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Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel
Biochemical Engineering Journal 45 (2009) 201–208
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel Hüseyin Alkan a , Nilay Bereli b , Zübeyde Baysal a , Adil Denizli b,∗ a b
Department of Chemistry, Dicle University, Diyarbakir, Turkey Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 15 January 2009 Received in revised form 10 March 2009 Accepted 27 March 2009 Keywords: Protein A Cryogels PHEMA Antibody purification IgG
a b s t r a c t Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 m size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Human immunoglobulin G (IgG) is an important plasma protein with many applications in therapeutics, immunodiagnostics and immunochromatography [1]. These applications generally require highly pure IgG [2]. For medical applications, immunoglobulins (IgGs) have been thoroughly purified using a combination of various methods, mainly precipitation and chromatographic techniques including ion exchange, hydrophobic interaction, histidine affinity, metal-chelate affinity and dye-affinity [3–8]. Fractionation by ethanol precipitation is the most used process to purify IgG from human plasma on industrial scale (27 ton/year) [9]. But this technique is not specific and can give partially denatured proteins [10]. Conventional chromatography technique uses gel beads column chromatography, which have certain limitations such as a high pressure drop, low flow-rates and hence low productivities and difficulties in efficient scale up [11]. It has also a major limitation; incapability of highly viscous fluids, for example human
∗ Corresponding author at: P.K. 1, Samanpazarı, 06242, Ankara, Turkey. Tel.: +90 312 2992163; fax: +90 312 2992163. E-mail address: [email protected] (A. Denizli). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.03.013
blood. The most obvious progress has been made in the development of alternative chromatography formats such as membrane adsorbents, monoliths and cryogels [12–15]. Cryogels provides a potential solution due to their low pressure drop and lack of diffusion resistances due to the macropores compared to traditional column chromatography [16–18]. Cryogels allow high flow-rates enabling the processing of large volumes within short process times. Whole blood can be applied to cryogels without any pretreatment [19]. Cryogels are also cheap materials and they can be used as disposable avoiding cross-contamination between batches [20]. Protein A is a cell wall associated protein domain exposed on the surface of the Gram-positive bacterium Staphylococcus aureus [21]. Immunoglobulin G (IgG) binds protein A via its Fc region [22]. The interaction appears to be characterized by hydrophobic interaction together with some hydrogen bonds and salt bridges [23,24]. Protein A consists of five homologous IgG binding domains [25]. Each of five domains in protein A is arranged in an anti-parallel there ␣-helical bundle and the three-dimensional structure is stabilized via a hydrophobic core [21]. In this study, we used poly(hydroxyethyl methacrylate) [PHEMA] cryogel carrying protein A as a model supermacroporous monolithic matrix capable of purification of IgG from human plasma.
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2. Experimental
2.4. CNBr activation
2.1. Materials
PHEMA cryogel was activated with cyanogen bromide in order to prepare active attachment sites for protein A. Prior to activation process, PHEMA cryogel was kept in distilled water for about 24 h and washed with 0.5 M NaCl solution and water. 2 ml of 0.5 M sodium carbonate buffer (pH 10.5) was added and stirred slowly. The mixture was placed in a fume hood and the glass pH electrode was immersed into this solution. CNBr was weighed carefully and added to the solution. The pH of this solution was quickly adjusted to 11.5 with 4 M NaOH and the pH was maintained between 10.5 and 11.5 during the activation reaction. The CNBr solution was recirculated through the column at 1.0 ml/min at room temperature (25 ◦ C) and activation procedure was continued for 60 min. After the activation reaction, in order to remove the excess activation agent, the PHEMA cryogel was washed with 0.1 M NaHCO3 and any remaining active groups (e.g., isourea) on the cryogel surface were blocked by the treatment with ethanol amine (pH 9.1) and FeCl3 solution for 1 h. Then, the CNBr-activated PHEMA cryogel was washed four times with distilled water containing 0.5 M NaCl. In the last stage the cryogel was washed with cold sodium citrate buffer (0.1 M; pH 6.5).
