Approaches for the Isolation and Purification of Fermentation Products

34 Approaches for the Isolation and Purification of Fermentation Products J.C. de Carvalho*, A.B.P. Medeiros, L.P.S. Vandenberghe, A.I. Magalhães, Jr., C.R. Soccol BIOPROCESS ENGINEERING AND BIOTECHNOLOGY DE PARTMENT, FEDERAL UNIVERSITY OF PARANÁ (UFPR), CURITIBA, PR, BRAZIL

34.1 Introduction The steps following the biosynthesis of a desired product, termed downstream processes, consist of separating the product of interest from several other substancesdwhich at this point are undesirable and are termed contaminants or, at best, by-products. In a broader sense, downstream processes also include formulation and packaging, but in this chapter, we will be concerned with the critical steps of isolation and purification, also termed bioseparation. Bioseparation is an essential part of the production of high-value-added biomolecules. At the end of a fermentation process, what is usually present is a mixture of hundreds of substances, both inside cells and dissolved in the liquid. Even in more controlled processes, such as biotransformations, the result is a mixture of precursor and product, as well as by-products. Fig. 34.1 gives an idea of the large amount of proteins present in the cell lysate of Escherichia coli, cultivated in minimal media (based on data from Ref. [1]). Separating the desired molecule from several others may be challenging, but it is essential for several applications: alcoholic beverages must frequently be clear (precipitate-free) and free of microorganisms; biofuels must be relatively pure to burn cleanly; food and drug acidulants must be colorless, and active pharmaceutical ingredients must be of high levels of purity and of definite composition. Downstream processes are, therefore, the part of the process that guarantees standardization of the product. The impact of bioseparation on the overall cost of the process depends largely on the product, but is significant: it can be as high as 40e80% of the total processing costs [2,3]. *

Corresponding Author.

Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products http://dx.doi.org/10.1016/B978-0-444-63662-1.00034-8 Copyright © 2017 Elsevier B.V. All rights reserved.

783

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Molecular weight (kDa)

70 60 50 40 30 20 10 4.5

5

5.5

6

6.5

7

7.5

8

pI FIGURE 34.1 Relative mass abundance (circle size) of proteins from an Escherichia coli sodium dodecyl sulfate/heat lysate. Based on data from Link AJ, Robison K, Church GM. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 1997;18:1259e313. http://dx.doi.org/ 10.1002/elps.1150180807.

This is especially important for products with high purity, or of difficult isolation and purification: the efficiency of each step and the number of steps affect the overall product recovery. The global yield is the product of all efficiencies in the separation sequence. Therefore, a single low-efficiency operation will reduce the overall yield. For example, the yield of a process with five steps having 95% separation efficiency and one with 65% efficiency will amount to a global yield of 50%. Enhancing the efficiency of each operation will increase the production and reduce the downstream processing costs, relative to the overall process. Bioseparation affects the amount of product recovered, and ultimately the gross profit for the process. Therefore, the overall product developmentdincluding upstream processesdmust be thought of with separations in mind. For example, the use of agroindustrial residues, which usually represent a lower impact on the fermentation step, compared to chemically defined media, may be impractical in the production of biomolecules of difficult separation: a more expensive culture medium may provide a fermented slurry with fewer contaminants. Conversely, a robust or flexible downstream might allow a variable upstream, as might be necessary in biofuel production. The positioning of the bioindustry or its product in the market also affects the downstream process. A product may be competitive in terms of quality, price, innovation, or sustainability, among other factors. Ideally, the product will be of high quality, cheap, unique, and “green”; in practice, one may sacrifice purity to have a lower price, or be generic (such as a therapeutic agent competing with more sophisticated alternatives), but invest in a robust process that gives consistent purity and low price. The only way to use near-optimal processes is to evaluate several alternatives and keep informed about new technologies. Regarding technologies, there is an important question of scale-up and process alternatives. Suppose that researchers succeeded in expressing and purifying a new

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biomolecule at the lab: a human protein produced in an E. coli. strain. Now they want to produce it on a larger scale, to start phase I testing in animals. The separation process in the laboratory scale consists of (1) using ultrasound for cell disruption, while the culture is in an ice bath; then, (2) the mixture is centrifuged and rinsed two times, and (3) the inclusion bodies that concentrate in a pellet are resuspended in an adequate buffer. (4) The solution is filtered with a membrane, again in a centrifuge, and (5) the filtered solution is frozen and lyophilized for concentration. After concentration, the material is (6) resuspended in a low amount of buffer and (7) separated by gel electrophoresis. (8) Finally, the region that contains the desired enzyme is cut from the gel, mixed with a suitable buffer, and purified by chromatography. Parts of the process just described may be impracticable or at least too expensive in larger scales. Step (1) heats the material too much, step (2) may lead to the loss of a lot of enzyme, step (5) may be too expensive for intermediate processing, and step (7) has very low capacity. Alternative processes that can be scaled up should be tested in small scale, e.g., other chemical or mechanical cell disruption methods and chromatographic methods. Conceptually, if the protein was already developed with an added histidine tag, the downstream process could be simplified and consist of cell disruption, inclusion body dissolution, and capture and purification in a metal-ion-affinity column. If a molecule is being studied to develop a bio-product, it is essential to think, from the beginning of the laboratory tests, of possible bioseparation steps. Although the physicochemical principles involved in each kind of separation process are the same, irrespective of scale, there may be a marked difference in resolution in small-scale or analytical processes, compared to large-scale processes; the latter tend to have lower resolution. For example, direct (dead-end) filtration of small volumes may be feasible because the accumulation of solids on the membrane surface is small, and the membrane may even be discarded after use. A similar process in large scale would be a tangential filtration, and would have either a higher loss of material (with the retentate, a liquid stream) or a lower loss but a more diluted filtrate (permeate). Another example is chromatography, in which columns have large theoretical plate numbers in small scale, whereas process-scale chromatography sacrifices resolution for higher column loads. Large and small scale is, of course, a matter of product: whereas therapeutic biomolecules may be produced in subkilogram batches, enzymes and antibiotics are produced in kilogram batches, and organic acids, solvents, and biofuels involve the processing of several tons per hour. These production scales affect the type of operation that can be used (chromatography, for example, has practical limits in column height and diameter), as well as the cost permissible for the operation. The smaller scale operations can have a higher resolution and are adequate for specialty bio-products, which in turn tend to have a higher selling price. Commodity bio-products frequently compete with cheap synthetic equivalents, and must be processed in large scale to maintain a low price. Finally, purity is also of importance in process scale: a very high purity bio-product may require several separation steps, which is consistent with higher selling prices and smaller scales.

