Table of Contents

Cover

Title Page

Dedication

Preface

About the Companion Website

Chapter 1: An Overview of Bioprocess Technology and Biochemical Engineering

1.1 A Brief History of Biotechnology and Biochemical Engineering
1.2 Industrial Organisms
1.3 Biotechnological Products
1.4 Technology Life Cycle, and Genomics- and Stem Cell-Based New Biotechnology
Further Reading
Problems

Chapter 2: An Introduction to Industrial Microbiology and Cell Biotechnology

2.1 Universal Features of Cells
2.2 Cell Membranes, Barriers, and Transporters
2.3 Energy Sources for Cells
2.4 Material and Informational Foundation of Living Systems
2.5 Cells of Industrial Importance
2.6 Cells Derived from Multicellular Organisms
2.7 Concluding Remarks
Further Reading
Problems

Chapter 3: Stoichiometry of Biochemical Reactions and Cell Growth

3.1 Stoichiometry of Biochemical Reactions
3.2 Stoichiometry for Cell Growth
3.3 Hypothetical Partition of a Substrate for Biomass and Product Formation
3.4 Metabolic Flux Analysis
3.5 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 4: Kinetics of Biochemical Reactions

4.1 Enzymes and Biochemical Reactions
4.2 Mechanics of Enzyme Reactions
4.3 Michaelis–Menten Kinetics
4.4 Determining the Value of Kinetic Parameters
4.5 Other Kinetic Expressions
4.6 Inhibition of Enzymatic Reactions
4.7 Biochemical Pathways
4.8 Reaction Network
4.9 Regulation of Reaction Rates
4.10 Transport across Membrane and Transporters
4.11 Kinetics of Binding Reactions
4.12 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 5: Kinetics of Cell Growth Processes

5.1 Cell Growth and Growth Kinetics
5.2 Population Distribution
5.3 Description of Growth Rate
5.4 Growth Stage in a Culture
5.5 Quantitative Description of Growth Kinetics
5.6 Optimal Growth
5.7 Product Formation
5.8 Anchorage-Dependent Vertebrate Cell Growth
5.9 Other Types of Growth Kinetics
5.10 Kinetic Characterization of Biochemical Processes
5.11 Applications of a Growth Model
5.12 The Physiological State of Cells
5.13 Kinetics of Cell Death
5.14 Cell Death and the Sterilization of Medium
5.15 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 6: Kinetics of Continuous Culture

6.1 Introduction
6.2 Kinetic Description of a Continuous Culture
6.3 Continuous Culture with Cell Recycling
6.4 Specialty Continuous Cultures
6.5 Transient Response of a Continuous Culture
6.6 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 7: Bioreactor Kinetics

7.1 Bioreactors
7.2 Basic Types of Bioreactors
7.3 Comparison of CSTR and PFR
7.4 Operating Mode of Bioreactors
7.5 Configuration of Bioreactors
7.6 Other Bioreactor Applications
7.7 Cellular Processes through the Prism of Bioreactor Analysis
7.8 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 8: Oxygen Transfer in Bioreactors

8.1 Introduction
8.2 Oxygen Supply to Biological Systems
8.3 Oxygen and Carbon Dioxide Concentration in Medium – Henry's Law
8.4 Oxygen Transfer through the Gas–Liquid Interface
8.5 Oxygen Transfer in Bioreactors
8.6 Experimental Measurement of K_La and OUR
8.7 Oxygen Transfer in Cell Immobilization Reactors
8.8 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 9: Scale-Up of Bioreactors and Bioprocesses

9.1 Introduction
9.2 General Considerations in Scale Translation
9.3 Mechanical Agitation
9.4 Power Consumption and Mixing Characteristics
9.5 Effect of Scale on Physical Behavior of Bioreactors
9.6 Mixing Time
9.7 Scaling Up and Oxygen Transfer
9.8 Other Process Parameters and Cell Physiology
9.9 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 10: Cell Culture Bioprocesses and Biomanufacturing

10.1 Cells in Culture
10.2 Cell Culture Products
10.3 Cellular Properties Critical to Biologics Production
10.4 Nutritional Requirements
10.5 Cell Line Development
10.6 Bioreactors
10.7 Cell Retention and Continuous Processes
10.8 Cell Culture Manufacturing – Productivity and Product Quality
10.9 Concluding Remarks
Further Reading
Problems

Chapter 11: Introduction to Stem Cell Bioprocesses

11.1 Introduction to Stem Cells
11.2 Types of Stem Cells
11.3 Differentiation of Stem Cells
11.4 Kinetic Description of Stem Cell Differentiation
11.5 Stem Cell Technology
11.6 Engineering in Cultivation of Stem Cells
11.7 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 12: Synthetic Biotechnology: From Metabolic Engineering to Synthetic Microbes

