Modeling and Optimization of Rare Earth Element Chromatography

The Rare Earth Elements are a group of metals, that are of growing technical and economical importance. Current separation techniques can be detrimental to the environment, and a clean technology for separation has been devised, based on analytical chromatography of the REEs. The thesis presents column preparation and impregnation to give desired properties of the separation system, experimental and model based optimization for operating points and design of a chromatography step for separating REEs

Robust Multi-objective Optimization of Rare Earth Element Chromatography

Rare earth elements comprise the metallic elements known as lanthanides as well as scandium and yttrium. They are extensively used in modern technological industries and are considered as strategic commodities in many countries. Rare earth element minerals with varying compositions are found at deposits throughout the world, though most of the global REE supply comes from only a few sources. The current industry standard is to employ liquid-liquid extraction methods to separate the elements and upgrade them to suitable purity levels for commercial applications. Chromatography has historically mainly been used as a final purification method, but it is developing to become an alternative separation method with benefits such as achieving higher purity levels, reducing the number of separation steps and utilizing less extractants compared to liquid-liquid extraction. This study is intended as a contribution to the work of developing chromatography as a rare earth element separation method, and focuses on optimization of chromatographic separation on a preparative scale. This has been done through experimental work, and to a large extent by applying optimization methods in conjunction with experimentally validated mathematical chromatography models.In the experimental optimization work, an overloaded one-step separation of the rare earth elements samarium, europiumand gadolinium was accomplished through preparative ion-exchange high-performance liquid chromatographywith an bis(2-ethylhexyl) phosphoric acid impregnated column and nitric acid as eluent. The main focus was to optimize the productivity rate, subject to a yield requirement of 80% and a purity requirement of 99% for each element, by varying the flow rate and batch load size. The optimal productivityrate was found to be 1.32 kg samarium/(m3 column,h), 0.38 kg europium/(m3 column,h) and 0.81 kg gadolinium/(m3 column,h).The model based optimizations have involved the separation of europium from a mixture of the middle rare earth elements samarium, europium and gadolinium as well as the separation of thulium from a heavy rare earth element mixture containing most of the elements. The results from the thulium batch separation showed that a productivity ranging between 0.1-0.45 kg/(m3 column,h) for yields between 73-99% can be expected under a purity constraint of 99%. The findings from the europium batch separation optimization wereused to provide with a general strategy for achieving desirable operation points, resulting in a productivity ranging between 0.61−0.75 kg europium/(m3 column,h) and a pool concentration between 0.52−0.79 kg europium/m3, while maintaining a purity above 99% and never falling below an 80% yield for the target component. In addition to this, a comparative study indicated that the performance of the batch separations can be improved by employing continuous multicolumn countercurrent solvent gradient purification chromatography due to its nature of being a continuous process and its ability to lower the solvent consumption through internal recycling.Finally, the impact of process disturbances was investigated for the europium batch separation process in conjunction with a robust optimization study. The results from the robust optimization were used to chart the required operation point changes for keeping the amount of failed batches at an acceptable level when a certain level of process disturbance was introduced. It was found that the process is very sensitive towards disturbances and a productivity loss in the range of 10-20% can be expected when accounting for robustness

Advanced control strategies for bioprocess chromatography: Challenges and opportunities for intensified processes and next generation products

Recent advances in process analytical technologies and modelling techniques present opportunities to improve industrial chromatography control strategies to enhance process robustness, increase productivity and move towards real-time release testing. This paper provides a critical overview of batch and continuous industrial chromatography control systems for therapeutic protein purification. Firstly, the limitations of conventional industrial fractionation control strategies using in-line UV spectroscopy and on-line HPLC are outlined. Following this, an evaluation of monitoring and control techniques showing promise within research, process development and manufacturing is provided. These novel control strategies combine rapid in-line data capture (e.g. NIR, MALS and variable pathlength UV) with enhanced process understanding obtained from mechanistic and empirical modelling techniques. Finally, a summary of the future states of industrial chromatography control systems is proposed, including strategies to control buffer formulation, product fractionation, column switching and column fouling. The implementation of these control systems improves process capabilities to fulfil product quality criteria as processes are scaled, transferred and operated, thus fast tracking the delivery of new medicines to market

Optimal design and operation of compact simulated moving bed processes for enantioseparations

