Adsorption and separation of proteins by a synthetic hydrotalcite

Colloids and Surfaces B: Biointerfaces 87 (2011) 217–225

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Adsorption and separation of proteins by a synthetic hydrotalcite Kathrin Ralla a,∗ , Ulrich Sohling b , Kirstin Suck b , Friederike Sander c , Cornelia Kasper c , Friedrich Ruf b , Thomas Scheper c a b c

Institut für Biotechnologie, FG Bioverfahrenstechnik, Technische Universität Berlin, Ackerstr aße 71-76, 13355 Berlin, Germany Süd-Chemie AG, Ostenrieder Str. 15, 85368 Moosburg, Germany Institut für Technische Chemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167 Hanover, Germany

a r t i c l e

i n f o

Article history: Received 22 February 2010 Received in revised form 11 May 2011 Accepted 12 May 2011 Available online 19 May 2011 Keywords: Hydrotalcite Layered double hydroxide Chromatography Separation Protein adsorption

a b s t r a c t In this study, the potential use of a synthetic Mg/Al hydrotalcite (layered double hydroxide) as a novel chromatography material for protein purification was investigated. The hydrotalcite is present in its carbonate form and is characterized by an Al/Mg-ratio of 1.85. Zetapotential measurements confirm a positive surface potential up to pH 10 suggesting applicability as anion exchanger. The binding of model proteins covering a broad range of isoelectric points and molecular weights was performed at different pH-values under batch conditions to evaluate the binding behaviour of the hydrotalcite. Furthermore, static binding capacities were exemplarily determined for hemoglobin and human serum albumin. Additionally, the adsorption and elution of hemoglobin was studied under dynamic conditions. The binding behaviour of the hydrotalcite was compared to commercially available anion exchangers and was found to be a function of pH, depending on the model protein. Variant adsorption behaviour is explained by further interactions like hydrogen bonds and by an unequal charge distribution over the protein surfaces. The hydrotalcite reveals high adsorption capacities under static (260 mg/g) as well as under dynamic conditions (88 mg/g at 34 cm/h; 61 mg/g at 340 cm/h). With appropriate buffers like 500 mM carbonate (pH 10) the adsorbed proteins can be nearly completely desorbed making regeneration possible. Due to the binding and elution properties it is concluded, that the hydrotalcite can serve anion exchange material for chromatographic protein separations. © 2011 Elsevier B.V. All rights reserved.

1. Introduction (Agro-) industrial by-products and raw materials are easily available in large amounts and do often contain proteins and enzymes with functionalities of potential industrial interest [1–3]. An intelligent downstream of those proteins offers the possibility to gain value from those natural sources. Among different isolation and fractionation methods for proteins from raw materials like whey or potato fruit juice, chromatographic approaches are the most promising ones, because methods like ultrafiltration or precipitation are often not readily amenable to commercial scaleup or to the isolation of tonne quantities [2]. In protein purification, ion exchangers are very commonly used as stationary phases. The sorbents are optimized towards selectivity and performance; furthermore, the regeneration, cleaning and sterilization after each separation are of relevance regarding product purity and lifetime of the sorbent. This requires high amounts of water, buffers or solvents. Furthermore, the sorbents have to be checked continuously concerning their performance or microbial contaminations [4]. This

∗ Corresponding author. E-mail address: [email protected] (K. Ralla). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.05.021

results in the fact that chromatographic purification methods are an important cost aspect of a whole production process. For the large-scale purification of proteins from industrial by-products, the utilization of cost intensive chromatographic processes is an economical difficulty. The benefit is not comparable to processes for proteins of for instance pharmaceutical interest. Low cost and/or disposable chromatographic systems reducing the costs for cleaning, inspection as well as lifetime and storage validations could be an interesting and economical alternative for the purification of proteins from industrial by-products. Important criteria for singleuse chromatography media are a reproducible high performance, low costs, and availability on large scale. High capacities and fast binding kinetics for the protein of interest or interfering contaminants are further relevant properties. Hydrotalcites, which are also referred to as anionic clays [5] exhibit a positive surface charge as well as exchangeable anions [6]. They are available on large scale and could due to their adsorption capacities for proteins serve as anion exchangers as cost-efficient alternative for commercially available anion exchange resins. It is known, that binding of enzymes to hydrotalcite like compounds leads to a negligible alternation of the secondary structure [7]. Hydrotalcites are naturally occurring compounds but can as well easily be synthesized [8,9]. All commercially relevant

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K. Ralla et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 217–225 Table 1 Molecular weights, isoelectric points (pI) and hydrodynamic radiuses (Stokes radiuses) of the model proteins calf alkaline phosphatase, bovine ␣-chymotrypsin, chicken lysozyme, human serum albumin, bovine hemoglobin and bovine trypsinogen.