12.1 Introduction
12.2 Generalized Pathways for Biochemical Production
12.3 General Strategy for Engineering an Industrial, Biochemical-Producing Microorganism
12.4 Pathway Synthesis
12.5 Stoichiometric and Kinetic Considerations in Pathway Engineering
12.6 Synthetic Biology
12.7 Concluding Remarks
Further Reading
Problems

Chapter 13: Process Engineering of Bioproduct Recovery

13.1 Introduction
13.2 Characteristics of Biochemical Products
13.4 Unit Operations in Bioseparation
13.5 Examples of Industrial Bioseparation Processes
13.6 Concluding Remarks
Further Reading
Nomenclature
Problems

Chapter 14: Chromatographic Operations in Bioseparation

14.1 Introduction
14.2 Adsorbent
14.3 Adsorption Isotherm
14.4 Adsorption Chromatography
14.5 Elution Chromatography
14.6 Scale-Up and Continuous Operation
14.7 Concluding Remarks
Further Reading
Nomenclature
Problems

Index

End User License Agreement

List of Illustrations

Chapter 1: An Overview of Bioprocess Technology and Biochemical Engineering

Figure 1.1 Milestones in biotechnology and historical advances in biochemical engineering.
Figure 1.2 Expression of recombinant DNA (rDNA) proteins in host cells.
Figure 1.3 Examples of posttranslational modifications that necessitate the expression of heterologous therapeutic proteins in mammalian cells. The posttranslational modifications frequently encountered include disulfide bond formation and glycosylation.
Figure 1.4 A typical manufacturing process of microbial product.
Figure 1.5 A typical manufacturing process of therapeutic proteins by mammalian cells. The duration of each expansion stage is longer and the size ratio of consecutive bioreactors is smaller than those in microbial fermentation.
Figure 1.6 Subjects of studies in biochemical engineering as bioprocessing advances. In the manufacturing of industrial products, technological development evolved from reactor design to aspect control (to reduce the contamination rate in the 1950–1970s) to implementing current Good Manufacturing Practice (cGMP) and establishing process development infrastructure in the 1980s–1990s. More recently, emphasis has been on developing Process Analytical Technology (PAT), Quality by Design (QbD), and platform technology that will be suitable for different products. Current interests include the search for transformative technology that may further cut the process development timeline and reduce the cost of goods.
Figure 1.7 Structure of an immunoglobulin-G (IgG) molecule. A major class of therapeutic proteins that has emerged in the past 15 years is antibodies, in particular IgG. For example, anti-VEGF (vascular endothelial growth factor) suppresses blood vessel formation in some tumors. Each IgG molecule has two heavy chains and two light chains that are linked by disulfide bonds and segregated into a constant region and variable region. The variable region recognizes antigen. Each IgG has two antigen-binding sites.
Figure 1.8 Classical and contemporary scheme of strain improvement for the producing microorganisms of industrial chemicals. The classical way is to introduce mutations in the producing organism through mutagenesis to increase the frequency of mutations on the genes that may affect the synthesis of the target metabolite. Sometimes, a selective strategy is available to enrich the mutated higher producing cells. For example, an analog (a compound that has a similar structure to a native compound, has some similar chemical properties such as exerting inhibition on an enzyme, but cannot be metabolized) may be used to cause inhibition of biosynthetic enzyme. Consequently, its biosynthesis in the wild-type microorganism is suppressed, causing cell growth to be retarded. Any mutant that is no longer inhibited by the analog will survive. Such an analog can thus selectively enrich feedback inhibition deregulated mutants. In any case, extensive screening of higher producers after mutagenesis is necessary. The modern method uses direct cloning to introduce, enhance, or eliminate a gene (or genes) to enhance the high-productivity trait. It can speed up the process of obtaining a high producer dramatically compared to the classical method.
Figure 1.9 The evolvement of penicillin as a technological product. Initial low productivity and production level were quickly enhanced by orders of magnitude due to the high demand created by the revolutionary nature of the product. This is followed by a slower but steady growth driven by the reduced price and wider affordability in different regions of the world. The latter slower growth period entails a much larger total volume of goods and sales. Eventually, the commercial value of penicillin is too low to be profitable for major pharmaceutical companies. The history of penicillin is a general reflection of natural products in classical biotechnology. The decline in classical biotechnological products is supplanted by recombinant DNA (rDNA) technology. The same life cycle will be repeated for rDNA technological products in the years to come.
Figure P.1.1 A metabolic pathway producing G from A with feedback inhibition (solid curve) and feedback repression (dashed curve).

