Protein adsorption isotherm behavior in hydrophobic interaction chromatography

Journal of Chromatography A, 1165 (2007) 67–77

Protein adsorption isotherm behavior in hydrophobic interaction chromatography Jie Chen, Steven M. Cramer ∗ Department of Chemical and Biological Engineering, RPI, NY 12180, USA Received 19 April 2007; received in revised form 18 July 2007; accepted 20 July 2007 Available online 24 July 2007

Abstract The adsorption behavior of proteins in hydrophobic interaction chromatography (HIC) was evaluated by determining the isotherms of a wide range of proteins on various HIC resin systems. Parallel batch experiments were carried out with eleven proteins on three hydrophobic resins with different ligand chemistries and densities. The effects of salt concentration, resin chemistry and protein properties on the isotherms were also examined. The resulting isotherms exhibited unique patterns of adsorption behaviors. For certain protein-resin combinations, a “critical salt behavior” was observed where the amount of protein bound to the resin increased significantly above this salt concentration. Proteins that exhibited this behavior tended to be relatively large with more solvent accessible hydrophobic surface area. Further, calculations indicated that under these conditions the occupied surface area of the adsorbed protein layer could exceed the accessible surface area. The establishment of unique classes of adsorption behavior may shed light on our understanding of the behavior of proteins in HIC systems. © 2007 Elsevier B.V. All rights reserved. Keywords: Hydrophobic interaction chromatography (HIC); Adsorption isotherm; Parallel batch experiment

1. Introduction Hydrophobic interaction chromatography (HIC) has been shown to have significant utility for the separation of proteins from complex mixtures [1–7]. There have been significant efforts towards understanding the mechanism of protein retention in HIC systems [4,8–22]. The solvophobic theory [8] is based on the association and solvation of the participating species and relates molal surface tension increment of the salt to retention [10,17,18]. Fausnaugh and Regnier [19] studied the adsorption of several proteins in the presence of different types of salt and found that the solvophobic theory alone could not adequately explain retention differences. The preferential interaction theory [11] has been shown to successfully capture salt type effects [14,21] and has been applied to study salt type effects on solute binding and selectivity [22] as well as the effects of pH [20]. Jungbauer and co-workers [4] have evaluated the effect of resin hydrophobicity on selectivity for several model proteins. ∗

Corresponding author. Tel.: +1 518 276 6198; fax: +1 518 276 4030. E-mail addresses: [email protected] (J. Chen), [email protected] (S.M. Cramer). 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.07.038

Van’t Hoff plots have been extensively employed to study protein adsorption in HIC systems (primarily by Hearn and coworkers) and the results have been employed to determine when conformational changes may take place [15,23–26]. Although HIC involves mildly hydrophobic surfaces [27], protein denaturation can still be an issue [28]. Fernandez and co-workers [29,30] have studied the effects of mobile phase, stationary phase and loading on protein conformation in HIC systems using hydrogen-deuterium exchange and mass spectrometry (HXMS). Protein properties, such as the quantity of aromatic amino acid residues, have also been used to explain resin selectivity in HIC systems [31]. In this paper, protein adsorption isotherms are investigated using parallel batch experiments with a variety of proteins on HIC resins with different ligand chemistries and densities. The effects of salt concentration, resin chemistry and protein properties on protein adsorption are evaluated. The resulting isotherms are classified into different categories based on their adsorption behavior. The correlation of “critical salt behavior” with protein size and solvent accessible hydrophobic surface area is examined. Finally, calculations on occupied surface area of the adsorbed protein layer and accessible surface area are carried out.

