**4.1.1 Purification of proteins and enzymes by HIC**

Recently, many strategies that involve a HIC step to purify proteins and enzymes of industrial and/or biomedical interest have been reported. For instance, Liu et al. (2010) developed a purification process to isolate and characterize an antifungal protein from *Bacillus subtilis,* which can be used as a bio-control agent. The process consisted of a preliminary precipitation with ammonium sulfate at 30-70% saturation, followed by HIC (using Phenyl Sepharose as stationary phase) and finally an IEC step. The process gave an overall recovery of 1.2% of total protein in the cell extract. The antifungal protein showed ribonuclease, protease and hemagglutinating activities.

On the other hand, Teng et al. (2010) purified and characterized an endo-β-1,4-glucanase from the giant snail (*Achatina fulica frussac*) by means of a process consisting of three chromatographic steps: size exclusion chromatography (SEC), anion exchange chromatography (AEC), and finally hydrophobic interaction chromatography. A 29-fold purity increase was achieved, and an overall recovery of 14.7% was reached. In addition, this

(11) based on amino acidic composition was used to predict chromatographic behavior in HIC, resulting in a performance 5% better than that observed in the model based on the

three-dimensional structure of proteins (equation (10)) (Salgado et al., 2008).

Fig. 5. Methodology for predicting protein retention time in HIC based on surface hydrophobicity. Using a PDB file as input to the program GRASP, the total and partial accessible areas of the exposed amino acids is determined. Using an amino acid

**4. Applications in biomedical engineering** 

**4.1.1 Purification of proteins and enzymes by HIC** 

ribonuclease, protease and hemagglutinating activities.

**4.1 General applications** 

described below.

hydrophobicity scale and equation (12), the average surface hydrophobicity can be obtained. Then, through simple mathematical correlations the DRTof the protein can be estimated.

Currently, many proteins of pharmacological and industrial interest are obtained through highly optimized purification processes, typically consisting of two or three chromatographic separation stages. Usually these processes involve one or two IEC steps followed by a HIC step (Asenjo & Andrews, 2004). In addition, most recombinant proteins can be obtained at therapeutic grade of purity, by processes of the same structure (Asenjo & Andrews, 2008). Then, HIC often forms part of processes to yield a purified macromolecule of biomedical interest, such as therapeutic proteins (Seely & Richey, 2001), DNA vaccines (Diogo et al., 2000), and enzymes (Teng et al., 2010), among others. Besides, the use of HIC to purify protein complexes (McCue et al., 2008), as well as to study protein folding from a thermodynamic point of view (Geng & Wang, 2007), have been reported. Some applications of HIC for purifying enzymes and protein complexes, and to studying protein folding are

Recently, many strategies that involve a HIC step to purify proteins and enzymes of industrial and/or biomedical interest have been reported. For instance, Liu et al. (2010) developed a purification process to isolate and characterize an antifungal protein from *Bacillus subtilis,* which can be used as a bio-control agent. The process consisted of a preliminary precipitation with ammonium sulfate at 30-70% saturation, followed by HIC (using Phenyl Sepharose as stationary phase) and finally an IEC step. The process gave an overall recovery of 1.2% of total protein in the cell extract. The antifungal protein showed

On the other hand, Teng et al. (2010) purified and characterized an endo-β-1,4-glucanase from the giant snail (*Achatina fulica frussac*) by means of a process consisting of three chromatographic steps: size exclusion chromatography (SEC), anion exchange chromatography (AEC), and finally hydrophobic interaction chromatography. A 29-fold purity increase was achieved, and an overall recovery of 14.7% was reached. In addition, this novel enzyme has a particularly high stability at a broad pH range, acidic pH optimum, and a very high thermostability, and therefore it would have a great potential use in industry.

Lavery et al. (2010) reported the purification of a peroxidase from horseradish roots (*Armoracia rusticana*) by means of a three-step strategy, consisting of ultrasonication, ammonium sulfate precipitation, and HIC (using Phenyl Sepharose). In this strategy, the only high-resolution purification step corresponded to HIC. An overall yield of 71% and a 291-fold purification were achieved, thus demonstrating the high efficiency of this technique. The purified peroxidase was extremely stable in different media, and therefore its commercialization seems promising. Bhuvanesh et al. (2010) used a single-step method to purify a filarial protein (expressed heterologously in *E. coli*) with great potential as a vaccine for preventing human lymphatic filariasis. The purification method consisted of a HIC step. An overall recovery of 60% and 100% purity were achieved.

### **4.1.2 Purification of protein aggregates by HIC**

The use of HIC to separate product-related impurities in the biopharmaceutical industry is well documented (Queiroz et al., 2001). This method is also used to separate multimers from monomeric forms of proteins of biomedical interest, since these conformations often differ in surface hydrophobicity. This difference owes to the fact that the stabilization of quaternary structures occurs due to hydrophobic interaction between the monomers, resulting in protein aggregation. In this way, the hydrophobic patches of a multimer are somewhat hidden, and therefore less accessible to the hydrophobic ligands of a HIC support, unlike the monomer whose hydrophobic patches are exposed to the solvent and, accordingly, accessible to the HIC ligands. The adsorption mechanism of protein aggregates in HIC is complex and not fully understood so far.

