Precipitation

Precipitation, which is the process of coming out of solution as a solid, is an important method in the purification of proteins that usually comes early in the purification process. Precipitation is frequently used in the commercial separation of proteins. The primary advantages of precipitation are that it is relatively inexpensive, can be carried out with simple equipment, can be done continuously, and leads to a form of the protein that is often stable in long-term storage. Since precipitation is quite tolerant of various impurities, including nucleic acids and lipids, it is used early in many bioseparation processes. The goal of precipitation is often concentration to reduce volume, although significant purification can sometimes be achieved. For example, all the protein in a stream might be precipitated and redissolved in a smaller volume, or a fractional precipitation might be carried out to precipitate the protein of interest and leave many of the contaminating proteins in the mother liquor. In this chapter the focus is first upon protein solubility, which is the basis of separations by precipitation. Then we discuss the basic concepts of particle formation and breakage and the distribution of precipitate particle sizes. The specific methods that can be used to precipitate proteins are treated next. The chapter concludes with methodology to use for the design of precipitation systems. After completing this chapter, the reader should be able to do the following: • Explain the various factors that influence protein solubility. • Use the Cohn equation to predict solution equilibria (precipitation recoveries). • Identify the distinct steps in the development of a precipitate. • Calculate mixing times in an agitated precipitator, the kinetics of diffusion-limited growth of particles, and the kinetics of particle-particle aggregation. • Perform particle balances as a function of particle size in a continuous-flow stirred tank reactor (CSTR). • Explain the methods used to cause precipitation. • Outline the advantages and disadvantages of the three basic types of precipitation reactor: the batch reactor, the CSTR, and the tubular reactor. • Implement simple scaling rules for a precipitation reactor.

Analytical Methods and Bench Scale Preparative Bioseparations

The development of efficient and reliable processes for bioseparations is dependent on the availability of suitable analytical methods. This means it is important that work on analytical methodology for the bioproduct of interest starts at the very beginning of process development. Analytical studies are important throughout the development and scale up of the process, as changes can occur either to the product or to its associated impurities from what may be thought of as minor changes in the process. This chapter gives access to the vocabulary and techniques used in quality control and analytical development activities, starting with a description of specifications typically set for a pharmaceutical and the rationale behind them. Then, before discussing the assays themselves, we describe assay attributes, which can be measured and used to help not only the assay developer but also the biochemist and engineer responsible for developing downstream processes determine the usefulness and meaning of the assay. Finally, we turn to assays that are commonly applied in biotechnology, as they apply to biological activity, identity, and purity. These assays are the ultimate yardsticks by which the process is measured. Purification methods are developed for their ability to remove a contaminant from the product of interest, whether it is a related molecule, a contaminant related to a host organism, such as DNA or endotoxin, or a process contaminant, such as a residual solvent or water. Critical to understanding process performance is an understanding of how the assays that measure these contaminants have been developed, what the assay strengths and limitations are, and what they indicate and why. Electrophoresis and magnetic separation are two methods that are now used for the bench scale preparative purification of bioproducts, including living cells. The electrophoresis systems with the highest capacity are free-flow electrophoresis, density gradient electrophoresis, recycling free-flow isoelectric focusing, and rotating isoelectric focusing, and the principles of operation of these are discussed. The physical principles of magnetic separations are presented, as well as magnetic reagents and applications of magnetic separators.

