Adsorption and Chromatography

2.47

Adsorption and Chromatography

Y Sun, Q-H Shi, L Zhang, G-F Zhao, and F-F Liu, Tianjin University, Tianjin, China © 2011 Elsevier B.V. All rights reserved.

2.47.1 2.47.2 2.47.2.1 2.47.2.2 2.47.2.3 2.47.2.4 2.47.2.5 2.47.2.6 2.47.2.7 2.47.3 2.47.3.1 2.47.3.1.1 2.47.3.1.2 2.47.3.1.3 2.47.3.1.4 2.47.3.1.5 2.47.3.2 2.47.3.3 2.47.3.4 2.47.4 2.47.4.1 2.47.4.2 2.47.4.3 2.47.5 2.47.5.1 2.47.5.1.1 2.47.5.1.2 2.47.5.1.3 2.47.5.2 2.47.5.2.1 2.47.5.2.2 2.47.5.2.3 2.47.5.3 2.47.5.4 2.47.5.5 2.47.5.5.1 2.47.5.5.2 2.47.5.5.3 2.47.6 References

Introduction Molecular Interactions in Adsorption Hydrogen Bond Hydrophobic Interaction Electrostatic Interaction van der Waals Interaction Coordination Bond Covalent Bond Conformational Entropy Chromatographic Methods Packed-Bed Chromatography Size-exclusion chromatography Ion-exchange chromatography Hydrophobic interaction chromatography Affinity chromatography Displacement chromatography Expanded-Bed Adsorption Chromatography Electrochromatography Radial-Flow Chromatography Theoretical Aspects of Adsorption and Chromatography Adsorption Equilibria Uptake Kinetics Theoretical Considerations of Chromatography Development of Adsorption and Chromatography Innovation of Chromatographic Matrices Flow-through media Membrane Monolith Selection and Design of Affinity Ligands Combinatorial library approach Rational design Combination of rational design and synthetic combinatorial library Mixed-Mode Ligands Displacer Screening and Design Molecular Insight into Protein Adsorption Modeling and visualization Adsorption process Protein conformational transition Conclusions

Glossary adsorption The adhesion or retention of molecules of gas, liquid, or dissolved solids to a surface resulting from the force field at the surface or the molecular interactions between the molecules and the ligand attached on the surface. affinity chromatography A liquid chromatography that uses a stationary phase with immobilized biologically related groups (affinity ligands), in which biomolecules are separated based on a highly specific biological

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interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. chromatography The separation technology of solutes dissolved in a mobile phase as they pass down a column due to the differential distribution of the solutes between the mobile phase and the stationary phase in the column. hydrophobic interaction chromatography A liquid chromatography with a stationary phase bonded with weakly hydrophobic ligands, by which molecules are

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separated in the column according to their differences in hydrophobicities. ion-exchange chromatography A liquid chromatography in which ionic solutes are separated according to their charges by binding to the cationic or anionic sites of the stationary phase known as ion exchangers.

size-exclusion chromatography A type of liquid chromatography, also called gel-filtration chromatography, which uses porous particles as the stationary phase, in which solutes are separated by the difference in their molecular sizes.

2.47.1 Introduction Adsorption is a phenomenon of solute attachment to a solid surface, and adsorption operations are widely applied in chemical and biochemical processes as well as in the living activities of human beings for the recovery or removal of specific substances. Solid materials, usually referred to as adsorbents, are used for adsorption operations. Because adsorption takes place at the solid surface, adsorbents must be fabricated to be of high specific surface area for high adsorption capacity. For this reason, adsorbents are usually porous materials. Chromatography is the primary mode of adsorption operations. It is a multistage separation technology and offers high-resolution separation of different solutes of high similarities. Bioseparations mainly involve liquid feedstocks, so adsorption operations in biotechnology usually occur at liquid–solid interfaces. Over the past three decades, biotechnology has developed rapidly as marked by the advances in genetic engineering and cell fusion techniques. In this process, chromatographic technology has played the most important role in the separation and purification of biomolecules. More importantly, chromatography is a separation methodology of great diversity; it is based on various interactions between target solutes and the ligands coupled to a solid surface, and also links to various other separation methods Hence, chromatographic separations can be achieved on the basis of different separation mechanisms, equipment configurations, and operation modes. As a result, the technology is so powerful that various substances, including small molecules, biopolymers, and particulate materials such as viruses and whole cells, can be purified by the combination of different chromatographic steps. Hence, chromatography is the most widely used separation method in biotechnology. This article is devoted to an overview of the science and technology of chromatography. Various molecular interactions involved in adsorptions are briefly described first. This is followed by the introduction of various chromatographic methods for biosepara­ tions. In this section, size-exclusion chromatography (SEC) is first mentioned as an important chromatographic technique, although it does not involve adsorptions. Only the fundamentals of the methods are outlined and readers can refer to other monographs on chromatography (e.g., References 1–3) for more detailed information. Next, the theoretical aspects of adsorption and chromatography are summarized, including adsorption equilibria, uptake kinetics, and the fundamental theories of chroma­ tography. Finally, the emphases are on the recent advances in studies of adsorption and chromatography to provide insight into the future development of biochromatography.

2.47.2 Molecular Interactions in Adsorption Adsorption of a solute on a surface involves various interactions between the solute and the surface or the chemical groups attached to the surface. The interactions that contribute to the adsorption of biomolecules include hydrogen bonding, hydrophobic interaction, electrostatic interaction, van der Waals interaction, coordination bonding, covalent bonding, and conformational entropy [4].

2.47.2.1

Hydrogen Bond

A hydrogen bond, described as D–H…A, is an interaction in which a hydrogen atom (H) is attracted simultaneously by two electronegative atoms (D and A). The electronegative atom (D) covalently bonded to H is named as donor, while the other (A) is named as acceptor. Hydrogen bonding is a driving force for adsorption with an energy range of 13–30 kJ mol–1 and increases with electronegativity of the participants, D and A. Hydrogen bonding energy decreases with increasing temperature and ion strength, as well as by the presence of chaotropic agents such as urea and guanidine hydrochloride.

2.47.2.2

Hydrophobic Interaction

Redistribution of ordered water molecules around single apolar solutes back into bulk solution causes the association of apolar molecules in water and the decrease of the Gibbs energy of the system, which is termed as hydrophobic interaction. This interaction exists between hydrophobic groups such as benzene rings or hydrocarbon chains and the hydrophobic region in biomolecules, with an energy range of 12–20 kJ mol–1. Both the salt type and concentration can affect hydrophobic interaction. Kosmotropic/lyotropic salts, for instance, (NH4)2SO4, Na2SO4, NaCl, KCl, and CH3COONH4, promote hydrophobic interaction, while chaotropic agents, for instance, KSCN, NaI, KClO4, and urea, reduce hydrophobic interaction. Stronger promotion or reduction effect by the agents is

Adsorption and Chromatography

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usually observed at higher concentrations. Hydrophobic interaction is enhanced by increasing temperature. Adsorption based on hydrophobic interaction is called hydrophobic adsorption.

2.47.2.3

Electrostatic Interaction

When a solute and a solid surface are both charged, electrostatic interaction, attractive or repulsive, can occur between them. The strength of electrostatic interaction depends on the charge numbers, so it is significantly affected by both pH and ion strength. The pH at which the charges on the solute and the surface just compensate each other gives rise to the maximum electrostatic adsorption, while the increase of ion strength will weaken or even completely screen the electrostatic interaction. The increase of temperature reduces electrostatic effect due to the enhanced thermal motion of molecules and atoms at elevated temperature. Adsorption based on electrostatic interaction is usually called electrostatic adsorption or ion exchange.