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2. Experiment

3. Results and discussion

2.1. Materials

Protein adsorption isotherms under a range of salt concentrations were investigated using parallel batch experiments with eleven proteins on three HIC resins as described in the experimental section. The resulting isotherms for these proteins on these three resins are presented in Figs. 1–11. As expected, the amount of protein bound to the resin increased with increasing salt concentration (especially at higher salt concentrations) in all

Bulk resin (e.g. butyl sepharose, low-sub Phenyl Sepharose 6 Fast Flow and high-sub Phenyl Sepharose 6 Fast Flow) were purchased from GE Healthcare (Piscataway, NJ, USA). Sodium phosphate (monobasic), sodium phosphate (dibasic), ammonium sulfate, sodium nitrate and blue dextran (Mw = 2,000,000) were purchased from Sigma (St. Louis, MO, USA). The following proteins were purchased from Sigma (St. Louis, MO, USA): ribonuclease A (bovine pancreas), trypsinogen (bovine pancreas), human serum albumin (HSA), ␤-lactoglobulin B (bovine milk), lysozyme (chicken egg white), ␣-chymotrypsin (bovine pancreas), protease carlsberg (bacillus licheniformis), ␣-lactalbumin (bovine milk), cellulase (Trichoderma reesei), catalase (bovine liver) and lectin (Arachis hypogaea, peanut) (note: protein purity by gradient HIC analysis was a prerequisite for including these proteins in our data set). 2.2. Apparatus Batch protein adsorption isotherms were determined using a 96-well membrane plate system (Millipore, Billerica, MA, USA). Protein concentrations were scanned using a Model HTS 7000 plate reader (Perkin-Elmer, Wellesley, MA, USA). A Fisher Vortex Genie 2 mixer (Fisher Scientific, Houston, TX, USA) was employed to assure that each plate was well mixed. 2.3. Procedures Eleven proteins and three resins (butyl sepharose, low-sub phenyl sepharose and high-sub phenyl sepharose) were utilized to obtain protein adsorption isotherms under different salt conditions. All of the resins were first washed with de-ionized water and then the carrier buffer, 25 mM sodium phosphate pH 7.0, containing different salt concentrations, ranging from 0.5 to 1.5 M ammonium sulfate. A 50% (v/v) resin slurry was prepared and 50 ␮l aliquots of the resin slurry were added in parallel to a 96-well membrane plate. The supernatant was removed by vacuum and 120 ␮l of protein solution in the same buffer was then added to the resin in each well. The initial protein concentrations used in this work were 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 7.0 and 10.0 mg/ml. The batch adsorption reactors were continually mixed using a vortex mixer and after 6 h incubation at 20 ◦ C, the supernatants were removed and the protein concentrations were analyzed using the plate reader at 280 nm with appropriate calibration curves for each protein (note: preliminary adsorption experiments were carried out at various times and the results indicated that after 6 h, the amount adsorbed did not measurably change). Protein adsorption on the stationary phase was then determined by mass balance. A control experiment was conducted by equilibrating 50 ␮l resin slurry with 120 ␮l of sodium nitrate and blue dextran to determine the dilution effect due to intraparticle and interstitial porosities. All experiments were performed in triplicate.

Fig. 1. Ribonuclease A isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

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Fig. 2. Typsinogen isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

of the resin systems. This is in agreement with many reports in the literature which have repeatedly confirmed that increasing kosmotropic salt concentration can enhance protein adsorption in HIC systems [14,32]. In addition, as can be seen from the figures, saturation of the isotherms was rarely achieved. While this result might be due to the limited range of protein concentrations employed in these experiments (due to solubility

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Fig. 3. Human serum albumin (HSA) isotherms. (a) Butyl sepharose; (b) lowsub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

constraints), similar results have been obtained by Pinto and coworkers [33,34]. In that paper, the authors concluded that the lack of saturation behavior at high concentrations was due to increasing repulsive interactions as surface converge increased and/or a change in conformation or orientation of the absorbed proteins. In addition to these expected trends in the isotherms, a wide range of adsorption behavior was exhibited by the proteins under different mobile phase and stationary phase conditions.

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Fig. 4. ␤-Lactoglobulin B isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

As seen in Fig. 1, ribonuclease A exhibited minimal binding to all three HIC resins. Furthermore, the effect of salt on protein binding was not very pronounced. This was the only protein that exhibited an initial decrease in protein binding followed by an eventual increase at higher salt concentrations for adsorption on low-sub phenyl sepharose in this study. This is to be expected for weakly hydrophobic proteins where electrostatic interactions may play an important role at low salt concentrations [35]. Trypsinogen exhibited moderate binding to all three HIC resins (Fig. 2). On the butyl and low-sub phenyl sepharose

