Optimal design and operation of the preparative chromatography of bioproducts

Abstract

The Simulated Moving Bed (SMB) is a continuous countercurrent chromatographic separation process, that allows to split a fccd mixture into two fractions. The principle of SMB is the simulation of thc continuous movemeni of the solid by periodically switching the inlet and outlet ports of thc unit; this is equivalent to the True Moving Bed (TMB) when a set of relationships is fulfilled. SMB yields to larger productivities and lower solvent eonsumptions when eompared to column chromatography, Furthermore. high purities may be achieved even at relatively low resolution. As a result, SMB has become an established technology for the separation of hydrocarbons, sugars and enantiomers. This thesis explores the use of the 8MB technology in the bioseparations field wherc it has been considered only rarely up to the current time; indeed, column (batch) chromatography is extensively used for the downstream processing of biocompounds. In bioprocesses, the target species is generally contained in a cornplex mixture of many species having a broad range of physical and chemieal properties; in this mixture, the target species may elute from the selecte d chromatographic media at an intermediate time arid therefore, a central cut of the chromatograrn is required. However, SMB has been traditionally used to split feed mixtures into two fractions, often pure, and this has been one of the main concerns regarding the applieation of SMB in biotechnology. Another important open question is related to eleaning issues, which are crucial in bioprocessing. In batch processes, a cleaning in place (CIP) step is performed after every cycle and often several colurnns are used in parallel. This is a crucial step in bioseparations and therefore, the use of 8MB's in the field is compromised by the need of an adequate solution for continuous (online) CIP. The first part of this work presents solutions that cnable the use of the SMB technology in bioseparations. Namely, the Equilibrium Theory Model is used to analyze new SMB configurations that allow to recover the target species in a ccntral cut, and to perform online cleaning. These are the three fraction 8MB (3F-8MB) and the integrated cleaning in placo SMB (CIP-SMB); the third SMB configuration is the enriched extract SMB (EE-SMB), which is indicated for binary separations. In the second part of the work, the theorctical findings described above are validated experirnentally for abioseparation of interest. The application investigated concerns the purification of plasmid DNA (pDNA) which is of interest in the areas of gene therapy and nucleie acid vaccination. The experimental CIP-SMB unit uscd is a modified version of the commercial unit ÄKTA Explorer from GE Healthcare. Finally, the third part of this work covers the cornparison of the optimized SMB and column chromatography processes for the pDNA purification step investigated experimentally. The list bclow provides sorne details about these investigations: Enriched Extraet SMB. A variation of the SMB operation called Enriehed Extract SMB (EE-SMB) is investigated, in whieh part of the extract product is concentrated and the resulting enriehed stream is re-injected into the SMB at that same point, i.e. at the inlet of section 2. This operation has been recently patented (M. Bailly, R.-M. Nieoud, A. Philippe, and O. Ludemann Hombourger. Patent No. W02004039468, 2004). In the study here presented, the Equilibrium Theory is used to obtain the constraints on the operating parameters and to identify the operating regimes for the EE-8MB operation. This analysis is carried out for a binary mixture whose adsorption behavior is described by a Langmuir isotherm. The operating regimes are translated into regions on the (m2, m3) plane representation, that can be easily compared with the corresponding ones of the standard SMB given by the Triangle Theory, In particular, for the EE-SMB operation, the region of complete separation turns out to be a line segment, i.e. a one dimensional locus. The theoretical findings are confirmed through detailed simulations of the process, and the value of the new EE-SMB operation mode is assessed. Three fraetion SMB and cleaning in place. A Three Fraction simulated Moving Bed (3F-SMB) unit for separating fced mixtures into three fractions is investigated; this responds to the requirement of many bioseparations which involve complex multicomponent feed mixtures (in opposition to the usual two component SMB feed mixtures, e.g. racemic mixtures). At linear chromatographie conditions, criteria for the selection of the flow rate ratios in the four sections of the unit are developed. These criteria translate into a region of three fractions complete separation in the (m2, m3) plane. The performance of the 3F-SMB unit is investigated through detailod simulations using as a model system the separation of the foul' nucleosides: 2'-Deoxyadenosine, 2'Deoxythymidine, 2'-Deoxyguanosine and 2'-Deoxycytidine. At non-linear chromatographic conditions, sirnulations on the same model system allow to draw qualitative conclusions on the perforrnance of the 3F-SMB. Further on, the possibility of using the 3F-8MB unit to implement a cleaning in place step (CIP) into the standard SMB is shown and validated through simulations, using as a model systern a mixture of two nucleosides and one highly retained impurity. This leads the so-called CIP-SMB configuration. Purification of plasmid DNA in a CIP-SMB unit. Interest in producing large amounts of plasmid DNA (pDNA) has increased in the last decade as a result of the advances in gene therapy and DNA vaccines. The last stage of the manufacturing process involves a puriIication scherne guaranteeing a safe arid stable product, considering the authorities recommendations. This part of the work investigates the first polishing step of plasmid DNA carried out in a eIP-SMB unit based on size exclusion chromatography. The feed mixtute consisted on a clarified oleared lysate, and the experiments were designed using the simple approach of Triangle Theory. The results indicate that total recovery of pD NA is possible and that the productivity can be tuned considering the trade-off with purity. Detailed simulations are used to validate these results, after establishment of a cleared lysate model. Optimization and eomparison of column chromatography and SMB. In this part of the work, the performances of the SMB and the column chromatography processes for two different case studies are analyzed: the first stage of the plasmid DNA polishing, and the Tröger's base enantiomer separation, in which the adsorption isotherrns are linear and non-linear respectively, Simulation tools are used together with an optimization routine (Non-Sorting Genetic Algorithm (NSGA)) in order to find the optimum operating conditions leading to maximum productivity arid minimum solvent consumption; the optimum solution for each of the processes is a curve on the productivity solvent consumption plane, the so-called Pareto set. The comparison between the column and the 5MB processes is based on the relative position of the two Pareto sets calculated at equal conditions and for the same final purity and rccovery of the target species. The results show that 5MB is superior to column chrornatography in the two case studies investigated, i.e, in the case of the linear isotherm (plasmid DNA), the productivity gain is up to a factor two for a given value of the solvent consumption. Furthermore, the flexibility of the SMB operation is larger, since the Pareto sets are flatter and they prolong into regions of the productivity-solvent consumption plane that are not accessible with the column chromatography process.