Article pubs.acs.org/Langmuir
Microcalorimetric Study of the Adsorption of PEGylated Lysozyme and PEG on a Mildly Hydrophobic Resin: Influence of Ammonium Sulfate Albert Werner, Tim Blaschke, and Hans Hasse* Laboratory of Engineering Thermodynamics, University of Kaiserslautern, Kaiserslautern, Germany ABSTRACT: Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl PPG-600M, a mildly hydrophobic resin is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements in sodium phosphate buffer at pH 7.0 and 25 °C. For PEGylation two different PEG sizes are used (5 and 10 kDa) which leads to six different forms of PEGylated lysozyme all of which are systematically studied. Additionally, the adsorption of five pure PEGs is explored. The ammonium sulfate concentration is varied from 600 to 1200 mM. The molar enthalpy of adsorption Δhads p is determined from the calorimetric and the adsorption equilibrium data. It is found to be endothermic in all experiments. The comparison of the adsorption of different PEGylated forms shows that the adsorption of PEGylated lysozyme is driven by the adsorption of the PEG chain. The results provide insight into the adsorption mechanisms of polymermodified proteins on hydrophobic chromatographic resins.
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INTRODUCTION PEGylation means a covalent binding of one or more polyethylene glycol (PEG) molecules to a protein. For the pharmaceutical industry, this chemical modification is of increasing interest. PEGylation modifies properties of proteins in order to improve, for example, in vivo half-life, chemical stability, immunogenicity and solubility. PEGylation chemistry and strategies, as well as their influence on protein behavior, have been described elsewhere.1−5 Upon PEGylation, different forms of PEGylated protein are usually obtained. They differ in the site to which the PEG is attached and in the number of sites that are PEGylated.5 The PEGylation reaction changes the hydrophobicity of the protein due to the fact that PEG has a different hydrophobicity than the protein itself. It has been shown that hydrophobic interaction chromatography (HIC) is a promising technique for the separation of PEGylated from unPEGylated protein. Further, separation of different PEGylation products is possible.6−8 However, process development of hydrophobic interaction chromatographic steps is very complex due to the many different process parameters like temperature, type of salt, ionic strength, pH, etc. There is a lack of fundamental understanding of the hydrophobic interactions between the proteins and the resins. Hence, the process design is often based on high throughput experiments.9 Blaschke et al.5,10 and Dieterle et al.11 have shown that calorimetric techniques can contribute to a better understanding of the retention mechanism in ion exchange chromatography. Further, it was shown that calorimetric techniques are helpful for understanding the hydrophobic interactions between proteins and hydrophobic resins.12−14 Ü berbacher et al.14 have illustrated © 2012 American Chemical Society
that hydrophobic interactions are not necessarily endothermic, as generally assumed. Lin et al.13 have shown that the adsorption enthalpy depends on ligand chain length and density. Blaschke et al.5 have studied the adsorption of PEGylated lysozyme on a strong cation exchange resin. In the present study this work is extended to a hydrophobic resin. The production process for PEGylated lysozyme was adopted from Moosmann et al.15 and adjusted to our needs. Lee and Park16 have shown that only three out of the six lysine residues of the lysozyme react in a considerable amount. Therefore, the following different forms of PEGylated lysozyme were obtained: mono-, di- and tri-PEGylated lysozyme for 5 and 10 kDa PEG each. For mono- and di-PEGylated lysozymes three isoforms are obtained, due to the three free lysine residues. As the first chromatographic step cannot separate the individual isoforms, each mix of isoforms is treated as one substance here. The adsorption of the substances (6 PEGylated lysozymes and native lysozyme) on Toyopearl PPG-600M, a mildly hydrophobic resin,17 was studied with microcalorimetric and adsorption equilibrium measurements. The polypropylene glycol (PPG) ligands of that adsorbent are similar to the structure of PEG. Additionally, adsorption and calorimetric measurements were carried out for 4, 5, 6, 12, and 18 kDa pure PEG. The molar enthalpies of adsorption, Δhads p , of different PEGylated lysozyme forms, native lysozyme, and pure PEG were determined. The experiments were carried out for ammonium sulfate concentration between 600 and 1200 mM. The molecular adsorption mechanism is discussed. Received: March 21, 2012 Published: July 25, 2012 11376