2.47.2.4

van der Waals Interaction

van der Waals interaction, originating from the interactions between fixed and/or induced dipoles, often contributes to adsorption. It is very sensitive to the separation distance (r) between the dipoles, diminishing as r–6. Therefore, it operates only over a limited range of intermolecular distance (around 0.2 nm). Moreover, van der Waals interaction is weaker than most of the other molecular interactions, usually with energy range of 4–8 kJ mol–1.

2.47.2.5

Coordination Bond

Transitional metal ions, such as Cu2+, Zn2+, Ni2+, and Co2+, can form coordination bonds with the imidazolyl group of histidine. Once a protein and a solid surface form coordination bonds with the same metal ion, a protein–metal ion-surface sandwich structure is observed and leads to the indirect binding (adsorption) of the protein to the surface. Increasing temperature can weaken coordination bonding, and the presence of chelating agents (e.g., ethylinediaminetetraacetic acid) can diminish the bonding due to the competitive binding of the chelating agent to the transition metal ions.

2.47.2.6

Covalent Bond

Reversible covalent bonds, for instance, disulfide bond, can be applied in the adsorption of biomolecules. The chromatographic method based on covalent bonding is called covalent chromatography.

2.47.2.7

Conformational Entropy

Adsorption results in the reduction of conformational entropy, so conformational entropy is thermodynamically unfavorable for adsorption. Therefore, adsorption takes place only if the loss in conformational entropy is compensated by sufficient attraction between the solute molecules and the surface. In general, solute adsorption involves one or more interactions described above. Moreover, in the adsorption of biomacromole­ cules such as proteins, molecular conformational transition is an important phenomenon for consideration. This is not only due to its relevance to the biological functioning of the molecules, but also due to the significant role it plays in the adsorption process. The structural flexibility of an adsorbed protein molecule strongly affects the interactions between the protein and the solid surface, such as electrostatic and hydrophobic interactions, and then affects its adsorption phenomena. So, protein adsorption is a complex process that is controlled by a number of subprocesses at the synergistic and antagonistic effects of the interactions mentioned above.

2.47.3 Chromatographic Methods 2.47.3.1

Packed-Bed Chromatography

Packed bed is an essential mode of chromatographic operations [2, 3]. In this mode, a chromatographic column for preparative separations is usually packed with porous beads (adsorbents) that serve as the stationary phase. Most stationary phases consist of two functional parts – porous matrix and ligand attached to the pore surface. The porous matrix possesses sufficient mechanical strength to endure the pressure across the column at fast mobile phase flow and provides high specific surface area for the coupling of ligands and then the adsorption of target molecules. The ligands can bind solutes based on different interactions and discriminate the solutes in feedstock. Various chromatographic techniques related to the difference in ligands will be introduced in the following sections. Chromatographic matrices for bioseparations are usually hydrophilic materials that do not interact with biomolecules and related solutes; so a mild environment is provided for maintaining the native structure of biomolecules. Pore size and particle diameter are two important parameters for the matrices. The pore size usually ranges from 10 to 100 nm, which is large enough for the accessibility of biomolecules and small enough to provide high specific surface area. The use of small-sized particles can offer high column efficiency, but gives rise to high pressure drop across the column. So, considering the tradeoff between column

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Table 1

Some commercial matrices for biochromatography

Commercial name

Chemical composition

Particle diameter (μm)

Manufacturer

Sepharose Superose Sephadex Superdex Sephacryl Minibeads Monobeads Source HyperD Hypercel Trisacryl HA Ultrogel Ultrogel AcA Toyopearl Bio-Gel A Bio-Gel P UNOsphere Bio-Gel HT Fluoroapatite Poros

Agarose Agarose Dextran Dextran and agarose Allyl dextran and N,N′-methylene bisacrylamide Polystyrene/divinyl benzene with hydrophilic coatings Polystyrene/divinyl benzene with hydrophilic coatings Polystyrene/divinyl benzene with hydrophilic coatings Polystyrene-mineral composite filled with hydrogel Cellulose N-acryloyl-2-amino-2- hydroxymethyl-1,3-propane diol Hydroxyapatite and agarose Acrylamide and agarose Hydroxylated methacrylic polymer Agarose Polyacrylamide Polyacrylamide Hydroxyapatite Fluoroapatite Polystyrene with hydrophilic coatings

34, 90, and 200 13 and 30 17–520 13 and 34 47 and 65 5 10 15 and 30 50 90 40–80 60–180 60–140 35, 65, 75, 100, and 200 38–75, 75–150, and 150–300 45–90, 90–180 80 and 120 20, 40, and 80 40 20 and 50

GE Healthcare GE Healthcare GE Healthcare GE Healthcare GE Healthcare GE Healthcare GE Healthcare GE Healthcare Pall Life Sciences Pall Life Sciences Pall Life Sciences Pall Life Sciences Pall Life Sciences Tosoh Bio-Rad Bio-Rad Bio-Rad Bio-Rad Bio-Rad Applied Biosystems

efficiency and operation pressure, the matrices for preparative chromatography are mostly in the range of 20–200 μm. Some commercial media are listed in Table 1. They are modified with different ligands for use as different adsorbents. Separation by adsorptive chromatography is usually achieved in five steps: column equilibration, feed loading, washing, elution, and regeneration. First, the column is equilibrated with a loading buffer. Second, after equilibration, the feedstock for separation is loaded onto the column and the target molecules are adsorbed to the stationary phase. Third, a washing step with the loading buffer is followed to remove any unbound materials from the column. Fourth, the target molecules are eluted with an elution buffer. A regeneration solution is then applied to remove any strongly bound substances from the column. Finally, the column is re-equilibrated with the loading buffer for the next separation.

2.47.3.1.1

Size-exclusion chromatography

SEC, also described as gel-filtration, steric exclusion, or gel chromatography, is a partition chromatography that separates molecules according to their molecular sizes. The separation of protein mixtures according to their sizes was first reported in 1959 using cross­ linked polydextran gels devoid of ionic groups for the fractionation of water-soluble substances. In SEC, molecules are eluted in the decreasing order of their sizes. Because the column is packed with a gel filtration matrix with a definite pore-size distribution, the molecules go through the column in different paths according to their sizes. A large molecule whose size is larger than the biggest pore goes through the interspaces of the gels and is eluted at the void volume of the column. Smaller molecules penetrate into the interior of the gels, leading to greater retention time. The smaller the molecular size, the more pores the molecule can penetrate into. Therefore, the largest molecules pass through the column first, while the smallest ones come last, leading to the size separation. Because SEC is a size-selective separation method, the pore-size distribution of an SEC medium is crucial for the separation performance. The difference of pore-size distributions of different SEC media is represented by the difference in their fractionation range. Initially, cross-linked gels of either dextran (Sephadex) or polyacrylamide (Bio-Gel P) were used. Furthermore, agarose gels (Sepharose) were used for the separation of solutes of even higher molecular mass (e.g., nucleic acids and viruses). Alternatively, porous glass beads provide an exclusion matrix that avoids the problems of column compaction often encountered with soft polysaccharide gels. At present, SEC media cover a fractionation range from 102 to 8 × 107. Most of the matrices listed in Table 1 have been made to SEC media for bioseparations. The SEC has advantageous of mild condition, simple operation, isocratic elution, and easy scaling up. It has been extensively applied in biotechnology, including the separation and analysis of proteins, peptides, lipids, antibiotics, sugars, nucleic acids, and viruses (50–400 nm), desalting of bioproduct solutions, molecular mass estimation, and characterization of molecular interactions.

2.47.3.1.2

Ion-exchange chromatography

Ion-exchange chromatography (IEC) is one of the most frequently used techniques for the purification of proteins and other biomolecules. It is based on the different degrees of electrostatic interactions between the stationary phase and solutes. Various cation- and anion-exchange chromatography media have been developed for protein purifications. Nucleic acids have low isoelectric point (pI) values and are usually purified by anion-exchange chromatography.

