Matrix assisted refolding of proteins by ion exchange chromatography

Journal of Biotechnology 117 (2005) 83–97

Matrix assisted refolding of proteins by ion exchange chromatography Christine Machold a , Robert Schlegl b , Wolfgang Buchinger b , Alois Jungbauer a, ∗ a Department of Biotechnology, Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, Vienna 1190, Austria b Boehringer Ingelheim Austria, Vienna, Austria

Received 5 July 2004; received in revised form 20 December 2004; accepted 7 January 2005

Abstract Two different approaches of matrix assisted refolding have been evaluated and compared to conventional refolding by dilution. Bovine ␣-lactalbumin was used for the studies as model protein. It was adsorbed under denaturing conditions on an ion exchange matrix and refolding was completed on the column prior to elution or, depending on the buffer system, in the eluate. Agarose based chromatography matrices showed high capacities for the denatured ␣-lactalbumin. A positive effect on the yield of refolded protein by the matrix could be observed for Fractogel® EMD DEAE and a negative for Toyopearl DEAE 650M, DEAE Sepharose FF and Q Sepharose FF. In the case of Fractogel® EMD DEAE the ion exchange surface might act as a folding helper. This property may be caused by the grafted polymers. For Source 30Q only a marginal negative influence on the refolding kinetics was observed, thus the ion exchanger is only a mean for removal of chaotropic agents. Refolding on the column is characterized by a low yield but high productivity due to significant reduction of refolding time. © 2005 Elsevier B.V. All rights reserved. Keywords: ␣-Lactalbumin; On column refolding; DEAE Sepharose; Folding kinetics; Matrix assisted refolding

1. Introduction Among many different approaches to protein refolding with high yield, matrix assisted refolding (MAR) using chromatography media as a matrix is one of the ∗ Corresponding author. Tel.: +43 1 360066226; fax: +43 1 3697615. E-mail address: [email protected] (A. Jungbauer).

0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.01.004

most promising methodologies (Creighton, 1990; Middelberg, 2002). MAR enables refolding at high protein concentrations. Additionally, purification and refolding can be achieved simultaneously. Such a strategy is desirable for processing of recombinant proteins expressed as inclusion bodies. Dilute solutions can be processed with adsorptive chromatography and the target protein can be concentrated during the refolding process instead of dilution. The versatile applicability

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of chromatography permits the development of refolding strategies for very different proteins. A large number of examples can be found in the literature, ranging from research applications such as using artificial chaperons as affinity ligands (Altamirano et al., 1997; Gao et al., 2003) to industrial scale applications like expanded bed technology (Cho et al., 2001). MAR was successfully applied with different chromatographic techniques, such as hydrophobic interaction chromatography (HIC) (Geng and Chang, 1992; Geng and Bai, 2002), affinity chromatography (Shi et al., 2003), size exclusion chromatography (SEC) (Shalongo et al., 1987; Werner et al., 1994; Gu et al., 2001; Schlegl et al., 2003) and ion exchange chromatography (IEX) (Suttnar et al., 1994). Each technique has its own demands and is more or less suitable for a given refolding process. When HIC is used for MAR the feed solution has to be supplemented with salt to promote binding of the protein to the surface. In case the protein is present in chaotropic agents such as urea or guanidine hydrochloride, solubility problems may arise. The most preferable technique is affinity chromatography due to its high specific interaction. However, suitable ligands are often not available or they are only marginally stable (Jungbauer et al., 2004). Also leakage has to be considered. In SEC, chaotropic agents are gradually removed by the column because they are stronger retarded than the protein. Furthermore, aggregates formed during refolding are separated simultaneously from the native monomer in one step. Chromatographic velocities and sample volume are the limiting factor in SEC (Jungbauer et al., 2004). Ion exchange chromatography is the most frequently used technique for chromatographic processes (Janson and Ryd`en, 1998). High binding capacities, widespread applicability, simplicity as well as controllability of this technique are the reason therefore. Creighton et al. (Creighton, 1990) published refolding of proteins by ion exchange chromatography in 1990. A selection of model proteins as well as of recombinant proteins from dissolved inclusion bodies was subjected to different ion exchangers and transferred to the native state after removal of denaturing and reducing agents. He claimed that the chaotropic agent, urea in which the sample is dissolved, has to be decreased linearly in order to maximize the refolding yield. Elution of the native protein is then achieved by a linear increase in

salt concentration. Elution profiles of an ion exchange chromatography for native and artificially denatured, reduced and then refolded model proteins were compared and found to be identical. It was therefore postulated that the denatured protein was refolded. However, similar retention times of native and renatured proteins in ion exchange chromatography are not a proof for native conformation. The eluate must be further analyzed for aggregate content, refolding kinetic and native three-dimensional structure. It was also shown, that ion exchange chromatography can be successfully applied for refolding of recombinant proteins. Simultaneous purification can be achieved and sample volumes are kept low due to the concentrating effect of the ion exchanger. In the work of Creighton the eluate was analyzed by SDS-Page. This method does not provide information about the protein conformation as SDS is a strong denaturing agent and therefore, the protein is again transferred to its unfolded state. Creighton provided many examples for the applicability of ion exchange chromatography in order to adsorb the target protein and to remove the denaturing agents. In contrast to conventional refolding by dilution, buffer volumes can be kept low. MAR with ion exchange chromatography is thus a promising alternative. Certainly, this methodology entails development of a complex system, where many factors have to be considered: First, the buffer system has to be optimized to enable refolding at high yield and it also has to fulfill the requirements for ion exchange chromatography. The ionic strength of the feed and the equilibration buffer must be low. The pH and the nature of the displacing ions for elution have to be chosen appropriately. Secondly, the influence of the chromatographic matrix on the refolding yield has to be investigated and high binding capacity of the sorbent for the denatured protein is desired. Third, the chromatographic conditions such as flow velocity, gradient slope, and residence time on the column may have an influence on the refolding yield and should be investigated. The protein concentration of the sample and its volume could also influence the refolding yield. Finally, it was found that the protein is not completely folded after elution and that refolding proceeds in the eluate. It is therefore important to determine conditions for the eluate, such as addition of folding additives, time required for complete refolding and final protein concentration. All these factors are