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34.2 From Molecule Properties to Process Selection The basis of a separation is that the substance or material of interest has some characteristic that differs from contaminants, such as volatility, charge, or size. Therefore, to develop a downstream process, it is essential to know the physicochemical properties of the product of interest and of the contaminants. From the analysis of these properties, it will be possible to identify the most relevant differences, and therefore separation techniques, that can be explored. The classical sources for chemical properties (compendia such as the Merck Index, CRC Handbook of Chemistry and Physics, etc.) have already migrated to databases and property prediction software. Table 34.1 suggests relevant sources for physicochemical property data. Well-known substances, such as insulin or cephalosporin C, will have both experimental and predicted data recorded; lesser-known substances whose chemical structure is defined may have their properties predicted through several techniques; these values must be used carefully, but the quality of predictions is growing better.

34.3 Separation Principles In addition to a difference in the properties of molecules in a mixture, there must be a driving force for the separationda space-distributed property that affects the behavior of the molecule, such as an electric, gravitational, or magnetic field, or a chemical potential gradient, etc. Table 34.2 lists typical downstream operations, their driving force, the Table 34.1

Selected Sources for Data About Biomolecules

Database

What Can Be Found

BRENDA brenda-enzymes.org Chemspider chemspider.com DrugBank drugbank.ca KEGG http://www.genome.jp/kegg/

Enzyme nomenclature, ligand, functional and organism-related information, structure, molecular properties Aggregated database: References, physical properties, interactive spectra, chemical suppliers for millions of compounds; includes predictive tools from ACD/Labs and EPA Drug-oriented chemical and pharmacological information, as well as drug target information Kyoto Encyclopedia of Genes and Genomes: Knowledge base for predicting biodegradation and biosynthesis of biomolecules at the cellular level; more useful in upstream development (metabolism and expression), but still insightful for downstream Chemical properties and biological activities of small molecules; points to several other important data sources Aggregates the Beilstein, Gmelin, and Patent Chemistry Database in one platform; links to original references; subscription-based Digital version of the Chemical Abstracts Service, curated by ACS; has properties and reactions for millions of substances, with links to literature; subscription-based This supplier has information and protocols for thousands of substances and is a good source for preliminary information

PubChem pubchem.ncbi.nlm.nih.gov Reaxys http://www.reaxys.com/ SciFinder Scholar scifinder.cas.org SigmaeAldrich page sigmaaldrich.com

Chapter 34
Isolation and Purification of Fermentation Products Table 34.2

787

Common Process-Applicable Bioseparations and Common Uses

Operation

Typically Used to Separate

Driving Force

Observation

Centrifugation Sedimentation Filtration Flocculation Microfiltration Ultrafiltration Nanofiltration Reverse osmosis Dialysis

Suspended solids or immiscible liquids from another liquid Solids from liquid/gas Aggregates suspended solids Suspended solids Macromolecules Small molecules Ions Operational mode for the membrane separations described above Volatile molecules Solutes dissolved in liquid

Gravity or centrifugal acceleration Pressure differential Electrostatic interactions Pressure differential Pressure differential Pressure differential Pressure differential Pressure differential

There must be a difference in density between phases SB: Filter medium, solids cake Ionic radius is reduced to facilitate aggregation SB: Membrane, pore size w0.1e1 mm SB: Membrane, pore size w0.1e0.001 mm SB: Membrane, pore size w0.001e0.0001 mm SB: Membrane, pore size w0.0001 mm (10 Å) SB: Membrane; solvent is added during the filtration

Vapor pressure Chemical potential

Membrane, usually hydrophobic Immiscible solvents create two phases

Pervaporation Liquideliquid extraction Solideliquid extraction Affinity chromatography Gel filtration (size-exclusion chromatography) Ion-exchange chromatography Adsorption

Solutes distributed in a solid matrix Solutes with different ligand affinities (antigeneantibody, enzymeesubstrate, etc.) Solutes with different sizes

Solutes with different charges

Drying

Same as chromatography, but without dynamic solute migration Water from solids or liquids

Evaporation

Water from liquids

Lyophilization Crystallization

Same as drying, but from frozen solids Pure solids from solution

Precipitation

Insolubilizes solutes

SB, separation barrier.