Adsorption and Chromatography

Table 2

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A list of ion-exchange ligands

Ligand

Structure

Comments

Sulfopropyl (SP) Methyl sulfonate (S) Carboxymethyl (CM) Quaternary ammonium (Q) Diethylaminoethyl (DEAE) Diethylaminopropyl (ANX)

–O–CH2CHOHCH2OCH2CH2CH2SO3– –O–CH2CHOHCH2OCH2CHOHCH2SO3– –O–CH2COO– –O–CH2N+(CH3)3 –O–CH2CH2NH(CH2CH3)2 –O–CH2CHOHCH2NH(CH2CH3)2

Strong cation exchanger Strong cation exchanger Weak cation exchanger Strong anion exchanger Weak anion exchanger Weak anion exchanger

Some of the frequently used ion-exchange ligands are summarized in Table 2. Based on the dissociation properties, these ligands are classified as ‘strong’ or ‘weak’ ligands. Strong ion-exchange ligands can retain their charges in a wide range of pH. By contrast, the ionic states of weak ion-exchange ligands are pH dependent, which can in some cases offer extra selectivity. Coupling of the ligands to the matrices listed in Table 1 leads to the production of ion-exchange adsorbents. The adsorption in IEC is usually achieved at low ionic strengths (typically 20–50 mmol l−1). The pH values that are 0.5–1 unit away from the isoelectric point of the target molecule are preferable for sufficiently high capacity. Elution is often achieved by an increasing salt gradient. At high salt concentrations, the salt ions compete with biomolecules in binding with the ligands and thus the biomolecules are eluted. Elution by pH change is also optional but is less often used, because this may involve crossing the isoelectric points of proteins and lead to precipitation. Hydroxyapatite (Ca5(PO4)3OH) is a special medium of chromatography that involves both anion- and cation-exchange interactions. The Ca2+ functional groups can interact with carboxylate residues at the protein surface, while PO42– can interact with basic residues. Proteins are usually adsorbed on hydroxyapatite chromatography at low phosphate concentrations and eluted by increasing phosphate gradient. The NaCl and (NH4)2SO4 do not influence the adsorption of proteins on hydroxyapatite, so samples eluted from common ion-exchange columns can be directly applied to a hydroxyapatite column for further purification.

2.47.3.1.3

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a liquid chromatography to separate and purify biomolecules by their hydrophobic interaction with the hydrophobic ligands coupled to porous media. The HIC was proposed for the first time by Tiselius in 1948, using the term ‘salting-out chromatography’. The name hydrophobic interaction chromatography was introduced by Hjerten in 1973. The HIC exploits stationary phase with weakly hydrophobic ligands such as short chain alkyl and phenyl immobilized on a hydrophilic matrix. Usually, there are some exposed hydrophobic amino acids on biomolecule surface. Thus, adsorption occurs due to the hydrophobic interaction between the hydrophobic surface patches on a solute and the ligands at moderately high salt concentra­ tions (ion strength), usually 1–2 mol l−1 ammonium sulfate or 3 mol l−1 NaCl. Because kosmotropic salts such as (NH4)2SO4 and Na2SO4 promote hydrophobic interactions, the adsorption increases with salt concentration in the mobile phase, and vice versa. Therefore, elution is usually performed via a gradient or stepwise reduction of salt concentration. Ligands are crucial for the bioseparations by HIC. Ligand chemistry can affect HIC selectivity for different proteins. Moreover, because hydrophobic interaction is proportional to ligand hydrophobicity and coupling density on the surface, ligand density should be varied according to the ligand hydrophobicity. Generally, immobilized ligand density in commercial HIC adsorbents is in the range of 10–40 µmol ml−1. Some of the commonly used hydrophobic adsorbents are provided in Table 3. The HIC can directly deal with a sample containing high salt concentration, so it is promising for the processing of samples obtained from salting-out precipitation or IEC elution. Because hydrophobic interaction strength can be readily adjusted by altering salt concentration in mobile phase, HIC is an important method in the bioseparations of therapeutic proteins, DNA vaccines, and hydrophobically tagged proteins. Table 3

Some commercially available hydrophobic adsorbents

Ligand

Adsorbent name

Manufacturer

Methyl Ether

Methyl HIC SOURCE ETH Toyopearl Ether-650 Toyopearl PPG-600 SOURCE ISO SOURCE PHE, Phenyl Sepharose Toyopearl Phenyl-600, Toyopearl Phenyl-650 Butyl Sepharose Toyopearl Butyl-600, Toyopearl Butyl-650, Toyopearl SuperButyl-550 t-Butyl HIC Toyopearl Hexyl-650 Octyl Sepharose

Bio-Rad GE Healthcare Tosoh Tosoh GE Healthcare GE Healthcare Tosoh GE Healthcare Tosoh Bio-Rad Tosoh GE Healthcare

Polypropylene glycol Isopropyl Phenyl Butyl t-Butyl Hexyl Octyl

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2.47.3.1.4

Affinity chromatography

In biological systems, biomolecules can bind specifically and reversibly to their complementary substances. The specific and reversible binding effect is called affinity interactions and the complementary substances are termed affinity ligands. Affinity chromatography (AC) is an adsorptive chromatography based on the affinity interactions with immobilized affinity ligands on a solid matrix. Table 4 lists the affinity systems that are often used in AC. The binding constants of the affinity pairs for AC should be in the range 104–108 l mol−1, that is, stable enough for high recovery in affinity adsorption and not too tight for convenient desorption in the elution process. Moreover, the ligands should be immobilized to the matrix surface via a spacer to avoid steric hindrance for the accessibility of target molecules. The importance of spacing between low-molecular-mass ligand and the surface was recognized in the early development of AC. Furthermore, to maintain the affinity, macromolecular ligands (e.g., proteins) must not be deformed by immobilization. The bioaffinity ligands such as hormones and monoclonal antibodies bind complementary receptors and antigens, respectively, in a highly specific manner, so bioaffinity chromatography is advantageous because of its extremely high selectivity. However, bioaffinity chromatography can only be used to purify a specific product or a small group of related biomolecules. By contrast, AC based on metal ions and synthetic dyes are less specific ligands that can bind to a variety of proteins, so they can be widely used in bioseparations. The AC based on metal ions and dyes are respectively called immobilized metal AC (IMAC) and dye-ligand AC. Recombinant deoxyribonucleic acid (DNA) technology has made it easy to express fusion proteins with polyhistidine tags; so IMAC is an important and cost-effective technique for the purification and/or refolding of recombinant proteins. Bioaffinity interactions are highly specific because they often combine steric complementarities and different interactions, including electrostatic, hydrophobic, hydrogen bonding, coordination bonding, and van der Waals interactions. This makes bioaffinity chromatography the most selective technique for protein purification. For the AC with ligands of moderate specificities, additional separation selectivity can be achieved by a selective elution method, such as applying a gradient of ionic strength, organic co-solvents, or competitive ligands that dissociate the bound biomolecules by competitively binding to the immobilized ligands or the bound biomolecules. The latter is referred to as specific elution, which is an important feature of AC that can be employed for the improvement of separation performance.