Chapter 2: An Introduction to Industrial Microbiology and Cell Biotechnology

Figure 2.1 A phospholipid molecule. A hydroxyl group of the glycerol backbone is linked to a phosphate. A saturated fatty acid and an unsaturated fatty acid are attached to the other two hydroxyl groups.
Figure 2.2 Lipids forming micelles in aqueous phase. A lipid bilayer allows both the interior and exterior of the lipid droplet to be aqueous phase.
Figure 2.3 Lipid bilayer membrane in a fluid state.
Figure 2.4 Classification of microorganisms according to their energy source, electron receptor, and carbon.
Figure 2.5 Gene expression from DNA to protein in prokaryotic cells.
Figure 2.6 Gene expression from DNA to protein in eukaryotic cells.
Figure 2.7 Organisms in the three domains of life.
Figure 2.8 Positions of representative industrial organisms in the tree of life.
Figure 2.9 Cell wall organization in bacteria.
Figure 2.10 Periplasmic and cytoplasmic overexpression of proteins in E. coli.
Figure 2.11 Electron transport across bacterial cytoplasmic membrane and generation of ATP.
Figure 2.12 Differentiation of Streptomycetes from vegetative cells to spore.
Figure 2.13 Animal (eukaryotic) cells with organelles.
Figure 2.14 A mitochondrion and major energy generation activities taking place in the inner membrane.
Figure 2.15 Expression and secretion of an extracellular protein in a eukaryotic cell.

Chapter 3: Stoichiometry of Biochemical Reactions and Cell Growth

Figure 3.1 Glycolysis pathway. The stoichiometric reaction equations are shown in Panels 3.1–3.3. The corresponding reaction number is shown beside each reaction step.
Figure 3.2 Energy metabolism: oxidation of pyruvate, tricarboxylic acid (TCA) cycle. PDH: pyruvate dehydrogenase; CS: citrate synthase; ACON: aconitase; IDH: isocitrate dehydrogenase; αKGDH: α-ketoglutarate dehydrogenase; SCS: succinyl CoA synthetase; SDH: succinate dehydrogenase; FHMU: fumarase; MDH: malate dehydrogenase.
Figure 3.3 Material inputs and outputs in a cell cultivation system.
Figure 3.4 Biomass yield coefficient.
Figure 3.5 Pentose phosphate pathway and its biochemical reactions.
Figure 3.6 Flux distribution through a pentose phosphate pathway under different cellular needs.
Figure 3.7 Stoichiometric coefficients of reactions in a pentose phosphate pathway.
Figure 3.8 Stoichiometric equations of a pentose phosphate pathway for solving flux distribution.

Chapter 4: Kinetics of Biochemical Reactions

Figure 4.1 Mechanism of peptide bond cleavage by chymotrypsin.
Figure 4.2 Amino acid identity in the substrate binding site determines the substrate specificity of an enzyme. Comparison of two serine proteases, chymotrypsin and trypsin.
Figure 4.3 The dependence of the enzyme reaction rate on the substrate concentration for an enzyme with Michaelis–Menten kinetics.
Figure 4.4 Using a Lineweaver–Burk plot to determine the kinetic parameters for the Michaelis–Menten type of enzyme kinetics.
Figure 4.5 Activity profile of an enzyme as a function of an environmental parameter such as temperature or pH.
Figure 4.6 Examples of enzymatic, bi-substrate, bi-product reactions.
Figure 4.7 Effect of inhibitor concentrations on enzyme reaction rate. (Left) Competitive inhibitor; (right) noncompetitive inhibition.
Figure 4.8 Enzymatic reactions forming a linear pathway.
Figure 4.9 Enzymatic reactions forming a branched pathway.
Figure 4.10 Competition of reactions sharing the same substrate. Regulation by binding affinity of the substrate.
Figure 4.11 Illustration of allosteric regulation of an enzyme.
Figure 4.12 Regulation of the flux of biochemical pathways by feedback inhibition and repression. Biosynthesis of aspartate family amino acids as an example.
Figure 4.13 Kinetics of a single-substrate/single-product reversible reaction.
Figure 4.14 Diffusion across cell membrane.
Figure 4.15 Different types of membrane transporter: a channel protein, a facilitated diffusion transporter, and two transporters for active transport. One uses an energy source such as ATP; the other uses the concentration gradient of Na+ and the electropotential gradient that helps drive a positively charged Na+ into cytosol.
Figure 4.16 The kinetic behavior of two glucose transporters, Glut1 and Glut2, with a low and a high Km, respectively.
Figure 4.17 Examples of binding reactions important in biological systems.
Figure 4.18 The organization of a lactose operon.
Figure 4.19 The organization of a gene under negative control. The repressor protein binds to the operator to suppress transcription. Binding of an inducer to the repressor releases it from the operator and induces the transcription.
Figure 4.20 The organization of a gene under positive control. Binding of the regulatory protein to the operator is necessary for starting the transcription. In this example, the binding of an inducer to the regulatory protein enhances the binding affinity of the inducer–regulatory protein complex to the regulatory region of the DNA, and thus induces the transcription.
Figure 4.21 The effect of inducer concentration on the transcription rate of an inducible gene.
Figure P.4.1 Lineweaver–Burk plot of an enzymatic reaction.
Figure P.4.2 Lineweaver–Burk plot of an enzyme with an allosteric regulation.
Figure P.4.3 A pathway with branched reactions.