Alkaline phosphatase ␣-Chymotrypsin Lysozyme Human serum albumin Hemoglobin Trypsinogen

Fig. 1. Schematic structure of hydrotalcites. (Adapted from [8]).

hydrotalcite-like compounds are synthetic [5]. There exist various synthesis procedures for hydrotalcite-like compounds including precipitation methods [10,11], hydrothermal processing [12], the urea method [13,14], sol–gel techniques [15] or exchange methods [16]. The composition of hydrotalcites can be described x+ x− by the general formula [MII1−x MIII [Xn− · nH2 O] . MII are x (OH)2 ] X/q divalent cations like Mg2+ whereas MIII are trivalent cations such as Al3+ . Xn− can be an organic or inorganic anion. The structure of hydrotalcites is similar to that of the mineral brucite (Mg(OH)2 ), where octahedra of Mg2+ (6-fold coordinated to OH− ) share edges to form infinite sheets. The sheets are stacked on top of each other and are held together by hydrogen bonds [17]. A schematic structure of hydrotalcites is given in Fig. 1. The divalent cations MII like Mg2+ can be partially substituted by trivalent cations MIII such as Al3+ , what results in positively charged hydroxyl sheets. This positive charge is balanced by exchangeable interlayer anions [10]. Furthermore, water molecules are present between the sheets. A very wide range of interlayer anions can be intercalated between the brucite-like sheets to compensate the positive charge. There is nearly no limitation to the kind of anions between the sheets [18]. Some examples are phosphates [19], OH− [20,21], (NO3 )− [6,22,23], (SO3 )2− and (SO4 )2− [24,25], (CO3 )2− and Cl− [6,26], Br− , I− or F− [6] as well as organic acids like adipic, oxalic, succinic, malonic, chlorocinnamic acid [27] and metallorganic complexes [28]. Moreover, the intercalation of biomolecules such as nucleic acids [29,30], or proteins [31,32] was reported. The activity of enzymes is remained after adsorption or intercalation [32–34]. Depending on the composition of the hydrotalcite-like compound and the nature of the protein, there exists the possibility for intercalation or adsorption at the outer surfaces of the hydrotalcite sheets. The intercalation of very large biomolecules like proteins is generally difficult [35,36]. Much less has been reported about the adsorption of biomolecules on hydrotalcites without intercalation. It is expected that intercalation of target molecules into the hydrotalcite is as less suitable for a successive recovery of target molecules as needed in bioseparation processes. This has been shown already by Vial et al. [37] where urease was immobilized on the chloride form of a Zn-hydrotalcite by a co-precipitation process and adsorption. Whereas the adsorbed urease could be desorbed, the urease intercalated between the sheets of the Zn-hydrotalcite could only be recovered to a low extent. The focus of our investigations was on the adsorption of proteins and enzymes on the outer surface of the hydrotalcite without or at least with negligible intercalation. For this purpose Mg/Al hydrotalcite in the carbonate form was chosen. The carbonate form of hydrotalcites is exceptionally stable because of a high affinity of carbonate to hydrotalcites [6,38]. Thus, other anions are difficult to be incorporated [39]. An intercalation of proteins leading to an irreversible adsorption that hinders the recovery of proteins is undesirable if the hydrotalcite is intended

MW (kDa)

pI

Stokes radius (Å)

140 25.3 14.3 66.4 64.5 23.9

6.0 8.1–8.6 10.5–11.0 4.9 6.8 9.3

77 [48] 20.0–22.0 [64] 14.6–19.5 [65–67] 31.5–35.6 [68,69] 24.0 [70] 19.6 [71]

for separation purposes. In this study, model proteins were employed with respect to their isoelectric points and molecular weights. The adsorption was investigated under static conditions concerning the dependence of the pH, and the desorption properties. Moreover, the adsorption was investigated under dynamic conditions and was compared to the adsorption of commercially available anion exchangers. In order to characterize the surface properties of the hydrotalcite, zeta potential measurements were performed as a function of the pH value and the porosity and specific surface area were characterized by nitrogen adsorption. 2. Materials and methods 2.1. Proteins, anion exchangers and ingredients for buffers The model proteins (alkaline phosphatase from calf intestinal mucosa (EC 3.1.3.1), bovine hemoglobin, human serum albumin, chicken lysozyme (EC 3.2.1.17), bovine ␣-chymotrypsin (EC 3.4.21.1) and trypsinogen (EC 3.4.21.4) from bovine pancreas) as well as chemicals for buffer preparation were purchased from Sigma–Aldrich Chemie GmbH, Steinheim, Germany and were of per analysis quality. All buffers and protein solutions were prepared in demineralized water (Arium, Sartorius AG, Göttingen, Germany). The molecular weights, the isoelectric points and the hydrodynamic radiuses of the model proteins are given in Table 1. The Süd-Chemie AG, Moosburg, Germany, provided the hydrotalcite with the commercial name Syntal 696. The commercial anion exchangers Macro-Prep Q® (hydrophilic macroporous polymeric beads functionalized with –(N+ (CH3 )3 ) and UNOsphere QTM (hydrophilic acrylamide-based beads functionalized with –(N+ (CH3 )3 ) were purchased from Bio-Rad Laboratories, Munich, Germany. 2.2. Characterization of Syntal 696 2.2.1. Particle size Dry sieve residue of the hydrotalcite was determined with commercially available metal sieves with mesh sizes of 25 ␮m, 45 ␮m and 63 ␮m and commercially available sieving equipment. Each sieve residue determination was performed with 25 g of the material. The calculation of the dry screening residue in percentage was as follows: actual weight of the residue on the sieve is multiplied with 100 and divided by the initial weight. Average particle size d50 of Syntal 696 was determined by laser diffraction using a Malvern Mastersizer, Malvern Instruments, Worcestershire, UK. Measurements were performed in air suing the dry powder feeder of the equipment. 2.2.2. Chemical composition The chemical composition of the hydrotalcite was determined by dissolving it in hydrochloric acid followed by ICP-AES measurements. In detail, 1 g sample was added to 10 mL concentrated hydrochloric acid in a 100 mL measuring beaker and the mixture