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interconnected and therefore, it is difficult to evaluate the influence on refolding yield by a single parameter. In this paper we have investigated MAR of a model protein with special emphasis on matrix selection, selection of buffer additives and productivity. Two modes of operation were compared. In the first mode, chromatography is used to remove the chaotropic agents and final refolding is completed in the eluate. In the second approach, the protein is refolded on the column.

2. Materials and methods 2.1. Protein and chemicals Ca2+ depleted bovine ␣-lactalbumin (Vienna, Austria). Guanidine hydrochloride (Gdn-HCl) was purchased from Fluka (Buchs, Switzerland), and for CD measurements, Gdn-HCl ultra pure from Sigma–Aldrich was used. Urea ultra pure grade was from Amresco (OH, USA). Dithiothreitol was from AppliChem (Darmstadt, Germany) and monothioglycerol was from Sigma–Aldrich. Cysteine, cystine, Tris–HCl, acetonitrile HPLC grade and trifluoroacetic acid (TFA) were from Merck (Darmstadt, Germany). NaCl was obtained from Salinen Austria (Salzburg, Austria). DEAE Sepharose FF, Q Sepharose FF and Source 30Q were purchased from Amersham Biosciences (Uppsala, Sweden). Toyopearl DEAE 650M was from TosoHaas (Stuttgart, Germany). Fractogel® EMD DEAE was obtained from Merck (Darmstadt, Germany). 2.2. Analysis of the folding conformation 2.2.1. Determination by HPLC All HPLC runs were performed with an Agilent 1100 HPLC system (Waldbronn, Germany). The folding conformation of ␣-lactalbumin was analyzed by RP-HPLC using a Vydac (Worms, Germany) C4 214TPB5 column (250 × 4.6 mm I.D.). Eluent A was deionized and 0.22 ␮m filtered water, supplemented with 5% acetonitrile and 0.1% TFA, eluant B was acetonitrile supplemented with 0.1% TFA. Different folding forms of the protein were eluted with a linear gradient from 34 to 45% eluant B in 20 min, regeneration of the column was effected by

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a step gradient to 90% B. Size exclusion HPLC was performed on a TSKgel SuperSW2000 (300 × 4.6 mm I.D.) column, equipped with a TSKgel SuperSW guard column (35 × 4.6 mm I.D.) from Tosoh Biosciences (Stuttgart, Germany). As a buffer, 0.1 M NaCl in 50 mM Tris–HCl, pH 7 was used. 2.2.2. CD spectroscopy Far UV CD spectra of native, refolded and denatured ␣-lactalbumin were recorded on a Jasco J-600 spectropolarimeter (Easton, MD, USA). The CD spectra of native and refolded protein were measured in 2 mM phosphate buffer pH 7.7 containing 1 mM CaCl2 at 25 ◦ C. Protein concentrations were 90 ␮g ml−1 . FarUV wavelength scans were performed in a 1 mm quartz cuvette using 5 repeats with an averaging time of 4 s at each wavelength and a spectrometer bandwidth of 1.0 nm. All spectra were averaged and smoothed taking the mean of the five data points. Due to the high amount of Gdn-HCl in the denatured protein sample, significant data could not be recorded at wavelengths below 215 nm. 2.2.3. Dynamic light scattering A PD2000DLS dynamic light scattering detector combined with a PDDLLS/Batch platform was used for determination of the hydrodynamic radius of native and denatured ␣-lactalbumin (Precision Detectors Inc., Franklin, MA). The measurement was done at a fixed angle of 90◦ . The digital correlator calculated the time correlation function and these data were further analyzed with the software PrecisionDeconvolve or PrecisionDeconview. DLS data were displayed in form of molecular weight normalized concentration distribution. 2.3. Matrix assisted refolding ¨ AKTA-Explorer 100 system (Amersham Biosciences, Uppsala, Sweden) consisting of a compact separation unit and a personal computer running a control system (UNICORN, version 3.1) was used for all matrix assisted refolding experiments. HR 5/5, HR 5/10, HR 10/5 and HR 10/10 columns from Amersham Biosciences were packed with selected sorbents. Other chromatographic conditions used for the single experiments are cited in the respective figure.