Chemical potential Pressure differential, chemical potential

Operation mode uses adsorption and migration to discriminate molecules

Pressure differential, concentration

Operation mode uses diffusion and migration to discriminate molecules

Pressure differential, electrostatic interaction

Operation mode uses charge and migration to discriminate molecules

Vapor pressure differential (proportional to temperature and concentration) Vapor pressure differential (proportional to temperature and concentration)

Gives a solid product

Gives a liquid concentrate

Must use vacuum to facilitate sublimation Chemical potential difference between liquid and solid phase Chemical potential, via solubility

Must maintain supersaturation

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molecular or material property explored, and the barrier or phase that enhances separation. Understanding driving forces and properties to explore for a molecule is paramount for selecting operations to be used in a downstream strategy. After a separation step is selected for evaluation, it is necessary to evaluate the size of the equipment. Even if the equipment is to be purchased from a specialty manufacturer, which is often the case for complex machines such as centrifuges or chromatographic columns, a preliminary sizing calculation will show the feasibility of using a selected operation. These calculations are based on two kinds of equations: a material balance (in which the user defines what is to be separated, which concentrations are to be achieved) and a process-specific equation (by which the physicochemical properties of the system will be used to predict operational parameters). The evaluation may be done fixing the throughput, and then the size of the equipment will be found, or fixing the size and verifying how long the separation will take. For these calculations, the use of computerized models is valuable; the equations may be implemented into a spreadsheet or programmed in a computer language, or a process simulator can be used. Of course, a critical analysis of the calculations is necessary, and this equipment sizing should be interactive. The sizing actually gives the designer a better understanding of the process and can lead to modifications in other operationsdfor example, protein centrifugation, which is a physically possible operation, may prove to be unfeasible because of low throughput.

34.4 Process Development for Bioseparations The development of downstream processing is a creative process that involves several steps of analysis and synthesis. Experience plays a key role in this development: a specialist may quickly define a sound strategy for the separation of a biomolecule. For the rest of us, there are some guidelines for process development, which are collectively termed heuristics (from the Greek words for “finding” and “discovering”), and designing methodologies such as the use of computational models and systems. Heuristics consist of evaluating several aspects of the process and the product, and making choices from these aspects. Because the evaluation may be done from several perspectives (energy input, purity, residue generation, molecule integrity, etc.), there are several heuristic approaches for process development. Some relevant approaches, the RIPP scheme [4,5] and method, design, species, and composition heuristics [6,7], are summarized in Table 34.3 and briefly discussed. The use of process simulators and computational models has became widespread. It is possible to develop a process using the properties of the substances to be separated and the basic equations underlying separation principles, to evaluate in silico the feasibility of the process. It is conceptually possible to feed to an expert system the composition of the broth and the desired product and let it develop an entire process, but in practice the number of combinations is prohibitive (for a separation train of just five operations chosen from 20 possibilities, there are almost 2 million possibilities).

Chapter 34
Isolation and Purification of Fermentation Products

Table 34.3

789

Some Process Heuristics Types

Heuristic

Basis

RIPP scheme

RIPP stands for removal of insolubles, isolation or concentration of the product, purification, and polishing. Most downstream processes have this sequence, and it became a way of thinking in the development of new processes. These develop the process regarding specific separation method preferences, such as microfiltration over centrifugation, or favor affinity separations. This may be based in experience and similarity to other processes or on available/accessible equipment. These focus on overall process characteristics. The RIPP scheme is arguably a design heuristic; others are the preference for the cheapest separation options, or the less energyintensive process, or yet have the focus on maximizing integration. These focus on the chemical species, for designing specific operations. For example, the fractionation of a protein with a distinctive molecular weight points to a size-based separation, such as gel filtration. These focus on the feed composition  separation costs, for example, the reduction of volumes at the beginning of a process.

Method heuristics

Design heuristics

Species heuristics

Composition heuristics

A basic process must be developed first. Current simulators (such as Aspen Plus or SuperPro Designer) are actually oriented toward aiding in equipment sizing and analyzing process throughput, rather than suggesting operation sequences. Therefore, it is necessary to start considering approaches for processes that already exist and work well (from laboratory scale, from previous experience, or from patent literature); then define an operational sequence using process heuristics, and finally evaluate it in silico. After a few iterations of designesimulationeanalysis, a candidate process may be tried in the lab or a pilot plant, and new iterations of process development will occur, until a sound production process is developed.

34.5 Typical Separation Steps for Selected Classes of Biomolecules Although each bio-product has its specificities, there are broad classes of products that involve more or less the same kinds of operations. For example, macromolecules may be separated using ultrafiltration, whereas volatile molecules might be separated by distillation. From several classes of biomolecules, after exposing the product (which is oftentimes intracellular), a short sequence of operations is enough to obtain partially purified products. Table 34.4 shows the process outlines for a few extracellular bio-products (adapted from Refs. [2,8]).

34.6 Alternative Separations In migrating from lab scale to pilot scale, some operations can be easily scaled up with similar results (especially those involving intrinsic properties of the system and not of

CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

Polyssacharides

Size

<200Da

Acid / base

Yes

No

No

Yes

Yes

No

No

Yes

No

polarity

High

High

Low

High

Low

Low

Low

High

High

Process step Solid-liquid separation Distillation Solid-liquid extraction Liquid-liquid extraction Precipitation Adsorption Acidification Ion exchange Evaporation Crystallization Drying Milling / classification Final product

200-1000Da

Proteins

Lipids

Erythromycin-A

Cephalosporin-C

Citric acid

γ-decalactone

Process Outlines for Selected Extracellular Bio-products

Lysine

Table 34.4

Ethanol

790

>1kDa

Chapter 34
Isolation and Purification of Fermentation Products

Table 34.5

791

Operation Correspondence and Possible Substitutions in Scaling Up

Common Lab-Scale Operation

Common Pilot/Large-Scale Equivalent or Substitute

Simple filtration Cell disruption by sonication, French press, or bead beaters Difficult direct filtrations Centrifugation in tubes

Scalable; dead-end filtration, centrifuging, filter centrifuges High-pressure homogenizer, microbead mills

Large relative centrifugal force, long centrifugations Extraction Evaporation Dialysis in bags Precipitation Solid-phase extraction Chromatography Electrophoresis Distillation Crystallization Vacuum or freeze-drying

Filtration with filter aids, tangential filtration Scalable; tubular or disk centrifuges; can be substituted by filtration or microfiltration Tangential micro- or ultrafiltration Scalable (perhaps multistage and/or with the aid of a centrifugal separator); can be substituted by adsorption Scalable; tangential filtration is an option for large molecules; can be substituted by cryoconcentration Tangential filtration, diafiltration mode Scalable, but the mixture of precipitating agent is critical Column adsorption Scalable (maybe with less resolution) Chromatography Scalable; in some cases, extraction may be an option Scalable, but the kinetics must be carefully adapted Scalable, but may take a long times

the equipment), such as precipitation, extraction, and even adsorption. However, some operations have a design or equipment that cannot be efficiently scaled up, for example, electrophoresis, whose practical dimensions are limited by the gel resistance and the heat generation. However, other operations exploring the same characteristics of the molecules, or that achieve the same effect, can be used. Table 34.5 suggests operation correspondences worth trying.

