Lars Aumann

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. Biotechnol. Bioeng. 2008;100: 1166–1177.

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.

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.

Model simulation and experimental verification of a cation-exchange IgG capture step in batch and continuous chromatography

The cation-exchange capture step of a monoclonal antibody (mAb) purification process using single column batch and multicolumn continuous chromatography (MCSGP) was modeled with a lumped kinetic model. Model parameters were experimentally determined under analytical and preparative conditions: porosities, retention factors and mass transfer parameters of purified mAb were obtained through a systematic procedure based on retention time measurements. The saturation capacity was determined through peak fitting assuming a Langmuir-type adsorption isotherm. The model was validated using linear batch gradient elutions. In addition, the model was used to simulate the start-up, cyclic steady state and shut down behavior of the continuous capture process (MCSGP) and to predict performance parameters. The obtained results were validated by comparison with suitable experiments using an industrial cell culture supernatant. Although the model was not capable of delivering quantitative information of the product purity, it proved high accuracy in the prediction of product concentrations and yield with an error of less than 6%, making it a very useful tool in process development.

Role of Cleaning-in-Place in the Purification of mAb Supernatants Using Continuous Cation Exchange Chromatography

Abstract Monoclonal antibodies (mAbs) are among the most important therapeutic proteins for the treatment of cancer. They also find wide application in the field of diagnostics. The market for mAbs was US

Closed loop control of the multi-column solvent gradient purification process

5.4 billion in 2002 and is expected to triple by 2010 (1). MAbs are mainly produced in cell culture and purified by chromatography. Apart from the mAb, the downstream processing is confronted with a multitude of impurities from the upstream process. Some of these impurities bind strongly to the chromatographic stationary phase and can only be removed by separate cleaning solutions. Depending on the type of media used, e.g. affinity or ion exchange, differing cleaning regimes may have to be applied. In addition, sanitization is required to prevent microbial contamination. Both steps are closely tied to the mAb production step and need to be repeated in regular intervals. In this work, the impact of irreversible impurity adsorption on the separation performance is investigated experimentally; first for a batch column and then for a continuous chromatographic process. At first, the retention time of a model substance is established as a suitable measure to describe the degree of irreversible adsorption. The impact of the presence and the absence of cleaning-in-place (CIP) on the retention time of the model substance is demonstrated. Since the issue of column cleaning is particularly important if processes are operated in continuous mode, in the second part of this work the introduction of a CIP step in the continuous multicolumn countercurrent solvent gradient purification (MCSGP) process is investigated. The process purifies a mAb from a cell culture supernatant that also contains irreversibly adsorbing impurities—or impurities that desorb in a later cycle of the process as a contamination. Following the same approach as for the batch experiments, the MCSGP process is operated in the presence and the absence of CIP in order to track prospective changes of the process performance caused by changes in the product retention time. Instead of the model substance, a cell culture supernatant, providing a real case impurity profile in an industrial application is used for the process. Although an impact of the product retention time change on the process yield is not observed it is shown that CIP is required for a stable long-term operation in terms of purity and system pressure drop. With the introduction of a CIP step the process was successfully operated for 9000 min (6 days) without interruption.

Role of recycling in improving the performance of chromatographic solvent gradient purifications.

A PID controller able to support the operator in the operation of the Multi-column Countercurrent Solvent Gradient Purification (MCSGP) process which is a continuous, countercurrent chromatographic process has been developed. As measurement, only the online UV signals at each column outlet are used. This guarantees a simple and cheap control implementation and a fast control action. Accordingly, the controller does not guarantee any purity or yield value, but simply that the withdrawn window of the product is centered in a specific region of the UV chromatogram where the purity specifications are expected to be satisfied. This can be determined by the operator based on the batch chromatogram selected for designing the MCSGP operating conditions. Thus the controller provides a reliable and efficient tool for the operator to run properly a MCSGP unit in combination with suitable offline analytics for the quantification of purity and yield. The applications are discussed involving the purification of a model protein and a peptide. It is shown that the developed controller is effective in driving the unit to steady state during start up and in keeping a stable steady state while rejecting external disturbances.

Experimental design of a twin-column countercurrent gradient purification process

With significant advancement in the upstream processing technology, downstream processing of large bio-molecules is becoming the bottle-neck in the production chain. To face this challenge, design and development of efficient separation processes has become crucial. As a step towards boosting the performance of a chromatographic separation process through improved design, we investigated the potential of recycling as a process option. The most important advantage of recycling is that it can be implemented in an existing batch system without any major investment and consultation. Although impure products are recycled in industries, it is done as additional batch, and only then, when the recoverable product is valuable enough to surpass the loss of productivity in running the additional batches. In our study, on the other hand, it was found that a well-designed recycle can not only improve the yield, but also the productivity of a multi-component purification. A series of multiobjective optimization studies were carried out on multi-component separation to comprehend the role of recycling with reference to an industrially relevant problem, i.e. the chromatographic purification step of the production process of calcitonin.

Improvement in industrial re-chromatography (recycling) procedure in solvent gradient bio-separation processes

As typical for separation processes, single unit batch chromatography exhibits a trade-off between purity and yield. The twin-column MCSGP (multi-column countercurrent solvent gradient purification) process allows alleviating such trade-offs, particularly in the case of difficult separations. In this work an efficient and reliable procedure for the design of the twin-column MCSGP process is developed. This is based on a single batch chromatogram, which is selected as the design chromatogram. The derived MCSGP operation is not intended to provide optimal performance, but it provides the target product in the selected fraction of the batch chromatogram, but with higher yield. The design procedure is illustrated for the isolation of the main charge isoform of a monoclonal antibody from Protein A eluate with ion-exchange chromatography. The main charge isoform was obtained at a purity and yield larger than 90%. At the same time process related impurities such as HCP and leached Protein A as well as aggregates were at least equally well removed. Additionally, the impact of several design parameters on the process performance in terms of purity, yield, productivity and buffer consumption is discussed. The obtained results can be used for further fine-tuning of the process parameters so as to improve its performance.

The role of ion-pairing in peak deformations in overloaded reversed-phase chromatography of peptides.

Re-chromatography or recycling impure products obtained from the batch runs of solvent gradient chromatography is commonly practiced in industry to improve product yield. However, as the re-chromatography steps are carried out at the expense of running fresh batches, any improvement in the yield comes as a trade-off with the production time, and hence productivity. In recent studies, on the other hand, it has been suggested that with a properly designed recycling process one can not only improve the yield, but the productivity as well. That study, however, considered a steady-state recycling process, a technology yet to be implemented with bio-chromatographic systems. In the present paper we are reporting a study made on non-steady-state recycling or re-chromatography, as it is typically done in industrial practice. The results point out an amendment to the standard way of designing solvent gradients, which is necessary to improve both the yield and the productivity of an industrial run with recycle. Although the test case used here was the separation of an industrial peptide, Calcitonin, in a reversed-phase column, the general methodology of gradient manipulation, needless to say, is also valid for other solvent gradient processes like ion-exchange, HIC, etc.