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2.3.1. Determination of refolding kinetics in the eluate from an anion exchanger Denatured and reduced ␣-lactalbumin was loaded onto 5 different IEX resins: DEAE Sepharose FF, Q Sepharose FF, Source 30 Q, Toyopearl 650 DEAE and Fractogel EMF DEAE. The amount loaded corresponded to 90% of the dynamic binding capacity for this particular protein, which was estimated beforehand. Columns were equilibrated with a buffer consisting of 20 mM Tris–HCl, 2 M urea, 2 mM CaCl2 at pH 8.0. After loading the columns they were washed with the same buffer for at least 3 CV. Then the protein was eluted in a step with buffer B, consisting of 0.5 M NaCl in equilibration buffer. Finally the column was regenerated with 0.5 M NaOH. The flow velocity was 1 CV min−1 and the column dimensions were 0.5 cm i.d. at a variable bed height, giving column volumes between 1 and 2 ml. The redox pair cysteine/cystine was added to the elution pool within 15 min after elution to a final concentration of 2 mM each. Then the refolding reaction in the eluate was stopped by adding HCl to final pH 2 after 5, 20, 60, 120, 180, 240, 300, 480, 720 min and when equilibrium was not reached sometimes after 1800 min. The amount of native proteins in aliquots of the respective samples was then determined by RP-HPLC. Yield was then calculated as the ratio between the concentrations of the native protein to total protein in the eluate. This ratio was plotted versus refolding time. 2.3.2. Determination of refolding kinetics in batch mode In order to determine the effect of the ion exchange resin, refolding experiments at similar protein concentrations as in the ion exchange eluate were carried out in a batch mode. The model protein was dissolved at high concentration in 6 M urea and denatured with 100 mM monothioglycerol. Refolding was initiated by a 30-fold dilution with the same buffer as used as equilibration buffer in the column experiments. Because the protein concentration after dilution should be the same as the eluate from the ion exchanger, a very high initial protein amount had to be dissolved. In case the protein could no longer be dissolved, the refolding kinetics for the respective protein concentration was calculated using the kinetic constants k2 and k3 . In case refolding kinetics were determined experimentally, the reaction was

stopped by addition of HCl and subjected to RP-HPLC analysis. 3. Results and discussion Matrix assisted refolding was studied in detail with the single model protein, ␣-lactalbumin, to assess relevant parameters for this refolding process and their interplay. By careful selection of the buffer system, adequate treatment of the eluate and screening for the best suited sorbent, the refolding yield can be remarkably increased. A simple variation in the buffer system allows choosing between two refolding strategies of MAR: the protein may either be eluted from the column in a non-native state and refolds completely in the eluate or refolding is already initiated during adsorption and almost completed after migration through the column. In the latter case, the protein elutes already in native conformation. Prior to the description of the refolding in the column, we give an overview how we have assessed the native and denatured model protein. Analysis of the folding conformation was usually performed with RP-HPLC. In this case, chromatographic analysis is a proof for the protein conformation. The native protein elutes earlier from the hydrophobic stationary phase as the denatured and reduced one, because surface hydrophobicity increases upon unfolding, as hydrophobic regions of proteins tend to be located in the interior of folded proteins (Dill, 1990). Even intermediates can be separated by this methodology. Analysis of denatured but not reduced ␣-lactalbumin showed similar retention in RP-HPLC, although different protein conformation of native and of denatured protein was confirmed by circular dichroism spectroscopy (Chaudhuri et al., 2000). Either is the resolving power of the column not sufficient to separate these conformations, or which is more likely the native protein is denatured due to the harsh conditions of the HPLC (McNay and Fernandez, 1999). For these reasons, CD spectra were recorded for representative samples and we found native structure of the refolded protein (Fig. 2). An independent verification was also made by Schlegl et al. (2003). Thus we assumed based on these extensive studies, if the protein eluted at the position of the native protein, that it had gained native conformation. During refolding protein aggregates are always simultaneously formed. This undesired reaction is often

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Fig. 1. Increase in peak area of the native peak during refolding analyzed by RP-HPLC. Aliquots of the same sample of refolded ␣-lactalbumin were taken and the refolding reaction was stopped by acidification of the sample at different time intervals. —: 0 min refolded; – – –: 90 min refolded; . . .: 180 min refolded; - - - -: 720 min refolded.

the main reason for low refolding yields. Aggregates formed during refolding of ␣-lactalbumin were qualitatively analyzed by SEC-HPLC. The monomer could be separated from the multimers. An increase of high molecular mass multimers but also of the monomer was observed during refolding. From this findings it can be concluded that initially formed di-and trimers may either subsequently aggregate to molecules of high order or may dissolve again to monomers. Peak areas of the multimers did not correlate with protein concentration,

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thus SEC-HPLC is not suited for quantification of native or denatured protein. In order to quantify the aggregate fraction, the refolded sample was again denatured by dilution with 8 M urea or 6 M Gdn-HCl and reduced with dithiothreitol or monothioglycerol. The protein concentration was then determined by RP-HPLC and the aggregate fraction was calculated by subtracting the concentration of the native protein from the total protein concentration. Aggregates cannot be quantified by RP-HPLC directly, as they either adhere to filters used to clear the sample prior to HPLC analysis or to the column inlet filters. It was also observed that aggregates bind tightly to the RP column and that the column has to be regenerated by injection of denaturing and reducing agents. Dissolution of aggregates during refolding was also detected indirectly by RP-HPLC analysis. In Figs. 1 and 2 an overlay of chromatograms of the same sample is shown for different refolding times. Acidification with HCl stops the refolding reaction, because the cysteine residues, prone to form intermolecular native disulfide bonds become fully protonated and disulfide bond formation is inhibited. Retention time of the native protein was 11.5 min, and 18.2 min for reduced ␣-lactalbumin, respectively for the applied gradient. The native peak increases with time, as shown in Fig. 1. Denatured protein was not detected, so increase of native protein can only be due to dissolution of soluble aggregates. Aggregate forma-

Fig. 2. CD-spectra of native, denatured and refolded ␣-lactalbumin.

