Biotechnology Advances 17 (1999) 595–597
0734-9750/99/$–see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 4 - 9 7 5 0 ( 9 9 ) 0 0 0 1 2 - 9
Book review
A handbook of chromatography?
Bioseparation and Bioprocessing: A Handbook G. Subramanian, editor, Wiley-VCH, New York, 1998
Volume 1 (xxii 1 537 pages) ISBN 3 527 28876 7
This volume is focused on four subjects: chromatographic separations; membrane separa-tions; modeling; and validation. (Volume 2 of this handbook is reviewed separately in this is-sue.) The book has 17 chapters. The section on chromatographic processing is by far the largest, with nine chapters which discuss every imaginable aspect: simulated moving bed chromatography; perfusion chromatography; hydrophobic interaction chromatography of proteins; displacement chromatography; affinity chromatography; design and operation of large-scale chromatography systems; radial flow chromatography; enhanced diffusion chro-matography media design; and expanded bed chrochro-matography. All this is apparently not enough and some of the chapters in the other sections treat themes related to chromatogra-phy. For example, there are chapters on membranes modified for biochromatography; com-puter modeling of chromatographic bioseparations; and validation of chromatographic pro-cesses. Surprisingly, the book has nothing on size exclusion chromatography or gel filtration which is used widely in protein purification, particularly for desalting.
The handbook is undoubtedly a good single source coverage of nearly all kinds of chro-matographic separations and that emphasis is well placed. For really difficult separations, the resolving power of chromatography is generally unmatched by any other purification method; nevertheless, several factors continue to limit use of chromatographic separations to particular niches. In pharmaceutical processing though, chromatographic separations are well accepted because of the need for exceptionally high purity. In addition, the high value of many biopharmaceuticals allows use of purification methods that would be otherwise too ex-pensive. Manufacture of every high-value bioproduct requires one or more chromatographic steps. Designing large-scale chromatographic separations demands a substantial understand-ing of fundamentals and practical skills. Yet, trainunderstand-ing of bioprocess personnel does not em-phasize chromatography enough. For example, bioprocess engineers are generally more fa-miliar with distillation and liquid–liquid extraction operations than with chromatographic processes. Similarly, of some half-dozen books that are now available on bioseparations, most devote only a chapter or two to chromatography. Compared to other bioseparations, de-sign and scale-up of chromatography are more problematic, but purification capabilities of a successfully implemented chromatographic separation usually exceed the combined perfor-mance of all other separation steps in a typical recovery train.
596 Biotechnology Advances 17 (1999) 595–597
fractionations of sugars and xylenes, but it is little used in purifying biopharmaceuticals, es-pecially proteins. The principles and applications of SMB chromatography are clearly pre-sented in chapter 1. Merits of the SMB method notwithstanding, it is unlikely to displace batch chromatography in most biopharmaceutical processing. Implementing SMB is not easy, particularly when the starting mixture is highly variable.
Chromatographic separations are slow. The maximum permissible flow rate through a column is limited by the need to allow a sufficient solid–liquid contact time for intraparticle diffusion to take place. Pores within particles provide most of the adsorption surface and, hence, the separation capacity in a column. The time needed for pore diffusion is shortened by reducing the particle size and this approach is used in HPLC columns; however, diffusion still limits. One way of getting around this problem is to provide flow-through pores within particles in addition to shorter diffusive pores that branch off the through pores. This is done in hyperdiffusion or perfusion media. As with any chromatographic method, effective use of perfusion media requires a careful and systematic approach to development of the separation process. A good overview of the developmental methodology is provided in one of the chap-ters. Further complementary treatment of enhanced diffusion media occurs later in the book in a chapter that considers some of the fundamentals of intraparticle transport, models of en-hanced diffusion, morphological features of hyperdiffusion media, and other aspects. The chapter on hydrophobic interaction chromatography (HIC) of proteins is also well written. The binding mechanism of HIC is fundamentally similar to that of the reversed phase chro-matography (RPC), but the two methods differ in important ways. Whereas HIC is applica-ble to a wide range of bioactive proteins, the RPC is generally not satisfactory because of de-naturing conditions. The HIC uses a hydrophobic matrix that has a low surface density of hydrophobic ligands. Binding of the protein to stationary phase is encouraged by a high con-centration of salt in the aqueous mobile phase. A stepwise or linear gradient of reducing ionic strength is used to mobilize the bound protein. The processing environment of the HIC is generally mild or stabilizing to most proteins. In contrast, the RPC uses a highly hydropho-bic support matrix that has many hydrophohydropho-bic ligands attached. The stationary phase adsorbs proteins even in pure water. The bound solute is eluted with a low polarity organic solvent such as acetonitrile. These conditions are denaturing to most bioactive proteins.
The chapter on affinity chromatography is much too brief and clearly no reflection of the widespread use of the many variants of this powerful method. Similarly perfunctorily treated are some of the design considerations for large-scale chromatography columns and other hardware. Some of the limitations of the traditional vertical columns have led to develop-ment of radial flow chromatography as discussed in one of the chapters. Radial flow columns have lower pressure drop relative to comparable vertical columns. In addition, radial col-umns are easier to scale up; the depth (i.e. the diameter) and the superficial flow velocity are kept constant and only the height is increased. The performance of the scaled-up device is fully predictable. Like some vertical columns, the radial columns are slurry packed and un-packed in situ, without dismantling.
Biotechnology Advances 17 (1999) 595–597 597
broth or cell homogenate with the chromatographic medium. Once adsorption is complete, the cellular solids are washed away using an up-flow of water. Then, the adsorption matrix is allowed to settle into a packed bed. Elution is done in the usual manner with a downflowing stream of buffer. The book has a good chapter on the principles and applications of expanded bed adsorption chromatography. Another approach to enhancing speed is reflected in mem-brane chromatography. Various modifications of polymer memmem-branes for use in chromato-graphic separations are explained in one of the chapters, but the current and future prospects of this method remain unclear.
Modeling of chromatographic separations continues to occupy many scientists and engi-neers. Modeling is essential to a better understanding of the mechanisms of separation, pre-diction and control of performance, and rational scale up. A chapter on computer modeling of chromatographic separations provides useful insights into factors that impact on perfor-mance and the many uncertainties. A model needs to consider convective and diffusive transport and the kinetics of the solute–surface interactions. The kinetic and transport param-eters change with the eluent. Variations may occur also along a column because of the axial changes in the composition of the mixture. All this complicates modeling. The complexity of the models and the need for information on parameters and how they may vary, has meant that the practice of chromatography remains highly empirical. Separation protocols are de-veloped by knowledgeable trial and error.
With a substantial focus on chromatography and limited space, this volume has but little on non-chromatographic bioseparations. This is no drawback, however, and much of the left out material is readily found in other books. The book’s five non-chromatography chapters discuss a somewhat motley collection of topics: membrane separations in the food and bev-erage industry; recovery of bioproducts by liquid emulsion membranes; use of neural net-works in fermentation processes; advances in modeling for bioprocess supervision and con-trol; and validation of viral safety for pharmaceutical proteins.
Overall, this is an excellent book. It is hardbound and durably produced on acid-free and chlorine-free paper. A good 25-page index and a detailed table of contents assure easy access to information.