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was heated to boiling for 3 min. Then, 50 mL of distilled water were added and the mixture was again heated to boiling for further 5 min. Finally, distilled water was added to obtain a final volume of 100 mL. For the ICP-AES measurements, the acidic solution was diluted by a factor 10 with distilled water. Calibration of the ICP-AES measurements was performed with single element standards for Mg and Al. The results for the contents of Al and Mg were recalculated as MgO and Al2 O3 . 2.2.3. X-ray diffraction The crystallographic characterization was performed with X’pert Pro diffractometer from PANalytical using standard powder diffraction sample holder and standard measuring set-up with Cu K␣ -radiation: Assignment to the mineral phases was made using the X’pert Pro software, which is based on a comparison of the detected reflections with a data base based of reference diffraction patterns from the International Centre of Diffraction Data (ICDD). 2.2.4. Determination of the BET surface and the cumulative pore volume The specific surface and the pore volume were determined by the BET-method (single-point method using nitrogen, according to DIN 66131) with an automatic nitrogen-porosimeter (Micrometrics, type ASAP 2010). The pore volume was determined using the BJH-method [40]. Pore volumes of defined ranges of pore diameter were measured by summing up incremental pore volumes, which were determined from the adsorption isotherm according to BJH. The total pore volume refers to pores having a diameter of 2–300 nm. The measurements provide as additional parameters the micropore surface, the external surface and the micropore volume. 2.2.5. Determination of the zeta potential The electrophoretic mobilities of the hydrotalcite and the anion exchanger Macro-Prep Q® were determined with a Zetasizer HSA3000 (Malvern Instruments Ltd., Malvern, Worcestershire, UK) followed by a calculation of the zeta potential using the Helmholtz–Smoluchowski approximation [41–43]. All measurements were performed thrice. 2.3. pH-dependence of the protein adsorption under batch conditions The anion exchange materials (25 mg) were equilibrated by shaking in 5 mL of the adequate buffer (100 mM) for 30 min at room temperature. After centrifugation (10 min, 4000 g) the buffer was carefully discarded. The anion exchange materials were then resuspended in 5 mL buffered (50 mM) protein solution (1 mg/mL) and were gently shaken with the protein solution for 2 h at room temperature. The period of 2 h was found to be sufficient to attain the adsorption equilibrium from preliminary experiments. The protein concentration in the supernatant was determined subsequent to centrifugation (10 min, 4000 g) at 280 nm using a standard calibration. The calibrations were done separately for each protein at each pH. Each experiment was done thrice. Following buffers were applied: acetate for pH 5, MES for pH 6, HEPES for pH 7, TRIS for pH 8 and pH 9 and CAPS for pH 10.

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supernatants after adsorption were determined subsequent to centrifugation (10 min, 4000 g) at 280 nm using a standard calibration of hemoglobin in 50 mM TRIS at pH 9. Each experiment was done thrice. The amount of adsorbed hemoglobin was then calculated by the mass balance equation: adsorbed protein amount [mg/g] =

(C0 − CE )V W

C0 is the initial protein concentration [mg/mL], CE is the equilibrium protein concentration, W is the amount of the anion exchanger and V is the volume of the protein solution. From the adsorbed protein amount at the different equilibrium concentrations (q) the maximum capacity qmax can be estimated from the part of the isotherm, where the adsorbed amount stays constant with increasing concentration. 2.5. Batch elution Elution under batch conditions was exemplarily studied with hemoglobin. Approximately 140 mg/g hemoglobin were bound by the hydrotalcite (at pH 8 in 50 mM TRIS) after equilibration as described in Section 2.3. Subsequent to adsorption, the remaining hemoglobin concentration in the supernatant after centrifugation was determined at 280 nm as described before. The supernatant was then discarded and the hydrotalcite was resuspended in buffer for washing (50 mM TRIS, pH 8). After 30 min washing in binding buffer, the protein concentration is again determined in the supernatant after centrifugation at 280 nm. The hydrotalcite is then resuspended in different elution buffers (500 mM carbonate (pH 10), 50 mM phosphate (pH 12), 50 mM TRIS with 20% polyethylene glycol 200 (pH 8), 50 mM phosphate (pH 7), and 50 mM acetate (pH 5)). Desorption was allowed to proceed for 2 h and again the protein concentration was determined in the supernatant after adsorption at 280 nm using standard calibration of hemoglobin in each buffer used in this experimental setup. The amount of protein determined in the washing fraction was subtracted from the protein amount that is found after desorption assuming that the washing fraction contains protein, which is in the buffer around the wet hydrotalcite and not adsorbed on the surface. Each experiment was done thrice. 2.6. Binding and elution under dynamic conditions For determining binding and elution properties under dynamic conditions, 250 mg of the adsorbents were suspended in binding buffer (50 mM TRIS, pH 8). The suspended materials were pipetted into an empty column (Assy FPLC column 15 × 50 mm, Omnifit, Cambridge, UK). The column was closed with a plunger and linked to a pump (FPLC-System BioLogic DuoFlow, Biorad, UK). Flow rates between 16.8 and 339.6 cm/h were applied. While equilibrating the materials with 25 mL binding buffer, the plunger was tightened slowly. After equilibration, 25 mL buffered protein solution (1 mg/mL hemoglobin or human serum albumin, respectively in 50 mM TRIS, pH 8) were pumped through the column. The column was washed with 25 mL buffer and then 25 mL of elution buffer were passed through. Carbonate buffer (500 mM, pH 10) was chosen exemplarily for elution. The protein concentration in each flow fraction was determined at 280 nm again using a calibration of each protein separately. Each experiment was done thrice.