2.47.3.1.5

Displacement chromatography

Most of the adsorptive chromatographic separations described above are carried out in elution-mode operations in which the bound solutes are eluted by decreasing their binding strengths via adjustment of mobile phase compositions (e.g., ionic strength in IEC and HIC). Displacement chromatography (DC) is an operational mode different from the commonly used elution chromato­ graphy. In DC, following the feed loading, the column is flushed with the solution of a substance (displacer) that binds to the stationary phase more strongly than any of the components in the feedstock. Due to the competition of the displacer in binding with the stationary phase, the adsorbed solutes will be desorbed (displaced) and move toward the column outlet with the mobile phase. The solutes with higher affinity to the stationary phase will in turn serve as the displacer for the low-affinity solutes, and the final pattern will be a series of adjacent bands of different solutes moving at the velocity of the displacer. This is called isotachic displacement train. After all the solutes in feedstock are displaced, the displacer bound to the stationary phase will be washed off with a regenerant and the column re-equilibrated for the next operation. A distinct difference between DC and elution chromato­ graphy is that displaced solutes migrate in the column before the displacer, while the eluant penetrates all the solutes. Moreover, the binding strength of the solutes to the stationary phase does not change distinctly in DC. The DC has several advantages. The solute bands in DC are closely connected with each other; so the column is more effectively utilized. Moreover, solutes can be simultaneously concentrated and separated by DC, while in elution chromatography a compro­ mise often has to be made between concentration and purity. Research has also shown that DC can offer much finer discrimination between similar substances.

Table 4

Affinity pairs for affinity chromatography

Ligand

Target molecule (receptor)

Antigen/Antibody Hormones (vitamins) Enzyme inhibitors (substrates or cofactor analogs) Coenzyme (NAD, NADP, AMP, ADP) Protein A DNA Heparin Lectins Metal ions (Cu2+, Ni2+, Zn2+, Co2+) Dyes (Cibacron Blue 3GA, Procion Red HE-3B, etc.) Histidine

Antibody/antigen Receptor proteins, carrier proteins Enzymes Enzymes (dehydrogenases, kinases) Antibody Polynucleotide, polynucleotide-binding proteins Proteins Glycoprotein, polysaccharide Histidine-rich proteins, metal-binding proteins Proteins Proteins

ADP, adenosine diphosphate; AMP, adenosine monophosphate; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate.

Adsorption and Chromatography

Table 5

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Displacers for bioseparations

Displacer

Application

Streptomycin A Neomycin B N-α-benzoyl-L-arginine ethyl ester Expell™ SP and Isolis™ SP* p-Toluene sulfonic acid sodium salt Ethyleneglycolbis(-aminoethylether)-N,N,N ′,N ′-tetraacetic acid Amaranth Expell™ Q, Isolis™ Q, and Propel™ Q* Benzyl tributyl ammonium chloride N,N-bis-(3-D-glucoamidopropyl)cholamide (Big Chap)

Cation exchange Cation exchange Cation exchange Cation exchange Anion exchange Anion exchange Anion exchange Anion exchange Hydrophobic interaction Hydrophobic interaction

*

Expell™, Isolis™ and Propel™ displacers are products of Sachem Inc. (Austin, Texas, USA).

The DC can be performed in almost all kinds of adsorption chromatography, provided suitable displacers are available. Therefore, availability of displacers is essential for the application of DC. Some displacers, including several specially designed commercial products, are listed in Table 5.

2.47.3.2

Expanded-Bed Adsorption Chromatography

Expanded bed is a stable liquid–solid fluidized bed in which the stationary phase with controlled particle size and/or density distribution is fluidized in a liquid stream directed upward [5]. The distribution of particle size and/or density within the expanded-bed system results in a distribution of terminal velocities (as calculated by Stokes’ equation), leading to a solid-phase classification within the expanded bed. The particles with larger settling velocities are found at the bottom of the bed while those with smaller settling velocities are at the top end. Thus, lower liquid dispersion level is obtained in expanded bed because this classification can reduce the mobility of the adsorbents. Hence, compared to conventional fluidized bed, there is a stable particle size and/or density classification in the axial direction, so expanded bed is a low-mixing fluidized bed with minimized solidphase mobility and reduced axial mixing of liquid phase. As a result, the chromatographic performance of an expanded-bed adsorption (EBA) can be comparable to a packed-bed adsorption. The increase in the upward flow velocity leads to bed expansion and bed-voidage increase, thus allowing particulates in a liquid stream to pass through the bed, so EBA is particularly suitable for application in the primary isolation of bioproducts from crude feedstock-containing particulate materials such as whole cells and/or cell debris. Moreover, EBA can be integrated into cell disruption or batch fermentation processes for direct product sequestration. Hence, using the EBA technology, a reduction in the number of process steps is achieved with particular advantages in terms of processing time and product yield, thus facilitating the establishment of a cost-effective bioseparation process. The EBA has been extensively studied in various aspects such as media development, column design, as well as process fundamentals and applications [6]. So, EBA for single-step purification of proteins has made great progresses, and the technology is expected to find more applications in other areas such as recovery of nanoparticles (e.g., plasmid DNA and viruses) and protein refolding. As the principal pillar supporting the development of the EBA technology, diversity of matrices is required to meet various needs in different applications. It is essential to design small-sized dense microspheres of appropriate size distribution, hopefully in a pellicular structure to reduce mass transfer resistance. Moreover, EBA matrices should be designed to minimize the interactions with particulate contaminants such as cells and cell debris in biological feedstreams. The efforts would offer more robust adsorbents for selection in the purification of different biomolecules in repeated use, making the integrative separation technology more sophisticated for widespread applications.

2.47.3.3

Electrochromatography

Electrochromatography is a liquid chromatography coupled with an external electric field (eEF). In an electric field, two electro­ kinetic phenomena occur, that is, electrophoresis of charged solutes and electroosmosis at a charged surface. In a typical electrochromatography, two electrodes are located at the two ends of a chromatographic column, so the eEF is applied at the longitudinal direction of the column. Therein, charged solutes flow through packed columns or open tubes via three possible modes, convection driven by pressure, electrophoresis, and electroosmosis. Both electrophoresis and electroosmosis in an eEF can promote mass transfer, thus lead to the increase of chromatographic performance. In addition, by the influence of eEF, mass transfer flux in bulk liquid phase is larger than that within particles due to the effect of diffusional resistance and size exclusion. Such a difference of fluxes can lead to more solute deposit on the surface of porous matrix, resulting in electrically induced concentration polarization (CP). The CP can change retention behavior of charged solutes in electrochromatography with porous media. Various types of chromatography that are operated at low ionic strength, for instance, SEC, IEC, and AC, can be coupled with an eEF [6]. However, difficulties are often encountered in the scale-up and application of electrochromatography in bioseparations due

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to Joule heating and electrolysis gases accompanied with the eEF. So, more detailed studies for its column design and scale-up capability for bioseparations are required.

2.47.3.4

Radial-Flow Chromatography

Normal chromatography belongs to axial-flow chromatography in which mobile phase flows through a packed column in the axial direction. In this normal column configuration, the scale-up by increasing the length of the column causes a significant increase of hydrostatic pressure needed to flow mobile phases along the column. Radial-flow chromatography (RFC) provides an efficient option for eliminating or minimizing this problem. An RFC column consists of two concentric porous cylindrical frits between which adsorbents are packed. In this configuration, liquid phase flows radially from the outer cylinder into the column, through the column and collects at the inner cylinder. As the flow path of a radial column can be much shorter than that in an axial column, the operating pressure of a radial column can be much lower, and higher flow rate can be utilized in chromatographic operations. In scale-up, the column radius can be kept unchanged, so the increase of column height gives rise to the increase of processing capacity without increasing the operating pressure. Therefore, RFC is particularly suitable for soft stationary phases, which are prone to collapse at higher hydrostatic pressure. Separation in RFC is achieved in the radial direction, so the chromatographic height is the radial thickness of the packing stationary phase. Accordingly, RFC offers fewer theoretical plates than an axial chromatography. Hence, RFC is useful for adsorptive chromatography such as IEC, HIC, and AC, but not for SEC that has low selectivity and needs larger column height for highresolution separations.