34.7 Sizing Guide for Operations After a downstream operation is defined, the sizing of the operation (i.e., the characteristic area or volume of the equipment) is done using a material balance and an equilibrium or segregation condition characteristic of the operation. Some characteristics that are overlooked in the lab scale, such as viscosity, are important here. In the following pages, the most common options for batch bioseparations are reviewed, to highlight the most important aspects for a preliminary process design.

34.7.1

Filtration

In this operation, a feed containing suspended solids is forced through a porous filter medium, which retains the suspended solids; the liquid passes, carrying solutes and

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smaller particles. The solids build up, forming a cake, which adds to the resistance to flow. Either flow is maintained, but pressure is increased during operation, or pressure is maintained, but the flow drops. The flow through a filter varies according to the following equation, one of the forms of Darcy’s law [9]: V ¼

1 dV K $ DP $ ¼ A dt ml

[34.1]

where V is the fluid velocity, dV/dt is the volumetric flow, l is the cake layer thickness, K is the cake porosity, A is the filter area, and DP is the pressure across the filter. A and DP may be held constant in a typical filtration, whereas K may be estimated from the CarmaneKozeny equation [10]: K ¼

ε3 dp2 180 $ ð1  εÞ2

[34.2]

where ε is the bed porosity, i.e., the ratio between void volumes and total volume, and dp is the particle diameter. Although the equation was developed for spherical particles, it works well for incompressible beds such as packed columns [9]. For filtration of incompressible cakes, K is constant; but for deformable, compressible cakes, K may decrease with pressure increase. There are two common types of large-scale filtration operations: direct, pressure filtration and rotary vacuum filtration. Pressure filtration has numerous designs, most of which operate in a discontinuous mode, whereas rotary vacuum filtration can be operated in a continuous mode. Key parameters to look at include suspended solids concentration, suspended solids size, cake porosity and permeability, cake water retention, liquid viscosity, and the need for washing the cake.

34.7.2

Centrifugation

In this process, a liquid stream containing solids is subject to a centrifugal force, which will accelerate the separation of particulate solids. It is heavily dependent on particle size, as can be seen by the general equation for sedimentation, vg ¼

d 2 $ ðrs  rÞ g 18 $ m

[34.3]

where vg is the settling velocity, d is the particle diameter, rs is its density, r is the density of the liquid, m is the viscosity, and g is the gravity acceleration. The dependence on particle size is the reason centrifuges will not work for dense, but small, particles such as globular proteins. However, the operation works well for separating immiscible liquids and relatively large solidsdeven bacteria. In centrifugation, vg is multiplied by the relative centrifugal force (RCF), making a faster separation. RCF can be estimated using the general equation for centrifuges, RCFðx $ gÞ ¼ 0:0000112N 2 $ R

[34.4]

where N is the rotational speed of the centrifuge, in rpm, and R is its average radius, in cm.

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793

Process centrifuges may separate volumes that are much larger than the internal volume of the separating chamber (bowl or disk stack, for example). In this type of equipment, the throughput may be estimated by the sigma analysis, which separates geometric factors inherent to the centrifuge from process-specific factors such as particle size and broth density: Q ¼ vg $ S

[34.5]

For the two most common kinds of centrifuges, tubular [11] and disk-stack [12]:
 pu2 $ L R22  R21 $  2R2  S¼ g ln 2 2 2

[34.6]

R2 þR1

X

¼


2p $ u2 ðN  1Þ r23  r13 3g $ tan q

[34.7]

where R2 and R1 are external and internal bowl or disk-stack radii, u is the angular velocity, g is the acceleration of gravity, and L is the length of the bowl, whereas N is the number of disks and q is the angle of the disks. There are several process centrifuge designs, both discontinuous and continuous. Continuous centrifuges can have large throughputs, but will not give a solid pellet (as obtained in the lab), but rather a concentrated stream and a clarified stream. Discontinuous centrifuges will give a much more concentrated mass of solids, but with the need of stopping the process and discharging the solids. Key parameters to look at include suspended solids concentration, suspended solids size and distribution, suspended solids density, and liquid viscosity.

34.7.3

Cell Disruption

Cell disruption is completely dependent on the type of cell, and is discussed in depth in Chapter 35. It consists of exposing an intracellular product of interest, by permeating the cell membrane (for smaller molecules) or creating fissures in the cell wall (for large molecules). Chemical disruption is easily scaled up: it is just a question of using proportionally larger volumes and maintaining the lysis conditions (pH, temperature, enzyme or solvent concentration). Mechanical disruptions may have to be altered from lab to pilot or plant scale; there are a few cost-effective and efficient options. Data obtained using a French pressure cell can be used as a guide for cell disruption in a highpressure homogenizer, whereas bead beater or microbead mill data may be used for estimating the conditions to be used in a larger microbead mill. Mechanical disruptions systems cause heating, and an external refrigeration loop may be necessary. The degree of disruption must be considered, because it affects the purification steps by releasing an enormous amount of intracellular molecules. If a selective permeation of the cell is enough to release the product of interest [13], it may be a good alternative. Key parameters to look at include target product thermal and shear stability, resistance to lysis buffers or enzymes, and disruption extent.