Mc Cue et al. (2010) developed a chromatography model to predict the separation of monomer and aggregate species. Equation (14) shows the Langmuir isotherm that describes equilibrium between the protein adsorbed to the resin and the protein that remains in solution. Here, C is the protein concentration in the mobile phase, q is the protein concentration in the stationary phase, qm is the resin maximum capacity and k is the equilibrium constant. Equation (15) shows the mass balance used to describe the protein concentration profiles. Mass conservation was assumed and the intra-particle mass transfer was considered to be driven by homogeneous diffusion. In equation (15), Deff is the effective diffusivity of the protein from the mobile phase bulk to the inner of the porous resin bead, t is time and r is the radial coordinate. The validity of the model was assessed by experimental determinations. A fraction of the aggregate proteins bound irreversibly to the HIC resin, becoming the major factor governing the process. This phenomenon was adequately described by the model.

$$C = \frac{q}{k \cdot (q\_m - q)}\tag{14}$$

$$\frac{\partial q}{\partial t} = D\_{eff} \cdot \left( \frac{\partial^2 q}{\partial r^2} + 2 \cdot \frac{\partial q}{r \cdot \partial r} \right) \tag{15}$$

### **4.1.3 Protein folding in HIC**

Protein folding is relevant from a process point of view, since most recombinant proteins produced in bacteria such as *E. coli* are accumulated as inclusion bodies, and therefore protein refolding constitutes an additional stage in the production and purification process in order to yield a "functional" product (especially in the case of enzymes). Hydrophobic interaction chromatography has been used to study thermodynamics aspects of protein folding. For instance, Geng et al. (2005) performed calorimetric determinations on the enthalpy change (ΔHfolding) of denatured lysozyme during its adsorption to a hydrophobic surface, with the simultaneous protein refolding. The surface consisted of PEG-600 made of a silica base HP-HIC (High Performance- Hydrophobic Interaction Chromatography) packing. At 25°C, ΔHfolding was found to be - 34 439 KJ/mol, involving adsorption, dehydration and molecular conformation enthalpies changes.

Later, Geng & Wang (2007) used the concept of "Protein Folding Liquid Chromatography" (PFLC), to describe a chromatographic process aiming to either raise the efficiency, or shortening the time of protein folding. Besides, an optimal PFLC should be able to simultaneously remove denaturant substances, separate contaminant proteins, promote refolding of the target protein, and ease denaturant recovery. Any type of chromatography can be used in PFLC, mainly Size Exclusion Chromatography, Ion Exchange Chromatography, Affinity Chromatography, and Hydrophobic Interaction Chromatography.

In HIC, the process is governed by thermodynamic equilibrium and so does the protein folding. PFLC provides the chemical equilibrium that favors the conversion from aggregate to desorbed protein, resulting in a higher refolding efficiency and shorter refolding time. The unfolded proteins, at a high ionic strength, are driven by hydrophobic interactions from the mobile phase to the HIC stationary phase, and the hydrophobic patches on the proteins surface get attached to the hydrophobic ligands, while the hydrophilic zones of the unfolded molecules remain in contact with the solvent. As a consequence, unfolded molecules are not able to aggregate. The unfolded molecules desorb from the HIC support as ionic strength in the mobile phase decreases. Protein molecules with incorrectly folded domains would be corrected by the spontaneous disappearance of the domains in the mobile phase due to their thermodynamic instability. After many HIC runs, the incorrectly folded domains will decrease, while the correctly folded molecules will predominate, resulting in protein refolding at high efficiency.

### **4.2 Applications in biomedical engineering**

Biomedical applications of HIC are broad, since this technique offers some advantages over other chromatographic techniques, such as Affinity Chromatography (AC) and Reverse-Phase Chromatography (RPC). The use of AC depends on the availability of a specific ligand for the protein or group of proteins to be separated, thus limiting their applicability and raising its cost. The main disadvantage of RPC relies on the nature of the solvent in which the purified protein is recovered, usually an organic solvent not suitable for human or animal use. Then, HIC constitutes a purification tool suitable for biomedical applications, such as vaccines, therapeutic proteins, plasmids and mainly antibodies. In addition, the use of chromatography in high-throughput studies, such as proteomics and protein interactions, is increasing. Some of these Biomedical Engineering applications of HIC are discussed below.