Simulated moving bed (SMB) chromatography is attracting more and more attention since it is a powerful technique for complex separation tasks. Nowadays, more than 60% of preparative SMB units are installed in the pharmaceutical and in the food in- dustry [SDI, Preparative and Process Liquid Chromatography: The Future of Process Separations, International Strategic Directions, Los Angeles, USA, 2002. http://www. strategicdirections.com]. Chromatography is the method of choice in these ¯elds, be- cause often pharmaceuticals and ¯ne-chemicals have physico-chemical properties which di®er little from those of the by-products, and they may be thermally instable. In these cases, standard separation techniques as distillation and extraction are not applicable. The noteworthiness of preparative chromatography, particulary SMB process, as a sep- aration and puri¯cation process in the above mentioned industries has been increasing, due to its °exibility, energy e±ciency and higher product purity performance. Consequently, a new SMB paradigm is requested by the large number of potential small- scale applications of the SMB technology, which exploits the °exibility and versatility of the technology. In this new SMB paradigm, a number of possibilities for improving SMB performance through variation of parameters during a switching interval, are pushing the trend toward the use of units with smaller number of columns because less stationary phase is used and the setup is more economical. This is especially important for the phar- maceutical industry, where SMBs are seen as multipurpose units that can be applied to di®erent separations in all stages of the drug-development cycle. In order to reduce the experimental e®ort and accordingly the coast associated with the development of separation processes, simulation models are intensively used. One impor- tant aspect in this context refers to the determination of the adsorption isotherms in SMB chromatography, where separations are usually carried out under strongly nonlinear conditions in order to achieve higher productivities. The accurate determination of the competitive adsorption equilibrium of the enantiomeric species is thus of fundamental importance to allow computer-assisted optimization or process scale-up. Two major SMB operating problems are apparent at production scale: the assessment of product quality and the maintenance of long-term stable and controlled operation. Constraints regarding product purity, dictated by pharmaceutical and food regulatory organizations, have drastically increased the demand for product quality control. The strict imposed regulations are increasing the need for developing optically pure drugs.(...

Development of Capillary-Channeled Polymer (C-CP) Fiber as Stationary Phase for Biological Sample Application

Capillary-channeled polymer (C-CP) fibers as an alternative support/stationary phase for biomacromolecule separations on analytical and preparative scales have been developed by Marcus research group. The combination of the fiber micro- and macro-structures and in-column orientation allows operation at comparably high linear velocities (~100 mm s-1) without excessive system backpressure. The ability to move fluid efficiently through the structure is complemented by the fact that the fiber physical structure is effectively nonporous with respect to the size of proteins, therefor there is no significant intrafiber diffusion. Ultimately, these factors combine for protein separations that are devoid of appreciable van Deemter C-term broadening. Polypropylene C-CP fibers have been employed for fast protein separations in reversed phase (RP) mode. For hydrodynamic studies, important factors (injected mass and volume) affecting overload in terms of peak height, width and shape (asymmetry) were evaluated. For LC-MS application, a model mixture of ribonuclease A, cytochrome c, myoglobin and lysozyme were prepared in phosphate buffered saline (PBS) and urine matrices. The efficiency of the matrix removal was reflected in the near-identical qualitative and quantitative responses in both UV-Vis and MS detection. Later, a new, trilobal-shaped C-CP fiber was under developed to address the issue of poor A-term performance of the previous eight-channeled form. The trilobal geometry provides better packing homogeneity due to the fewer potential orientations of the symmetric fiber geometry. Comparisons of separation efficiency and peak shape were made between the two fiber shapes through several dynamic parameters. Poly(ethylene terephythalate) (PET) C-CP fiber was used as the stationary phase for the separation of a synthetic protein mixture based on hydrophobic interaction chromatography (HIC). Optimum resolution and fast analysis times were achieved employing a steep gradient using higher linear velocities. The use of PET C-CP fibers in a HIC protocol was examined to isolate exosomes from a human plasma sample. Initial results demonstrated the ability to isolate exosomes with comparable yields and size distributions and on a much faster time scale when compared to traditional isolation methods, while also alleviating concomitant proteins and other impurities. And, 2D-HPLC method for characterizing mAb concentration and aggregation level, combining ProA purification, a novel injector-loop capture step (HIC processed by PET C-CP fiber), and aggregation determination by SEC is described. Advantages are seen in terms of limiting in-system mAb aggregation due to reduced low-pH solvent exposure, improved 2D chromatographic resolution, better monomer/aggregate ratio fidelity, and enhance quantitative figures of merit

High-throughput and modeling technologies for process development in antibody purification

This cumulative thesis evaluates opportunities, to integrate high-throughput and model-based technologies in biopharmaceutical protein purification

Use of high-throughput tools to optimise polishing-chromatography sequences for complex feed mixtures