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34.7.4

Tangential Filtration

In tangential filtration, the bulk flow is parallel to the filtration medium (usually a polymeric porous membrane), therefore reducing the buildup of a cake. The process is so effective that it goes beyond filtering suspended solids (in microfiltration) to segregating colloidal suspensions (in ultrafiltration) and true solutions (in nanofiltration and reverse osmosis). The flow of liquid through the membrane depends (in addition to pressure and membrane resistance, as in traditional filtration) on the concentration of particles retained over the membrane, which add to the resistance. However, these particles either are hydrodynamically dragged back or have enough mobility to diffuse back to the bulk of the liquid. Hence, an equilibrium may be reached, in which the resistance to flow over the membrane is constant: J¼

Cm  Cp D ln d Cb  Cp

[34.8]

where D is the diffusion coefficient and d the boundary layer thickness. D/d can be thought of as a mass transfer coefficient, k. The ratio Cm/Cb is the concentration over the membrane divided by that in the bulk, and is known as the concentration polarization modulus. This ratio increases with increasing J and with decreasing k and can be easily determined from experimental data. Cp is the concentration in the permeate, and it is important for complex mixtures of proteins or for membranes with a low rejection for the selected proteindthe cutoff of a membrane is not sharp across a molecular weight point. Tangential filtration systems have had a great development in the past decades, and large filtration areas can be used in relatively compact, modular systems. For scaling up, a membrane with a cutoff similar to that used in the laboratory should be used, and similar conditions of bulk and retentate concentrations should be tested. A preliminary evaluation of membrane flux  transmembrane pressure will point to the optimal conditions; the results obtained with one filtration module are scaled up by using several modules in parallel. This operation is also a suitable substitute for operations that will not make it from lab to pilot plant, such as ultracentrifugation. Key parameters to look at include solutes’ and contaminants’ molecular weight, viscosity, membrane molecular weight cutoff, and concentration polarization.

34.7.5

Broth Conditioning

Some properties of the liquids to be processed affect the molecules dissolved in it, and are routinely tweaked in operations such as flocculation or precipitation (pH or ionic strength) and extraction (pH or the dielectric constant). These strategies may also be used for broth conditioning [14], facilitating a subsequent operation: with larger particles or a modified surface charge (and therefore a lower viscosity), separations such as centrifugation and filtration have enhanced throughputs. Three other properties that can be altered, to the benefit of some separations, are density, viscosity, and temperature.

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Liquid density can be reduced by the addition of solvent (pure water or other solvents). Although this addition increases the volume to be processed, it may enhance the settling of particles with density similar to that of the liquid. Density can also be changed to facilitate phase separation in liquideliquid extraction. Viscosity is a good target for broth conditioning: as it affects the flow of the liquid, lower viscosities give better separations. This reduction may be achieved by dilution (again, increasing the volume) or by heating the broth. It is important to know what causes a broth to be viscous (exopolysaccharides, proteins, DNA after disruption, etc.). If the culprit is not the bio-product, an enzyme (an endohydrolase) can be used to partially break the polymers, with dramatic effects on the viscosity [9]. Temperature affects several particle and bulk properties: lower temperatures stabilize proteins, higher temperatures may denature and precipitate proteins or enhance the solubility of suspended low-solubility biomolecules. Temperature actually affects all equilibria, from charge distribution to pH to solubility; hence, its effects must be evaluated case-wise.

34.7.6

LiquideLiquid Extraction

This is an equilibrium-based separation, and the most important aspectsdchemical composition and volume proportion of the phasesdcan be maintained in scaling up. However, kinetics plays an important role and that depends on contact area and mixing; phase separation after extraction is also critical, and will be facilitated if a centrifugal separator is used. The equilibrium between phases is linear for low solute concentrations, and is given by K ¼

x y

[34.9]

where K is the partition coefficient, x is the equilibrium concentration of the solute in the solvent phase, and y is its concentration in the aqueous phase. Most substances have experimental or predicted Kow (the octanolewater partition coefficient, sometimes expressed as its logarithm, log P) easily found in databases, and this value is indicative of the suitability of extraction as a downstream operation. Even for modest K values, the operation may be feasible if several stages or a differential extractor is used. The overall efficiency of a countercurrent process is given by  En  1 ; E nþ1  1



h¼1

with E ¼ K

S A

[34.10]

where h is the efficiency or solute recovery (%), E is the extraction factor, S is the volumetric flow of solvent used, A is the volumetric flow of aqueous phase, and n is the number of stages. At small scales, usually reagent-grade solvents are used. At larger scales, there are three major concerns: recycling, volatile organic compound (VOC) emission (for volatile solvents), and safety. Recycling the solvent is important because it reduces emissions

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CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

and cuts costs; using solvents that have limited volatility will reduce VOC emissions, which are both a labor and an environmental issue; and flammability and toxicity are obvious things to avoid. Solvents with similar solubility parameters may substitute for others used in the laboratory. Classical solvent extraction is most efficient for separation and concentration of nonpolar or weakly polar molecules, such as antibiotics and lipids. However, two strategies may be used for enhancing extraction: the use of ion pairs and the use of aqueous distinct phases. The use of ion pairs may allow the extraction of formally charged particles, such as acids or amines, by using low-polarity counterions and forming ion pairs [15], e.g., a fatty amine salt of an organic acid. Distinct aqueous phases may form when using high concentrations of a polymer such as PEG and a salt, or another polymer such as dextran. This phenomenon allows the selective partition of protein mixtures without denaturation, in aqueous two-phase extraction systems. The partition coefficients are modest, but selectivity is good and the process is scalable. Preliminary information about distribution coefficients is important, and it can be extrapolated from laboratory and database information [16]. Kinetics of solute distribution and phase separation is much harder to predict in larger scale, because it depends on solvent viscosity, surface tension, and broth composition; impurities such as proteins and phospholipids may stabilize emulsions, and again preliminary tests must be done. Key parameters to look at include distribution (partition) coefficients, pH influence, candidate ion pairs, solute and solvent solubility parameters, and extraction kinetics.