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desalting columns from GE-Healthcare (Uppsala, Sweden) in series. A 50 mM sodium phosphate buffer with an additional 150 mM sodium chloride was used at a flow rate of 1.5 mL/min and a detector temparature of 35 °C. For calibration, solutions with different PEG concentrations were prepared. The maximum PEG concentration for calibration was 2 mg/ mL. The injection volume was 50 μL. The determined area of the peak in the refractive index signal corresponding to the eluting PEG was correlated with mass of injected PEG. A linear relationship was found. The linear calibration factor was 5.0 mRIUs/mg for PEG-4 kDa and 5.1 mRIUs/mg for PEGs with chain lengths of 5, 6, 12, and 18 kDa. Isothermal Titration Calorimetry. The microcalorimetric measurements were carried out using a Thermal Activity Monitor 3 (TAM3) from TA-Instruments (Eschborn, Germany). The TAM3 was equipped with the ″Nanocalorimeter″ isothermal titration unit. The 1 mL sample ampule was filled with 50.9 μL of adsorbent using a MediaScout ResiQuot device (Atoll GmbH, Weingarten, Germany). Then, the resin was suspended in 600 μL of degassed buffer solution. The content was continuously stirred at 200 rpm with a propeller stirrer. Preliminary experiments showed that all resin particles are suspended in solution at this stirring rate. After thermal equilibration the degassed protein solution was titrated into the cell stepwise using a 250 μL glass syringe. The injections were driven by a computer controlled syringe pump. Twenty-five 10 μL injections were carried out in 40 min intervals, after which the baseline was reached again. The temperature fluctuations indicated by the instrument are below 0.00001 K. The error in the molar enthalpy of adsorption is difficult to assess. The repeatability of the calorimetric experiments is excellent as can be seen from the rather small scattering of the data around the trend lines in the figures for this work. There are, however, different sources of the systematic errors. The most important of these are (a) the integration of the calorimetric signal, which is not unambiguous, and (b) the determination of the amount of adsorbed protein from the adsorption isotherm. Also it is well-known, called “first peak anomaly” in literature, that the signal obtained in the first injection of an ITC run usually only gives unreliable results.18 Therefore these results are not shown in the figures below. They deviate from the trend lines typically by about 10%. As all data taken in this work were treated the same way, systematic errors should mostly cancel out when data points are compared, that is, all trends and comparisons of isotherms should be reliable. The absolute numbers of Δhads p are subject to an uncertainty which we estimate to be of the order of 20%, if the number is not close to zero.
MATERIALS AND EXPERIMENTAL METHODS
Chemicals. Hen egg white lysozyme (Mp = 14.3 kDa) was obtained from Sigma-Aldrich (St. Louis, MO, USA, L6876). MethoxyPEG-aldehyde was obtained in two average molecular masses (5 kDa and 10 kDa) from NOF Corp. (Tokyo, Japan). The hydrophobic resin Toyopearl PPG-600 M was obtained from Tosoh Bioscience GmbH (Stuttgart, Germany, lot 60PPGMB501F). All nonactivated polyethylene glycols (4, 6, 12, and 18 kDa) were obtained from Polymer Standard Service (Mainz, Germany). The salts used for the buffer preparation, namely monosodium phosphate (NaH2PO4·2H2O), disodium phosphate (Na2HPO4·2H2O) and ammonium sulfate ((NH4)2SO4) were obtained from Carl-Roth GmbH (Karlsruhe, Germany). All other chemicals were of analytical grade obtained from Carl-Roth GmbH (Karlsruhe, Germany). Production of PEGylated Lysozyme. The production process for the preparation of PEGylated lysozyme was adopted from Moosmann et al.15 and optimized for the present equipment. Process analytics were carried out exactly as described from Moosmann et al.15 The production process and the separation of the isoforms were described in detail recently by Blaschke et al.5 That reference also contains the chromatograms of the separation. Note that the separation was not carried out with a hydrophobic resin as the one that was studied in the present study, but rather with an ion-exchange resin, which was preferred as the separation protocol was already available.5 Adsorption Equilibrium Isotherms. Protein. For the adsorption experiment 50.9 μL of adsorber were filled into a 96 × 1.2 mL deepwell