2.47.4 Theoretical Aspects of Adsorption and Chromatography 2.47.4.1

Adsorption Equilibria

Adsorption equilibria on adsorbents are described by the relations between free solute concentration (C) and the adsorbed solute concentration (Q). In liquid–solid adsorption systems, the relations are usually determined at constant temperature, so they are called adsorption isotherms. Adsorption equilibrium data and isotherms are of importance for adsorbent evaluation, as well as process analysis, design, and optimization of adsorption and chromatography. There have been a great deal of efforts on the development of adsorption equilibrium theories, but empirical or semi-empirical equations are still largely employed to express adsorption equilibria of biomolecules [1, 2]. Of the various formulas, the Langmuir equation (eqn 1) is the most widely used isotherm: Q¼

Qm Ka C 1 þ Ka C

½1

where Qm is the adsorption capacity and Ka is the association constant. For n-component adsorption, the isotherm of component i is expressed by Qi ¼

Qmi Kai Ci n

1 þ ∑ Kaj Cj

ði ¼ 1; 2; 3; ……; nÞ

½2

j¼1

n

At low solute concentrations, if one has ∑ Kaj Cj ≪ 1, eqn 2 can be reduced to a linear isotherm, j¼1

Qi ¼ mi ci

½3

where mi is a constant. Originally developed to represent gas adsorption, the Langmuir theory is based on three essential assumptions, that is, monolayer coverage, binding sites equivalence, and binding sites independence. In general, the assumptions do not hold for the adsorption of biomolecules such as proteins, so the Langmuir equation is regarded as an empirical expression when applied to liquid–solid adsorption systems. Nevertheless, the equation can be used to express the adsorption equilibria of a variety of solutes in a wide concentration range, including both small molecules and biomacromolecules. The Langmuir isotherm is advantageous because of its simplicity and wide applicability, but a distinct drawback of the expression is the lack of its link to the effect of liquid-phase modulators (e.g., salt concentration in ion exchange) on adsorption. Hence, research efforts have been made to develop sophisticated models taking into account the effect of liquid-phase modulators. Most of the researches have focused on the effect of salt concentration on the ion-exchange equilibria of proteins. Compared with small molecules, proteins are typical of polyelectrolyte characteristics and their adsorption behaviors are more complex. Protein adsorption depends on several small regions of the protein surface (e.g., the regions rich of charges or hydrophobic patches) termed as contact regions, rather than the whole protein surface. These contact regions in liquid phase will associate with solvent or counterions by solvation and electrostatic interaction. In ion-exchange adsorption, these solvent molecules or counterions bound to the protein are displaced, which is assumed to obey a stoichiometric relationship. Some isotherm models have been developed in terms of the stoichiometric displacement law, of which the steric mass action (SMA) model [7] has been recognized for better description of protein adsorption equilibria. In addition to the stoichiometric

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displacement, the SMA model accounts for the steric shielding effect of binding sites by the bound protein, as described below, 0 1zi
 Qi B Cs C Ci ¼ ½4 @ A ði ¼ 1; 2; 3; ……; nÞ n

Kai Λ− ∑ ðzi þ σi ÞQi

i ¼ 1

where Cs is the salt concentration, zi is the characteristic charge of protein i, Λ is the ionic capacity of the ion exchanger, and σi is the steric factor of protein i. The SMA model offers a concise form to express the effect of ionic strength on protein adsorption equilibria to ion-exchange adsorbent and has proved to well describe nonlinear adsorption chromatography of proteins at the condition of varying salt concentrations. Moreover, the model can be extended to other adsorption systems such as hydrophobic and affinity adsorptions. Ion exchange is really based on electrostatic interaction, and the long-range electrostatic interaction does not follow the stoichio­ metric law. Therefore, besides the empirical and semi-empirical equations described above, many efforts have been made to develop theoretical models for the ion-exchange equilibria of proteins since the 1980s (see Reference 8 and references cited therein). In the models, both the protein and adsorbent are defined as charged bodies (e.g., a sphere for protein and a planar surface for adsorbent) surrounded by electrical double layers. By the theoretical approaches, the retention behavior or the nonlinear adsorption isotherm of protein in IEC can be predicted. Although these models offer a strictly theoretical framework to elucidate the adsorption equilibria, it is in general difficult to correctly estimate the model parameters, which limits the applicability of the models in protein chromatography.

2.47.4.2

Uptake Kinetics

Solute uptake to porous adsorbent beads experiences several sequential steps: (1) the solute migrates through the stagnant layer of liquid-film adjacent to the surface of adsorbent, (2) the solute penetrates into the pore and moves toward the adsorption site, and (3) the solute binds to the surface and bound solute keeps in equilibrium with the free solute in the pore at the same radial position. If the solute is not tightly bound on the surface, it may also migrate on the surface along the bound concentration gradient, which is called surface diffusion. Surface diffusion is in parallel to the pore diffusion. Solute binding to a surface is generally very fast as compared with the diffusive mass transfer processes, so the uptake rate usually depends on the mass transfer behaviors. Liquid-film mass transfer rate (TR) is related to the thickness of stagnant layer and properties of liquid phase, expressed by the product of a liquid-film mass transfer coefficient and a linear driving force as defined below. TR ¼ kf aðCb −Cs Þ

½5

where kf is the liquid-film mass transfer coefficient, a is the specific outer surface area of adsorbent, and Cb and Cs denote the solute concentrations in bulk liquid phase and on the adsorbent surface, respectively. The liquid-film mass transfer and pore diffusion are sequential mass transfer processes, and the slower one determines the process rate. In general, the pore diffusion within porous adsorbents is the rate-limiting step in biochromatography. Hence, liquid-film resistance can be ignored particularly for protein adsorption in porous media. However, the importance of liquid-film mass transfer increases with decreases in adsorbent size and solute molecular mass (i.e., the decrease in intraparticle mass transfer resistance). Mass transfer inside porous materials is driven by pore diffusion, surface diffusion, and sometimes intraparticle convection. Because normal adsorbents have a pore diameter comparable to the mean free path or molecular size of a solute, the intraparticle diffusion is hindered by the porous structure of the matrix, especially for macromolecules. This is an important reason, for that, intraparticle diffusion dominates the uptake rate of proteins. Considering the liquid-film mass transfer, the general diffusional mass transfer model for spherical adsorbents is given by: 
 ∂C ∂Q 1 ∂ 2 ∂C ∂Q εp þ ¼ 2 r εp Dp þ Ds ½6 ∂r ∂r ∂t ∂t r ∂r t ¼ 0; Q ¼ 0; C ¼ 0 r ¼ 0; r ¼ rp ; εp Dp

∂Q ¼0 ∂r

∂C ∂Q þ Ds ¼ kf ðCb −CÞ ∂r ∂r

½6a ½6b ½6c

where εp is the porosity of the adsorbent, C is solute concentration in the pore fluid, r is the radial direction, rp is the particle radius, and Dp and Ds are the diffusivities in pore fluid and adsorbed phase, respectively. Equation 6 reduces to a pore diffusion model if Ds=0, and it reduces to a surface diffusion model if Dp=0. One can also lump the intraparticle diffusions to a single parameter, effective diffusivity, De. In this case, the differential equation is expressed as, ∂Q De ∂ 2 ∂Q ¼ 2 ðr Þ ∂r ∂t r ∂r

½7

In combination with an adsorption isotherm and a mass conservation equation for an adsorption operation (e.g., well-mixed contactor or packed-bed chromatography), the kinetic equations can be solved by numerical techniques.