Protein A (from S. aureus, Cowan Strain I; Product No.: P6031), immunoglobulin G (IgG), N,N -methylene-bis(acrylamide) (MBAAm) and ammonium persulfate (APS) were supplied by Sigma (St. Louis, MO, USA). N,N,N ,N -tetramethylene diamine (TEMED) was obtained from Fluka A.G. (Buchs, Switzerland). All other chemicals were of reagent grade and were purchased from Merck AG (Darmstadt, Germany). All water used in the adsorption experiments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure® organic/colloid removal and ion exchange packed-bed system. Laboratory glassware was kept overnight in a 5% nitric acid solution. Before use all the glassware was rinsed with deionised water and dried in a dust-free environment. 2.2. Preparation of PHEMA cryogel Preparation of the PHEMA cryogel is described below. Briefly, monomers (1.6 ml HEMA and 0.3 g N,N -methylene-bis(acrylamide) (MBAAm) were dissolved in deionised water (5 ml) and the mixture was degassed under vacuum for 5 min to eliminate soluble oxygen. Total concentration of monomers was 12% (w/v). The cryogel was produced by free radical polymerization initiated by TEMED and APS. After adding APS (25 mg, 1% (w/v) of the total monomers) the solution was cooled in an ice bath for 2–3 min. TEMED (20 L, 1% (w/v) of the total monomers) was added and the reaction mixture was stirred for 1 min. Then, the reaction mixture was poured into a plastic syringe (5 ml, id. 0.8 cm) with closed outlet at the bottom. The polymerization solution in the syringe was frozen at −16 ◦ C for 24 h and then thawed at room temperature. After washing with 200 ml of water, the cryogel was stored in buffer containing 0.02% sodium azide (NaN3 ) at 4 ◦ C until use. 2.3. Characterization of PHEMA cryogel The swelling degree of the cryogel (S) was determined as follows: cryogel was washed with water until washing water was clear. Then it was sucked dry and then transferred to pre-weighed vial and weighed (mwet gel ). After drying to constant mass in the oven at 60 ◦ C, the mass of dried sample was determined (mdry gel ). The swelling degree was calculated as: S=
mwet gel − mdry gel mdry gel
(1)
The morphology of a cross-section of the cryogel was investigated by scanning electron microscope (SEM). The sample was fixed in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer overnight, post-fixed in 1% osmium tetroxide for 1 h. Then the sample was dehydrated stepwise in ethanol and transferred to a critical point drier temperated to 10 ◦ C where the ethanol was changed for liquid carbon dioxide as transitional fluid. The temperature was then raised to 40 ◦ C and the pressure to ca. 100 bar. Liquid CO2 was transformed directly to gas uniformly throughout the whole sample without heat of vaporization or surface tension forces causing damage. Release of the pressure at a constant temperature of 40 ◦ C resulted in dried cryogel. Finally, it was coated with gold–palladium (40:60) and examined using a JEOL JSM 5600 scanning electron microscope.