plate from Ritter GmbH (Schwabmünchen, Germany) using a MediaScout ResiQuot device (Atoll GmbH, Weingarten, Germany). All following steps were carried out on a fully automated modular laboratory robot station EVO200 from Tecan Group Ltd. (Maennedorf, Switzerland). A 500 μL aliquot of protein solution was added to every sample, mixed from a protein stock solution and the corresponding buffer. After 6 h of equilibration on a tempered shaker (shaking rate 1320 rpm) from Inheco GmbH (Martinsried, Germany) with a special plate adapter for better tempering, the samples were pipetted into a 0.22 μm AcroPrep 96-well filter plate from Pall Corporation (Port Washington, USA). After filtration into a 1.3 mL U96 DeepWell plate from Nalge Nunc International (Rochester, USA) using a microplate centrifuge from Agilent Technologies (Santa Clara, USA) the protein concentration in the liquid phase cp was determined by UV adsorption at 280 using an Infinite M200 plate reader from Tecan Group Ltd. (Maennedorf, Schweiz) and UV-Star microplates from Greiner Bio-One International AG (Kremsmünster, Austria). The UV extinction coefficients of all PEGylated forms of lysozyme were calculated from the theoretical molecular masses under the assumption that PEG does not influence UV-absorption of lysozyme and are given in ref 5. Error of the isotherm measurements is hard to assess, as the spectroscopic analysis has a large absolute error, due to uncertainties of the extinction coefficient of native lysozyme which had to be taken from the literature. This uncertainty of the calibration leads to an uncertainty of the concentration measurements of about 10%. Note, however, that this comparatively large systematic uncertainty of the calibration affects all measurements in the same way, so that comparisons between data points, trends, etc. are subject only to smaller errors. The magnitude of the random error can be assessed directly from the adsorption isotherm data where they show up as a scattering around the trend line. They are also of the order of 10% in some cases (see figures). Polyethylene Glycol (PEG). Adsorption experiments with pure PEG were carried out exactly as those with PEGylated lysozymes and native lysozyme. As PEG does not absorb light between 200 nm and 1000 nm wavelength, analytics were carried out as follows. PEG samples were analyzed using a 1100/1200 HPLC system from Agilent Technologies (Böblingen, Germany) equipped with a quaternary pump (G1311A), a tempered autosampler (G1329A), and a refraction index detector (G1362A, Agilent Technologies, Böblingen, Germany). Separation of PEG from salt was accomplished using two HiTrap
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THEORY
Equilibrium Model. To calculate the mass of protein adsorbed upon each injection in the ITC experiments, the experimental results of the adsorption equilibrium isotherms have to be correlated with a model. Note that this model does not have to be physically justified and any suitable equation can be used for the correlation. Nevertheless, a semiempirical model, the colloidal model of Oberholzer and Lenhoff19,20 was chosen, which correlates experimental isotherms well. The colloidal can be written in the following form: cp =
qp K pads
⎡ exp⎢⎢β qp/q0 exp ⎣
⎤ −γ ⎥ qp/q0 ⎥⎦
(1)
where Kads p is the equilibrium constant which is equal to the initial slope of the equilibrium isotherm.5 β and γ are lumped fitting parameters20 which are kept dimensionless by the introduction of q0 = 1 mg mL−1. Thermodynamic Analysis. Blaschke et al.5 recently presented a new way to thermodynamically analyze the results of combined caloric and adsorption equilibrium measurements 11377
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as they were carried out in the present work. This method allows gaining insight into the adsorption mechanism by answering the question if the adsorption process is enthalpy or entropy driven for all loadings along the isotherm. The thermodynamic analysis is presented in detail in the online supporting information from Blaschke et al.5 Evaluation of ITC Experiments. The evaluation of the ITC data and the calculation of the molar enthalpy of adsorption were carried out as previously described,5,11 and are not discussed here in detail. Preliminary experiments showed that the contribution of the adsorption of buffer ions and the heat of dilution of buffer ions to the measured signal can be neglected here. Hence, the enthalpy of adsorption of the proteins was determined from the adsorption experiments by subtracting only the enthalpy of mixing, which was determined in a blank experiment. The molar enthalpy of adsorption was then determined from Δhpads