34.7.7

Adsorption

Adsorption, often confused with chromatography, is a very common way of separating biomolecules, especially tagged proteins. It consists of contacting a solution containing the biomolecule of interest with adsorbent particles, which will bind the target molecule according to charge, hydrophobicity, or specific interactions such as antibodyeantigen or proteinseheparin. The adsorbent is a solid particulate material, and although it can be in contact with the solutions in agitated vessels, it is more common and efficient to put it in a packed column and move only the solutions. The operation consists of adsorption or binding (passing the mother liquor through the column, with the solute attaching to the adsorbent) and then changing the mobile phase to conditions favorable for desorption, or release of the solute, which is more concentrated and has fewer contaminants. Intermediary steps for washing may be used. Some common separations in the laboratory, such as solid-phase extraction or immobilized metal-ion-affinity chromatography (IMAC), are frequently adsorption operations. The capacity of a resin, i.e., how much solute it can hold, is a function of the equilibrium concentration and is thus affected by the amount of solute in the beginning of the operation, following an isotherm such as that of Langmuir: q ¼ qmax

y kþy

[34.11]

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797

where qmax is the nominal resin capacity, q is the effective concentration on the adsorbent, y is the equilibrium concentration in the solution (not the initial concentration), and K is an affinity constant. The maximal capacity of a resin depends largely on its structure and chemical composition, the ligand, and the conditions (pH, ionic strength) used. Here are a few examples of affinity adsorption:
an avidin resin may bind from 1 to 10 mg/mL of biotinylated bovine serum albumin (Thermo Pierce HCSA),
a heparin resin has a binding capacity of 2 mg/mL for coagulation factors (Heparin Sepharose 6 Fast Flow, GE),
a NieIMAC protein may bind 20e22 mg/mL of a 70-kDa histidine-tagged protein (Bio-Rad Profinity), and
a typical protein A resin may bind 27e60 mg/mL of monoclonal antibodies [17]. As in extraction, adsorption equilibrium depends on mixture and adsorbent composition, being readily replicated in larger scales. The difficulty in scaling up is much more the question of maintaining proper flow through the column: resistance increases with the increase in bed height, whereas the homogeneous distribution of liquids on a large column is difficult. If a commercial column of the same adsorbent has enough capacity for the amount of solute to be processed, the flow through the column and pressure to be applied can be predicted (from filtration theory). For larger columns, this must be coupled to an equilibrium equation and mass transport data (both obtainable from the laboratory) to simulate column efficiency for different dimensions. In addition, the process must be developed bearing in mind that washing and eluent solutions will not be applied manually, but rather be pumped from specific reservoirs. Their volumes and disposition must be considered. Key parameters to look at include equilibrium data, resin (adsorbent) properties, adsorption, and elution profiles.

34.7.8

Chromatography

Chromatography is similar to adsorption in terms of binding chemistry, but is dynamicdsolutes and contaminants bind and release from the adsorbent in a column bed, while they are also carried by the mobile phase. The operation has a high resolution, but is more complicated for scaling up. Chromatography relies on weaker adsorption, selected so that similar moleculesdthe biomolecule of interest and its contaminantsd can be discriminated. The dynamics of the separation depends both on the adsorption equilibrium and on the flow. The easiest way of scaling up is increasing its crosssectional area and volumetric flow, but maintaining the bed height and liquid linear velocity tested in small scale. Process columns usually have a lower resolution than the laboratory equivalents (Fig. 34.2), partly because of the inherently higher problems with flow injection and bed packing, and partly because process columns are loaded with more solute than lab-scale columnsdthis increases the throughput but sacrifices resolution.

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FIGURE 34.2 Small- and large-scale size-exclusion chromatography of a mixture of proteins. Notice the loss of resolution, highlighted for the last protein (in the inset, with the individual peaks).

Even if the resolution in large scale will be reduced, the process is still capable of greatly enhancing the purity of biomolecules. In general, the concentration of the solute will be reduced in comparison with the feed, because of dispersion. The operation can be simulated from equilibrium and transport data, but the evaluation of column performance in large scale can be inferred by loading laboratory or pilot columns with higher volumes (e.g., 10% of the column length) and evaluating conditions for acceptable separation efficiency. Key parameters to look at include column elution profiles, permissible surface velocity, and resolution for different column loads.

34.7.9

Precipitation

Precipitation consists of insolubilizing a solute. This is important especially for salt-forming compounds, proteins, and polysaccharides, and may be done by adding a solvent (e.g., precipitating polysaccharides with ethanol), a counterion (e.g., calcium compounds as precipitant for organic acids), or a salt (e.g., ammonium sulfate for protein precipitation). Other forms of precipitation are by altering the pH or quickly cooling a solution. In any case, it is easy to replicate the operation in larger scales, if mixing conditions are similar to those used in small scale. In addition, precipitate separation is a natural sequence for this operation and must be evaluated; centrifuging precipitates in small-scale, high-RCF centrifuges may not be scalable. Precipitate aging (with gentle agitation) may enhance solids separation. The precipitant to be used must be cheap [4] and preferably recyclable, for large amounts will be used. Although this is a simple separation, it is reliable and has multiple uses:
Broth conditioning, precipitating colloids, which reduces viscosity, aids in centrifugation, filtration, and membrane operations

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Concentration of solutes, such as a desired protein or organic acid
Elimination of undesirable contaminants, such as cold-precipitable proteins or tannins in beverages
Fractionation of proteins, using both salt and isoelectric point Precipitation of proteins is fairly represented by the Cohn equation [5,18]: X ci zi 2 with I ¼ 1 2 =

S ¼ S0 $ eKI ;

[34.12]

where S is the concentration of the solute, S0 is its saturation concentration, K is the salting-out concentration, and I is the ionic strength, calculated from the molality of each ion (ci) and its charge (zi). Low-concentration species are ignored. The limit concentration S0 depends highly on initial pH, and adjusting pH to the isoelectric point of the desired protein enhances precipitation. For other solutes and precipitants, a similar empirical equation can be used, with I substituted by the concentration of the precipitant or the dielectric constant of the mixture, and using a recalculated K. Key parameters to look at include precipitanteyield curves, alternative precipitants, mixing strategy to avoid high concentrations of precipitant, and precipitate aging for enhanced separation.