Introduction to Bioproducts and Bioseparations

Bioproducts—chemical substances or combinations of chemical substances that are made by living things—range from methanol to whole cells. They are derived by extraction from whole plants and animals or by synthesis in bioreactors containing cells or enzymes. Bioproducts are sold for their chemical activity: methanol for solvent activity, ethanol for its neurological activity or as a fuel, penicillin for its antibacterial activity, taxol for its anticancer activity, streptokinase (an enzyme) for its blood clot dissolving activity, hexose isomerase for its sugar-converting activity, and whole Bacillus thuringiensis cells for their insecticide activity, to name a few very different examples. The wide variety represented by this tiny list makes it clear that bioseparations must encompass a correspondingly wide variety of methods. The choice of separation method depends on the nature of the product, remembering that purity, yield, and activity are the goals, and the most important of these is activity. This first chapter therefore reviews the chemical properties of bioproducts with themes and examples chosen to heighten awareness of those properties that must be recognized in the selection of downstream processes that result in acceptably high final purity while preserving activity. The final part of this chapter is an introduction to the field of bioseparations, which includes a discussion of the stages of downstream processing, the basic principles of engineering analysis as applied to bioseparations, and the various factors involved in developing a bioproduct for the marketplace. The pharmaceutical, agrichemical, and biotechnology bioproduct industries account for many billion dollars in annual sales—neglecting, of course, commodity foods and beverages. By “bioproduct” we mean chemical substances that are produced in or by a biological process, either in vivo or ex vivo (inside or outside a living organism). Figure 1.1 indicates a clear inverse relationship between bioproduct market size and cost. Owing to intense competition, cost, price, and value are very closely related, except in the case of completely new products that are thoroughly protected by patents, difficult to copy, and of added value to the end user.

Crystallization

Crystallization is the process of producing crystals from a homogeneous phase. For biochemicals, the homogeneous phase from which crystals are obtained is always a solution. Crystallization is similar to precipitation in that solid particles are obtained from a solution. However, precipitates have poorly defined morphology, while in crystals the constituent molecules are arranged in three-dimensional arrays called space lattices. In comparison to crystallization, precipitation occurs at much higher levels of supersaturation and rates of nucleation but lower solubilities. These and other differences between crystallization and precipitation are highlighted in Table 9.1. Because of these differences and because the theory of crystallization that has been developed is different from that for precipitation, crystallization is considered separately from precipitation. Crystallization is capable of producing bioproducts at very high purity (say, 99.9%) and is considered to be both a polishing step and a purification step. Polishing refers to a process needed to put the bioproduct in its final form for use. For some bioproducts, such as antibiotics, this final form must be crystalline, and sometimes it is even necessary that a specific crystal form be obtained. In some instances, the purification that can be achieved by crystallization is so significant that other more expensive purification steps such as chromatography can be avoided. There are actually two very different applications of crystallization in biotechnology and bioproduct engineering: crystallization for polishing and purification, and crystallization for crystallography. In the latter case, the goal is a small number of crystals with good size (0.2–0.9 mm) and internal quality. Although it has become common to crystallize proteins for characterization of their three-dimensional structure by x-ray diffraction, this is performed only at small scale in the laboratory, and the knowledge about how to crystallize proteins at large scale in a production process is less developed. However, many antibiotics and other small biomolecules are routinely crystallized in production scale processes. This chapter is oriented toward the use of crystallization in processes that can be scaled up.

Filtration

Filtration is an operation that has found an important place in the processing of biotechnology products. In general, filtration is used to separate particulate or solute components in a fluid suspension or solution according to size by flowing under a pressure differential through a porous medium. There are two broad categories of filtration, which differ according to the direction of the fluid feed in relation to the filter medium. In conventional or dead-end filtration, the fluid flows perpendicular to the medium, which generally results in a cake of solids depositing on the filter medium. In crossflow filtration (which is also called tangential flow filtration), the fluid flows parallel to the medium to minimize buildup of solids on the medium. Conventional and crossflow filtration are illustrated schematically in Figure 4.1. Conventional filtration is typically used when a product has been secreted from cells, and the cells must be removed to obtain the product that is dissolved in the liquid. Antibiotics and steroids are often processed by using conventional filtration to remove the cells. Conventional filtration is also commonly used for sterile filtration in biopharmaceutical production. Crossflow filtration has been used in a wide variety of applications, including the separation of cells from a product that has been secreted, the concentration of cells, the removal of cell debris from cells that have been lysed, the concentration of protein solutions, the exchange or removal of a salt or salts in a protein solution, and the removal of viruses from protein solutions. Filtration often occurs in the early stages of bioproduct purification, in keeping with the process design heuristic “remove the most plentiful impurities first” (see Chapter 12, Bioprocess Design and Economics). At the start of purification, the desired bioproduct is usually present in a large volume of aqueous solution, and it is desirable to reduce the volume as soon as possible to reduce the scale and thus the cost of subsequent processing operations. Filtration, along with sedimentation and extraction (see Chapters 5 and 6), is an effective means of accomplishing volume reduction.