=
Table 1. Overview of the Experimental Program for Adsorption Equilibrium and Caloric Experiments in 25 mM Sodium Phosphate Buffer pH 7.0, 25 °C on Toyopearl PPG600M for Different Ammonium Sulfate Concentrations (c(NH4)2SO4 [mM]). All Ivestigated Combinations Are Marked with a Check Mark adsorption isotherms experiment lysoyzme mono-PEGlysoyzme-5kDa di-PEG-lysoyzme5kDa tri-PEG-lysoyzme5kDa mono-PEGlysoyzme-10kDa di-PEG-lysoyzme10kDa tri-PEG-lysoyzme10kDa PEG-4kDa PEG-5kDab PEG-6kDa PEG-12kDa PEG-18kDa
ΔHpads ΔmpadsM p
(2)
where Δmads p is the amount of protein adsorbed upon each injection and Mp is its molar mass. Δmads p was determined from the adsorption equilibrium isotherm. Experimental error of caloric data is hard to assess as it depends on a variety of coupled parameters which can vary from point to point and with experimental setup. For high protein concentrations, and hence, high adsorber loadings Δmads p becomes small due to flat isotherms. Coupled with a low caloric signal at high loadings, calculation of Δhads p using equation eq 2 is difficult (division of zero by zero). A general statement on the error would be inappropriate. The scatter of the data shown in the figures gives the best indication for the random error.
600
800
1000
1200
√ √
√ √
√ √
√
√
√
√
√
√
ITC 800
1000
1200
√
√a √
√a √
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√ √ √ √ √
√ √
600
√
√ √
√ √ √ √ √
√ √
Results are not discussed in detail here, as Δmads p is very small due to very flat isotherms. bNote that PEG-5kDa is methoxy-PEG-aldehyde, all the other PEGs are unmodified. a
PEG-lysozyme-10kDa has the same PEGylation degree as diPEG-lysozyme-5kDa. Native lysozyme has a very flat isotherm which indicates very weak interactions between the protein and the adsorbent. For PEGylated lysozymes, the slopes of the isotherms at low protein concentrations increase with increasing PEGylation degree. This is supported by the equilibrium constants, Kads p , shown in Table 2, which are a measure of the binding strength between PEGylated lysozyme and the adsorbent. Isotherms of PEGylated lysozymes with attached PEG-5kDa chains show a clear behavior. Tri-PEG-lysozyme has the highest slope, followed by di-PEG-lysozyme and mono-PEG-lysozyme. An explanation for this behavior is, that upon adsorption of lysozymes with a higher PEGylation degree, more PEGmonomers interact with the adsorbent. For very high PEGylation degrees, the differences among the differently PEGylated lysozymes become small, as can be seen from Figure 1. The small differences between di-PEGlysozyme-10kDa and tri-PEG-lysozyme-10kDa do not significantly exceed the experimental uncertainty. As lysozyme hardly binds to the adsorbent, adsorption of the PEGylated lysozymes must be based on the adsorption of the PEG-chains. At low protein concentrations, the adsorption is favored by higher PEGylation degrees, but the tendency is reversed for high protein concentrations; for example, the adsorption isotherm for tri-PEG-lysozyme-5kDa is above that for diPEG-lysozyme-5kDa for low protein concentrations (cf. Figure 1) but at high protein concentrations the isotherm for di-PEGlysozyme-5kDa is above that for tri-PEG-lysozyme-5kDa. At the low protein concentrations the adsorption of lysozyme with higher PEGylation degrees is better than for PEGylated lysozyme with a lower PEGylation degree due to a more favorable interaction of the PEG and the adsorbent as described
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RESULTS In this work, microcalorimetric experiments and corresponding equilibrium experiments were carried out for lysozyme, six PEGylated forms (mono-, di-, tri-) for two PEG chain lenghts (5 kDa, 10 kDa) and five pure PEGs (4, 5, 6, 12, and 18 kDa). Three different ammonium-sulfate concentrations were investigated for each molecule. An overview of the experimental program is given in Table 1. The temperature for all experiments was 25 °C and the pH value was held constant at pH 7.0 using a 25 mM sodium phosphate buffer. As this is a large body of experiments, we will only discuss the major findings and results using representative examples. The full data set is presented by Werner.21 The following discussion is mostly based on the results obtained with 800 mM ammonium sulfate, as all substances were investigated at this ammonium sulfate concentration. The investigated range of salt concentrations was chosen so that at low salt concentration sufficient loading still occurred and that at high salt concentrations the protein was still soluble.
