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%.     

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.     

Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP)

A two‐step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP‐captured material in batch mode. Using multi‐modal anion exchange in bind‐elute mode, mAb was produced consistently within the purity specification. The benchmark was a state‐of‐the‐art 3‐step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2‐step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase. Biotechnol. Bioeng. 2010;107: 974–984. © 2010 Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

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     

Countercurrent tangential chromatography for large-scale protein purification

Recent advances in cell culture technology have created significant pressure on the downstream purification process, leading to a "downstream bottleneck" in the production of recombinant therapeutic proteins for the treatment of cancer, genetic disorders, and cardiovascular disease. Countercurrent tangential chromatography overcomes many of the limitations of conventional column chromatography by having the resin (in the form of a slurry) flow through a series of static mixers and hollow fiber membrane modules. The buffers used in the binding, washing, and elution steps flow countercurrent to the resin, enabling high-resolution separations while reducing the amount of buffer needed for protein purification. The results obtained in this study provide the first experimental demonstration of the feasibility of using countercurrent tangential chromatography for the separation of a model protein mixture containing bovine serum albumin and myoglobin using a commercially available anion exchange resin. Batch uptake/desorption experiments were used in combination with critical flux data for the hollow fiber filters to design the countercurrent tangential chromatography system. A two-stage batch separation yielded the purified target protein at >99% purity with 94% recovery. The results clearly demonstrate the potential of using countercurrent tangential chromatography for the large-scale purification of therapeutic proteins.     

Advanced model‐based control strategies for the intensification of upstream and downstream processing in mAb production

Current industrial trends encourage the development of sustainable, environmentally friendly processes with minimal energy and material consumption. In particular, the increasing market demand in biopharmaceutical industry and the tight regulations in product quality necessitate efficient operating procedures that guarantee products of high purity. In this direction, process intensification via continuous operation paves the way for the development of novel, eco‐friendly processes, characterized by higher productivity and lower production costs. This work focuses on the development of advanced control strategies for (i) a cell culture system in a bioreactor and (ii) a semicontinuous purification process. More specifically, we consider a fed‐batch culture of GS‐NS0 cells and the semicontinuous Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) for the purification process. The controllers are designed following the PAROC framework/software platform and their capabilities are assessed in silico, against the process models. It is demonstrated that the proposed controllers efficiently manage to increase the system productivity, returning strategies that can lead to continuous, stable process operation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:966–988, 2017     

高速逆流色谱技术在生物大分子分离纯化中的应用

高速逆流色谱是一种连续液-液色谱技术,具有无固相载体、样品无需严格预处理等优点。近10年来,在设备结构和溶剂体系等方面进行了大量的研究开发,已推广应用于生物技术、医药、天然产物、环境监测、食品等领域。为适应生物大分子和活性细胞的分离,采用条件温和的双水相体系,研究开发相应的高速逆流色谱设备已成为热点。针对双水相体系的特点,已经开发出了多种具有较高固定相保留率的新型高速逆流色谱设备,通过优化实验条件,成功地进行了多种蛋白质的分离纯化。本对该领域的最新进展进行了综述与评价。     

正相和反相柱层析组合分离纯化紫杉醇

采用正相氧化铝柱层析和反相C₁₈柱层析从东北红豆杉培养细胞浸提物中分离纯化了紫杉醇。优化了氧化铝柱层析和反相柱层析的操作条件。实验发现,经过氧化铝柱层析后,测得的紫杉醇量大大增加。经两步层析,使紫杉醇的含量从小于1.0%提高到95%,样品中微量杂质继以重结晶步骤除去,即可获得纯度超过98%的紫杉醇晶体。采用13-CNMR对晶体分析,所得产物结构与文献上紫杉醇的结构一致。     

The application of preparative batch HPLC, supercritical fluid chromatography, steady-state recycling, and simulated moving bed for the resolution of a racemic pharmaceutical intermediate

This article discusses the chromatographic resolution of a racemic pharmaceutical intermediate. Preparative batch high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), steady-state recycling (SSR), and simulated moving bed (SMB) were used to resolve a total of 12.2 kg of a racemic pharmaceutical intermediate. In this study, a first batch of 0.8 kg of racemate was separated on the preparative batch HPLC and SFC, and subsequently another 5.9 kg of racemate was separated on the SSR. Lastly, a third batch of 5.5 kg was separated on the SMB. The separation conditions and results of these techniques are discussed. The productivities and solvent costs of SFC versus HPLC are compared. The productivities and solvent costs of SMB, SSR, and HPLC are also compared. The analytical method development and process optimization of these processes are also discussed in this article.     

