UV-based dynamic control improves the robustness of multicolumn countercurrent solvent gradient purification of oligonucleotides.

Therapeutic oligonucleotides (ONs) have great potential to treat many diseases due to their ability to regulate gene expression. However, the inefficiency of standard purification techniques to separate the target sequence from molecularly similar variants is hindering development of large scale ON manufacturing at a reasonable cost. Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a valuable process able to bypass the purity-yield tradeoff typical of single-column operations, and hence to make the ON production more sustainable from both an economic and environmental point of view. However, operating close to the optimum of MCSGP can be challenging, resulting in unstable process performance and in a drift in product quality, especially when running a continuous process for extended periods where process parameters such as temperature are prone to variation. In this work, we demonstrate how greater process robustness is introduced in the design and execution of MCSGP for the purification of a 20mer single-stranded DNA sequence through the implementation of UV-based dynamic control. With this novel approach, the cyclic steady state was reached already in the third cycle and disturbances coming from fluctuations in the feed quality, loading amount and temperature were effectively compensated allowing a stable operation close to the optimum. In response to the perturbations, the controlled process kept the standard deviation on product recovery below 3.4%, while for the non-controlled process it increased up to 27.5%.

Experimental design of a twin-column countercurrent gradient purification process.

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.

The paper reports a study on the role of ion-pairing behind peak deformations, e.g. peak splitting and even peak disappearance, during the elution of a peptide at highly overloaded conditions in reversed-phase chromatography. Deformation of component peaks is not uncommon in chromatography. There are reports which discuss their occurrence, but mostly at analytical scale, while their occurrence is quite common also in the preparative scale, as in the case discussed in this work. This paper first describes the conditions leading to peak splitting and peak disappearance of an industrial peptide, then explains the plausible reasons behind such behaviour, and finally with experimental analysis demonstrates the role of ion-pairing in causing such behaviour.

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.

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

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.

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

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.

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.

Improvement of an overloaded, multi-component, solvent gradient bioseparation through multiobjective optimization.

Solvent gradient chromatography is quite often used in analytical studies for decreasing the analysis time of samples having components with widely different retention behaviour. Several studies, both theoretical and experimental, have been reported on the optimization of gradient profiles in improving analytical separation performance, suggesting various linear and non-linear gradients. In preparative chromatography, on the other hand, though solvent gradient is being increasingly used (especially in bioseparation) to improve the product yield and productivity, there is a dearth of literature and clearer understanding of the effect(s) of modifier gradients on the separation performance. For this, the gradients used in applications are of relatively simple profiles like step or linear gradients, obtained through hand optimization based on experience and intuition. Significant improvements, however, can be expected using the state-of-the art modelling of chromatographic processes and optimization routines running on widely available hi-speed desktop computers. In this work we are reporting such an optimization procedure to improve the purification of an industrial multi-component mixture, containing 65.8% of Calcitonin as the main product, in an overloaded reversed-phase column. The work comprises both theoretical simulations and their experimental validation using multilinear gradients as optimization variable. The study produced interesting insights for modifier gradient design, like using peak deformation of the target peptide to increase yield and productivity, and improved our understanding of the effect of modifier gradients in non-linear separations.

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.

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%.

A continuous, counter-current multi-column chromatographic process incorporating modifier gradients for ternary separations.

The existing literature of continuous chromatographic process indicates the scarcity of processes which allow the continuous separation of a ternary mixture and which also exploit the power of modifier gradients and counter-currency. A new process filling this gap is presented and analyzed using an equilibrium theory model and linear isotherms. The analysis leads to the explicit formulation of an operating region, where 100% purity and yield can be obtained. A normalized flow rate ratio is introduced, which allows an easy description and understanding of the process behavior. The gained insights are verified using a detailed simulation model, where the influence of changes in the normalized flow rate ratios on the unit performance is investigated.

Experimental verification of sample-solvent induced modifier-solute peak interactions in biochromatography.

In a previous theoretical analysis based on equilibrium theory it has been shown how differences in the sample and elution modifier concentrations can lead to unexpected behavior of the solute eluted peaks such as retention time distortion, peak deformation and peak doubling. All these features are verified experimentally in this work using the polypeptide calcitonin and a variant of a specific monoclonal antibody as chromatographic model systems. For both experimental systems, the retention time distortion can be predicted with high accuracy by the solution of the equilibrium theory model. For the polypeptide, the predictions from the theory about the occurrence of peak deformation and double peaks has been successfully verified by a series of tailored experiments with positive as well as negative modifier perturbations.