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DISCUSSION Equilibrium Adsorption Isotherms. PEGylated Lysozyme. Figure 1 shows the equilibrium adsorption isotherms for native lysozyme and all PEGylated forms obtained in experiments with an ammonium sulfate concentration of 800 mM. We define the PEGylation degree here as the total molecular mass of the PEG chains attached to the protein; that is, mono11378
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Table 2. Parameters of the Colloidal Model for the Adsorption Equilibrium Isotherms of PEGylated and Native Lysozyme in 25 mM Sodium Phosphate Buffer pH 7.0, 25 °C on Toyopearl PPG-600M. R2 is the Coefficient of Determination lysoyzme
mono-PEGlysoyzme-5kDa di-PEG-lysoyzme5kDa tri-PEGlysoyzme-5kDa mono-PEGlysoyzme10kDa di-PEG-lysoyzme10kDa tri-PEGlysoyzme10kDa
c(NH4)2SO4 [mM]
Kads p [−]
β [−]
γ [−]
R2 [−]
800 1000 1200 800 1000 1200 600 800 1000 600 800 1000 600 800 1000 600 800 1000 600 800 1000
0.87 1.54 3.92 7.44 82.7 546.7 1.97 104.1 539.1 18.9 137.3 772.2 0.91 401.6 598.9 23.6 1096.9 1428.5 65.1 681.2 747.1
0.00 1.99 0.13 1.93 3.83 4.21 10.00 1.18 5.74 0.97 12.75 10.00 47.58 1.07 17.28 4.29 10.00 4.66 4.74 7.84 4.39
10.00 1.86 0.00 5.63 7.79 11.21 9.68 1.97 10.00 0.00 10.83 14.51 92.88 0.00 16.75 3.90 8.60 10.00 4.17 10.00 10.00
0.92 0.94 0.96 0.99 0.99 0.99 0.99 0.99 0.91 0.99 0.98 0.91 0.85 0.98 0.98 0.99 0.96 0.92 0.98 0.85 0.80
Figure 1. Equilibrium adsorption isotherms for native lysozyme and all studied PEGylated lysozymes in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate on Toyopearl PPG-600M. The adsorbed phase concentration qp is plotted versus the liquid phase concentration cp. (Top) PEGylated lysozymes 5 kDa and native lysozyme; (bottom) PEGylated lysozymes 10 kDa. The adsorption depends on degree of PEGylation.
above. An explaination for the different capacities of the lower and higher PEGylated lysozymes could be a steric hindrance during the adsorption. The bigger molecules cannot diffuse into the smallest pores of the adsorbent particle. These results are consistent with the findings of Müller et al.22 and Pinholt et al.23 Pure PEG. In Figure 2 the isotherms of pure PEGs with chain lengths of 4, 5, 6, 12, and 18 kDa are shown. The conditions are the same as in the experiments with PEGylated lysozyme described above. With increasing PEG chain length, interactions between PEG and adsorbent get stronger. The adsorption isotherms for longer PEGs are always higher than those for shorter PEGs and also the initial slopes monotonously increase with increasing molecular mass of the PEGs (cf. Table 3). It is remarkable that the differences between PEG-5kDa and PEG-6kDa are smaller than could be expected from the rest of the data. The reason could be the different end groups of the PEGs as the used PEG-5kDa is modified with an aldehyde group, whereas the PEG-6kDa is unmodified, as all other studied PEGs. Comparison of Pure PEG and PEGylated Lysozyme. A comparison of the adsorption isotherms of PEG-5kDa and mono-PEG-lysozyme-5kDa is shown in Figure 3. The isotherm of PEG-5kDa is above that of the mono-PEG-lysozyme-5kDa. The attached lysozyme obviously slightly weakens the
Figure 2. Equilibrium adsorption isotherms for pure PEGs with chain lenghts of 4, 5, 6, 12, and 18 kDa in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate on Toyopearl PPG600M. The adsorbed phase concentration qp is plotted versus the liquid phase concentration cp. The adsorption depends on PEG chain length.