Polishing chromatography is a critical element of a bioprocess, because it is currently the only scalable separation technique that can remove process-related impurities, thereby achieving the high purity required of a biotherapeutic. Optimising the polishing chromatography of complex feeds has not been systematically addressed in the literature. This thesis identified a novel, academically affordable ternary protein mixture and systematically developed an optimal two-column polishing train for it. The ternary protein feed mixture was selected using many criteria, but had no special feature to aid identification, such as a chromophore, making it more difficult to characterise. The resulting analytical chromatogram could not be fully resolved, which is typical of industrially relevant products, such as glycoproteins. The selected HPLC column produced fast separations, resulting in a comparatively rapid quantification of preparative chromatograms. Many chromatographic resins and operating conditions were screened, resulting in the non-obvious sequence a hydrophobic interaction (HIC) followed by an anion-exchange (AX) adsorbent. Systematic experimental studies optimised the sequence with respect to yield, purity and amount recovered. Although the loading exceeded the binding capacity of the HIC column, runs at extremely high loadings (60 — 150 g/L) gave very efficient separation in an unusual combination of flow-through and bind-and-elute modes. It was found to achieve >200 mg of acceptably pure product from a single run. A variety of problems were encountered during the development of this polishing train, to which solutions were developed. While these problems are not uncommon, the literature does not contain systematic solutions to them. Examples include decisions about sequence design, protein solubility issues, and the detailed characterisation of samples from preparative runs (not achieved by analytical HPLC). In particular, a system-specific deconvolution methodology was developed that allowed complete characterisation of the mixture; the approach is likely to be widely applicable to industrially relevant biological feed mixtures

Downstream processing development of enveloped viruses for clinical applications: innovative tools for rational process optimization

Dissertation presented to obtain a Ph.D. degree in Engineering and Technology Sciences, Biotechnology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaViral vectors and virus-like particles hold a tremendous potential in various clinical applications in the areas of gene therapy and/or vaccination, drawing the attention of biotechnology and pharmaceutical companies. The majority of these products are manufactured in animal cell cultures, inherently making the process costly. A great deal of effort is taking place to generate optimized biological and engineering strategies to find scalable and cost-effective processes, easily transferable to cGMP facilities. However, the implementation of robust downstream processes generating this type of biopharmaceuticals in the amounts required for pre-clinical and clinical trials is still lacking and lagging. By including a labile lipid membrane layer harboring glycoproteins (often critical for infection) over the viral capsid, enveloped viruses bring extra challenges in terms of their bioprocessing particularly downstream. The work developed during this thesis aimed at improving the state-of-the-art purification processes for these types of viral particles. The rationale was to integrate process understanding with product characterization, still scarce in such biological systems.(...

Theoretical Analysis and Experimental Investigation of Simulated Moving Bed Chromatography for the Purification of Protein Mixtures

Stepwise-Elution Simulated Moving Bed Chromatography (SE-SMB) is a promising method for ‘intensification’ of polishing chromatographic processes in downstream bioprocessing. This is because SE-SMB systems are continuous, capable of high-resolution separations, efficient in their utilization of chromatographic resins, well-suited to non-isocratic proteinaceous separation problems operated under high feed-loading conditions, and highly productive. However, there are a number of theoretical and practical problems which have impeded industrial interest in the adoption of SE-SMB separations into downstream processes. Fundamental phenomena, such as the modulator dynamics of SE-SMB systems, have yet to be theoretically analysed. Consequently, important practical questions – such as how productive and high-resolution separations may be best achieved through SE-SMB systems – remain unanswered. Furthermore, the complexity and operational fragility of SE-SMB systems require much improvement in their ‘robustness’ before any consideration of their application to industrial purification of therapeutic proteins may be entertained. This thesis constitutes an initial investigation of the theoretical and practical issues which arise concerning the application of SE-SMB to industrial bioseparations. Regarding the theoretical issues, an analysis of modulator dynamics in SE-SMB systems is presented. This provides new insights into how such systems – both for binary and ternary separations - should be designed for productive and robust operations. Furthermore, the behaviour of SE-SMB systems under high feedloading conditions is also investigated. Regarding practical issues, experimental SMB separations of a challenging proteinaceous mixture are demonstrated, and simulated comparisons are used to investigate the comparative performance of various intensified processes. Finally, an exploration of SE-SMB fault detection and diagnosis methods is undertaken. The results suggest that SE-SMB chromatography may be ‘de-risked’ to such an extent that, with future development, it becomes an attractive option for incorporation into industrial bioprocesses