Fig. 5. Lysozyme isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

(Fig. 2a and b) there was minimal binding at low salt concentrations. In fact, there was no measurable increase in binding when the salt concentration was increased from 0.5 to 1.0 M. At higher salt concentrations, protein binding started to increase gradually with the increasing salt concentration. This behavior corresponds to a system with weak hydrophobic interactions

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Fig. 7. Protease carlsberg isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M. Fig. 6. ␣-Chymotrypsin isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

between the proteins and the resin surface. Only when the salt is increased above a certain level is there any measurable protein adsorption. The observation that proteins only exhibited measurable binding to HIC resins above a minimal salt concentration has been reported previously [10,36]. This phenomenon might also be related with the electrostatic interactions that can

occur under low salt conditions as discussed above. In contrast to the behavior on the butyl and low-sub phenyl resins, trypsinogen exhibited stronger binding to the high-sub phenyl sepharose material (Fig. 2c) with the adsorption increasing continually as the salt concentration was increased from 0.5 to 1.4 M ammonium sulfate. This pattern of continual increase of protein adsorption with increasing salt concentration corresponds to a system with moderate hydrophobic interactions between the proteins and the resin surface. This conventional salt depen-

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Fig. 8. ␣-Lactalbumin isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.5 M.

dent adsorption isotherm will be classified as “Type 1” in this work. Human serum albumin, ␤-lactoglobulin B and lysozyme (Figs. 3–5) exhibited a similar adsorption pattern as trypsinogen on these resins. The salt concentration that resulted in measurable binding to both the butyl and low-sub phenyl

Fig. 9. Cellulase isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

sepharose materials was 1.0 and 0.8 M for ␤-lactoglobulin B and lysozyme, respectively. Interestingly, lysozyme exhibited quasilinear isotherms at higher salt concentrations. This might be due to the limited range of protein concentrations employed in some of these experiments (due to solubility constraints) resulting in only the linear part of the lysozyme isotherms being observed.

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Fig. 10. Catalase isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M.

While ␣-chymotrypsin required 0.8 M salt to have any measurable binding on butyl sepharose, this protein showed a continual increase in protein adsorption with salt concentration on low-sub and high-sub phenyl sepharose (Fig. 6). For protease carlsberg (Fig. 7), a continual increase with salt concentration was observed on all three HIC resins. Furthermore, the protease carlsberg isotherms at higher salt concentrations

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Fig. 11. Lectin isotherms. (a) Butyl sepharose; (b) low-sub phenyl sepharose; (c) high-sub phenyl sepharose () 0.5 M; () 0.8 M; () 1.0 M; (♦) 1.2 M; () 1.4 M. All experiments were carried out with 25 mM sodium phosphate pH7.0 buffer containing different concentration of ammonium sulfate at 20 ◦ C.

were also quasi-linear, which may again be due to the limited protein concentration range employed in these experiments. ␣-Lactalbumin exhibited typical salt dependent adsorption behavior on butyl and low-sub phenyl resins (Fig. 8a and b). However, on high-sub phenyl sepharose, it exhibited a more dramatic increase in protein adsorption when going from 1.0 to 1.2 or 1.5 M ammonium sulfate. We will define protein adsorp-