Chapter 5: Kinetics of Cell Growth Processes

Figure 5.1 Cell cycle in eukaryotes.
Figure 5.2 Cell number, protein, and DNA content change during growth in a synchronized population or in a single cell.
Figure 5.3 Distribution of cellular DNA content and cell size of a eukaryotic cell population. (a, b) Separate measurements of DNA and size; (c) distribution of simultaneous measurements of DNA content and cell size; (d) contour plot of the same data in (c).
Figure 5.4 Growth phases and variation in cellular properties in a batch culture.
Figure 5.5 Dependence of growth rate on substrate concentration. The relationship can be described by the Monod model.
Figure 5.6 A double reciprocal plot (1/μ vs. 1/s) of the Monod model for estimating μ_max and K_m.
Figure 5.7 Maintenance requirements and specific substrate consumption rate.
Figure 5.8 Effect of substrate and product inhibition on cell growth rate.
Figure 5.9 Relationship between specific product formation rate and specific growth rate.
Figure 5.10 Derivation of cells from tissues and surface attachment–dependent growth.
Figure 5.11 Diauxic growth of E. coli on a mixture of glucose and lactose.
Figure 5.12 Fermentative and aerobic metabolic states of yeast cells.
Figure 5.13 Schematic of a bioprocess system. Model variables include abiotic reactor phase and biotic intracellular reactions.
Figure 5.14 Effect of temperature on the killing of E. coli cells.
Figure 5.15 Effect of temperature on the killing of Bacillus stearothermophilus spores.
Figure P.5.1 Kinetics of cell growth and substrate consumption of a microorganism in culture.
Figure P.5.2 Double-reciprocal plot of the specific growth rate and substrate (glucose) concentation.

Chapter 6: Kinetics of Continuous Culture

Figure 6.1 Comparison of batch and continuous processes.
Figure 6.2 A continuous culture system.
Figure 6.3 Steady-state biomass and limiting substrate concentrations in a continuous culture at different dilution rates.
Figure 6.4 Steady-state cell and limiting substrate concentrations at different feed substrate concentrations. Note that all different feed concentrations give the same substrate concentration curve in the reactor until the washout dilution rate. Beyond the washout dilution, the residual substrate concentration is the same as the feed (not shown).
Figure 6.5 The throughput of biomass in a continuous culture.
Figure 6.6 A continuous culture with cell recycling.
Figure 6.7 The concentrations of biomass and substrate in a continuous culture with cell recycling. A simple continuous culture of the same microorganism is also shown for comparison.
Figure 6.8 A two-stage continuous culture system.
Figure 6.9 An activated sludge waste-water treatment plant with recycling of flocculated cells.
Figure 6.10 Different scenarios of the communal relationship between two microorganisms in a mixed culture.
Figure 6.11 The transient responses of cell concentration and rate-limiting substrate concentration to pulse and step perturbations.
Figure P.6.1 The relationship between a microorganism's specific glucose consumption rate and the specific growth rate.

Chapter 7: Bioreactor Kinetics

Figure 7.1 Solid-state fermentation for soy sauce production in a centuries-old production plant in China.
Figure 7.2 An idealized plug flow continuous reactor and the concentration profile resulting from a step change of concentration in the feed.
Figure 7.3 (a) An idealized stirred-tank reactor and (b) the concentration profile in the reactor resulting from a step change of concentration in the feed.
Figure 7.4 Idealized reactors with reactions, with symbols used in material balance.
Figure 7.5 The concentration profile of the reactant along a plug flow reactor, for first-order and zero-order kinetics.
Figure 7.6 The concentration profile of the reactant for an enzyme reactor with Michalis–Menten kinetics in a plug flow bioreactor.
Figure 7.7 The concentration profiles of cell and substrate in a plug flow and a batch bioreactor.
Figure 7.8 The distribution of the residence time of fluid elements in a plug flow bioreactor and a continuous stirred-tank bioreactor.
Figure 7.9 Growth kinetics in a stirred-tank bioreactor operated at (a) batch, (b) intermittent fed-batch, and (c) continuous feeding fed-batch mode.
Figure 7.10 Different type of bioreactors: (a) a stirred tank for microbial culture, (b), a stirred tank for cell culture, (c) an air-lift fermenter, and (d) a hollow-fiber bioreactor.
Figure 7.11 The protein secretion pathway in the Endoplasmic Reticulum and the Golgi apparatus in a eukaryotic cell.