Evaporation

Evaporation is a process that involves the removal by vaporization of part of the solvent from a solution, with the objective being to concentrate the solution. In the evaporation of solutions containing biological compounds, the volatile solvent can be water or an organic solvent. Organic solvents are frequently used for antibiotics, steroids, and peptides. Often the solution is under a moderate vacuum, at pressures down to about 0.05 atm absolute [1], which is especially important for heat-sensitive biologicals where the temperature should be as low as possible to minimize degradation. The energy source for evaporation is usually steam at a low pressure, below 3 atm absolute [1]. Evaporation processes typically occur after the processes used for the removal of insolubles. They are often used to concentrate a solution just prior to the bioproduct being crystallized or precipitated. Evaporation can often be coupled with extraction: for example, a bioproduct is extracted from an aqueous stream with an organic solvent, and the extract is sent to an evaporator for concentration. In this chapter, the basic principles of evaporation are discussed, followed by a description of the most common types of evaporators for heat sensitive biological products and a discussion of scale-up and design methods. After completing this chapter, the reader should be able to do the following: • Explain the different types of resistances to heat transfer in an evaporator. • Take into account the boiling point elevation in heat transfer calculations for evaporators. • Calculate the heat transfer resistances and residence time for the concentration of a heat-sensitive bioproduct in a falling film evaporator. • Estimate the fouling factor in an evaporator. • Calculate the maximum allowable vapor velocity from an evaporator. • Select an appropriate type of evaporator to use based on the specific operational and product characteristics. • Size evaporators based on specific operating conditions and the expected overall heat transfer coefficient. The main principles to consider for evaporators are heat transfer and vapor-liquid separation. The theoretical basis of these principles will be discussed.

Cell Lysis and Flocculation

If a product is synthesized intracellularly and not secreted by the producing cell, or if the product is to be extracted from plant, animal, or fungal tissue, it is necessary to remove the product from the cell or tissue by force. The choice of procedure is highly dependent on the nature of the product and the nature of the cell or tissue. It was seen in Chapter 1 that bioproducts represent a wide variety of chemical species. In this chapter, we also see that the sources of bioproducts—cells and tissues—are widely varied. For this reason, there exists a wide variety of methods for breaking, or lysing, cells and tissues, broadly classified as “chemical” and “physical” methods. Once cells have been suspended and/or broken open, the resulting suspension of solids must be separated from the liquid in which it is suspended. This separation process, filtration and/or sedimentation (the subjects of the next two chapters), is enhanced by having larger particles. Larger particles can be achieved by flocculation, a process whereby particles are aggregated into clusters, or flocs. In recent years, it has become desirable to isolate specific cell types from mixtures of suspended cells and to deliver the resulting cell subpopulation(s) to a process for which they, and only they, are required. Most examples come from in vivo sources such as blood and dispersed tissue cells. This aspect of cell processing, namely, cell purification, places special demands on separation processes that are capable of handling particulate matter under conditions that allow cells to remain alive. This chapter presents two major elements of cell processing: the science and engineering of cell rupture by physical and chemical methods and the flocculation of cells and subcellular particles in aqueous suspension. First, however, it is helpful to develop a broad appreciation for the variety and compositions of cells that are likely to be encountered in downstream bioprocessing.