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Fig. 3. Estimation of the hydrodynamic radius of native (A) and denatured/reduced (B). ␣-Lactalbumin by dynamic light scattering.

tion was always postulated to be irreversible (Goldberg et al., 1991). Critical analysis of lysozyme refolding kinetics by labeling native lysozyme with fluorescein isothiocyanate showed, that native protein is incorporated in aggregates (Buswell and Middelberg, 2002). The native protein may again diffuse from the aggregate fraction, resulting in an increase of native protein. RP-HPLC is a fast and well-suited method for analysis of the folding conformation of ␣-lactalbumin. The limit of quantification is low, approximately 20 ␮g ml−1 . Native protein conformation can be further distinguished from the unfolded conformation by dynamic light scattering. The diffusion coefficient is measured as a function of the amount of scattered light in a small time period. From the diffusion coefficient the hydrodynamic radius can be calculated. The hydrodynamic radius includes the protein and its hydrate shell. Dynamic light scattering measurements of native and unfolded ␣-lactalbumin were performed. In Fig. 3 the normalized molecular weight distribution of the hydrodynamic radii of native (A) and denatured (B) protein is shown. It should be noticed that the hydrodynamic radius is not a single value but rather a distribution of measured values in a certain range. The bars in Fig. 3 should be understood as the distribution of species within the range of these hydrodynamic radii. The diffusion coefficient for ␣-lactalbumin dissolved in water was 1.18 × 10−6 cm2 s−1 , whereas for ␣-lactalbumin dissolved in 8 M urea, it was 5.5 × 10−7 cm2 s−1 . The higher viscosity of the 8 M urea solution, having an influence on the diffusion coefficient was accounted for the calculation. The hydrodynamic radius ranged from 1.74 to 2.09 nm for the native protein and from 2.13 to 2.55 nm for the unfolded protein. Similar values were found by Gast et al. (1998). As light scattering measure-

ments are highly sensitive to impurities and absorbing buffer substances, it was refrained to measure the refolding samples by DLS. Prior to set up of matrix assisted refolding experiments, refolding by dilution was performed to screen for optimized refolding conditions. A huge number of additives exist to improve the refolding kinetics and reduce aggregate formation. Chaotropic agents in low concentration, l-arginine, detergents or sometimes salt can be used for this purpose (Clark, 1998). For the formation of correct disulfide bonds, redox reagents such as oxidized and reduced glutathione, cysteine or DTT at very low concentrations are used (Clark et al., 1998). Gdn-HCl cannot be used as buffer additive in ion exchange chromatography due to its high ionic strength. Urea is well suited since it even decreases the conductivity and allows binding of proteins to the ion exchanger. Refolding experiments by dilution have been performed with 0, 2, 4 and 6 M urea in the refolding buffer. The increase in native protein was determined at distinct time intervals and results are shown in Fig. 4. The refolding yield is defined as concentration of the native protein divided by the total protein concentration. cN Y= (1) c0 Y is the yield, cN the concentration of the native protein (mg ml−1 ) and c0 the concentration of the initial, denatured protein. For a final protein concentration of 0.17 mg ml−1 , approximately 80% of native protein was obtained, when the buffers contained 0 or 2 M urea, respectively. The refolding reaction was drastically decreased when the buffer contained 4 M urea. The protein could not

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Fig. 4. Influence of urea on the refolding kinetics. Denatured with 8 M urea and reduced with 200 mM monothioglycerol, ␣-lactalbumin at a protein concentration of 8.5 mg ml−1 was diluted 50-fold in a Tris–HCl buffer, supplemented with 2 mM cysteine and 2 mM cystine, pH 8.0. The refolding buffers additionally contained 0 M urea,
; 2 M urea, ; 4 M urea, ♦; and 6 M urea, .

be refolded in 6 M urea. As the protein concentration prior to refolding by dilution was 8.5 mg ml−1 , 200 mM monothioglycerol was used in order to ensure complete reduction. The final concentration was 4 mM monothioglycerol, since refolding was started by a 1:50 dilution. A mixture of a thiol reagent, cysteine and its disulfide counterpart, cystine was added to the dilution buffer at a concentration of 2 mM each. Such a system should provide rapid reshuffling of incorrect disulfide bonds and oxidation of monothioglycerol (Rudolph et al., 1997). In a second refolding experiment by dilution, the influence of concentration and ratio of the thiol and disulfide reagents, cysteine and cystine, was investigated. Fig. 5 shows the increase in refolding yield for

Fig. 5. Influence of the redox reagents cysteine and cystine on the refolding kinetics. ␣-Lactalbumin at a protein concentration of 7.5 mg ml−1 was diluted 50-fold. The final concentration of monothioglycerol was 1 mM. The refolding buffer contained 2 M urea and varying concentrations of cysteine/cystine: 0/0 mM,
; 5/0.5 mM, ; and 2/2 mM ♦.

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Fig. 6. Refolding by dilution of ␣-lactalbumin at a final protein concentration of 1.1 mg ml−1 at increasing concentrations of urea from 0 M to 2 M.
, 0 M urea; , 0.5 M urea; ♦, 1 M urea; , 2 M urea.

