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Process analytical technology (PAT) for the manufacture of monoclonal antibodies (mAbs) is defined by an integrated set of advanced and automated methods that analyze the compositions and biophysical properties of cell culture fluids, cell-free product streams, and biotherapeutic molecules that are ultimately formulated into concentrated products. In-line or near-line probes and systems are remarkably well developed, although challenges remain in the determination of the absence of viral loads, detecting microbial or mycoplasma contamination, and applying data-driven deep learning to process monitoring and soft sensors. In this review, we address the current status of PAT for both batch and continuous processing steps and discuss its potential impact on facilitating the continuous manufacture of biotherapeutics., Highlights Process analytical technology (PAT) has evolved from hardware-based analyses for defined biological, biomolecular, and biochemical analytes to a toolbox that encompasses data analytics and soft sensors to monitor and control monoclonal antibody (mAb) manufacture. Engineered cell lines used in batch processes and continuous manufacturing have helped improve qualities and production rates for mAbs. Data analytics has become increasingly important as sensors become smaller, more robust, and increasingly ubiquitous, with soft sensors enabling determination of a rolling baseline of process conditions and consequences during the production of biologics. In-line sensors utilized for downstream processes provide a template for how such sensors might be used as part of PAT in the real-time monitoring of the manufacture of biotherapeutic proteins in both upstream and downstream unit operations.

The use of recombinant DNA methods to produce large quantities of protein for therapeutic uses has revolutionized medicine. Industrial challenges for manufacture of biotherapeutic proteins are related to the characteristics of these proteins and the increasing quantities required to address needs of patients, worldwide. A brief overview of therapies in which proteins are employed helps to frame some of the challenges facing their industrial production. The number of molecules and their applications have significantly expanded over the last 15-20 years, together with the quantities used to address specific indications. Challenges addressed include achieving cell density, protein expression, separations of cells and protein, protein purification, and segmentation of protein production into smaller quantities with the evolution of personalized medicine and products designed for increasingly small patient populations. This chapter highlights some of the current challenges.

The use of recombinant DNA methods to produce large quantities of protein for therapeutic uses has revolutionized medicine. Industrial challenges for manufacture of biotherapeutic proteins are related to the characteristics of these proteins and the increasing quantities required to address needs of patients, worldwide. A brief overview of therapies in which proteins are employed helps to frame some of the challenges facing their industrial production. The number of molecules and their applications have significantly expanded over the last 15–20 years, together with the quantities used to address specific indications. Challenges addressed include achieving cell density, protein expression, separations of cells and protein, protein purification, and segmentation of protein production into smaller quantities with the evolution of personalized medicine and products designed for increasingly small patient populations. This chapter highlights some of the current challenges.

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.

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.

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.

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.

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.

This chapter teaches students and practicing engineers the fundamentals of bioprocess design with an emphasis on bioseparation processes. It combines the information presented in earlier chapters for use in the context of integrated processes. The ultimate objective is to enable the reader to efficiently synthesize and evaluate integrated bioseparation processes. Given a product and a desired annual production rate (process throughput), bioprocess design endeavors to answer the following and other related questions: What are the required amounts of raw materials and utilities needed for a single batch? What is the total amount of resources consumed per year? What is the required size of process equipment and supporting utilities? Can the product be produced in an existing facility or is a new plant required? What is the total capital investment? What is the manufacturing cost? What is the optimum batch size? How long does a single batch take? How much product can be generated per year? Which process steps or resources constitute scheduling and throughput bottlenecks? What changes can increase throughput? What is the environmental impact of the process (i.e., amount and type of waste materials)? Which design is the “best” among several plausible alternatives? After completing this chapter, the reader should be able to do the following: • Initiate a process design and choose the appropriate sequencing of processing steps. • Set up a process flowsheet using the unit procedure concept. • Become familiar with batch process simulators. • Schedule batch processes. • Estimate capital and operating costs. • Perform profitability analysis. • Assess the environmental impact of a process. • Perform process sensitivity analyses. Process design is the conceptual work done prior to building, expanding, or retrofitting a process plant. It consists of two main activities, process synthesis and process analysis. Process synthesis is the selection and arrangement of a set of unit operations (process steps) capable of producing the desired product at an acceptable cost and quality. Process analysis is the evaluation and comparison of different process synthesis solutions.

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.

