Search

The purification of the product, the so-called downstream process (DSP), tends to be one of the most costly aspects of modern bioprocessing, especially in the case of proteins. In such cases, chromatography is still the major tool on all levels of the DSP from the first capture to the final polishing step. In this chapter, we will first outline the commonly used methods and their setup, in particular ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography (AC), and gel filtration (GPC, SEC), but also some less-known alternatives such as hydroxyapatite chromatography (HAC). Then the rational design of a downstream process, which usually comprises three orthogonal chromatographic steps, is discussed. Finally, process variants deviating from the usual batch-column/gradient elution approach will be presented, including expanded bed, displacement, and continuous chromatography, but also affinity precipitation. A most recent trend observable in the biotechnical DSP is the drive towards disposable elements (single-use technologies). Some options for this will be discussed as well.

Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The principle of continuous annular chromatography (CAC) has been known for several decades. CAC is a continuous chromatographic mode, which lends itself to the separation of multi-component mixtures as well as of bi-component ones. In CAC, the mobile and stationary phases move in a crosscurrent fashion, which allows transformation of the typical one-dimensional batch column separation into a continuous two-dimensional one. With the exception of linear gradient elution, all chromatographic modes have at present been applied in CAC. This review focuses on the capacity of CAC for preparative bioseparation. The historical developments and the predecessors of modern CAC are briefly summarized. The state-of-the-art in the theoretical prediction and simulation of CAC separations is discussed, followed by an overview of current CAC instrumentation and example applications, especially for the isolation of proteins and other bio(macro)molecules. In this context, issues of scale up as well as method development and transfer from batch to continuous CAC columns are discussed using recent bioseparation efforts as pertinent examples.

A conventional anion exchange column packed with porous particles (BioScale Q2), and a novel continuous-bed column (UNO Q1) were compared for displacement separation of dairy whey proteins with polyacrylic acid as displacer. The steric mass action model was investigated as a means to aid and accelerate this development. Characteristic charges and steric factors were measured for the proteins and the displacer according to the model, and used together with the affinity constant derived from the adsorption isotherms for simulations, as well as for the construction of the affinity and operating regime plots. If possible, the latter two were used to select conditions for the actual experiments. In the case of the particle-based column, experimental results and simulations did not agree. In addition, the operating regime plot could not be constructed. The affinity plot did predict the order in the displacement train correctly, but gave misleading information concerning the possible effect of a change in displacer concentration. This is taken to be a result of the porous nature of the particles, which handicaps, to some extent, the interaction of the proteins and the displacer molecules with the adsorptive surface. Results were considerably better in case of the continuous-bed column, where there is no intraparticulate surface.

Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the three-albeit isolated-bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 x 52 mm, 10-micron porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 x 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.

Chromatography techniques are essential for the isolation and purification of most of the high value products of modern biotechnology. The economically sensible and technically satisfactory downstream processing of a therapeutic protein, usually involves a number of chromatographic steps. Its development and optimization require considerable knowledge of the various physico-chemical and engineering aspects of biochemical chromatography. This review addresses the various modes of chromatography and the design of chromatographic separation processes from a biotechnologist's point of view. Strategies for optimizing the structure of the downstream process are outlined and scaling up consideration are discussed. The importance of the different chromatographic methods in research and development is estimated in an analysis of protein purification schemes recently published in the literature. Finally, examples of the application of chromatographic procedures for process scale product purification in the biotechnological industry are given.