34.7.10

Crystallization

Crystallization consists of maintaining a low supersaturation with respect to the bioproduct of interest, which will form a solid phase much more ordered than precipitates: crystals. This is usually done by removing solvent (in evaporators) or by lowering the temperature (in agitated vessels). Process-scale macromolecule crystallization is difficult; the process is much more suitable for small molecules. The kinetics of nucleation and crystal growth is complex and highly dependent on the supersaturation conditions and the interaction with other solutes. After an adequate crystallization strategy is developed in small scale, the conditions must be reproduced in large scale: the same cooling or evaporation rates should give the same results. The slurry containing crystals is then separated by centrifugation or filtration, and the crystals may be washed with a saturated solution or a nonsolvent and eventually reprocessed; one modern process for insulin production, for example, involves three crystallizations of the zinc complex. If the crystals are to be the final presentation form of the product, seed crystals can be used for starting the crystallization with a definite concentration of crystal centers, and therefore controlling the final crystal size. Natural nucleation and crystal growth from fragments lead to a broader size distribution, and that calls for a size classification step that can be part of the crystallizer (such as elutriation legs) or in an external loop. Dry crystals can be classified by sieving. The supersaturation is defined as Dc ¼ c  c*, where c is the concentration of the solute, and c* is its saturation concentration. If c as a function of time or antisolvent concentration is known, then a temperature (cooling) or antisolvent addition strategy can be defined [19] (e.g., maintaining Dc as a few percent of the saturation).

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Crystallization occurs in the metastable zone of solubility, which calls for feedback control of temperature or antisolvent and, if possible, of the solute concentration [20]. Key parameters to look at include solubility curves, metastable mixtures, crystal polymorphs, cooling rates, and supersaturation levels.

34.7.11

Evaporation and Drying

These are not bioseparations per se, being actually water- or solvent-removal steps. However, these operations can be important intermediary steps for concentrating a solution, conditioning biomass to be extracted by solvents, creating supersaturation for crystallization, and removing solvents from crystallized or precipitated solids. Evaporation has liquid input and output, whereas drying has liquid or solid input, but a solid output. In both processes, the mixture is heated either directly, by contact with a hot surface (trays or complex surfaces in tunnel dryers, heat exchanger tubes in evaporators), irradiation (infrared from flames or heating elements, microwave, or dielectric heating), or a dry, hot gas (in spray and tunnel dryers). The processes are facilitated by vacuum. This technology is well developed and the conditions used in laboratory and plant scales are usually replicated, and even improved, in industrial processes. In small scale, it is common to work with relatively little information about the material to be evaporated or drieddthe conditions that routinely “work” in a lyophilizer or rotary vacuum evaporator are applied to new mixtures. However, curves of temperature and concentration or weight  time are essential for defining important parameters for the operations, e.g., heat transfer rates, boiling points, viscosity, critical moisture, etc. For evaporation, crystallization or precipitation of multiple components of the mixture must be evaluated; for drying, the scaling up must be done with care, for the thickness of solids to be dried have a dramatic impact on drying time. Key parameters to look at include limiting temperatures for the target molecule, curves for evaporation (liquid temperature  time) or drying (weight or moisture content  time), solvent or water vapor pressure  temperature, and solvent flammability.

34.8 Niche Operations and Single-Use Systems It is actually quite rare for new operations to suddenly appear and gain space in the process-scale separation market. This is not because there is no space for innovation, but because the most common materials or molecular properties are already explored in classical operations, and therefore a “new” technology can usually be substituted by a well-known operation. It is much more common that an experimental technology is first applied in analytical and laboratory systems, and as the equipment design evolves, it finds niche applications and becomes eventually ubiquitous. The most important trend in downstream bioprocessing is the use of smaller-scale, flexible single-use systems.

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801

Niche Operations

There are several analytical, high-resolution separation systems that did not make it to the larger scale, such as electrophoresis or field-flow fractionation. Others are gaining markets, such as electrodialysis and pervaporation membrane processes. Three processscale bioseparation systems that have the technology well developed, and are still uncommon but can be considered in bioprocess development, are cryoconcentration, foam fractionation, and supercritical fluid extraction. Cryoconcentration is the crystallization of a solvent (usually water) from solutions, concentrating the solutes in the remainder of the solution. It has a low impact on biological activity [21]. The operation is energy-intensive, but less so than evaporation, and can be a good option for a concentration step. However, the formation of a solid layer over heat-exchanging surfaces makes it harder to scale up and competes with membrane technologies. Foam fractionation consists of passing a gas through a solution and creating foam. Several substancesdespecially proteinsddistribute in the gaseliquid interface, and may be carried by the foam. The operation can concentrate and segregate proteins and is worth a try. However, it can cause denaturation of proteins, and it demands quite large equipment. Although used in the metal industry and on fish farms, there are no largescale bioprocesses using foam fractionation yet [22]. The process can be substituted by membrane filtration. Supercritical fluid extraction is a well-known extraction process in which the solvent is usually supercritical CO2 (other gases are rarely used). Cosolvents may be added. The process relies on the high diffusivity of molecules in supercritical fluids to efficiently extract molecules of interest, mostly of molecular mass below 1 kDa. It is useful for natural product extraction [23], although the installation costs are considerable. An alternative process is the classical solvent extraction.