2.4. Adsorption isotherms 3. Results and discussion Isotherms were carried out for hemoglobin. 20 mg of the materials were equilibrated in 100 mM TRIS buffer (pH 9) as described in Section 2.3. The adsorption experiments were performed by incubating the equilibrated materials with a defined volume of the protein solution in 50 mM TRIS buffer (pH 9) with varying protein concentrations for 2 h. The protein concentrations in the

3.1. Characterization of Syntal 696 Important properties of Syntal 696 are summarized in Table 2 and Table 3. The material has an average pore diameter (BJH) of 24.6 nm. Although the composition, the synthesis route, and the

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Fig. 2. X-ray diffractogram of Syntal 696. A standard measuring set up with Cu K␣ -radiation was used. The detected reflections were compared with a database based of reference diffraction patterns from the International Centre of Diffraction Data (ICDD). Table 2 Physical properties of the synthetic hydrotalcite Syntal 696.

presence of the hydrotalcite phase Mg2 Al(OH)7 . In addition a small amount of Mg(OH)2 (Brucit) is present which was not quantified.

Chemical analysis Al2 O3 (wt.%) MgO (wt.%) Mole ratio Mg:Al Dry Sieve residue on 63 ␮m (wt.%) on 45 ␮m (wt.%) on 25 ␮m (wt.%)

3.2. The zeta potential of Syntal 696

20.8 33.8 1.85 5 6.5 25.5

preparation conditions influence the porosity of the hydrotalcite, the porosity data are shortly compared with literature values. Typical values of the specific surface area of hydrotalcites measured by the BET method are in a range from 20 to 85 m2 /g [8]. For Mg/Al hydrotalcites containing anions such as carbonate, chloride, or nitrate, the BET surface is mostly less than 100 m2 /g [9,44–47]. Comparable cumulative pore volumes are given in the literature with 0.5–0.6 [46]. Pore diameters (BJH) can be found in literature with 3–80 nm [46]. The pore diameter of Syntal 696 makes it suitable for protein separation, as the diameter of the pores should be several factors above the diameter of the protein to be adsorbed or separated. Stokes radiuses (hydrodynamic radiuses) of proteins with high molecular weights (140 kDa) like alkaline phosphatase are around 77 A˚ [48]. Within this investigation model proteins and enzymes are employed with stokes radius up to 35.6 A˚ (see Table 1). Hence, the pores of Syntal 696 should be large enough for the adsorption of the chosen proteins in the pores. Fig. 2 shows the X-ray diffractogram of Syntal 696. Reference patterns prove the

The most important factor affecting the zeta potential is the pH of the solution, which stays in contact with the particles. It also depends on the properties and the concentration of the electrolyte. The zeta potentials of Syntal 696 and the commercially available anion exchanger Macro-Prep Q® were measured in dependence of the pH. The values are given in Fig. 3. The strong anion exchanger Macro-Prep Q® shows a zeta potential, which is nearly constant up to pH 11 between 20 and 30 mV. This is attributed to the highly ionized quaternary ammonium groups. In comparison, the zeta potential of the hydrotalcite is stronger influenced by the pH reaching the point of zero charge at pH 11. Nevertheless, the hydrotalcite reveals a higher zeta potential than Macro-Prep Q® up to pH 9. From

Table 3 Comparison of physical properties of the anion exchangers Syntal 696, Macro-Prep Q® and UNOsphere QTM . Macro-Prep Q®

UNOsphere QTM

Syntal 696

Mean particle size (␮m)

50

120

Pore size (Å) Surface area (m2 /g) Pore volume (cm3 /g)

1000–1200 18–22 0.5–0.7

1000 n.s. n.s.