Laboratory Exercises in Bioseparations

Chapters 3 to 11 of this text are organized around the study of various unit operations in the approximate order of their customary application to bioseparations. In this chapter, five of these operations are singled out for further exploration in the laboratory. These are flocculant screening (Chapter 3), crossflow filtration (Chapter 4), centrifugation of cells and lysate (Chapter 5), aqueous two-phase partitioning of a protein (Chapter 6), and gradient-elution ion exchange chromatography of test proteins (Chapter 7). Each section of this chap­ter is thus an independent laboratory exercise. The instructions can be applied flexibly to the materials and equipment available at a particular laboratory or department. The calculations, reporting, and scale-up applications are applicable to any experiment that follows the generic paradigm of each of the sets of lab instructions. The pattern to be followed consists of becoming acquainted with the equipment and describing it as a unit operation in a report, execution of a predesigned experiment, recording of appropriate data, analysis of the data in the context of this textbook, presenting reduced data in a report, critically analyzing the quality of the results, and, finally applying the actual numerical results to a scale-up to production scale. Process economics may be applied where appropriate. In this laboratory exercise, a flocculant will be evaluated for its ability to flocculate cells or lysate particles. Lysate particles are smaller and require, typically, higher concentration of flocculant than that required to flocculate whole cells. The flocculant concentration required will be determined by observing the persistence of flocculation and clarity of supernatant, measured as a function of flocculant concentration. Flocculants are usually polymers with properties, such as charge, that cause them to interact with cells or lysate particles and bind them together. Their effectiveness depends on molecular weight, charge, solubility, and other properties, and their interactions with cells and particles depend, therefore, on pH, ionic strength, temperature, and dry solids concentration. Unless a great deal is known about the suspended material of interest, or extensive experience has been published, the choice of flocculating conditions usually depends on educated trial and error.

Drying

The last step in the separation process for a biological product is usually drying, which is the process of thermally removing volatile substances (often water) to yield a solid. In the step preceding drying, the desired product is generally in an aqueous solution and at the desired final level of purity. The most common reason for drying a biological product is that it is susceptible to chemical (e.g., deamidation or oxidation) and/or physical (e.g., aggregation and precipitation) degradation during storage in a liquid formulation. Another common reason for drying is for convenience in the final use of the product. For example, it is often desirable that pharmaceutical drugs be in tablet form. Additionally, drying may be necessary to remove undesirable volatile substances. Also, although many bioproducts are stable when frozen, it is more economical and convenient to store them in dry form rather than frozen. Drying is now an established unit operation in the process industries. However, because most biological products are thermally labile, only those drying processes that minimize or eliminate thermal product degradation are actually used to dry biological products. This chapter focuses on the types of dryer that have generally found the greatest use in the drying of biological products: vacuum-shelf dryers, batch vacuum rotary dryers, freeze dryers, and spray dryers [1]. The principles discussed, however, will apply to other types of dryers as well. We begin with the fundamental principles of drying, followed by a description of the types of dryer most used for biological products. Then we present scale-up and design methods for these dryers. After completing this chapter, the reader should be able to do the following: • Do drying calculations involving relative humidity using the psychrometric moisture chart and the equilibrium moisture curve for the material being dried. • Calculate the relative amounts of bound and unbound water in wet solids before drying. • Model heat transfer in conductive drying and calculate conductive drying times. • Interpret drying rate curves. • Calculate convective drying times of nonporous solids based on mass transfer.

Liquid Chromatography and Adsorption

Liquid chromatography and adsorption processes are based on the differential affinity of various soluble molecules for specific types of solids. In these processes, equilibrium is approached between a solid phase, often called the resin, or stationary phase, and the soluble molecules in a liquid phase. The solid phase is “stationary” because it is often packed in a fixed column. Since the liquid phase is often flowing past the solid phase, it is referred to as the mobile phase. Chromatography and adsorption are related unit operations. In chromatography, typically multiple solutes are separated from each other, with the target product solute being one of many that might be recovered at the end of the process step. In adsorption, there are typically only three groups of solutes: those that do not adsorb to the stationary phase (sometimes called “flow through”); secondly, those that adsorb and then are subsequently recovered by an elution step; and thirdly, those solutes that are nearly irreversibly bound and can only be removed from the adsor­bent by regenerating the adsorbent, which usually results in the chemical destruction of these solutes. The word “adsorption” is used both to describe the physical adherence of a solute to a stationary phase, and as the name of the unit operation described above. Adsorption is a subset of the “sorption” phenomena, absorption (transfer of a solute from one phase into another) and ion exchange (exchange of a counter-ion between two opposing co-ions) being the other two sorption phenomena. The unit operations chromatography and adsorption can rely on any of these three sorption processes individually or in combination. In chromatography and adsorption, a mixture of solutes in a feed solution is introduced at the inlet of a column containing the stationary phase and separated into zones of individual solutes over the length of the column. The solutes are carried by the convective action of an elution solvent that is continuously fed to the column after the feed solution has been introduced.