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tion patterns with moderate binding at lower salt concentrations and a more dramatic increase at higher salt concentrations as “Type 2” behavior. This “Type 2” behavior was also observed with cellulase and catalase on all of the resins (Figs. 9 and 10), with the significant increase in protein adsorption occurring at salt concentrations above 1.0 M ammonium sulfate. This dramatic increase in adsorption at higher salt concentrations may be due to elevated hydrophobic interactions and/or protein conformational changes which could result in the exposure of more hydrophobic surface area of the adsorbed proteins [24]. While lectin (Fig. 11) had quite low adsorption at lower salt values, this protein exhibited very high adsorption at elevated salt concentrations. For lectin, a dramatic increase in protein adsorption occurred at salt values above 1.0, 1.0, and 0.8 M on butyl, low-sub phenyl and high-sub phenyl sepharose, respectively. We will define adsorption patterns with low binding at lower salt concentrations and a dramatic increase in binding at higher salt concentrations as “Type 3” behavior. It is likely that the significant increase in protein binding at higher salt concentrations associated with “Type 3” behavior is related to partial unfolding of proteins occurring during the adsorption process in these systems [24]. 4. Resin effects on protein adsorption in HIC systems It is known that resin ligand type, density and backbone chemistry can play important roles in protein adsorption in HIC systems [4,37]. By comparing protein isotherms on butyl sepharose, low-sub phenyl sepharose and high-sub phenyl sepharose (Figs. 1–11), it was found that binding was significantly higher on high-sub phenyl sepharose, although many proteins showed similar affinity on butyl and low-sub phenyl sepharose. In general, for the same backbone and spacer arm chemistry, the aromatic ligand (e.g. phenyl) is expected to have higher affinity towards proteins than the alkyl ligand (e.g. butyl) because of the combination of hydrophobic and aromatic interactions between the aromatic ligand and the proteins (e.g. ␲–␲ interactions) [38,39]. In addition, it is well known that the higher the ligand density, the stronger the affinity for protein adsorption. In this study, the ligand density of butyl sepharose was

50 ␮mol/ml, while it was only 20 ␮mol/ml for low-sub phenyl sepharose. Thus, the similar behavior of butyl sepharose and low-sub phenyl sepharose is likely due to a combination of higher ligand density of weaker ligand groups and lower ligand density of the stronger hydrophobic ligands. In addition, with comparable ligand densities (50 ␮mol/ml for butyl sepharose and 40 ␮mol/ml for high-sub phenyl sepharose), protein binding appeared to be significantly higher on high-sub phenyl sepharose than on butyl sepharose, which agrees with a previous report [4]. 5. Protein properties effects on protein adsorption in HIC systems Table 1 summarizes the data for all of the evaluated proteins which includes isotherm class as well as the salt concentration where a significant increase in protein adsorption was observed above this concentration. Ribonuclease A exhibited minimal binding in these systems and only adsorbed when a more hydrophobic resin and higher salt concentrations were employed. Trypsinogen, HSA, ␤-lactoglobulin B, lysozyme, ␣chymotrypsin, protease carlsberg and ␣-lactalbumin exhibited qualitatively similar adsorption behavior (Figs. 2–8). Those proteins all exhibited “Type 1” behavior on all the three resins. Although ␣-lactalbumin exhibited “Type 1” behavior on butyl and low-sub phenyl sepahrose, on the more hydrophobic highsub phenyl sepharose, there was an significant enhancement of ␣-lactalbumin binding beyond 1.0 M ammonium sulfate (“Type 2”). Cellulase and catalase exhibited “Type 2” behavior on all three resin systems with a significant increase in protein adsorption at salt concentrations great than 1.0 M ammonium sulfate (Figs. 9 and 10). Lectin showed a dramatic effect of salt on adsorption behavior, exhibiting “Type 3” behavior in all three resins (Fig. 11). In order to provide more insight into this isotherm behavior, the effect of both protein hydrophobicity and size was examined. Protein surface hydrophobicities were determined by calculating the molecular descriptor “HydroSurfDE H1” which is defined as:  (hi ri ) (1) HydroSurfDE H1 =

Table 1 Protein classification result Proteins

Ribonuclease A Trypsinogen Human serum albumin ␤-Lactoglobulin B Lysozyme ␣-Chymotrypsin Protease carlsberg ␣-Lactalbumin Cellulase Catalase Lectin a

Butyl sepharose

Low-sub phenyl sepharose

High-sub phenyl sepharose

Isotherm class

Salt concentration (M)a

Isotherm class

Salt concentration (M)a

Isotherm class

Salt concentration (M)a

Low binding 1 1 1 1 1 1 1 2 2 3

N/A N/A N/A N/A N/A N/A N/A N/A 1.0 1.0 1.0

1 1 1 1 1 1 1 1 2 2 3

N/A N/A N/A N/A N/A N/A N/A N/A 1.0 1.0 1.0

1 1 1 1 1 1 1 2 2 2 3

N/A N/A N/A N/A N/A N/A N/A 1.0 1.0 1.0 0.8

The salt concentration that resulted in significant increase in protein binding on HIC resins.