Chapter 8: Oxygen Transfer in Bioreactors

Figure 8.1 Oxygen supply into a tubular flow bioreactor.
Figure 8.2 Oxygen transfer across a gas–liquid interface.
Figure 8.3 Oxygen concentration gradient across a gas–liquid interface and the driving force for oxygen transfer.
Figure 8.4 Oxygen supply through aeration in a bioreactor.
Figure 8.5 Measurement of overall mass transfer coefficient (K_La) by degassing.

Chapter 9: Scale-Up of Bioreactors and Bioprocesses

Figure 9.1 Two mixing patterns caused by two different classes of agitation mechanisms in a stirred tank.
Figure 9.2 Three types of impellers commonly used in stirred-tank reactors.
Figure 9.3 Important physical parameters for the description of a stirred-tank bioreactor.
Figure 9.4 The correlation between a dimensionless power number (N_p) and impeller Reynolds number (Re_I) for different types of impellers in a stirred-tank reactor.
Figure 9.5 (a) Measurement of average (bulk) mixing time using a tracer. (b) Measurement of mixing time distribution in a stirred-tank reactor.
Figure 9.6 Oxygen transfer from gas phase in a stirred-tank bioreactor.

Chapter 10: Cell Culture Bioprocesses and Biomanufacturing

Figure 10.1 Antibody and antibody-derived protein therapeutics.
Figure 10.2 Examples of N-glycans.
Figure 10.3 Protein N-glycosylation in the endoplasmic reticulum.
Figure 10.4 Extension of glycans in the Golgi apparatus.
Figure 10.5 A vector for the expression of a transgene using a DHFR amplification system.
Figure 10.6 Schematic of cell line development for the production of recombinant proteins.
Figure 10.7 A roller bottle for cell culture.
Figure 10.8 A stirred-tank bioreactor for cell cultivation.
Figure 10.9 Micrographs of cells growing on microcarriers. Cells were stained with crystal violet to increase the contrast.
Figure 10.10 Estimated timeline for the development of originator biologics and biosimilars.

Chapter 11: Introduction to Stem Cell Bioprocesses

Figure 11.1 Intestinal epithelium stem cells.
Figure 11.2 Development of hematopoietic stem cells into blood cells of different lineages.
Figure 11.3 Derivation of embryonic stem cells from inner cell mass on a feeder layer of mouse embryonic fibroblasts, and morphology of the derived embryonic stem cells on the feeder layer.
Figure 11.4 Derivation of induced pluripotent stem cells.
Figure 11.5 Directed in vitro differentiation of stem cells toward different lineages.
Figure 11.6 A 3D space of marker gene expression (transcript or protein) levels depicting cells at a pluripotent state, endodermal state, and hepatic state. Each dot represents a cell with the corresponding levels of marker gene expression. The three envelopes represent regions of the pluripotent, endodermal, and hepatic states.
Figure 11.7 A typical manufacturing process for the production of therapeutic proteins.
Figure 11.8 A possible manufacturing process for the production of stem cells for regenerative medicine.

Chapter 12: Synthetic Biotechnology: From Metabolic Engineering to Synthetic Microbes