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2.47.4.3

Theoretical Considerations of Chromatography

The performance of chromatography is dependent on the adsorption equilibria, mass transfer and adsorption (if any) kinetics, and dispersion behavior of the mobile phase. There are various theoretical descriptions of chromatography [1], and the most widely applied ones are the plate model and general rate model. The plate model is developed for linear chromatography in which the equilibrium isotherm is linear (eqn 3). In linear chromatography, the influence of thermodynamics on chromatographic profiles vanishes. Namely, the linear isotherm of a solute controls only the position of its peak (retention time), while the kinetics of mass transfer and axial dispersion controls the peak shape (band width). The plate model depicts a continuous column by a discrete number of identical well-mixed cells. The wellmixed cells are called equilibrium stages, or theoretical plates, because the mobile and the stationary phases in each of these successive plates are in equilibrium. Thus, the kinetics of mass transfer and axial dispersion are lumped by the plate number, to which the band width or profile shape is directly related. By the plate model, column efficiency is characterized by the plate number or the height equivalent of a theoretical plate (HETP). The general rate model is a sophisticated chromatographic theory, which can simultaneously consider all the possible contribu­ tions to the chromatographic performance, including axial dispersion, liquid-film mass transfer, intraparticle diffusions, and the rate of adsorption–desorption. Certainly, some unimportant phenomena (such as adsorption kinetics and/or liquid-film mass transfer) can be ignored in the general rate model for simplicity. By an axial dispersion assumption, the mass balance equation in the mobile phase is written as � ∂C @Q ∂2 C ∂C þF ¼ Dz 2 −u ∂z ∂t ∂t ∂z

½8

where F= (1 – ε)/ε is the phase ratio (ε is the column voidage), u is the interstitial velocity, Dz is the axial dispersion coefficient, and � is the average value of adsorbed solute concentration over the entire particle. Combining an equilibrium isotherm, a kinetic Q expression (e.g., eqn 7), and proper initial and boundary conditions, the model can be solved by numerical techniques to obtain chromatographic profiles. For linear chromatography, analytical solutions can be derived. By considering the homogeneous diffusion of solute in the stationary phase (eqn 7), the column efficiency is derived from the general rate model as, 2

HETP ¼

2mFurp 2Dz þ u 15 ð1 þ mF Þ 2




1 5m þ De rp kf

 ½9

Equation 9 can be reduced to the classical van Deemter equation, HETP ¼A þ

B þ Cu u

½10

where A is the contribution of axial dispersion and dependent on the packing quality of the stationary phase as well as adsorbent shape and size distribution, B is the contribution of molecular diffusion, and C is the contribution of mass transfer resistances. Equation 10 indicates that there is a flow rate that gives rise to a minimum value of HETP (highest column efficiency). If the axial dispersion is negligibly small and the rate of mass transfer kinetics is infinite, the general rate model is simplified to equilibrium model. The equilibrium model describes an ideal condition of chromatography (ideal chromatography), in which the free and adsorbed solute concentrations are constantly at equilibrium at any time and position in the column. Under the ideal condition, the plate number of a finite-length column is infinite, and the elution peaks in linear chromatography are identical to the injection profiles. This situation is certainly unrealistic, and is usually of little importance. However, for nonlinear isotherms, ideal chromatography can qualitatively depict the influence of the isotherm shape on elution profiles.

2.47.5 Development of Adsorption and Chromatography 2.47.5.1

Innovation of Chromatographic Matrices

In liquid chromatography with a porous stationary phase, intraparticle mass transfer of macromolecules is significantly hindered; so the intraparticle diffusivity is much lower than that in bulk liquid phase and decreases more with increasing molecular size. Hence, it is recognized that intraparticle diffusive mass transport is the rate-limiting step in chromatographic processes of biomacromo­ lecules. Therefore, chromatographic matrices need evolution to overcome this problem. In the past two decades, various efforts were made to reduce mass transfer limitations for realizing high-performance preparative biochromatography [6].

2.47.5.1.1

Flow-through media

A direct way to the goal of elimination or alleviation of intraparticle diffusive mass transfer resistance is to open convective flow channels in size of submicron to microns in porous particles. Mobile phase can flow through the channels in chromatographic operations, so the intraparticle mass transport is greatly enhanced due to shortened diffusive path. This kind of chromatography is called flow-through chromatography or perfusion chromatography and the materials with intraparticle convection are called flowthrough or perfusion media. As compared with the diffusive pores of conventional media (usually, 10–200 nm), the flow-through pores are over 600 nm, one to two orders of magnitude larger than the diffusive pores. So, the wide pores through which mobile

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phase can flow are also called superpores or gigapores, and the corresponding particles are called superporous (or gigaporous) microspheres. Moreover, one characteristic of the material is the bimodal pore size distribution, so this kind of microsphere is also called biporous bead or bidisperse porous bead. The micropores (i.e., diffusive pores) offer large specific surface area for solute binding, so high adsorption capacity can be maintained for the adsorbents of biporous geometry. There are some superporous adsorbents commercially available, such as Poros, HyperD, and Source listed in Table 1. In recent years, continuous efforts have been made to develop different superporous media made by double emulsification and using solid granules as porogen. The convective flow of mobile phase through the superpores can lower the backpressure and the HETP value at a flow velocity up to 50 cm min−1, and the dynamic binding capacity of a biporous adsorbent can be much higher than that of a microporous one at high flow rates. So, the superporous adsorbents are promising for high-speed biochromatography.

2.47.5.1.2

Membrane

To maximize chromatographic throughput, mass transfer limitations need to be eliminated for the fast uptake of target substance, and the flow rate should be as high as possible at a given pressure drop across the bed. This leads to efforts to design short bed of large diameter and other column configurations such as RFC. In this perspective, it is obvious that an ideal chromatography column is a piece of filter because a porous membrane in thickness of 100-µm order is the shortest bed available in reality. Microfiltration membranes primarily contain flow-through pores, so the main feature of membrane-based chromatography is the absence of pore diffusion, which is the main transport resistance in conventional chromatography using porous particles. In membrane chromato­ graphy, the target solute binds to the ligands attached to the inner surface of the through pores when it flows through the pores with feedstock, so only the surface film diffusion resistance is left. For this reason, membrane chromatography can be operated at high flow rate and low pressure drop at maximum efficiency of ligand utilization. Hence, membrane chromatography offers high-speed purification of biomacromolecules such as proteins and plasmid DNA, and now it is particularly popular for antibody purification.

2.47.5.1.3

Monolith

A drawback of membrane chromatography is its low chromatographic efficiency and low binding capacity for proteins. A solution to these problems is to use monolithic columns. A monolith can be regarded as a piece of very ‘thick’ membrane. Besides larger plate number than membrane chromatography, monolithic column can offer higher binding capacity than membrane because it can be made to contain both flow-through pores and diffusive pores. Monolithic column has several advantages over packed bed of porous particles. With controlled pore structure, a monolith can offer lower mass transfer resistance. At present, monolithic materials have been widely studied for diverse applications, especially for the analytical and preparative chromatography of biomolecules such as proteins and DNA. Recently, an ideal chromatographic medium was suggested as a monolith with straightforward and evenly distributed uniform through pores (flow channels) separated by a skeleton full of diffusive pores of proper size that provide large surface area accessible for solute molecules [6]. With this structural design, high-performance chromatography (high adsorption capacity/column effi­ ciency and low back pressure at high flow rate) may be realized.

2.47.5.2

Selection and Design of Affinity Ligands

Due to the high selectivity and purification power, AC has become one of the best ways for purification of biomolecules. However, the difficulty in the use of AC is the lack of specific ligands for target molecules. So, the research focus on AC has recently been shifted toward selecting and designing ligands of high affinity and specificity. Currently, there are mainly three approaches to the discovery of or generating affinity ligands, as described below.