8 (laser diffraction) 246 61.4 0.48

Fig. 3. Zeta potential of the hydrotalcite Syntal 696 and the commercially available anion exchanger Macro-Prep Q® between pH 6 and 12. The zeta potential was recalculated from the electrophoretic mobility using the Helmholtz–Smoluchowski approximation. The data points are mean values from triple determinations with standard deviations.

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Fig. 4. pH-dependence of bovine hemoglobin; HEM (A) and human serum albumin; HSA (B) binding by the commercially available anion exchangers UNOsphere QTM and Macro-Prep Q® and by the hydrotalcite Syntal 696. The data points are mean values from triple determination with standard deviations. Binding conditions: 50 mM buffer (pH 5: acetate, pH 6 and 7: MES and pH 8 and 9: TRIS); amount of adsorbent: 25 mg; initial protein concentration: 1 mg/mL; volume: 5 mL; binding time 2 h.

the zeta potential as a function of the pH it could be expected that hydrotalcite could serve as a “weak anion exchanger” in the view of that the charges on the surface varies with the pH. This is comparable to the variation of the ionization state of functional groups with the pH in commercially available weak ion exchangers. The hydrotalcite Syntal 696 should be capable of binding negatively charged biomolecules in the pH range from 5 to 10. 3.3. Adsorption of model proteins and enzymes by Syntal 696 Fig. 4 shows the adsorption of hemoglobin and human serum albumin by the hydrotalcite in comparison to the commercially available anion exchangers Macro-Prep Q® and UNOsphere QTM . Because hydrotalcite is unstable in an acidic solution [19], protein binding was examined in a pH range starting from pH 5. Above the isoelectric point (pI) of the two model proteins the binding capacity increases (proteins are negatively charged). The binding behaviour of the synthetic hydrotalcite follows the behaviour of the commercially available anion exchangers. This adsorption behaviour indicates that the interactions between the proteins and the hydrotalcite are mainly of electrostatic nature and that the hydrotalcite behaves like an anion exchange material. In an idealized picture, it is frequently assumed that an anionic protein should only adsorb to a cationic surface at pH values above its isoelectric point. However, the charged groups are not equally distributed over the surface of the protein molecules. Consequently, electrostatic interactions can still take place, even if the net charge of the surface and the protein are the same [49,50]. This is an explanation for the adsorption behaviour of the three materials in Fig. 4 adsorbing proteins at pH values close to and below their pI. The adsorption of hemoglobin by Macro-Prep Q® shows a more ideal behaviour around the pI than by UNOsphere QTM . On the other hand, UNOsphere QTM shows the highest adsorption capacity for human serum albumin. For hemoglobin, the hydrotalcite reveals the highest adsorption capacity at pH 5 to 8, but still significant adsorption in the pH region close to the pI. In the case of UNOsphere QTM and Macro-Prep Q®

a stronger decrease of adsorption with decreasing pH and a much lower adsorption close to the pI is observed. Differences between the three adsorbents might be explained by a different charge distribution on the surfaces of the adsorbents in relation to the charge distribution on the protein’s surface, as well by additional contribution of other binding forces like hydrogen bridges or van der Waals interactions [49]. As additional model proteins/enzymes ␣-chymotrypsin, lysozyme, trypsinogen and alkaline phosphatase were employed to characterize the adsorption behaviour of the hydrotalcite (see Fig. 5). Maximum 10 mg lysozyme is adsorbed by 1 g of hydrotalcite in the range of pH 5–10. This low adsorption compared to the other proteins can be contributed to unspecific interaction as the examined pH range is below its pI. The adsorbed amounts of ␣-chymotrypsin and trypsinogen are very low at pH 5 with a strong increase starting at pH 6. Taking the pI of the two latter enzymes into consideration, the adsorption behaviour for both cannot only be explained by electrostatic interaction. Hydrophobic interactions and/or hydrogen bonds may additionally be responsible for adsorption. The adsorption can also be attributed to unequal charge distribution over the protein surfaces. Furthermore it is known, that proteins with pI of 6–8 often tend to dissociate from anion exchangers at higher pH than their pI because of an inherent flat nature of the protein titration curve from pH 6 to pH 9 [51]. 3.4. Adsorption isotherms The effect of increasing concentration of hemoglobin on the adsorbed amount was investigated to estimate the maximum static adsorption capacities for Syntal 696, UNOsphere QTM and Macro-Prep Q® . The adsorption isotherms were performed at pH 9 in 50 mM TRIS buffer. The experimental data were fitted to the Langmuir model and to the Freundlich model. The Langmuir and the Freundlich model can generally be used to describe adsorption processes of proteins [52–54] with the constraint that the Langmuir isotherm as the simplest model cannot describe all phenomena occurring during protein adsorption [55]. For the Langmuir

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isotherm, following assumptions are important: The adsorbate is adsorbed in a mono layer, all surface sites are energetically equivalent, and that the surface is homogeneous. Furthermore, no interactions between adjacent adsorption sites or adsorbed molecules do occur. The Langmuir equation is given by: q=

K qmax Ceq 1 + K Ceq

The Freundlich isotherm can also be used to model adsorption processes on heterogeneous surfaces. Because of the exponential character of the Freundlich isotherm it is not possible to represent a maximum adsorption on surfaces [55,56]. The Freundlich equation is given by: m q = K Ceq

Fig. 5. pH-dependence of binding of ␣-chymotrypsin, trypsinogen, lysozyme and alkaline phosphatase by the synthetic hydrotalcite Syntal 696. The data points are mean values from triple determination with standard deviations. Binding conditions: 50 mM buffer; (pH 5: acetate, pH 6 and 7: MES, pH 8 and 9: TRIS, and pH 10: CAPS); amount of adsorbent: 25 mg; initial protein concentration: 1 mg/mL; volume: 5 mL; binding time 2 h. The arrows mark the pI of the proteins.