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Table 2 Protein accessible surface area and occupied surface area on high-sub phenyl sepharose Protein name

PDB

Ribonuclease A Trypsinogen Human serum albumin ␤-Lactoglobulin B Lysozyme ␣-Chymotrypsin Protease carlsberg ␣-Lactalbumin Cellulase Catalase Lectin

1rbx 1tgb 1ao6 1b0o 1aki 5cha 1af4 1f6s 1cel 4blc 2pel

Weight (Da)

HydroSurfDE H1

Length ˚ (A)

Aac (m2 /ml)

13690 23985 66439 18271 14314 25207 27292 14174 45965 230404 100757

−3.20 12.58 2.11 13.44 7.53 18.73 18.75 11.28 25.59 61.94 50.45

42 52 93 45 48 52 51 50 67 113 91

37.38 36.07 32.86 36.74 37.28 35.94 35.73 37.31 34.17 26.83 31.15

Aocc (m2 /ml)

q (mg/ml)

r

0.5 M

1.0 M

1.4 M

0.5 M

1.0 M

1.4 M

0.5 M

1.0 M

1.4 M

9.53 5.63 13.13 7.44 9.68 11.33 12.76 7.78 28.32 39.69 10.93

17.58 6.06 22.89 15.02 18.51 26.28 28.16 12.49 40.69 69.09 68.68

37.54 5.86 11.71 3.00 38.51 8.02 28.00 3.96 33.04 7.47 41.21 5.84 60.67 5.69 31.15a 6.42 118.63 12.95 175.64 10.45 102.43 4.23

10.80 3.23 13.99 8.00 14.29 13.55 12.55 10.31 18.61 18.19 26.58

23.07 6.23 23.53 14.91 25.50 21.24 27.05 25.71a 54.27 46.25 39.64

0.2 0.1 0.2 0.1 0.2 0.2 0.2 0.2 0.4 0.4 0.1

0.3 0.1 0.4 0.2 0.4 0.4 0.4 0.3 0.5 0.7 0.9

0.6 0.2 0.7 0.4 0.7 0.6 0.8 0.7a 1.6 1.7 1.3

Aac : The available accessible surface area per milliliter of settled resin volume. Aocc : The occupied surface area per milliliter settled resin volume. a Value was determined under salt concentration of 1.5 M ammonium sulfate.

where hi is the hydrophobicity value assigned to amino acid i based on Hearn 1 scale [40], ri is the absolute number of equivalent amino acid i exposed to the protein surface: ri =

si asai

(2)

This hydrophobic descriptor has previously been shown to play an important role in describing protein retention times in HIC systems [37]. The crystal structures of eleven model proteins used in this study were downloaded from the RCSB Protein Data Bank (PDB) (www.rcsb.org) for analysis [41]. Table 2 shows the protein names, corresponding PDB identities, molecular weights and the value of HydroSurfDE H1 for each protein. As seen in the table, catalase, lectin and cellulase had the highest values of HydroSurfDE H1. In addition, these proteins were the largest proteins (with the exception of HSA). Previous reports have indicated that protein size can affect the extent of adsorption in HIC systems [42,43]. The larger protein size may result in a larger contact surface area between the resin and proteins and therefore enhanced hydrophobic interactions. It is also possible that larger proteins may be more flexible, increasing the probability of undergoing conformational changes which could result in increased hydrophobic interactions in HIC systems. These results indicate that both protein size and surface hydrophobicity (as defined by HydroSurfDE H1) may play an important role in determining which proteins will exhibit “Type 2” and “Type 3” isotherm behavior. Since protein size is an important factor influencing adsorption in HIC systems, the available resin accessible surface area for each protein per unit mobile phase volume (aac ) was calculated for the high-sub phenyl sepharose data. Based on the work by DePhillips and Lenhoff [44], Fogle et al. [29] determined that a reasonable approximation for (aac ) on high-sub phenyl sepharose can be made by taking the average values previously reported for SP sepharose and CM sepharose using appropriate molecular weight dextran probes. In our current work we used data obtained from dextran probes of the same intrinsic radius as the proteins, rather than the same molecular weight. The protein’s intrinsic radius were calculated by fitting

Eq. (3) [45]: Rη = 0.051 × MW0.378

(3)