Figure 12.1 A general material flow in the production of biochemical products. Carbon and nitrogen sources, sometimes also precursors, are imported through transporters. Multiple biochemical pathways and/or multiple compartments (in eukaryotic cells) may be involved in the synthesis. Side products may also be formed. The product is secreted through a transporter.
Figure 12.2 A general strategy of engineering a microorganism for the production of a biochemical.
Figure 12.3 Transformation of a microorganism with plasmids. The plasmid carries a selective marker gene that is expressed to produce a protein that allows those cells that received the plasmid to have a growth advantage under selective conditions. The plasmid also contains the gene(s) that enable or enhance those cells to produce the product.
Figure 12.4 Bioassays for screening microorganisms that produce a product that suppresses or enables the growth of an assay organism. The former uses an assay organism that is sensitive to the inhibition of the product. The latter uses an auxotroph that is dependent on the metabolite for growth.
Figure 12.5 A replica plate method to detect conditional mutants whose growth is suppressed only under some conditions.
Figure 12.6 Feedback inhibition and repression. The binding of the metabolites (D, F) to the repressor protein (I) and the enzyme (a) causes the repression and allosteric inhibition, respectively. Protein engineering to impair their binding can alleviate feedback regulation.
Figure 12.7 Two pathways for nitrogen assimilation into amino acids are (A) the glutamate dehydrogenase pathway, and (B) the glutamine synthetase–GOGAT pathway.
Figure 12.8 Two different glucose uptake systems in bacteria.
Figure 12.9 The introduction of a xylose transporter system and a pathway leading to its utilization through the pentose phosphate pathway.
Figure 12.10 Time dynamics of biochemical metabolite production. Classical processes for the production of (a) a primary metabolite and (b) a secondary metabolite. Industrial fed-batch cultures for the production of (c) primary and (d) secondary metabolites, versus (e) the industrial production of metabolites using an inducible system.
Figure 12.11 Regulation of flux distribution in aspartate family amino acids biosynthesis.
Figure 12.12 The engineering of a glycolysis pathway for the synthesis of 1,3-propanediol.
Figure 12.13 A synthetic pathway for the production of a novel biochemical methyl-valerolactone in E. coli.
Figure 12.14 Metabolic engineering of a glucose metabolism pathway to produce 1,3-propanediol.
Figure 12.15 Metabolic fluxes in the metabolically engineered 1,3-propanediol pathway when the conversion yield is maximum.
Figure 12.16 Cell-free systems for protein and biochemical synthesis.
Figure 12.17 Concept of BioBricks and in vitro assembly of DNA fragments. (a) DNA parts, devices, and systems. (b) Lego-like assembly of DNA fragments in vitro. (c) Logic gates formed from BioBricks.
Figure 12.18 Synthetic circuit coupled signaling. (a) Intrapopulation signaling. The signal may be processed as it is secreted. When it accumulates to high levels, the signal triggers the expression of genes that cause the population density-dependent response, such as biofilm formation and antibiotic biosynthesis. (b) Interpopulation (community) signaling. An example of cooperative chemical synthesis from raw material S to product P.
Figure 12.19 Chemical synthesis of DNA of the genome, and generation of a semisynthetic cell by replacing the genome of a surrogate donor.

Chapter 13: Process Engineering of Bioproduct Recovery

Figure 13.1 Synthesis of recombinant protein in Escherichia coli as intracellular inclusion bodies and periplasmic soluble molecules.
Figure 13.2 Idealized filtration process, with the filter cake formed by uniform incompressible spheres.
Figure 13.3 Sedimentation and centrifugation in batch mode and continuous mode the dash line represents the trajectory of a particle settling to the “particle capturing surface”.
Figure 13.4 Continuous sedimentation and the effect of settling surface area.
Figure 13.5 Batch extraction: relationship between equilibrium and operating (mass balance) lines.
Figure 13.6 Depiction of countercurrent continuous extraction.
Figure 13.7 Stage-wise material balance in countercurrent continuous extraction.
Figure 13.8 Tangential-flow membrane filtration.
Figure 13.9 Solubility of a solute at different temperature or different pH, solvent, and salt concentration. Combining differential solubilities of two proteins (or solutes) in different temperatures and chemical conditions to achieve fractional precipitation.
Figure 13.10 Flowchart of recombinant antibody production.
Figure 13.11 Flowchart of penicillin G production.
Figure 13.12 Continuous rotary drum filter and continuous centrifugal extractor.
Figure 13.13 Flowchart of recovery process for monosodium glutamate production.
Figure 13.14 Flowchart of Cohn fractionation for blood protein production.

Chapter 14: Chromatographic Operations in Bioseparation

Figure 14.1 Two types of chromatographic operations: (a) affinity chromatography, a type of adsorption; and (b) elution chromatography.
Figure 14.2 Components of a chromatographic system.
Figure 14.3 Charge profile of ion exchange chromatographic adsorbent.
Figure 14.4 (a) Gel permeation (size exclusion) chromatography, (b) hydrophobic interaction chromatography, and (c) reverse phase chromatography.
Figure 14.5 Langmuir adsorption isotherm.
Figure 14.6 (a) Adsorption isotherm under different conditions (illustrated with pH) ranging from favoring adsorption to promoting desorption. (b) Material balances on adsorption and elution operations.
Figure 14.7 Discrete stage analysis of an adsorption process. (a) Operational cycle, and (b) an example of solute distribution in different stages over time.
Figure 14.8 Solute profile in adsorption. In-column view and end-of-column view.
Figure 14.9 Population density of a Gaussian distribution and its use to describe a breakthrough curve.
Figure 14.10 Solution profile in an adsorption column predicted by the Lapidus–Amundson equation.
Figure 14.11 Effect of dispersion on the shape of a breakthrough curve.
Figure 14.12 Discrete stage simulation of solute elution from an adsorption column; and the elution solute profile in an industrial adsorption chromatography.
Figure 14.13 Discrete stage model of an elution chromatography.
Figure 14.14 In-column view of solute profile in an elution chromatography distribution (a) in the column at four different times, and (b) in four different positions in the column (four different stages) over time.
Figure 14.15 van Deemter equation depicts the effect of fluid velocity on HETP.
Figure 14.16 A sequential-multicolumn system for continuous adsorption chromatography.
Figure 14.17 Continuous (simulated moving bed) chromatography. Solutes A and B are separated by an eight-subcolumn elution. Each subcolumn goes through a periodic operation. A fluid flow switching system directs the addition points of the feed, elution buffer, and the exit points of the raffinate (containing the faster moving solute) and extract (containing the slower moving solute) to different subcolumns. After each period, the addition and exit points are all advanced by one substage. The solute profile for each subcolumn, at the beginning and at the end of each period of operation, is shown.
Figure P.14.1
Figure P.14.2
Figure P.14.3