the first 20 min when the buffer was devoid of redox reagents, for a concentration of 0.5 mM and 5 mM cysteine/cystine and as well for a concentration of 2 mM and 2 mM cysteine/cystine. The dilution buffer also contained 2 M urea. Such a concentration increased the refolding yield remarkably for higher protein concentrations (e.g. 1 mg ml−1 ), which is shown in Fig. 6. From Fig. 5 it can be concluded that addition of redox reagents is essential in order to accelerate the refolding reaction. The ratio and concentration was not critical, so it was refrained to investigate other concentrations and ratios of the thiol and disulfide reagents. For simplicity reasons, the concentration of both cysteine and cystine was 2 mM. The buffer systems used for matrix assisted refolding contained therefore, urea at 2 M and 2 mM CaCl2 which is essential for refolding of ␣-lactalbumin (Ewbank and Creighton, 1993). The pH was 8, so as ␣-lactalbumin has a pI of 5.8, anion exchange chromatography could be performed. 2 mM cysteine and 2 mM cystine was used to enhance the refolding reaction after elution of the protein from the column. Once the buffer system was chosen and some preliminary refolding experiments were performed in order to test the applicability of this method, the binding characteristics of the model protein were investigated. The different adsorption characteristics of native and denatured model protein were exemplified by adsorption isotherm on the ion exchanger medium Source from Amersham Biosciences. The denatured and reduced protein was dissolved in 8 M urea and 100 mM monothioglycerol and applied at protein concentrations of 0.05, 0.1, 0.2, 1 and 2 mg ml−1 . Adsorption isotherms for unfolded

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Fig. 7. Adsorption isotherms for denatured and reduced ␣-lactalbumin at concentrations from 0.05 to 5.5 mg ml−1 (A) and native ␣-lactalbumin at concentrations ranging from 0.05 to 1 mg ml−1 (B).

and native ␣-lactalbumin are shown in Fig. 7A and B respectively. Data points were fitted with a Langmuir adsorption isotherm, according to Eq. (2) q=

Bqs C 1 + BC

(2)

where q is the protein concentration in the stationary phase, C the protein concentration in the mobile phase, qs the protein concentration in the stationary phase for maximum capacity. B is a constant related to the association constant. Adsorption isotherms were almost rectangular. The maximum capacity for denatured ␣lactalbumin (qs ) was 30 mg ml−1 , whereas for native protein it was doubled, namely 60 mg ml−1 . The big difference in qs may be due to decrease in electrostatic interaction caused by the high concentration of urea. Electrostatic interaction is indirectly related to the dielectric constant. The dielectric constant of an 8 M urea solution is 70.9 (Weast and Astle, 1981), whereas for water the constant is 78.25 at 25 ◦ C. The denatured protein is loaded in a solution containing 8 M urea and 100 mM monothioglycerol in order to maintain the denatured conformation. It has also been found that protein surface charge properties change as the protein conformation changes (Creighton, 1993). In addition

the larger diameter of unfolded proteins may also contribute to lower binding capacities, as they may not enter small pores of the chromatographic beads that are still accessible for folded proteins. Five different anion exchange sorbents were screened for their binding capacity for denatured and reduced ␣-lactalbumin. Industrial sorbents with a different base matrix were selected: DEAE Sepharose FF and Q Sepharose FF are based on cross-linked agarose. Source Q has a rigid backbone, made of polystyrene/divinyl benzene. Toyopearl DEAE is also a polymeric rigid material and Fractogel® EMD DEAE is a sorbent based on cross-linked methacrylate polymer where the functional groups are attached to flexible polyelectrolyte chains which should provide easier access to the solute molecule (Muller, 1990). Dynamic binding capacities (DBC) were estimated by frontal analysis. A denatured and reduced protein solution of 1 mg ml−1 was applied to approximately 0.5 ml of each sorbent until total breakthrough occurred. DBC was measured as the mass of protein corresponding to 10% of total breakthrough. Values for DBC are listed in Table 1. The agarose gels, DEAE Sepharose FF and Q Sepharose FF showed higher binding capacities than

Table 1 Dynamic binding capacity for denatured and reduced ␣-lactalbumin, yield of native ␣-lactalbumin for maximal load and productivity of five selected anion exchange resins Anion exchange resin

DBC (mg ml−1 )

Yield (%)

Productivity (mg ml−1 h−1 )

Source 30Q DEAE Sepharose FF Q Sepharose FF Toyopearl DEAE 650 M Fractogel EMD DEAE

30 45 42.5 12.3 22.4

63 53 52 57 84

1.09 4.16 1.80 1.36 5.07

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the sorbents made of synthetic polymers. Toyopearl showed lowest binding capacity for the protein, followed by Fractogel® EMD DEAE and Source 30Q. The binding capacity of ion exchange resins depends rather on the availability than on the number of charged groups, as ion exchangers are commonly highly substituted. The availability is in turn dependent on the molecular mass of the protein as well as on the porosity of the sorbent. Porosity of the agarose gels, DEAE Sepharose FF, Q Sepharose FF and of Source 30Q was not determined. The manufacturer estimated an aver˚ with a large distribution from age porosity of 350 A ˚ ˚ Porosity of Toyopearl 100 A up to more than 1000 A. ˚ DEAE 650 M is 1000 A, indicated by the manufacturer. ˚ Difference For Fractogel® EMD DEAE it was 800 A. in binding capacities cannot be explained solely by the porosity, the access of the protein to the pores as well as the pore geometry are also important features but the sorbents were not investigated in this respect. The five sorbents have been further compared concerning their impact on the refolding yield, when a protein mass corresponding to approximately 90% of the dynamic binding capacity for denatured and reduced ␣-lactalbumin was loaded onto the respective sorbent. The mass of denatured and reduced ␣-lactalbumin applied onto DEAE Sepharose FF was 17 mg, for Q Sepharose FF it was 18 mg, on Source 30Q, 10 mg ␣lactalbumin were loaded and to the Toyopearl 650 M and Fractogel® EMD DEAE sorbent, 4.5 and 9.0 mg were applied respectively. The running buffers contained 2 M urea and the redox reagents were added right to the protein eluate after elution. The refolding time in the eluate varied from 4 h for DEAE Sepharose FF, Fractogel® EMD DEAE and Toyopearl 650 M to approximately 10–11 h for Q Sepharose FF and Source 30Q. The protein was then analyzed by RP-HPLC. As the mass of protein loaded was very different for the selected sorbents, the productivity was calculated in order to account for the mass of protein that can be processed with the same quantity of chromatographic media. The productivity was calculated according to Eq. (3). P=