The available accessible surface area per ml of settled resin volume, Aac , was then determined by correcting aac for the phase ratio (0.9 for the three resins in this study). While the intrinsic radius of the proteins was determined in order to obtain the correct value of the accessible surface area from previous dextran based results [44], it was also important to use the crystal structure data from the Protein Data Bank (PDB) to determine the protein coverage on the resin surface. Assuming that the adsorbed protein horizontally sweeps a circular area on the HIC resin surface with its long axis (L), the occupied surface area per molecule (aocc ) can then be determined by the following: aocc =

NA · π · L2 4000 · Mw

(4)

where NA is Avogadro’s number (6.022 × 1023 ), L is approximate protein length and Mw is protein molecular weight (Da). Thus, the occupied surface area per ml of settled resin volume (Aocc ) at the highest protein adsorption observed from each isotherm experiment (q) can be determined from Eq. (5) and the coverage (r) can be determined by Eq. (6). Aocc = r=

q · NA · π · L 2 4000 · Mw

Aocc Aac

(5) (6)

Table 2 lists the accessible surface area (Aac ), occupied surface area (Aocc ) and coverage (r) of each protein on high-sub phenyl sepharose with 0.5, 1.0 and 1.4 M ammonium sulfate. While most of the proteins had coverage less than 1, cellulase, catalase and lectin exhibited surface coverages that exceeded 1 at sufficiently high salt concentrations. There are several possible explanations for this observation. If proteins undergo conformational changes or partial unfolding, it may be possible for the protein to adopt a more compact conformation under these conditions [24,29,46]. In addition, partial unfolding will result

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in the exposure of more hydrophobic surface area, leading to stronger interactions with the resin and higher coverage. In the calculation of surface coverage, it was assumed that the adsorbed protein horizontally sweeps a circular area on the HIC resin surface with its long axis (L). However, proteins might undergo changes in their orientation upon adsorption at high loadings. Several authors have indicated that a vertically aligned adsorption pattern can result in increased protein adsorbed on these surfaces [47–49]. Clearly, if there is a change in orientation the coverage r could be higher than 1 since it is based on the assumption of a horizontal orientation. An alternative coverage based on the assumption that the protein sweeps a circular area with its short axis (W) was also calculated for cellulase, catalase and lectin on high-sub phenyl sepharose at 1.4 M ammonium sulfate. The resulting values were 1.0, 1.4 and 1.1, respectively. Therefore, reorientation of adsorbed protein on the HIC resin may play a role in the elevated protein coverages achieved with these proteins at high salt conditions. Another possible explanation, could be that attractive proteinprotein interaction are present in these systems, leading to multilayer adsorption on the HIC surfaces [34]. In the current study, some of the protein coverage values are significantly higher than 1. For example, the coverage for catalase on highsub phenyl sepharose at 1.4 M ammonium sulfate is 1.7, which is greater than that expected for a single protein adsorption layer. It is important to note that these coverage values were obtained at the highest protein adsorption levels determined in these experiments and that even higher values may be attainable. Protein self-association at low salt concentrations (e.g. 1 M NaCl) [50] has been shown to form oligomers. In fact, some of the proteins in the current study (e.g. lectin and catalase) are oligomers. The accessible surface area for these proteins was calculated using the oligomer molecular weights. Thus, the relatively low binding of lectin at low salt concentrations in this study may in fact be a result of the limited accessible surface area for these proteins. At higher salt, it is possible that the adsorbed oligomers might dissociate due to the salt denaturation effect, resulting in higher adsorption due to an increased effective accessible surface area for these lower molecular weight species. Furthermore, more hydrophobic surface area may become available for the dissociated proteins, resulting in increased hydrophobic interactions of the proteins to the HIC surfaces. It is also possible that several of these mechanisms could be occurring simultaneously. Clearly, these explanations are purely conjecture at this point and future work will be required to elucidate these mechanisms. 6. Conclusions The effects of salt concentration, resin chemistry and protein properties on protein adsorption were evaluated using parallel batch HIC adsorption experiments. As expected, protein adsorption increased at higher salt concentrations. The resin chemistry effect was also investigated and the results indicated that resins containing phenyl ligands showed higher affinity than the butyl resins due to a combination of hydrophobic and aromatic interactions (e.g. ␲–␲ interactions) of the phenyl ligand to the proteins.