34.8.2

Single-Use Systems

The evolution of the mass fabrication of plasticware is leading to the development of disposable, single-use systems (SUSs) for relatively small-volume operations. This is relevant for specialty biomolecules, especially therapeutic products, and may increase in importance as the biopharmaceutical industry develops niche and/or tailored products, such as monoclonal antibodies for treatment of rare diseases. Although opting for singleuse filtration cartridges, membranes, or centrifugal systems seems a bit of a waste, the analysis should take into account two factors: the consistent quality of the SUS and the comparative cost of cleaning and sterilizing systems for reuse. As to their quality benefits, SUSs can be sealed and irradiation-sterilized by the manufacturer, increasing reproducibility and safety. The elimination of one step in in situ cleaning and sterilization also guarantees that no cross-contamination between batches occurs, limiting problems. The process also gives flexibility to the production

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Table 34.6

Selected Manufacturers of Process-Scale Single-Use Systems

Supplier

What is Disposable

GE Healthcare Life Sciences www. gelifesciences.com Merck Millipore www. merckmillipore.com Pall www.pall.com Sartorius Stedim www.sartorius.com

Bins, bags, connectors, filtration cartridges, tubing, chromatography flow paths, sensor assemblies, prepacked sterile chromatography columns All tubing, connectors, filtration cartridges and cassettes, collection bags, manifolds, chromatography flow paths, pump heads, sensors Mixers, bags, bioreactors, connectors, filters, filling systems Bags, containers, connectors, filters, tubing, sampling systems, mixers, bioreactors, filling systems, membrane adsorbers, freeze-and-thaw systems

facility [24] and faster batch turnaround [25]. It is actually possible that regulatory agencies will gravitate toward demanding that SUSs be the norm in specific production platforms, because of the inherent quality, flexibility, and quick setup, and possibly because of manufacturer strategies, which will understandably offer more SUS options. For cost comparison, SUSs reduce setup time and the use of heat and/or cleaning solutions in some steps of the process. This, together with batch consistency, product quality, and plant flexibility, means higher overall quality and productivity, ultimately being economically advantageous for the manufacturer. There are dozens of suppliers for SUS products and systems. Table 34.6 lists the suppliers with a broader range of products, each having its line of downstream platform solutions.

34.9 Selected Guidelines for Process Development In addition to the heuristics already described in Section 34.4, the following guidelines can aid in the development of a pilot- or large-scale separation and purification process:
If possible, consider the downstream processes in upstream development. Different expression platforms and culture media may lead to quite different downstream processes.
Find or determine the physicochemical properties of the target biomolecule. Some may be distinctive, such as volatility or low solubility at the isoelectric point; others may set limits for processing, such as stability toward pH and temperature.
Find or determine the physicochemical properties of the most important contaminantsdthose in high concentrations and those that must not appear in the final product. Again, look for distinctive properties.
What is the presentation form expected for the product? Solids are usually more stable; liquid formulations will preclude a final drying step.
What is the purity expected for the product? Therapeutic and diagnostic biomolecules must be very pure; for other uses, less puritydwith lower processing costsdmay be acceptable.

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the product intra- or extracellular? If it is extracellular, removing the biomass previously is beneficial; if it is intracellular, a previous biomass concentration step can reduce volumes.
When possible, concentrating the solutions is important for reducing processing volumes and equipment size for downstream operations.
The most abundant impurities should usually be removed early in the process, because reducing their level will aid in reducing the volume.
Separate the easiest-to-separate impurities early in the process, because the size of subsequent, high-resolution separations will be smaller.
Orthogonality: sequential operations should preferably rely on different separation properties. In fractionations with multiple salt precipitations/solids separation steps, go from the lowest precipitant concentration to the highest. Then use dialysis or gel filtration to remove salt.
For large processes, think of energy-efficient phase separations (e.g., ultrafiltration or precipitation instead of evaporation under vacuum for large molecules).
For any process, think of the residues generated and component reuse to enhance safety, sustainability, and cost: an operation with higher fixed (implantation) cost may be better in the long run than an easier, sloppier operation (e.g., adsorption  solvent extraction).
Buffer, conditioning, and sanitizing solution preparation, storage, and distribution to the operation must be taken into account from the start of the process development.
High-resolution, complex operations are usually expensive and more suitable for high-value-added products.
For highly pure, small-scale processes, consider the use of an SUS.
Take regulation and good management practices into account from the start of the process development, for the process will have to be compliant and developing it accordingly is better than adapting.
Do not rely solely on one draft of the separation train. Process development should be iterative, i.e., adapted, tweaked, and revised several times.

34.10 Conclusions and Perspectives Considering the recent trends, the evolution of fabrication techniques, the regulation pressure for immunobiologicals, and the product life cycles, the next decade will show the strengthening of technologies dependent on organism (platform) modification and the use of disposable systems. More specifically, there will be an increase in the use of traditional and new tagged-proteins systems, because the technology is mature, is powerful, and dramatically facilitates downstream processes. There will be also an increase in the use of niche operations, because of the inherent understanding of underlying principles and the development of cheaper instrumentation

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and control systems. On the other side, large-scale processes of bulk bio-products will see more integration, following biorefinery and environmental trendsdsolvent and water recovery, emission control, and by-product processing into diversified portfolios are possible. The use of multipurpose platforms relying heavily on SUSs will become more common. Platforms and product portfolios will continue to evolve into more options for powerful, cheaper processes; on the other side, regulatory authorities will aid in directing the industry toward SUSs, perhaps specifying types of sterilization (e.g., irradiation) or materials (special alloys in favor of plastics) for biologics. Finally, microfabrication techniques will allow the miniaturization of processes to produce tailored batches, meeting the tailored therapeutics demand, which will be continually growing.

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