List of Tables

Chapter 1: An Overview of Bioprocess Technology and Biochemical Engineering

Table 1.1 Microorganisms used in food processing
Table 1.2 Some primary metabolites and their producers
Table 1.3 Examples of additional metabolites as industrial chemicals
Table 1.4 Examples of industrial proteins produced in native producers and in recombinant hosts
Table 1.5 Some secondary metabolite producing organisms and their products as drugs
Table 1.6 Examples of pharmaceutically important biologics, including viral vaccines and recombinant proteins
Table P.1.1 Matching terms with close relationships.

Chapter 3: Stoichiometry of Biochemical Reactions and Cell Growth

Table P.3.1 Gas composition exiting from a bioreactor.
Table P.3.2 Cell composition of two microorganisms.

Chapter 4: Kinetics of Biochemical Reactions

Table P.4.1 Reaction kinetics of a common substrate A for two enzymes.
Table P.4.2 Kinetic parameter value of enzymes in the pathway.

Chapter 5: Kinetics of Cell Growth Processes

Table P.5.1 Specific glucose consumption rate of a microorganism at different specific growth rates.
Table P.5.2 Inactivation rate constant at different energy input levels.
Table P.5.3 Concentration profiles of a yeast batch culture using glucose as the carbon source.
Table P.5.4 Temperate and death rate constant profile during heat sterilization.
Table P.5.5 Percentage of bacteria carrying different number of plasmids.

Chapter 6: Kinetics of Continuous Culture

Table P.6.1 Batch culture data for a CHO cell line.
Table P.6.2 Steady-state data for continuous cultures of CHO cells, with a feed glucose concentration of 1.0 g/L.
Table P.6.3 Kinetic parameters of two microorganisms growing in a continuous mixed culture.

Chapter 8: Oxygen Transfer in Bioreactors

Table P.8.1 Gas composition at the inlet and outlet of a bioreactor.
Table P.8.2 Time course data of oxygen concentration in two experiments.
Table P.8.3 Gas composition at the inlet and outlet of a bioreactor.

Chapter 10: Cell Culture Bioprocesses and Biomanufacturing

Table 10.1 Major viral vaccines produced using tissues or animal cells
Table 10.2 Major cell strains and lines for human biologics production
Table 10.3 Some therapeutic proteins produced in mammalian cells
Table 10.4 Recombinant antibodies produced in mammalian cells
Table 10.5 Approximate concentrations of nutrients in cellular and culture environment
Table P.10.1 Dissolved oxygen level during a degassing period of K_La measurement.

Chapter 11: Introduction to Stem Cell Bioprocesses

Table P.11.1 Percentage of cells.

Chapter 13: Process Engineering of Bioproduct Recovery

Table 13.1 Concentrations of product in the bioreactor
Table 13.2 Properties used in different bioseparation methods
Table 13.3 Simulated concentration profile in IgG bioseparation
Table P.13.1 Composition of a feed stream into a product recovery process.
Table P.13.2 The composition of a process stream after different unit operation steps.
Table P.13.3 Relevant product purity data after different unit operation steps.

Chapter 14: Chromatographic Operations in Bioseparation

Table P.14.1 Concentrations of the dye, and the platelets in the testing of two materials.
Table P.14.2 Breakthrough curve of an adsorption process.
Table P.14.3 Total retention time and the peak width at half height.
Table P.14.4 Concentration profile.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

The right of Wei-Shou Hu to be identified as the author of this work has been asserted in accordance with law.

John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com.

Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats.