Yc0 V0 Vr t

(3)

P is the productivity in mg ml−1 h−1 , V0 the volume of the initial solution (ml), Vr the volume of the re-

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actor (ml), and t the refolding time (min). Vr in this case was the volume of the chromatographic bed. Values for yield and productivity are listed in Table 1, columns 2 and 3. Yield of native protein was highest for Fractogel® EMD DEAE, 84% of native protein could be recovered. With Source 30Q, a yield of about 63% could have been obtained, followed by the Toyopearl DEAE 650 M sorbent with about 57%. The Sepharose sorbents, DEAE Sepharose FF and Q Sepharose FF gave similar yield, approximately 50%. Productivity for matrix assisted refolding by ion exchange chromatography exceeds productivity achieved with refolding by dilution to a great extent. Even with matrix assisted refolding by size exclusion chromatography, such high productivity cannot be obtained because the sample volume that can be loaded is limited to 10% of the total column volume (Schlegl et al., 2003). Productivity is highest for these sorbents exerting high binding capacity, high yield and short incubation time. Thus, with Fractogel® EMD DEAE a productivity of about 5.07 mg ml−1 h−1 was obtained due to higher yield compared to other the sorbents. As DEAE Sepharose FF showed high binding capacity productivity was 4.16 mg ml−1 h−1 , which is higher than for Q Sepharose FF although similar yield and binding capacity was obtained. The refolding kinetic in the eluate of Q Sepharose FF was slower which is also illustrated in Fig. 8. Similarly, productivity of Source 30Q with 1.09 mg ml−1 h−1 was lower than for Toyopearl DEAE 650 M where a productivity of 1.36 mg ml−1 h−1 was obtained. Refolding kinetic in the eluate of Source 30Q was slower than compared to Toyopearl DEAE 650 M. However, yield has to be treated with caution. Yield is not only influenced by the matrix, but also by the environment after elution. It was observed that the refolding reaction is not completed after elution. In Fig. 1 RP-HPLC chromatograms of an ion exchange eluate at certain time intervals after elution are shown. No native peak was detected right after elution. Addition of the redox pair cysteine/cystine after elution increased the refolding yield, which underlines that the refolding reaction is not completed. Additionally, refolding yield decreases with protein concentration, because the formation of aggregate follows a reaction order of two or even higher, depending on the aggregation number. The increase of native protein and protein aggregates during refolding is described by the kinetic constants k2 and k3 , respectively. The constants can be estimated

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Fig. 8. Refolding kinetics in the eluate of DEAE Sepharose FF, Q Sepharose FF, Source 30 Q, Toyopearl DEAE 650M and Fractogel® EMD DEAE. 䊉, data points; —, fit according to Eq. (4); - - -, refolding kinetics by batch dilution.

from the following equation described by Kiefhaber et al. (1991):
 cN k2 c 0 k3 = ln 1 + (1 − e−k2 t ) (4) c0 c0 k 3 k2 k2 is the reaction constant for formation of the native protein, k3 the reaction constant for aggregate formation. The protein concentration in the eluate depends on the mass of initially loaded protein and the resulting elution volume. The eluates were collected manually and therefore the volume of the eluate varied. In some cases tailing was observed, especially when the protein load was high. A mass balance for each matrix assisted refolding experiment was calculated in order to estimate, if protein is accumulated in the matrix. In order to get correct values for the mass balance, it was important to collect the whole eluate. The influence of the matrix on the refolding kinetics was investigated by comparison of different chromatographic media. Refolding kinetics in the eluate of the five sorbents, DEAE Sepharose FF, Q Sepharose FF, Source 30Q, Toyopearl DEAE 650 M and Fractogel® EMD DEAE, respectively were determined. The refolding reaction

of aliquots of the eluate was stopped by acidification at different time intervals and followed by immediate RP-HPLC analysis to quantify to concentration of native protein. Eluates from experiments with the highest productivity were investigated (Table 1). All experiments were performed in replicates and approximately the same refolding kinetics was obtained. The refolding reactions to generate native protein are shown in Fig. 8. The concentration of native protein was plotted versus time and fitted by Eq. (4) using and the kinetic constants k2 and k3 as parameters. The values of k2 and k3 are listed in Table 2. As the kinetic constants differ slightly from each other, a certain influence of the resin on the refolding kinetics can be assumed. In order to estimate, if the contact of the denatured protein with the matrix had a positive or negative impact on the refolding kinetics, refolding by dilution with approximately the same final protein concentrations as observed in the eluate was performed and compared to refolding with matrix contact. Final protein concentrations of 2.59 and 4.8 mg ml−1 were investigated in the dilution experiments, since these values were the equivalent concentrations in the eluate. Only a dilution factor of 30 could be applied due to