连续流层析技术在亲和层析中的应用及生产放大评估

生物制药行业迅速发展,尤其是上游表达量的增加和规模的扩大,促使上游培养采用连续灌流方式,同时也推动了下游纯化生产工艺相应的采取连续纯化策略.以灌流培养的Fc融合蛋白为例,采用BioSMB PD设备,对比了下游工艺亲和层析捕获步骤中单柱批次纯化和连续流层析纯化的样品纯度和收率,并在此基础上进行小试工艺放大和生产实际用量成...     

疏水层析结合冷乙醇沉淀纯化人血清白蛋白

将层析技术与冷乙醇工艺相结合用于人血清白蛋白的纯化 ,对各过程所采用的层析介质及层析条件进行了探索 ,得到了一条从人血浆中制备血清白蛋白的新路线 :将一步冷乙醇沉淀后的血浆上清进行脱盐除乙醇 ,用阳离子交换介质CMSepharoseFF以透过式层析的模式吸附非白蛋白组分 ,最后选用ButylSepharoseFF一步疏水层析后所得样品经SDS-PAGE银染显示一条单带 ,分析其纯度大于 99% ,计算工艺收率为 81.2%。与传统冷乙醇工艺相比较 ,该工艺最终样品纯度更高 ,且层析可以在常温下操作 ,易实现自动化控制.     

Process chromatography: current constraints and future options for the adsorptive recovery of bioproducts

A contemporary review of adsorption chromatography must embrace aspects of fixed-bed, batch suspension and fluidised-bed contacting of complex feedstocks with adsorbents chemically derivatised with ligands with binding specificities for target bioproducts. Advances in the design of adsorbents, ligands and contactors have facilitated opportunities for integrated processing of unclarified feedstocks to benefit purity and yield of macromolecular products. In the face of competition from apparently simpler, yet productive, technologies (e.g. precipitation, crystallisation and aqueous solvent extraction), further advances in chromatographic purification of macromolecules and nanoparticulates demand close collaboration between inventors and/or manufacturers of new products and the suppliers of chromatographic hardware and consumables.     

铜藻岩藻黄素提取及纯化工艺研究

为了对岩藻黄素的提取、纯化进行系统研究,进而为高纯度岩藻黄素的工业化生产提供研究基础,筛选了适用于提取铜藻（Sargassum horneri）鲜藻中岩藻黄素的有机溶剂,并通过单因素实验和正交实验确定了最佳的提取溶剂浓度、提取温度、提取时间、料液比等工艺参数。随后采用硅胶柱层析法进行纯化,并通过单因素实验确定了最佳的硅胶柱床高度、上样量和洗脱流速。最后采用制备液相法对经层析纯化的岩藻黄素进一步纯化。结果表明,有机溶剂萃取的最佳工艺条件为：甲醇浓度90%,提取温度50 ℃,提取时间1 h,料液比1∶10,此条件下岩藻黄素提取率达到(0.258 9±0.003 6) mg·g-1鲜重（FW）［(1.078 8±0.015 0) mg·g-1干重（DW）］。硅胶柱层析的最佳工艺条件为：硅胶柱床高度10 cm,上样量6 g,洗脱流速10 mL·min-1,此条件下岩藻黄素得率为0.176 5 mg·g-1FW（0.735 3 mg·g-1 DW）,纯度为87.01%±0.88%。经制备液相进一步纯化后,岩藻黄素得率为0.127 1 mg·g-1 FW（0.529 4 mg·g-1 DW）,纯度为99.27%±0.22%。研究所用工艺简单,岩藻黄素得率高,为高纯度岩藻黄素的制备提供了实验基础。     

动态连续逆流提取绞股蓝皂苷的研究

本文在中试条件下,通过单因素试验和正交试验考察不同因素对绞股蓝皂苷提取得率的影响,从而探讨动态连续逆流提取绞股蓝皂苷的最佳工艺。结果表明:动态连续逆流提取绞股蓝皂苷最佳条件为:提取溶剂温度为80℃,料液比为1∶35(g/mL),提取时间为50 min。在此条件下,绞股蓝提取物平均提取得率为33.95%,皂苷得率为8.9%;动态连续逆流提取绞股蓝皂苷具有生产连续性好、皂苷提取得率高、产品纯度高等优点。     

Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned.     

Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins

Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn²⁺–CCB exhibited more specific adsorption capacity toward the target protein compared with Ni²⁺–CCB and Cu²⁺–CCB. The maximum adsorption of EGFP was 1.84?mg/g of Zn²⁺–CCB, with 90% purity under the optimized conditions (ionic strength (1.0?M NaCl), pH (7.2) and imidazole concentration (500?mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.