2.47.5.2.1

Combinatorial library approach

This approach focuses on the selection of ligands from large libraries constructed randomly by synthetic or biological display techniques. Using combinatorial synthesis, a huge number of structurally distinct organic molecules are synthesized at a time, which can provide a great many novel compounds for random screening. Several laboratories have recently reported the application of combinatorial synthesis methods to select affinity ligands from the libraries based on substituted triazine and peptides. Biological display is another widely used method to construct a biological combinatorial library (e.g., peptide, oligonucleotide, protein domain, and protein), and it has rapidly matured and evolved as a tool for discovering high-affinity ligands. It includes phage display, ribosome display, and systematic evolution of ligands by exponential enrichment (SELEX) methods. Phage display and ribosome display are used to construct many peptides, protein domains, or antibodies, whereas SELEX is used for oligonucleotide ligands. Phage display is basically achieved by inserting a randomized oligonucleotide sequence at an appropriate site in the structural gene of coat protein. Ribosome display utilizes a cell-free transcription, translation, and selection approach to display ligand. The SELEX exploits oligonu­ cleotide libraries constructed by solid-phase oligonucleotide synthesis, and cell-independent enzyme-based in vitro selection approaches. When a large library is generated, it is screened and ligands are selected against the target molecule in immobilized form. However, these approaches often suffer from the problems such as pseudo-positives, high cost, and long screening time.

2.47.5.2.2

Rational design

The method uses the information of the structure of natural ligands or its target protein to upgrade or create a new ligand. Many molecular simulation techniques (e.g., docking, molecular surface analysis, and molecular dynamics (MD) simulation) have been

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developed to calculate, visualize, formulate, and hypothesize about the energy and orientation of candidate ligands in the pocket of a target protein. Especially, with the rapid advances in computational tools and the availability of more 3-D structures of proteins obtained by X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and homology modeling technology, rational design of affinity ligands has become faster, more feasible, and more powerful. There are two distinct rational design methods, that is, structural template approach and functional approach. The first one is based on the knowledge of the target protein structure and the interactions between the target protein and its natural ligands or counterparts. The natural ligand-protein complex is investigated, and the conformation of the bound molecule is used as a template to design a new ligand. If there are no natural ligands or counterparts, and only the structure of the target protein is known, a candidate ligand library is first constructed and used for screening using docking softwares. Then, the affinity between candidate ligands and the target protein is evaluated using a suitable scoring function [6]. The second approach is employed when no sufficient structural data are available for the target protein and a reliable protein model cannot be built by homology modeling. The method is based on preexisting knowledge for the interactions of the target protein with the functional groups, moieties, and molecular shapes of natural ligands, for example, substrates and/or inhibitors of enzymes. Ligands can be designed by: (1) exploiting recognizable molecular shape and properties, such as hydrophobicity and electrostatic potential, (2) introducing a specific functional group, and (3) combining the above two functional features on the same ligands.

2.47.5.2.3

Combination of rational design and synthetic combinatorial library

Recently, the combination of rational design and synthetic combinatorial library emerged as a new and promising approach for ligand selection [9]. It can integrate the strengths of the above two approaches. For example, this method not only enables the selection and discovery of lead compounds or groups using molecular modeling but also reflects the chemical, geometrical, and steric constraints imposed by the complex 3-D solid support environment. The method involves the following steps: (1) selection of an appropriate site on the target protein, (2) design of a complementary ligand compatible with the candidate pocket using modeling techniques, (3) synthesis of a limited ligand library of structures resembling the rationally designed lead ligand, and (4) screening of the library against the target protein.

2.47.5.3

Mixed-Mode Ligands

As has been discussed in Section 2.47.3, most traditional chromatographic methods (except for AC) are dedicated to separate target molecules by the differences in a specific mode of interaction between the targets and the stationary phase. Mixed-mode interactions or the so-called nonspecific interactions are usually to be avoided, because they may counteract with each other or deteriorate resolution. However, recent researches have shown that a proper combination of different interactions can give rise to unique selectivity and facilitate the separation process. A number of mixed-mode ligands had been developed accordingly. Some of the commercially available mixed-mode media are listed in Table 6. Other mixed-mode ligands have been recently reviewed [10]. Streamline Direct HST I is a mixed-mode adsorbent specially designed for EBA. The proper balance of hydrophobic interaction, electrostatic interaction, and hydrogen bonding enables high adsorption capacity of proteins in a wide range of ionic strength, whereas the capacity of traditional ion-exchange and hydrophobic adsorbents are strongly salt dependent. Therefore, crude cell extract can be directly applied to the Streamline Direct HST I column without prior adjustment of salt concentration. Elution can be achieved by simultaneously increasing the salt concentration and adjusting the pH. The ‘salt-tolerance’ of mixed-mode adsorbents can greatly simplify the purification process and reduce production cost. The 4-mercapto-ethyl-pyridine (MEP) Hypercel is a new-generation hydrophobic adsorbent with electrostatic interaction functionalities. The ligand of MEP Hypercel has a chargeable aromatic pyridine ring with a pKa of 4.85. At physiological conditions (pH~7), the ligand is uncharged and adsorption is achieved by hydrophobic interaction. When the pH is reduced to 4 or lower, the ligand will take on a proton and become positively charged. Most proteins are also positively charged at those pH values, so elution is achieved by charge repulsion. This process is denoted as hydrophobic charge induction chromatography (HCIC) [11], in which proteins are adsorbed by hydrophobic interaction and desorbed by charge repulsion. This facile elution process enables easy recovery of proteins from adsorbents of high ligand densities. Moreover, because of the high ligand densities, high-capacity hydrophobic adsorption takes place at physiological salt concentrations, thus eliminating the need of high salt concentration usually employed in the adsorption stage of traditional HIC.

2.47.5.4

Displacer Screening and Design

Displacers are essential for the application and development of DC. In recent years, various approaches have been exploited to develop high-affinity displacers for protein purification, with a number of high-affinity displacers identified. High-throughput screening (HTS) is an efficient experimental approach to screen displacers from a number of existing compounds [12]. In the screening, a known amount of adsorbent is equilibrated with a protein solution, and the amount of protein adsorbed is calculated by the equilibrium concentration in the supernatant. Then, the medium is divided into small aliquots, which are incubated with solutions of different displacers. Protein concentrations in the supernatants are then assayed, and the efficacy of displacers are denoted by the percent protein displaced or the displacer concentration needed to displace 50% of the adsorbed protein. This approach has enabled the evaluation of many displacers in parallel, thus improving the screening efficiency.

Adsorption and Chromatography

Table 6

677

Some commercially available mixed-mode media

Medium

Ligand

Manufacturer

Modes of interaction

Capto™ MMC and Streamline Direct HST I

GE Healthcare

Cation exchange, hydrophobic interaction, and hydrogen bonding

Capto™ adhere

GE Healthcare

Anion exchange, hydrophobic interaction, and hydrogen bonding

MEP 4-mercapto-ethyl-pyridine Hypercel

Pall Life Sciences

Hydrophobic interaction and charge repulsion

HEA hexylamine Hypercel

Pall Life Sciences

Hydrophobic interaction and anion exchange

PPA phenylpropylamine Hypercel

Pall Life Sciences

Hydrophobic interaction and anion exchange

MBI 2-mercapto-5-benzimidazole sulfonic acid Hypercel

Pall Life Sciences

Hydrophobic interaction and cation exchange

Moreover, the efficacy data obtained in HTS can be used to establish quantitative structure–efficacy relationships (QSERs) with the assistance of molecular simulation software. The QSER models thus established can then be used for the virtual screening of displacers from a broad range of compounds. Another way to get high-affinity displacers is de novo design and synthesis. A common strategy of displacer design is to synthesize linear or dendrimeric polymers/oligomers from monomers that have affinity to the stationary phase. Monosaccharides can also be used as the base for high-affinity displacers, to which multiple affinitive moieties are attached via the hydroxyl groups. Recently, it was found that some displacers can selectively displace some proteins while leaving others with similar affinity on the column [13]. Experimental studies and MD simulations show that the mechanism of this ‘chemically selective displacement’ is that these displacers can selectively bind the proteins and retain them on the stationary phase. Based on this mechanism, specific chemically selective displacers can be designed and synthesized by combination of a protein-binding group and a stationary-phase­ binding group. The protein-binding group can be highly specific ligands used in AC or general protein-binding moieties (e.g., hydrophobic groups), and the stationary-phase-binding group is usually a known high-affinity displacer. Fluorescent hydrophobic groups have also been used as the protein-binding group for the synthesis of fluorescent chemically selective displacers for online monitoring of the displacement process.