Fig. 6A (Langmuir) and Fig. 6B (Freundlich) exemplarily show the adsorption of hemoglobin fitted with the Langmuir equation and the Freundlich equation, respectively, whereby a correlation of the measured data to both equations can be noticed. The regression coefficients R2 are in a range between 0.9953 and 0.9178. Within the experimental errors it is not possible to differentiate which model describes the data more accurately. The agreement of the experimental data especially with the Freundlich isotherm in the case of the synthetic hydrotalcite suggests the existence of different adsorption energies on the surface [54,57,58]. It may be that protein adsorption takes place in a monolayer [54] but also multilayer adsorption may be conceivable [59]. Nevertheless, the Langmuir model was applied to calculate maximum equilibrium adsorption capacities to enable a comparison of the three materials. For UNOsphere QTM a qmax value of 528.5 mg/g, for Macro-Prep Q® a qmax of 446.2 mg/g and for Syntal 696 a maximum value of 260.0 mg/g was calculated. The adsorption capacity of the synthetic hydrotalcite in the batch system is approximately as half as high than the capacities of the commercially available anion exchangers. An explanation for the differences in adsorption capacities may be a better pore

Fig. 6. Adsorption isotherms with standard deviations of hemoglobin for the synthetic hydrotalcite Syntal 696 and the commercially available anion exchangers UNOsphere QTM and Macro-Prep Q® fitted to the Langmuir (A) and to the Freundlich (B) model. The isotherms were performed in 50 mM TRIS buffer at pH 9. Binding conditions: amount of adsorbent: 20 mg; reaction volume: 5 mL; binding time 2 h. The amount of adsorbed protein was calculated from the remaining protein concentration in the supernatant after the binding and centrifugation of the adsorbent using the mass balance equation. The data points are mean values from triple determination with standard deviations.

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Fig. 7. Batch elution of hemoglobin bound to the hydrotalcite Syntal 696. The data points are mean values from triple determination with standard deviations. Binding of hemoglobin was done in 50 mM TRIS (pH 8) for 1 h, whereby approx. 140 mg/g hemoglobin was bound to the hydrotalcite. After a washing step (30 min in binding buffer), the hemoglobin was eluted using following buffers: 500 mM carbonate (pH 10), 50 mM phosphate (pH 12), 50 mM TRIS with 20% polyethylene glycol 200 (pH 8), 50 mM phosphate (pH 7), and 50 mM acetate (pH 5). The elution step was allowed to proceed for 2 h.

accessibility of the commercially available anion exchangers with ˚ The mean pore size of the synthetic pores as large as 1000–1200 A. ˚ Although the mean hydrotalcite is four times smaller (about 246 A). pore size of the hydrotalcite is large enough for protein adsorption, there are as well pores with smaller diameters with reduced access for proteins. 3.5. Batch elution The application of hydrotalcites as separation material requires the feasibility to elute bound biomolecules. Therefore, protein recovery was investigated under equilibrium conditions using bovine hemoglobin as model protein. The effect of a decrease of the pH to reduce electrostatic interactions between the protein molecules and the hydrotalcite surface was investigated (50 mM acetate, pH 5). Because of the fact, that carbonate reveals a high affinity to hydrotalcites composed of Al and Mg [6,38], high carbonate concentrations (0.5 M and 1.0 M at pH 10) were tested for capability of displacing bound protein molecules. Moreover, hydrotalcites expose an affinity for phosphate salts (PO4 3− ) [60]. Therefore, the effect of phosphate containing buffers (50 mM phosphate at pH 7 and pH 12) was investigated concerning desorption. Furthermore, the effect of polyethylene glycol (PEG 200) as buffer additive (20% (v/v) in 50 mM TRIS, pH 8) was tested. Polyethylene glycol could displace protein molecules from the hydrotalcite surface as suggested for protein desorption from zeolite surfaces [61,62]. The results of different desorption experiments are given in Fig. 7. It was observed, that carbonate is suitable for desorption of hemoglobin. With 500 mM carbonate around 52% of the bound protein was detected. With 1 M carbonate (pH 10) a similar value (53%; data not shown) was achieved. It is likely that in the case of carbonate a displacement of the hemoglobin molecules is responsible for desorption. As expected, the use of phosphate containing buffers as well leads to a release of hemoglobin. With 50 mM phosphate at pH 7 around 48% of the hemoglobin were desorbed and with 50 mM phosphate at pH 12 even 72% could be desorbed. The higher amount of desorbed protein at pH 12 compared to pH 7 can be attributed to the loss of positive charge of the hydrotalcite (see Fig. 3, zeta potential of Syntal 696) leading to a loss of electrostatic interactions combined with the loss of the ability to adsorb proteins by disturbing the hydrotalcite structure with adding the phosphate. A decrease of the pH beneath the isoelectric point of hemoglobin by applying 50 mM acetate (pH 5) results in a loss of electrostatic attraction redounding to desorption of hemoglobin. Polyethylene glycol 200 was as well suitable for desorption. Also glycerol at 20% (v/v) in 50 mM TRIS (pH 8) was capable of desorbing around 50% of bound hemoglobin (data not shown).