interactions between the PEG and the adsorbent. This finding holds not only for the example shown in Figure 3, but is a general finding from the experimental studies carried out in the present work. The comparison also supports the statement, that adsorption of the PEGylated lysozymes is based on PEG adsorption. Comparison of Different PEG Chain Lengths. A comparison between adsorption isotherms of mono-PEG-lysozyme10kDa and di-PEG-lyszoyme-5kDa is presented in Figure 4. It shows that adsorption of these two species is similar. Both molecules have the same PEGylation degree. The number and the size of attached PEG has obviously only little influence on the adsorption as long as the overall amount of added PEG 11379
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Ammonium Sulfate Concentration. Figure 5 shows the equilibrium adsorption isotherms for tri-PEG-lysozyme-5kDa at
Table 3. Parameters of the Colloidal Model for the Adsorption Equilibrium Isotherms of Pure PEGs in 25 mM Sodium Phosphate Buffer pH 7.0, 25 °C on Toyopearl PPG600M. R2 is the Coefficient of Determination PEG-4 kDa PEG-5kDa PEG-6 kDa
PEG-12kDa
PEG-18kDa
c(NH4)2SO4 [mM]
Kads p [−]
β [−]
γ [−]
R2 [−]
800 800 800 1000 1200 800 1000 1200 800
3.24 11.95 16.1 381.2 411.1 153.2 205.4 448.8 794.1
0.24 0.61 0.68 2.31 2.73 7.93 3.32 15.69 3.39
0.00 0.00 0.00 2.14 4.71 6.53 6.20 8.64 3.46
0.99 0.99 1.00 0.99 0.98 0.97 0.99 0.98 0.95
Figure 5. Equilibrium adsorption isotherms for tri-PEG-lysozyme5kDa in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with ammonium sulfate concentrations of 600, 800, and 1000 mM on Toyopearl PPG-600M. The loading depends on the salt concentration. With increasing salt concentration, the loading increases.
different ammonium sulfate concentrations. The loading of the adsorbent increases with increasing salt concentration. An increasing ammonium sulfate concentration makes the solvent less compatible with the tri-PEG-lysozyme. This salting out effect leads to better adsorption on the hydrophobic resin. Another argument is, that with increasing salt concentration, the hydration shells around the molecules are weakened and the interactions between PEGs and the PEG like groups on the adsorbent are favored. A similar behavior was observed for all PEGylated lysozymes, native lysozyme, and all pure PEGs. This behavior was expected for this system.22,24,25 As the solubility of the lysozymes with higher PEGylation degrees strongly decreases with increasing ammonium sulfate concentration, no measurements with higher ammonium sulfate concentrations were carried out. Molar Enthalpy of Adsorption. PEGylated Lysozyme. In Figure 6 the molar enthalpies of adsorption are shown for all PEGylated lysozymes at an ammonium sulfate concentration of 800 mM. The molar enthalpy is plotted versus the loading of adsorbent. The molar enthalpies of adsorption always refer to the entire molecule (lysozyme with PEG). The adsorption is endothermal (Δhads p > 0) for all experiments of the present work. This can be explained with the endothermal nature of the release of water from the hydration shell of the PEGylated lysozymes and the adsorbent upon adsorption. This water release also results in an increase in entropy. The experiments show that the enthalpy of adsorption increases with increasing PEGylation-degree. Lysozymes with a higher PEGylation degree have higher molar enthalpies of adsorption than lysozymes with a lower PEGylation degree. This effect can be explained by the fact that it is the PEG that adsorbs (see previous discussion). For higher PEGylation degrees, more PEG-monomers interact with the adsorbent. There is a higher water release upon adsoption of the lysozymes with a higher PEGylation degree, which results in larger endothermic effects. The molar enthalpies of adsorption of mono-PEG-lysozyme10kDa and di-PEG-lysozyme-5kDa are similar. This indicates again that for the same PEGylation degree, number and size of
Figure 3. Equilibrium adsorption isotherms for mono-PEG-lysozyme5kDa and pure PEG-5kDa in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate on Toyopearl PPG-600M. The attached lysozyme weakens the adsorption compared to pure PEG.
