staged configuration used to reduce the amount of wash buffer required and increase host cell protein removal. Long-term operation was achieved by operating the membrane modules below the critical filtrate flux to avoid fouling. Experimental results demonstrate the feasibility of this fully continuous integrated precipitation process at bench scale, with design calculations used to explore the key factors affecting the performance of this system for initial antibody capture.     

High-performance countercurrent membrane purification for host cell protein removal from monoclonal antibody products

The transition to continuous biomanufacturing has led to renewed interest in alternative approaches for downstream processing of monoclonal antibody (mAb) products. In this study, we examined the potential of using high-performance countercurrent membrane purification (HPCMP) for the removal of host cell proteins (HCPs) derived from Chinese Hamster Ovary cells in the purification of a mAb. Initial studies used several model proteins to identify appropriate operating conditions for the hollow fiber membrane modules. HPCMP was then used for mAb purification, with mAb yield >95% and more than 100-fold reduction in HCP. Stable operation was maintained for 48 h for feeds that were first prefiltered through the 3M^TM Harvest RC chromatographic clarifier to remove DNA and other foulants. In addition, the Process Mass Intensity for HPCMP can be much less than that for alternative HCP separation processes. These results highlight the potential of using HPCMP as part of a fully continuous mAb production process.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Affinity purification of monoclonal antibodies,using a bifunctional oligosaccharide hapten

A bifunctional hapten was synthesized consisting of a blood group A active tetrasaccharide (A-tetra) and a blood group Le^a active pentasaccharide. lacto-N-fucopentaose II (LNF II), linked to each other with a phenylaminothiourea spacer connecting the reducing ends (A-tetra-LNF II). The hapten was demonstrated to retain both blood group A and Le^a activity and could be easily bound to both monoclonal anti-A and anti-Le^a affinity columns. Due to the strong temperature dependence of the two antibodies in their binding to oligosaccharides, the bifunctional hapten could be utilized to achieve easy desorption in the final step of affinity purification of either monoclonal anti-Le^a or anti-A. The system is postulated to have general applicability in affinity purification of any ligate that binds with an avidity too high to achieve non-denaturing desorption.To whom correspondence should be addressed.     

One step purification of corilagin and ellagic acid from Phyllanthus urinaria using high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) has been successfully applied to the preparative separation of corilagin and ellagic acid in one step from the Chinese medicinal plant Phyllanthus urinaria L. by use of direct and successive injections of a crude methanolic extract. Some aspects concerning the practical use of this technique in the described application are considered.     

Integrated flow-through purification for therapeutic monoclonal antibodies processing

An integrated all flow-through technology platform for the purification of therapeutic monoclonal antibodies (mAb), consisting of activated carbon and flow-through cation and anion exchange chromatography steps, can replace a conventional chromatography platform. This new platform was observed to have excellent impurity clearance at high mAb loadings with overall mAb yield exceeding 80%. Robust removal of DNA and host cell protein was demonstrated by activated carbon and a new flow-through cation exchange resin exhibited excellent clearance of mAb aggregate with high monomer recoveries. A ten-fold improvement of mAb loading was achieved compared to a traditional cation exchange resin designed for bind and elute mode. High throughput 96-well plate screening was used for process optimization, focusing on mAb loading and solution conditions. Optimum operating windows for integrated flow-through purification are proposed based on performance characteristics. The combination of an all flow-through polishing process presents significant opportunities for improvements in facility utilization and process economics.     

Design and operation of a continuous integrated monoclonal antibody production process

The realization of an end‐to‐end integrated continuous lab‐scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter‐based cell‐retention, a continuous two column capture process, a virus inactivation step, a semi‐continuous polishing step (twin‐column MCSGP), and a batch‐wise flow‐through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity‐yield trade‐off of classical batch‐wise bind‐elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight‐through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end‐to‐end integration. The steady‐state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303–1313, 2017     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

G蛋白亲和色谱法纯化动物血清、抗体效果比较

比较 Hitrap G蛋白 Sepharose亲和色谱系统对不同动物血清或腹水的纯化效果 ,为抗体纯化提供依据。结果显示 ,G蛋白对不同动物血清 Ig G吸附能力不同 ,体现在单位体积抗体回收量明显不同 ,这一特点与 A蛋白亲和色谱系统相似。该方法简便快速 ,纯化抗体纯度高 ,免疫活性好 ,色谱柱可反复使用多次