3.6. Binding capacities under dynamic conditions and elution In order to investigate the suitability for chromatographic separation of proteins a buffered protein solution was loaded on a column packed with the synthetic hydrotalcite. Velocities between 33.9 and 339.6 cm/h (1–10 mL/min) were applied to investigate the dynamic binding capacities. Bovine hemoglobin and human serum albumin were exemplarily chosen as model proteins. The binding behaviour of Syntal 696 was compared to the commercially available anion exchanger Macro-Prep Q® . Fig. 8 shows the amounts [mg/g] of the model proteins, hemoglobin and human serum albumin, that are bound by the anion exchangers when using different flow rates. Because of a shorter contact time between the protein and the anion exchangers, a decrease of the amount of bound protein can be observed with an increasing flow velocity. In the case of human serum albumin, it was observed that the dynamic binding capacity of the hydrotalcite is lower than the capacity of the commercially available anion exchanger Macro-Prep Q® at low velocities, but at higher velocities (250 cm/h) the capacity of the hydrotalcite overtakes the capacity of the commercially available anion exchanger. In the case of hemoglobin it was observed, that the dynamic adsorption capacities at different velocities are even higher than for the commercially available anion exchanger. These data are differing from the results of the batch adsorption experiments, where the adsorption capacities of the commercially available anion exchangers are approx. two times higher than for the synthetic hydrotalcite (cp. Section 3.4). This could be explained by the fact that the adsorption of proteins is strongly influenced by diffusion. In the equilibrium experiments, protein adsorption is allowed to proceed for 2 h. Thus, there should be enough time for the protein molecules to diffuse through the laminar layer and into the pores to reach maximum adsorption in the case of all the tested materials. When the adsorption time is limited, as for the dynamic adsorption, film diffusion and internal pore diffusion become more important for the adsorption capacity. The diffusion process limits the adsorption rate, what is especially the case for high porosity adsorbents like Macro-Prep Q® media which comprise a small particle diameter [63]. The synthetic hydrotalcite exhibits smaller pores resulting in a reduced diffusion path length, so the internal pore diffusion does not limit the adsorption as much as in the case of Macro-Prep Q® . Thus, the dynamic adsorption capacities for the different materials are more equal assuming that protein molecules in the case of Macro-Prep Q® do not have the time to reach full adsorption capacity. This explanation is supported by the fact that at higher flow rates (250 cm/h) the capacity of the synthetic hydrotalcite for hemoglobin overtakes the capacity of the commercially available anion exchanger, whereas at lower

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Fig. 8. Binding of model proteins (A: bovine hemoglobin; HEM and B: human serum albumin; HSA) by the commercially available anion exchanger Macro-Prep Q® and by the synthetic hydrotalcite Syntal 696 at different flow velocities between 16.8 and 339.6 cm/h. The proteins were bound at pH 8 in 50 mM TRIS. The experiments were carried out with 250 mg of the adsorbents and 25 mL of buffered protein solution with a concentration of 1 mg/mL in a 15 × 50 mm column. The data points are mean values from triple determination with standard deviations. 100 Binding Elution 80

60

q [mg/g]

velocities the Macro-Prep Q® provides higher dynamic capacities (see Fig. 8B) arising from a longer contact time of adsorbent and protein molecules allowing more protein molecules to diffuse into the large pores of Macro-Prep Q® an to get bound. The dynamic capacity of the synthetic hydrotalcite is similar to the capacity of the commercially available anion exchangers. This furthermore accounts for the hydrotalcite’s suitability to serve as adsorbent for protein separation processes. An important aspect of the suitability of a material as stationary phase is the reversibility of binding. As tested under static conditions, different buffer systems are suitable for protein elution. In the following 500 mM carbonate (pH 10) was applied to elute hemoglobin at different flow velocities with the Syntal 696 as stationary phase. As it was shown in the batch elution experiments, phosphate-containing eluents are most suitable for protein desorption. However, phosphate salts modify the hydrotalcite structure and thereby cause all proteins to desorb. This would be a rather unspecific way to elute different adsorbed proteins. In addition, the use of acidic buffers for elution could result in partial dissolving releasing different adsorbed proteins at once and not specifically. Therefore, carbonate was chosen for elution assuming that due to the high affinity of carbonate to Mg/Al hydrotalcites different proteins could be eluted by displacement depending on the affinity of the distinct protein. The results are shown in Fig. 9. It was observed, that it is possible to recover bound hemoglobin with 500 mM carbonate (pH 10) up to 75% at 339.6 cm/h and up to 92% at 33.9 cm/h showing the suitability of this material that adsorbed proteins can also be recovered at a high percentage. The differences between the batch elution with 500 mM carbonate (52% recovery) and the column elution (up to 92% recovery at lower velocities) do arise because in the dynamic system proteins are mainly adsorbed at the outer surface in part of pores which are easy accessible. Therefore, also the elution can proceed easier. In contrast, in batch systems, proteins have more time to reach also adsorption sites deeper in