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Table 2 Comparison of refolding kinetics after contacting the denatured protein with an ion exchange surface and conventional refolding by simple dilution (refolding constants obtained and estimated from batch dilution experiments)

c0 k2 k3

DEAE Sepharose

Q Sepharose

Source

Toyopearl 650

Fractoge FMD

5.5 0.00016 0.88

5.62 0.0000915 0.50

3.89 0.00010 0.51

2.47 0.000134 1.19

4.38 0.000136 0.15

5.62 0.00014 0.33

3.89 0.00013 0.33

2.59 0.00011 0.34

4.8 0.00014 0.33

Refolding by batch c0 5.5 k2 0.00014 k3 0.33

the limited solubility of the respective stock solutions. Therefore, the remaining concentration of reducing agent was 3 mM, whereas in the eluate no reducing agent was found. However, such low concentration of monothioglycerol does not affect the refolding system (data not shown). The kinetic constants obtained from

these experiments are listed in Table 2. Refolding yield dependent on the total protein concentration, c0 can be calculated according to Eq. (4), using the kinetic constants listed in Table 2. These curves were then compared with kinetics observed by refolding in the eluate with former contact of the protein with

Fig. 9. Elution profiles of the model protein from DEAE Sepharose FF, CV: 1.6 ml, when 3 mg were loaded. (A): Refolding in the eluate — equilibration and elution buffer supplemented with urea; - - - - - no urea in buffers. (B): On column refolding.

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the different sorbents (Fig. 8). For the calculation batch refolding kinetics at protein concentrations of 5.5 and 5.6 mg ml−1 the same kinetic constants as for batch refolding at 4.8 mg ml−1 were taken. For a concentration of 3.89 mg/ml−1 , a mean value for k2 and k3 was assumed. Only for Fractogel® EMD DEAE a positive influence on the refolding kinetics could be observed. The aggregation constant k3 of Fractogel® EMD DEAE was lower than with all other sorbents and lower than the corresponding batch dilution. This is a strong evidence that the contact of the protein with Fractogel® EMD DEAE suppressed aggregation. All other matrices had a negative or no effect on the refolding kinetics. k3 was always higher than in the corresponding batch dilution. From the curves shown in Fig. 8 it can be seen that refolding equilibrium is obtained after approximately 7 h. However, such long reaction times are often undesirable because they reduce productivity and considerably contribute to production costs. Thus elution of an already folded protein is advantageous. By a simple modification of the buffer composition it was possible to elute the model protein in native conformation. The redox reagents were added to the equilibration and elution buffer. In Fig. 9, two chromatograms of a matrix assisted refolding experiment on DEAE Sepharose FF are shown. Fig. 9A represents an experiment without a redox pair in the running buffers, whereas in Fig. 9B, the buffers contained 2 mM cysteine and 2 mM cystine. All other parameters such as column dimensions, sample load and flow velocity were kept constant. A very different elution profile was obtained. Without a redox pair in the running buffer almost all protein could be eluted by salt (Fig. 9A) and only a very small fraction was eluted during regeneration of the column with a solution of 6 M Gdn-HCl and 100 mM monothioglycerol. However, most of the protein was found in the regenerate when chromatography was performed with a buffer system containing the redox pair (Fig. 9B). 2 M Gdn-HCl and 100 mM monothioglycerol was sufficient for complete regeneration of the column. 8 M urea was also tested for this purpose, but it was not as effective as Gdn-HCl solution. Owing to the chaotropic nature of the regeneration solution the protein in the regenerate was present in a denatured conformation. The elution peak with salt (Fig. 9B) contained completely folded protein and further incubation was not required. The light gray profile in Fig. 9A represents the elution profile of a similar

Table 3 Evaluation of three matrix assisted refolding experiments: 3 mg protein were loaded onto a DEAE Sepharose sorbent Experiment no. Buffers supplemented with

1 2 M urea

2 –

3 2 mM cysteine

Recovery (%) Yield (%) Regenerate (%) Productivity (mg ml−1 h−1 )

98 76 1 0.0036

97 49.5 32.5 0.0024

2 mM cystine 86 5.5 75 0.0036*

The column volume was 1.5 ml, the flow velocity was 306 cm h−1 . Equilibration and elution buffers were supplemented with different agents. ∗ R time: 30 min. f

refolding experiment except 2 M urea was not present in the running buffer. The peak containing the native protein was smaller and some protein was found in the regenerate. The mass balance and productivity of these three experiments are listed in Table 3. The yield of native protein is very low when the redox pair was added to the running buffers (Table 3). Protein precipitation at the top of the column was observed during loading. The local protein concentration close to the column inlet was very high due to the high binding capacities of the ion exchange sorbent. The layer of precipitated protein could not be removed by the salt gradient and was only dissolved by the regeneration solution. We assumed that this layer was made up of aggregates formed upon the first contact of the feed with the redox reagents. These folding additives accelerate the refolding reaction, thus aggregates are formed immediately and they bind more tightly to the matrix because of their larger surface. Urea in low concentration was used for suppression of the aggregate formation but as a consequence the refolding reaction was slowed. When urea was omitted in the buffer system, approximately 30% of the initially loaded protein were aggregates. Therefore, refolding yield was highest for a buffer system containing 2 M urea but being devoid of 2 mM cysteine and 2 mM cystine (experiment No 1, Table 3). Yield is not the only criterion in evaluation of a refolding process. The time required for completion of the reaction is also very important and is reflected by the productivity of the given system. In Table 3, pro-

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Table 4 Column dimensions, refolding yield and recovery of four different on column refolding experiments Dimensions

i.d. (mm)

Length (mm)

Bed volume (ml)

Yield (%)

Recovery (%)

HR5/5 HR5/10 HR10/5 HR10/10

5 5 10 10

29 80 27 100

0.57 1.57 2.1 7.85

1 6 15 16

85 90 93 82

3 mg of denatured and reduced ␣-lactalbumin were applied.