The resulting isotherms exhibited unique patterns of adsorption behaviors. For certain protein-resin combinations, the amount of protein bound to the resin increased significantly above a certain salt concentration. Proteins that exhibited this behavior tended to be relatively large with more solvent accessible hydrophobic surface area. Furthermore, it is probable that protein conformational changes upon binding may also play a role in this type of salt dependent adsorption behavior. Interestingly, the data also indicates that some proteins exhibited surface coverages exceeding monolayer coverage. Several possible explanations for this include conformational changes, reorientation on the surface, multilayer adsorption, and/or oligomer dissociation upon binding in HIC systems. Clearly, the data presented in this paper open up several potential avenues of future investigations. Future work will focus on investigations of the behavior of proteins under the conditions established in the current work using a range of experimental techniques (e.g. circular dichroism, Raman, amide hydrogendeuterium exchange and mass spectrometry). Acknowledgements The authors would like to thank Abigail Provine (Rensselaer Polytechnic Institute, Troy, NY, USA) for help on the isotherm determinations and Qiong Luo and Curt M. Breneman (Rensselaer Polytechnic Institute, Troy, NY, USA) for the calculation of the protein hydrophobic descriptor. The authors would also like to thank Shekhar Garde (Rensselaer Polytechnic Institute, Troy, NY, USA) for helpful discussions related to this work. This work was funded by the Grant number BES-9810794 from the National Science Foundation. References [1] M.M. Diogo, J.A. Queiroz, G.A. Monteiro, S.A.M. Martins, G.N.M. Ferreira, D.M.F. Prazeres, Biotechnol. Bioeng. 68 (2000) 576. [2] M.M. Diogo, J.A. Queiroz, D.M.F. Prazeres, Bioseparation 10 (2001) 211. [3] H. Husi, M.D. Walkinshaw, J. Chromatogr. B 736 (1999) 77. [4] C. Machold, K. Deinhofer, R. Hahn, A. Jungbauer, J. Chromatogr. A 972 (2002) 3. [5] K.C. O’Connor, S. Ghatak, B.D. Stollar, Anal. Chem. 278 (2000) 239. [6] K. Pomazal, C. Prohaska, I. Steffan, J. Chromatogr. A 960 (2002) 143. [7] K.M. Sunasara, F. Xia, R.S. Gronke, S.M. Cramer, Biotechnol. Bioeng. 82 (2003) 330. [8] C. Horvath, W. Melander, I. Molnar, J. Chromatogr. 125 (1976) 129. [9] H.P. Jennissen, Hoppe-Seylers Zeitschrift Fur Physiologische Chemie 357 (1976) 265. [10] W. Melander, C. Horvath, Arch. Biochem. Biophys. 183 (1977) 200. [11] T. Arakawa, S.N. Timasheff, Biochemistry 23 (1984) 5912. [12] B.F. Roettger, J.A. Myers, M.R. Ladisch, F.E. Regnier, Biotechnol. Progr. 5 (1989) 79. [13] X.D. Geng, L.A. Guo, J.H. Chang, J. Chromatogr. 507 (1990) 1. [14] T.W. Perkins, D.S. Mak, T.W. Root, E.N. Lightfoot, J. Chromatogr. A 766 (1997) 1. [15] F.Y. Lin, W.Y. Chen, M.T.W. Hearn, J. Mol. Recognit. 15 (2002) 55. [16] M.A.J. Chowdhury, R.I. Boysen, H. Ihara, M.T.W. Hearn, J. Phys. Chem. B 106 (2002) 11936. [17] W.R. Melander, D. Corradini, C. Horvath, J. Chromatogr. 317 (1984) 67. [18] W.R. Melander, Z.E. Rassi, C. Horvath, J. Chromatogr. 469 (1989) 3. [19] J.L. Fausnaugh, F.E. Regnier, J. Chromatogr. 359 (1986) 131. [20] F. Xia, D. Nagrath, S.M. Cramer, J. Chromatogr. A 1079 (2005) 229.

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