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.     

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.     

Preparative chromatographic separation: Simulated moving bed and modified chromatography methods

Chromatography has been the method of choice for the separation of complex biological mixtures for analytical purposes, particularly for the last fifty years. Its use has recently been extended to preparative separation where the productivity relative to the amount of resin and solvent used is a matter of concern. To overcome the inherent thermodynamic inefficiency of batch chromatography, as exemplified by the partial temporal usage of the resin and dilution of the product with the solvent, chromatography has been continually modified by separation engineers. Column switching and recycling represent some of the process modifications that have brought high productivity to chromatography. Recently, the simulated moving bed (SMB) method, which claims a high separation efficiency based on counter-current moving bed chromatography, has become the mainstay of preparative separation, especially in chiral separation. Accordingly, this paper reviews the current status of SMB, along with several chromatographic modification, which may be helpful in routine laboratory and industrial chromatographic practices.     

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.     

Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems

Selective desorption on ceramic hydroxyapatite (CHT) was implemented for the purification of monomeric monoclonal antibody (mAb) from associated aggregates and other post-protein A step impurities. A robotic liquid handling system was employed to carry out a parallel batch screen of selective desorbents on a post-protein A step mAb mixture. The effect of different phosphate concentrations was also investigated. Selective batch separations were achieved between monomeric mAb and associated aggregates/impurities. The batch screen results also established optimal mobile phase conditions for each selective desorbent. These initial batch results were then used to guide column separations, and baseline separation of monomeric mAb from associated aggregates and impurities was achieved, validating the screening results. Selective desorption also resulted in improved separations on CHT, with 100% yield of pure monomeric mAb as compared to 61% and 79%, respectively, for conventional linear and step gradient operations. This proof of concept study demonstrates selective desorption on CHT as an effective separation technique for the purification of monomeric mAb from associated aggregates and other post-protein A step impurities in a single process step.     

Immobilized metal affinity chromatography in open-loop simulated moving bed technology: Purification of a heat stable histidine tagged β-glucosidase

Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged β-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each of columns for load, wash, elution etc within the SMB. Only the wash and elution are operated with columns in sequence. The β-glucosidase was purified to almost single band purity with a purification factor of 15 and a recovery of 91%. SMB-performance showed reduced buffer consumption, higher purification fold, a better yield and higher productivity.     

Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.     

A simple and inexpensive sample-handling method for the semi-preparative RP-HPLC of polypeptides and non-polar peptide derivatives: pre-adsorption of samples

A simple method is described for the application onto HPLC columns of very crude or alternatively poorly soluble polypeptide samples prior to their chromatographic purification. The procedure involves the batch pre-adsorption of the crude polypeptide mixture from a dilute solution onto an appropriate preparative-grade chromatographic adsorbent, removal of the solvent by rotary evaporation or lyophilisation and then dry-packing the pre-adsorbed chromatographic material into guard column cartridges of suitable dimensions. The polypeptide products can then be eluted either by isocratic or gradient elution methods through the cartridge coupled in tandem with prepacked semi-preparative HPLC columns. This method has been successfully utilised for the routine RP-HPLC purification of polar and hydrophobic polypeptides prepared by solid phase peptide synthesis (SPPS) methods as well as peptide derivatives and intermediates used as part of SPPS procedures.     

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.     

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.     

Large scale protein separations: engineering aspects of chromatography

The engineering considerations common to large scale chromatographic purification of proteins are reviewed. A discussion of the industrial chromatography fundamentals is followed by aspects which affect the scale of separation. The separation column geometry, the effect of the main operational parameters on separation performance, and the physical characteristics of column packing are treated. Throughout, the emphasis is on ion exchange and size exclusion techniques which together constitute the major portion of commercial chromatographic protein purifications. In all cases, the state of current technology is examined and areas in need of further development are noted.

The physico-chemical advances now underway in chromatographic separation of biopolymers would ensure a substantially enhanced role for these techniques in industrial production of products of new biotechnology.     

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.     

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.     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

Continuous preparative liquid chromatography in the downstrem processing of biotechnological products

Continuous counter‐current chromatographic processes have been successfully used in the petrochemical and sugar industry over the last 30 years. Only recently has simulated moving bed (SMB)‐technology attracted widespread interest in the pharmaceutical industry, mainly as a very efficient system for chromatographic enantioseparation. The application of this technique to the downstream processing of biotechnological products requires some specific changes to meet the special demands of bioproduct isolation. Production processes are set up on an multi‐ton scale, for example, for the purification of fructose with both yield and purity higher than 90%. Examples for other mono‐ and oligosaccharides are reported. In the purification of fatty acids or fat soluble vitamins, SMB technology under supercritical fluid conditions gives additional benefits and increases the productivity by a factor of four when a pressure gradient is applied. Another field of operation is the isolation of drug compounds from natural sources where different batch‐ and SMB‐chromatographic steps could be successfully combined. First examples are reported for cyclosporine A and paclitaxel isolation. Finally, step‐gradient elution modes can be used continuously, as demonstrated for the isolation of monoclonal antibodies.     

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.     

Comparing the performance of one-column process and four-zone simulated moving bed by computer simulation

A new one-column chromatography process, analogous to a four-zone simulated moving bed (SMB), was presented. The basic principle of the process was identical to that of a four-zone SMB. The process consisted of one chromatographic column and four tanks, instead of the four columns in the four-zone SMB (1-1-1-1), and has been used for the separation of two amino acids, phenylalanine and tryptophan, using an ion exchange resin. The operating parameters for the one-column process and four-zone SMB were obtained from equilibrium theory. Computer simulations were used to compare the performances of the new one column process to that of the general four-zone SMB, using Aspen Chromatography^™ v 11.1. The differences between the one-column and SMB processes in terms of the purities and yields of phenylalanine and tryptophan were less than 4 and about 6%, respectively. The lower purities of the one-column process were due to the loss of the developed concentration profiles in the column when the liquid was stored in tanks. The one-column process gave great flexibility, and would be useful for reconstructing an existing conventional chromatography process to one of a SMB.     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014