## **4.2.1 Antibodies purification**

At the beginning of the antibody industry, purification was performed through AC. For instance, protein A - AC was used for purifying monoclonal antibodies (MAbs), due to the

protein refolding constitutes an additional stage in the production and purification process in order to yield a "functional" product (especially in the case of enzymes). Hydrophobic interaction chromatography has been used to study thermodynamics aspects of protein folding. For instance, Geng et al. (2005) performed calorimetric determinations on the enthalpy change (ΔHfolding) of denatured lysozyme during its adsorption to a hydrophobic surface, with the simultaneous protein refolding. The surface consisted of PEG-600 made of a silica base HP-HIC (High Performance- Hydrophobic Interaction Chromatography) packing. At 25°C, ΔHfolding was found to be - 34 439 KJ/mol, involving adsorption,

Later, Geng & Wang (2007) used the concept of "Protein Folding Liquid Chromatography" (PFLC), to describe a chromatographic process aiming to either raise the efficiency, or shortening the time of protein folding. Besides, an optimal PFLC should be able to simultaneously remove denaturant substances, separate contaminant proteins, promote refolding of the target protein, and ease denaturant recovery. Any type of chromatography can be used in PFLC, mainly Size Exclusion Chromatography, Ion Exchange Chromatography,

In HIC, the process is governed by thermodynamic equilibrium and so does the protein folding. PFLC provides the chemical equilibrium that favors the conversion from aggregate to desorbed protein, resulting in a higher refolding efficiency and shorter refolding time. The unfolded proteins, at a high ionic strength, are driven by hydrophobic interactions from the mobile phase to the HIC stationary phase, and the hydrophobic patches on the proteins surface get attached to the hydrophobic ligands, while the hydrophilic zones of the unfolded molecules remain in contact with the solvent. As a consequence, unfolded molecules are not able to aggregate. The unfolded molecules desorb from the HIC support as ionic strength in the mobile phase decreases. Protein molecules with incorrectly folded domains would be corrected by the spontaneous disappearance of the domains in the mobile phase due to their thermodynamic instability. After many HIC runs, the incorrectly folded domains will decrease, while the correctly folded molecules will predominate, resulting in protein

Biomedical applications of HIC are broad, since this technique offers some advantages over other chromatographic techniques, such as Affinity Chromatography (AC) and Reverse-Phase Chromatography (RPC). The use of AC depends on the availability of a specific ligand for the protein or group of proteins to be separated, thus limiting their applicability and raising its cost. The main disadvantage of RPC relies on the nature of the solvent in which the purified protein is recovered, usually an organic solvent not suitable for human or animal use. Then, HIC constitutes a purification tool suitable for biomedical applications, such as vaccines, therapeutic proteins, plasmids and mainly antibodies. In addition, the use of chromatography in high-throughput studies, such as proteomics and protein interactions, is increasing. Some of these Biomedical Engineering applications of HIC are discussed

At the beginning of the antibody industry, purification was performed through AC. For instance, protein A - AC was used for purifying monoclonal antibodies (MAbs), due to the

dehydration and molecular conformation enthalpies changes.

refolding at high efficiency.

**4.2.1 Antibodies purification** 

below.

**4.2 Applications in biomedical engineering** 

Affinity Chromatography, and Hydrophobic Interaction Chromatography.

extremely low MAb concentration in the initial solution (fermentation broth), and the high amount of contaminant proteins. Therefore, affinity chromatography was the most suitable technique, given its high selectivity and resolution. Unfortunately, this purification technique has a serious disadvantage given by the high affinity of the MAb for the ligand (such as protein A), making it difficult to release the MAb from the ligand, with the consequent economical detriment. Moreover, MAbs are highly hydrophobic macromolecules, and then the use of HIC has been suggested (Asenjo & Andrews, 2008). At the present time molecular biology advances have enabled reaching high concentrations of MAbs in the fermentation broth, making it possible to use less selective but cheaper purification techniques, such as HIC. Figure 6 depicts a monoclonal antibody (A) and the antibody attached to a HIC stationary phase (B).

Fig. 6. (A) Schematic representation of a MAb. The antigen binding sites of the MAb are highlighted. Since this zone is characterized by an extremely high hydrophobicity, MAbs exhibit a high attraction for the hydrophobic ligands used in HIC resins. (B) Schematic representation of MAbs attached to a HIC resin. The antigen binding site interacts directly with the hydrophobic ligands of the HIC resin.

HIC is used as a polishing step in the purification processes of immunoglobulin-related products, since it has the ability to remove aggregated forms of the antibody (Rinderknecht & Zapata, 2006). Despite the high resolution offered by HIC, there are some drawbacks for its use in MAbs purification, given by the relatively low binding capacity of HIC supports and the consequent low yield in MAb recovery, compared to AC. Besides, MAb elution is usually achieved at a relatively high salt concentration, which implies that the solution containing the purified MAb also contains a high amount of salt that hinders sample manipulation and transitions during large-scale production.

This has encouraged research on HIC optimization, mainly regarding chromatographic supports. Recently, Chen et al., (2008) showed that the optimization of pore size of a HIC support significantly improved Immunoglobulin G binding capacity and also increased HIC efficiency, maintaining the MAb stability. Optimizing pore size facilitates mass transfer from mobile phase bulk towards the hydrophobic ligand. Kostareva et al. (2008) purified a heteropolymer (a kind of MAb consisting of a dual antibody conjugate) by HIC. They found that using a Propyl-HIC resin the heteropolymer was efficiently separated from free MAbs, thus confirming the ability of HIC for separating aggregates from monomers, and also its suitability for purifying MAbs.