Figure 4. Equilibrium adsorption isotherms for mono-PEG-lysozyme10kDa and di-PEGlysozyme-5kDa in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate on Toyopearl PPG600M. The adsorbed phase concentration qp is plotted versus the liquid phase concentration cp. Adsorption isotherms are similar. For the same PEGylation degree, the distribution of the attached PEG only has a weak influence on the adsorption.
remains constant. Also this statement is supported by the other experimental data from the present study. Possible differences existing between isotherms with the same PEGylation degree cannot be resolved by our experimental methodology. 11380
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Figure 7. Thermodynamic analysis of the adsorption of mono-PEGlysozyme-10kDa on Toyopearl PPG-600 M in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate. The reference state Gibbs energy of adsorption Δgpads ref*, the ref *, and the molar enthalpy of corresponding contribution TΔsads p are plotted versus the adsorber loading qp. adsorption Δhads p
Figure 6. Molar enthalpy of adsorption of all studied PEGylated lysozymes on Toyopearl PPG- 600 M in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate. The molar enthalpy of adsorption Δhads p is plotted versus the adsorber loading qp. Δhads p depends on PEGylation degree.
attached PEG does not influence adsorption. A similar behavior was observed for all other investigated ammonium sulfate concentrations. The molar enthalpy of adsorption Δh pads is almost independent of the loading for the lysozymes with a low PEGylation degree. This indicates that for low PEGylation degree there is not much interaction between the adsorbed PEGylated lysozymes on the surface and that enough favorable binding sites are available on the adsorbent even at high loading. For PEGylated lysozymes with a high PEGylation degree this is different, for these proteins Δhads decreases p slightly with increasing loading of the adsorbent. This indicates interactions between the adsorbed proteins by their PEG chains at high loadings. Not all of the PEG segments find favorable binding sites as many of these are already occupied by other PEG chains. The findings are consistent with the results found by Blaschke et al.5 for the adsorption of the same molecules on a strong cation exchanger. Thermodynamic Analysis. The result of the thermodyamic analysis is shown in Figure 7 using mono-PEG-lysozyme10kDa, again with 800 mM ammonium sulfate. In Figure 7 the ref molar Gibbs free energy Δgads * (see ref 5 for definition and p derivation) as determined from the equilibrium isotherm of adsorption is shown together with the corresponding molar enthalpy of adsorption Δhads p , determined from the ITC experiments, and the resulting molar entropic contribution ref TΔsads *. The process is entropy driven. The strong entropy p gain is fully in line with the picture of the water release from the solvent shells upon adsorption. This finding holds for all investigated molecules. Pure PEG. In Figure 8 the molar enthalpies of adsorption for all investigated pure PEGs with 800 mM ammonium sulfate are plotted again versus the loading of the adsorbent. The molar enthalpy of adsorption increases with increasing PEG chain length. Longer PEG chains have higher molar enthalpies of adsorption than shorter PEG chains. This effect can be explained again with the water release upon adsorption. Upon adsorption of a longer PEG chain more water molecules are released from the water shells of the PEG and the adsorbent
Figure 8. Molar enthalpy of adsorption of all investigated pure PEGs on Toyopearl PPG-600 M in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate. The molar enthalpy of is plotted versus the adsorber loading qp. Δhads adsorption Δhads p p depends on PEG chain length.