While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Description: First edition. | Hoboken, NJ, USA : John Wiley & Sons, Inc., 2018. | Includes bibliographical references and index. |

Identifiers: LCCN 2017016663 (print) | LCCN 2017018764 (ebook) | ISBN 9781119159032 (pdf) | ISBN 9781119159049 (epub) | ISBN 9781119159025 (cloth)

Classification: LCC R855.3 (ebook) | LCC R855.3 (print) | NLM QT 36 | DDC 610.285-dc23

Preface

Bioprocesses use microbial, plant, or animal cells and the materials derived from them, such as enzymes or DNA, to produce industrial biochemicals and pharmaceuticals. In the past two decades, the economic output from bioprocesses has increased drastically. This economic growth was the result of the translation of numerous discoveries to innovative technologies and manufactured products. The success has brought together numerous scientists and engineers of different disciplines to work together to break new ground. The task of taking biotechnological discoveries to a successful product or process requires a multidisciplinary team consisting of engineers and chemical and biological scientists to work synergistically. The success of a project, a team, or even a company in biotechnology often hinges on the ability of scientists and engineers of different specialties to work effectively together. This book has been written with this important characteristic of the bioprocess industry in mind. A major goal of the book is to give students the necessary vocabulary and critical engineering knowledge to excel in bioprocess technology.

This textbook is based on a biochemical engineering course that has been offered at the University of Minnesota for a number of years. The contents are intended for a semester course of about 14 weeks of three lecture-hours a week. Although the majority of the students taking this course are senior undergraduate and graduate students from chemical engineering and bioengineering, nearly one-third are graduate students from a life science background. An emphasis of the content and writing of the book is thus the fundamental engineering principles, the quantitative practice, and the accessibility of analysis for students of different backgrounds. The target audience of the book is not only students taking the biochemical engineering or bioprocess engineering courses given in chemical engineering or bioengineering programs but also students in biotechnology programs that are outside of the chemical engineering disciplines, especially in countries outside North America.

In writing this book, I assumed that the students have had at least one biology course, and have fundamental knowledge of carbohydrates, DNA, RNA, proteins, and other biomolecules, as is the case for most engineering students nowadays. Nevertheless, students from both engineering and life science backgrounds will encounter new vocabulary and new concepts that will help them in cross-disciplinary communication once they join the biotechnology workforce.

Chapters 1 and 2 give an overview of organisms, cells and their components, how they become the product, and what the bioprocesses that produce them look like. Chapters 3 and 4 use basic biochemical reactions, especially the energy metabolism pathways, to familiarize engineering students with analysis of biochemical systems and to introduce the concepts of material balance and reaction kinetics to students with a life science background. For all students, these chapters introduce them to kinetic analysis of binding reactions, gene expression, and cellular membrane transport.

Chapters 5 and 6 cover the quantitative description of cell growth and the steady-state behavior in a continuous bioreactor. This paves the way for dealing with different types of bioreactors. Chapters 7, 8, and 9 are the core of bioreactor engineering, dealing with subjects important to process development. These chapters draw upon extensive practical interactions with industry to make them more relevant to bioprocess technology.

The next three chapters – 10, 11, and 12 – discuss three segments of bioprocesses. Cell culture processes, the subject of Chapter 10, currently produce goods valued over US$100 billion per annum. After introducing cell culture processes, the evolution of biomanufacturing and its life cycle is discussed. Chapters 11 and 12 look to the future on cell-based therapy and on the technologies arising from synthetic biology. In dealing with stem cells, the kinetic description of cellular differentiation is also introduced, and in discussing synthetic pathways the importance of using a stoichiometric relationship to determine the maximum conversion yield is reiterated. The last two chapters, 13 and 14, highlight the bioseparation processes. The overall strategy and the key concepts of various unit operations in bioseparation are covered briefly in Chapter 13. Chapter 14 focuses on the basic quantitative understanding of chromatography.

Writing this book has been a long undertaking. Many of my former and current graduate students have helped in formulating the problem sets and the examples. In preparing the book, I also took ideas from many textbooks on biochemical engineering, especially Bioprocess Engineering: Basic Concepts by Shuler and Kargi; Biochemical Engineering Fundamentals by Bailey and Ollis; Fermentation and Enzyme Technology by Wang, Cooney, Demain, Dunnill, Humphrey, and Lilly; and Biochemical Engineering by Aiba, Humphrey, and Millis. I extend my gratitude to my colleagues at the University of Minnesota, especially Arnold G. Fredrickson, Friedrich Srienc, Edward Cussler, Ben Hackel, Kechun Zhang, Samira Azarin, Efie Kokkoli, Yiannis Kaznessis, and Prodromos Daoutidis, for their stimulating discussion that helped shape the book. Finally, I thank Kimberly Durand for her editorial devotion to this book.