ductivity for experiment No. 1 and No. 3 are similar, because in experiment No. 1 high yield is obtained on the expense of a long refolding time and in experiment No. 3 for the short reaction time yield has to be sacrificed. For experiment No. 3 a refolding time of approximately 30 min was assumed, based on the residence time of the protein in the column. It depends on the value of the product, if high yield is preferred to short refolding time. However, as the regenerate represents again denatured and reduced protein, it might be recycled and again applied to the matrix. On column refolding was further investigated. Yield is dependent on the amount of aggregates formed. It seems that protein-aggregation occurred instantaneously with contact of the surface resulting in a layer of precipitate at the top of the column. Thus the influence of the column geometry and ratio between loaded protein and volume of ion exchanger sorbent was studied. Four different sized columns have been used in order to refold the same amount of ␣-lactalbumin. Two columns with an inner diameter of 5 mm (HR5/5 and HR5/10) but different bed height and two columns of an I.D. of 10 mm (HR10/5 and HR10/10) also with differ-

ent bed height were used. Chromatographic conditions were scaled on a column volume basis. The respective bed volumes were 0.57, 1.57, 2.1 and 7.85 ml. Refolding yield on the smallest column was only 1%. Increasing the column length led to an increase in refolding yield up to 6%. When the column diameter was 1 cm, the refolding yield increased to 15% for a bed volume of 2.1 ml and 16% for a bed volume of 7.85 ml. Column dimensions, refolding yield and recovery are listed in Table 4. The protein concentration in the eluate was in each experiment approximately 0.1 mg ml−1 , so refolding yield was solely dependent on the chromatographic set up. Since we hypothesized that the local concentration on the column inlet is very critical a larger column diameter must be beneficial for refolding on the column. By increasing the ratio of sorbent volume to amount of loaded protein the local protein concentration was lowered resulting in a higher yield of native protein. Similar observations for column refolding on a hydrophobic interaction matrix led to the construction of refolding columns with a large aspect ratio (Geng and Bai, 2002).

Table 5 Comparison of different refolding strategies to obtain 100 mg of native product: batch dilution, matrix assisted refolding where refolding is completed in the eluate and matrix assisted refolding on the column Starting protein concentration (mg ml−1 ) Starting volume (ml) Mass of initial protein (mg) Yield (%) Native protein concentration (mg ml−1 ) Final volume (ml) Mass of native protein (mg) Time (min) Dilution factor (Vfin /Vstart ) Column volume Productivity (mg ml−1 min−1 ) n.a = not applicable.

Dilution

MAR–refolding in eluate

MAR–refolding on column

30 3.5 105 95 0.15 667 100 420 190 n.a 0.0003

10 16.6 166 60 10 10 100 420 0.6 4 0.0600

10 66.6 666 15 5 20 100 30 0.3 700 0.0032

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We showed that ␣-lactalbumin could be either refolded with high yield or in short time on an ionexchanger. In order to judge the advantages of the different operation modes a comparison of refolding by conventional batch dilution and MAR in the two modes, refolding in the eluate and refolding on column, respectively was made. We assumed a scenario to produce 100 mg of native protein (Table 5). In batch dilution, the refolding yield is approximately 95% when the protein concentration in the refolding solution is 0.15. In order to obtain 100 mg of native product, 3.5 ml of a 30 mg ml−1 solution of denatured and reduced ␣lactalbumin has to be diluted 190-fold. The resulting productivity is 0.0003 mg ml−1 min−1 ; the reactor volume is the volume of the dilution-vessel. In MAR, the initial protein concentration and volume does not have a significant influence on the refolding yield. The protein concentration in the eluate was more important. A yield of 60% was estimated, when the protein concentration in the eluate is 10 mg ml−1 . An elution volume of 10 ml can be expected, when 165 mg protein are applied to a column with volume of 4 ml. The column volume is in turn dependent on the dynamic binding capacity of the sorbent. A productivity of 0.06 mg ml−1 min−1 was calculated. When the protein is completely refolded on the column, the volume of the column was the most critical parameter. For such an experiment a column with 700 ml would be required in order to obtain 100 mg of native product. 666 mg of denatured protein must be processed, because yield is only 15%. Due to the large column volume, the elution volume is expected to be in the range of 20 ml and the resulting concentration of native protein is 5 mg ml−1 . For this process productivity is 0.03 mg ml−1 min−1 , since further incubation is not required. In summary a large final volume but highest yield of refolding by batch dilution resulted though in lowest productivity. Thus a large refolding reactor is required for refolding process based on dilution. A large quantity of protein can be processed in a comparable small reactor using the strategy of MAR. Incubation time in the eluate is similar to refolding by dilution but the applicable concentration of protein is much higher. Thus productivity is highest for this approach. Although process times are shortest for complete refolding of the model protein on the column, the low yield and large column volume required reduces productivity 20 fold compared to refolding in the eluate but it is still ten times higher compared to refolding by dilution.

Conclusion MAR is a more complex refolding system than refolding by dilution. For the development of a successful refolding process, many parameters have to be considered. The influence of the matrix on the refolding kinetics should be further investigated. Once the chromatographic parameters are optimized and treatment of the eluate is well established MAR is certainly a promising alternative to conventional refolding by dilution, since the protein is concentrated and productivity is much higher. Using a chromatographic column as a refolding reactor, simultaneous purification and refolding can be achieved. This is advantageous when recombinant proteins from dissolved inclusion bodies have to be processed. Relevant parameters for MAR derived from preliminary studies with the model protein ␣-lactalbumin should be considered for the development of a refolding strategy for recombinant proteins. Acknowledgements The Austrian Industrial Research Promotion Fund (FFF) supported the project (project No. 80 3983).

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