40

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0 50

100

150

200

250

300

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Velocity [cm/h] Fig. 9. Elution of hemoglobin using different velocities. Hemoglobin is loaded onto the column packed with Syntal 696 at the same velocity applied for elution. Binding: 50 mM TRIS, pH 8; elution: 500 mM carbonate, pH 10. The experiments were done with 250 mg of the hydrotalcite in a 15 × 50 mm column and 25 mL of buffered protein solution with a concentration of 1 mg/mL. The data points are mean values from triple determination with standard deviations.

long pores and a subsequent elution can be hindered because of effects like gradients in long pores. 4. Conclusions According to zeta potential measurements, the synthetic Mg/Al hydrotalcite Syntal 696 reveals a positively charged surface in a pH range from 5 to 10 what makes it appropriate to bind negatively charged biomolecules by the means of electrostatic interactions.

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The suitability of the synthetic Mg/Al hydrotalcite Syntal 696 for protein binding was demonstrated with different model proteins. Depending on the protein, the interactions with the hydrotalcite are mainly of electrostatic nature. As protein adsorption was also noticed below the pI, as well hydrogen bonds or van der Waals forces need to be considered to explain the adsorption behaviour of the model proteins. Carbonate as interlayer anion with a very high affinity to this type of hydrotalcite prevents protein intercalation into the interlayer space of the Mg/Al hydrotalcite used in this study, what enables a reversible binding of proteins. The largest amount of protein could be eluted when applying pH values beneath the protein’s isoelectric point to decrease electrostatic interactions between the protein and the hydrotalcite surface. In addition, carbonate or phosphate buffers were suitable for replacement of bound protein molecules, and buffer additives (20% (v/v) in 50 mM buffer) like polyethylene glycol or glycerol. Those different possibilities for elution allow a choice of the elution buffer, which is best regarding the requirements of the process or the protein of interest. Syntal 696 shows similar pressure behaviour when packed into columns like the commercially available anion exchangers. Thus, it is possible to use this material not only for batch binding, but also in columns without having problems with pressure drop. The dynamic binding capacity of the synthetic hydrotalcite is comparable to the capacity of a commercially available anion exchanger. It is possible to recover bound protein up to 90% under dynamic conditions, depending on the flow velocity. Due to the binding and elution properties it is concluded, that the synthetic hydrotalcite Synthal 696 is suitable as anion exchange material for chromatographic protein separations. Acknowledgement The data in this work were partly performed in the scope of the BMBF project BIOCATALYSIS 2021 P7/P8. The authors thank the BMBF for financial support as well as Dr. habil. Lars Dähne (Surflay Nanotec GmbH, Berlin, Germany) for kindly performing zeta potential measurements. References [1] S. Lokra, R. Schuller, B. Egelandsdal, B. Engebretsen, K. Straetkvern, LWT-Food Sci. Technol. 42 (2009) 906. [2] D. Chatterton, G. Smithers, P. Roupas, A. Brodkorb, Int. Dairy J. 16 (2006) 1229. [3] D. Knorr, J. Food Sci. 45 (1980) 1183. [4] E. Boschetti, J. Chromatogr. A 658 (1994) 207. [5] A. Burzlaff, S. Brethauer, C. Kasper, B. Jackisch, T. Scheper, Cytometry Part A 62A (2004) 65. [6] S. Miyata, Clays Clay Miner. 31 (1983) 305. [7] M. Ahn, A. Zimmerman, C. Martinez, D. Archibald, J. Bollag, J. Dec, Enzyme Microb. Technol. 41 (2007) 141. [8] F. Bergaya, B. Theng, G. Lagaly, Handbook of Clay Science, Elsevier, Amsterdam, 2006. [9] J. Inacio, C. Taviot-Gueho, C. Forano, J. Besse, Appl. Clay Sci. 18 (2001) 255. [10] J. Boclair, P. Braterman, Chem. Mater. 11 (1999) 298. [11] E. Crepaldi, P. Pavan, J. Valim, J. Braz. Chem. Soc. 11 (2000) 64. [12] F. Labajos, V. Rives, M. Ulibarri, J. Mater. Sci. 27 (1992) 1546. [13] J. Oh, S. Hwang, J. Choy, Solid State Ionics 151 (2002) 285. [14] M. Adachi-Pagano, C. Forano, J. Besse, J. Mater. Chem. 13 (2003) 1988. [15] T. Lopez, P. Bosch, E. Ramos, R. Gomez, O. Novaro, D. Acosta, F. Figueras, Langmuir 12 (1996) 189.

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