Designed for undergraduates, graduate students, and industry practitioners, Bioseparations Science and Engineering fills a critical need in the field of bioseparations. Current, comprehensive, and concise, it covers bioseparations unit operations in unprecedented depth. In each of the chapters, the authors use a consistent method of explaining unit operations, starting with a qualitative description noting the significance and general application of the unit operation. They then illustrate the scientific application of the operation, develop the required mathematical theory, and finally, describe the applications of the theory in engineering practice, with an emphasis on design and scaleup. Unique to this text is a chapter dedicated to bioseparations process design and economics, in which a process simular, SuperPro Designer® is used to analyze and evaluate the production of three important biological products. New to this second edition are updated discussions of moment analysis, computer simulation, membrane chromatography, and evaporation, among others, as well as revised problem sets. Unique features include basic information about bioproducts and engineering analysis and a chapter with bioseparations laboratory exercises. Bioseparations Science and Engineering is ideal for students and professionals working in or studying bioseparations, and is the premier text in the field.

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.

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.

Sedimentation is the movement of particles or macromolecules in an inertial field. Its applications in separation technology are extremely widespread. Extremes of applications range from the settling due to gravity of tons of solid waste and bacteria in wastewater treatment plants to the centrifugation of a few microliters of blood to determine packed blood cell volume (“hematocrit”) in the clinical laboratory. Accelerations range from 1 × g in flocculation tanks to 100,000 × g in ultracentrifuges for measuring the sedimentation rates of macromolecules. In bioprocessing, the most frequent applications of sedimentation include the clarification of broths and lysates, the collection of cells and inclusion bodies, and the separation of fluids having different densities. Unit operations in sedimentation include settling tanks and tubular centrifuges for batch processing, continuous centrifuges such as disk centrifuges, and less frequently used unit operations such as field-flow fractionators and inclined settlers. Bench scale centrifuges that accommodate small samples can be found in most research laboratories and are frequently applied to the processing of bench scale cell cultures and enzyme preparations. Certain high-speed ultracentrifuges are used as analytical tools for the estimation of molecular weights and diffusion coefficients. The chapter begins with a description of the basic principles of sedimentation, followed by methods of characterizing laboratory and larger-scale centrifuges. Two important production centrifuges, the tubular bowl centrifuge and the disk-stack centrifuge, are analyzed in detail to give the basis for scale-up. Ultracentrifuges, important for analytical and preparative work, are then analyzed. The effect of flocculation of particles on sedimentation is presented, and sedimentation of particles at low accelerations is discussed. The chapter concludes with a description of centrifugal elutriation.

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.

An economic evaluation of a chromatographic separation is discussed. The effects of particle size, cycle time, solvent, and column costs are analyzed. With small particles ( 60 µm), resin costs are less than half of the total. A strong optimum is found between 20–40 µm for maximum productivity, using both Gaussian models and the mass transfer model of Lapidus and Amundson to generate peaks. A new compilation of resin costs, column costs, and pump costs is given.

Scale-up of aqueous two-phase extraction, which is useful in the isolation and purification of certain bioproducts, is limited by the slow demixing rates of the two aqueous phases. Electrokinetic demixing has been shown to increase by more than 5-fold the demixing rates of systems up to 100 mL in volume in a manner that depends on field strength, field polarity, pH, and phase composition. The present study is an attempt to relate demixing rates to droplet electrokinetic mobilities which were measured microscopically and inferred from demixing data. A clear dependence of demixing rate was observed on drop electrophoretic mobility and pH. The electrophoretic mobility of individual phase droplets suspended in the other phase was measured for poly(ethylene glycol)/Dextran systems using a microelectrophoresis unit and compared with mobilities predicted by electrokinetic theory. We confirmed earlier reports that the droplet electrophoretic mobility increased with increasing drop diameter and explained this increase on the basis of an internal electroosmotic flow model. Effective electrophoretic mobilities were estimated from electrokinetic demixing data in a 100-mL column and compared with predicted as well as experimentally measured values of electrophoretic mobility. The mobilties increased with increased phosphate ionization due to change in pH irrespective of the sign (or polarity) of the applied electric field. The electroosmotic flow model could explain satisfactorily the following two paradoxes: (1) the direction of migration of drops is the opposite of that predicted by colloid electrokinetics and (2) the phase demixing rate increased irrespective of the sign of the applied electric field.