2.47.5.5

Molecular Insight into Protein Adsorption

Adsorption of biomolecules at liquid–solid interfaces is of fundamental importance in chromatographic separation process, so a comprehensive understanding of the adsorption phenomena and particularly the molecular mechanism is crucial for the research and development of biochromatography. Many microscopic experimental examination techniques, for instance, atomic force microscopy, NMR, X-ray crystallography, surface plasmon resonance, hydrogen–deuterium isotope exchange, and confocal laser scanning microscopy, have been used to explore the microscopic information of the process. However, none of these techniques can detect the dynamic process and protein conformational transition within adsorbent pores, which restricts not only the exploration of adsorption mechanism, but also the ligand design and process optimization. Molecular simulation has been used to explore the molecular insights into protein adsorption [14, 15], including the description of adsorbed state, analysis of adsorption dynamics, and protein conformational transition at an interface. Molecular simulation is a

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Downstream Processing and Product Recovery

powerful tool with sufficiently small scale in both time and space, thus it can offer clear microscopic information in a direct manner. It has been widely used to understand protein conformational transition at molecular-level resolution, and is becoming a fundamental technique complementary to experimental and theoretical studies. In molecular simulations, both the adsorbate and surface can be visualized using coarse-grained models or all-atom models, depending on the description precision required and the computational power provided. Furthermore, the actual chromatographic process, especially the adsorption can be monitored through the models using Monte Carlo (MC) or MD simulation. To date, molecular simulations have been successfully used to examine adsorption processes, including modeling and visualization, adsorption process, and protein conformational transition on ligand surface.

2.47.5.5.1

Modeling and visualization

The adsorbent is usually modeled as a plate, cylinder, or sphere with immobilized ligands; the ligand conformation and surface morphology are visualized and examined through statistical mathematics. The changes caused by ligand parameters such as ligand length, composition (inclusion of embedded polar groups), and bonding density can be easily monitored and analyzed to explore the general rule, which is helpful for the rational design and fabrication of ligands. For example, a coarse-grained model was constructed to simulate porous dextran layers on the surface of a base matrix, using implicit flat and nonflat agarose surfaces, and the 3-D porous structures were characterized using MD simulation. An all-atom model of ligand immobilized to agarose with a spacer arm was proposed, and the conformation of ligand–spacer–agarose was visualized using MD simulation to examine the influence of spacer arm and the interaction between agarose and ligands.

2.47.5.5.2

Adsorption process

The adsorption behaviors, especially adsorbate-ligand interactions, adsorption and desorption processes, solvent partitioning and retention properties, can be described by molecular simulations. The effects of various chromatographic parameters can be investigated, including the composition of mobile phase, column pressure, and pore shape. For example, various chromatography behaviors in reversed-phase liquid chromatography have been examined using a constructed all-atom adsorbent model in contact with mobile phases of water/methanol mixtures. A 3-D stochastic simulation was performed to provide a detailed understanding of the mass transfer processes in liquid chromatography, including the kinetics of partitioning mechanism in both homogeneous and heterogeneous systems.

2.47.5.5.3

Protein conformational transition

As discussed in Section 2.47.2, conformational transition is of importance in protein adsorption because it affects the recovery yield of the native product. Furthermore, the orientation and extension of protein on ligand surface strongly affects the protein-surface interactions. Thus, protein conformational transition on the ligand surface has been widely examined, focusing on the proteinligand interaction, protein orientation and conformation. For instance, the interaction between a polymer chain and planar surface, as well as the adsorption and orientation of antibodies on charged surfaces have been examined by MC simulation. Moreover, MD simulations were used to study the interactions between lysozyme and the self-assembled monolayers in the presence of explicit water molecules and ions, and to investigate the initial stages of lysozyme adsorption at a charged solid interface through both allatom model and simplified uniformly charged sphere model. The equilibrium and flow properties of polymer liquid between two brush-covered surfaces were also investigated through a coarse-grained bead-spring model. Furthermore, MD simulation studies on HCIC have been reported and molecular insights into protein conformational transition within adsorbent pores were explored. Based on the applications summarized above, it can be concluded that molecular simulations can yield practically exact results and significantly new insights at the molecular level, and thus are suitable for exploring the mechanisms of protein adsorption. It is expected that combination of computational quantum chemistry at quantum level, molecular simulations at atomistic level, and experiments at macroscopic level can get more comprehensive understanding of protein adsorption and chromatography.

2.47.6 Conclusions Various chromatographic methods have been widely applied in the practice of downstream processing of peptides, proteins, nucleic acids, viruses, and many other biological substances because of their favorable characteristics in high resolution, wide availability, and mild operating conditions. At present, biochromatography is still a major area of bioseparation research activities, and new achievements are reported continuously. It is expected that more efforts will be denoted to the fundamentals of biomolecule adsorption and the innovation of chromatographic materials, including matrices, ligands, and displacers. In this process, molecular simulations will play an important role in understanding molecular interactions at liquid–solid interfaces, adsorption equilibrium, kinetics, and molecular transport phenomena in adsorbent pores, besides experimental and theoretical studies.

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Norde W (2003) Driving forces for protein adsorption at solid surfaces. In: Malmsten M (ed.) Biopolymers at Interfaces, 2nd edn. New York, NY: Dekker. Hubbuch J, Thömmes J, and Kula MR (2005) Biochemical engineering aspects of expanded bed adsorption. Advances in Biochemical Engineering/Biotechnology 92: 101–123. Sun Y, Liu FF, and Shi QH (2009) Approaches to high-performance preparative chromatography of proteins. Advances in Biochemical Engineering/Biotechnology 113: 217–254. Brooks CA and Cramer SM (1992) Steric mass-action ion exchange: Displacement profiles and induced salt gradients. AIChE Journal 38(12): 1969–1978. Su XL and Sun Y (2006) Thermodynamic model for nonlinear electrostatic adsorption equilibrium of protein. AIChE Journal 52(8): 2921–2930. Rupasinghe CN and Spaller MR (2006) The interplay between structure-based design and combinatorial chemistry. Current Opinion in Chemical Biology 10(3): 188–193. Zhao GF, Dong XY, and Sun Y (2009) Ligands for mixed-mode protein chromatography: Principles, characteristics and design. Journal of Biotechnology 144: 3–11. Burton SC and Harding DR (1998) Hydrophobic charge induction chromatography: Salt independent protein adsorption and facile elution with aqueous buffers. Journal of Chromatography A 814(1–2): 71–81. Mazza CB, Rege K, Breneman CM, et al. (2002) High-throughput screening and quantitative structure–efficacy relationship models of potential displacer molecules for ionexchange systems. Biotechnology and Bioengineering 80(1): 60–72. Rege K, Ladiwala A, Tugcu N, et al. (2004) Parallel screening of selective and high-affinity displacers for proteins in ion-exchange systems. Journal of Chromatography A 1033(1): 19–28. Mungikar AA and Forciniti D (2002) Computer simulations and neutron reflectivity of proteins at interfaces. ChemPhysChem 3(12): 993–999. Raffaini G and Ganazzoli F (2007) Understanding the performance of biomaterials through molecular modeling: Crossing the bridge between their intrinsic properties and the surface adsorption of proteins. Macromolecular Bioscience 7(5): 552–566.