which result in higher enthalpies of adsorption. It is remarkable that the molar enthalpy of adsorption Δhpads is almost independent of the loading for all PEGs except PEG-12kDa. We presently have no explanation for that finding. We assume that this is an artifact related rather to uncertainties of the adsorption equilibrium measurement or its fit than to an uncertainty of the calorimetric experiment. Comparison of Pure PEG and PEGylated Lysozyme. From the results of the molar enthalpy of adsorption Δhads p presented above, the molar enthalpy of adsorption at infinite dilution ∞ Δhads can be obtained from an extrapolation of the data. p These numbers were determined for the PEGs of different molar mass as well as for all studied forms of PEGylated lysozyme, that is, with 5 kDa PEGs (mono-, di-, tri-) and 10 kDa PEGs (mono-, di-, tri-). The results were then plotted as a function of the overall molecular mass of the PEGs attached to the lysozyme. The plot is presented in Figure 9. ∞ For pure PEGs Δhads displays a linear dependence on the p molar mass of the PEG. Hence, the adsorption enthalpy related to a PEG-monomer is constant. This implies that the interactions between PEG and the adsorbent are independent 11381
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increases with increasing ammonium sulfate conentration. This finding holds for all investigated PEGylated lysozymes, native lysozyme, and pure PEGs and can be explained with the dehydration of the hydrophobic groups. With increasing salt concentration the shells of ordered water around the hydrophobic groups are weakened and the interactions between the hydrophobic groups become stronger. This results in higher molar enthalpies of adsorption. Figure 10 also shows that Δhads p depends on the loading of the adsorbent as discussed.
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CONCLUSION This work demonstrates that microcalorimetric studies can contribute to a better understanding of the mechanisms of protein adsorption on chromatographic resins. The adsorption of PEGylated lysozyme isoforms on a mildly hydrophobic resin was found to be endothermic and entropy driven. The adsorption of PEGylated lysozyme with ammonium sulfate is mainly caused by the adsorption of the PEG chains. The PEG chains, attached to the protein, interact with the PPG groups of the PEG-like adsorbent that was studied here. The water release upon adsorption is endothermic but the entropy gain overcompensates this energetic penalty. Upon adsorption of higher PEGylated lysozymes, more water molecules are released from the hydration shells of the PEGylated lysozyme and the adsorbent, respectively. This higher water release results in higher molar enthalpies of adsorption for higher PEGylated proteins. For the same PEGylation degree, adsorption is similar. The adsorption mechanism does not change for PEGs with different chain lengths.
∞ Figure 9. Molar enthalpy of adsorption at infinite dilution Δhads of p pure PEGs and all PEGylated lysozymes, that is, mono-, di-, tri-5kDa and mono-, di-, tri-10kDa, on Toyopearl PPG-600 M in 25 mM sodium phosphate buffer, pH 7.0, 25 °C, with 800 mM ammonium sulfate, ploted versus the molar mass of the attached PEGs per ads ∞ are extrapolated values adsorbed molecule Mtotal PEG. The data of Δhp of experimental data on Δhads p to cp = 0. The lines are linear fits through the origin of the extrapolated data. PEG size does not influence the adsorption mechanism.
from length of the PEG chain but only dependent on the number of monomer interactions. Thus, the adsorption mechanism does not change with the molecular mass of PEG. ∞ Also Δhads of the PEGylated lysozymes can be linearly p correlated to the total molecular mass of the attached PEG chains. Again, the length and number of the attached PEGs is irrelevant. However, the slope of the regression for PEGylated lysozymes is smaller than for pure PEG. This is again in harmony with the results found for the adsorption equilibrium and confirms that the attached lysozyme weakens the interaction between PEG and the adsorbent. Note, that the statements made in this section hold only for infinite dilution and, hence, free surface of the adsorbent. Ammonium Sulfate Concentration. The molar enthalpy of adsorption of tri-PEG-lysozyme-5kDa is shown in Figure 10 for the three investigated ammonium-sulfate concentrations. Δhads p
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +49 (0)631 2053835. Phone: +49 (0)631 2053497. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge funding of this work by Bundesministerium für Bildung und Forschung (BMBF) and thank Egbert Müller from Tosoh Bioscience GmbH (Stuttgart) for his great preliminary work on the systems.
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Figure 10. Molar enthalpy of adsorption of tri-PEG-lysozyme-5kDa on Toyopearl PPG-600 M in 25 mM sodium phosphate buffer, pH 7.0, 25 °C with ammonium sulfate. Results from three ammonium sulfate concentrations are shown. The molar enthalpy of adsorption Δhads p is plotted versus the adsorber loading qp. The molar enthalpy of adsorption depends on the loading and the salt concentration. 11382
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