2.5. Protein A attachment Protein A attachment was carried out in a recirculating system equipped with a water jacket glass column for temperature control. The cryogel was washed with 100 ml of water. After washing procedure, the cryogel was treated with 50 ml of carbonate buffer (pH 10) at 1.0 ml/min for 1 h. Then, 50 ml of protein A solution (2.0 mg/ml, pH 6.5) was pumped through the column under recirculation at 1.0 ml/min for 2 h. The amount of protein A attachment on the cryogel was determined by measuring the decrease of protein A concentration and also by considering the protein A adsorbed nonspecifically (amount of protein A adsorbed on the PHEMA cryogel), by Lowry method. The protein sample (200–400 l) was diluted to 1.0 ml with phosphate buffered saline (PBS, 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4 , 8.1 mM Na2 HPO4 ). Thereafter, 1.0 ml of a freshly prepared Lowry reagent was added. After 30 min incubation at room temperature, 500 l of freshly prepared Folin-Ciocalteu’s reagent was added and the solution was mixed using a vortex. The blank solution was prepared analogously as the protein sample using 200–400 l of a 3.0% sodium dodecyl sulfate solution in PBS instead of the protein solution. After 30 min incubation at room temperature, the absorbance of the protein sample was measured at 730 nm against the blank solution. A calibration curve was established using protein A with known concentration to relate the protein concentration in the solution with the absorbance of the sample. The leakage of protein A from the cryogel was followed by treating the cryogel with PBS for 24 h at room temperature. Protein A released after this treatment was measured in the liquid phase spectrophotometrically at 280 nm. 2.6. IgG adsorption from aqueous solutions IgG adsorption studies were also performed in a glass column equipped with a water jacket for temperature control. The cryogel was washed with 50 ml of water and then equilibrated with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). Then, IgG solution was pumped through the column. In a typical adsorption system, IgG solution was passed through the cryogel, by a peristaltic pump. Dynamic adsorption capacity was calculated from IgG breakthrough curves. The adsorption was followed by monitoring the decrease in UV absorbance at 280 nm. Effects of flow-rate, initial concentration of IgG and temperature of the medium on the adsorption capacity were studied. The flow-rate of the solution was in the
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range of 0.5–4.0 ml/min. To observe the effects of the IgG concentration on adsorption, it was changed between 0.1 and 2.0 mg/ml. To determine the effect of temperature on the adsorption, temperature of the solution was changed between 4 and 37 ◦ C. 2.7. IgG purification from human plasma Human blood is collected from thoroughly controlled voluntary blood donors. Each unit separately controlled and found negative for hepatitis B surface antigen and HIV I, II and hepatitis C antibodies. No preservatives are added to the blood samples. Human blood was collected into EDTA-containing vacutainers and frozen at −20 ◦ C. Before application, the blood sample was diluted with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). Dilution ratios were 1/2 and 1/10. Human blood (50 ml) with a IgG content of 14.8 mg/ml was pumped through the cryogel at a flow-rate of 1.0 ml/min for 1 h. The amount of IgG adsorbed on the PHEMA cryogel was determined by ELISA. Human anti-IgG (Sigma, I-9384) diluted 1/1000 in 50 mM NaHCO3 , pH 9.6, was adsorbed to PVC microtitre plates at 4 ◦ C for 12 h. The plates were washed with PBS containing 0.05% Tween 20 (wash buffer) and blocked with PBS containing 0.05% Tween 20, 1.5% bovine serum albumin, and 0.1% sodium azide (blocking buffer). Samples (2.5 ml, neutralized with 0.5 ml of 1.0 M trisodium citrate) or controls containing known amounts of IgG were added and incubated at 37 ◦ C for 1 h. Attached IgG was detected with the anti-IgG labeled with biotin followed by peroxidase-conjugated streptavidin and o-phenylenediamine. The absorbance was measured at 492 nm. SDS-PAGE analysis of the serum samples was performed on 10% separating mini gels (9 cm × 7.5 cm) for 120 min at 200 V. Stacking gels (5%) were stained with 0.25% (w/v). Coomassie Brillant R 250 in acetic acid–methanol–water mixture (1:5:5, v/v/v) and destained in ethanol–acetic acid–water mixture (1:4:6, v/v/v). The SDS-PAGE gels were scanned using a Shimadzu dual-wavelength flying spot scanning densitometer (Shimadzu, Tokyo, Japan). 2.8. FPLC studies The selectivity of PHEMA–protein A cryogel (diameter: 1.0 cm, length: 5.0 cm) with respect to the competitive proteins was also applied by utilizing Fast Protein Liquid Chromatography (FPLC). FPLC separation was performed using an AKTA-FPLC (Amersham Bioscience, Uppsala, Sweden) system equipped with a UV detection system. The system includes M-925 mixer, P-920 pump, UPC-900 monitor, INV-907 injection valve and Frac920 fraction collector. Separation was carried out at GE Healthcare column (10/10, 195001-01) with PHEMA–protein A cryogel. FPLC mobile phases A and B were prepared using 25 mM phosphate buffer (pH 7.4) and 0.1 M glycine–HCl buffer (pH 3.5), respectively. The chromatographic separation was performed using a linear gradient at 3 ml/min flow-rate. After a 7-min starting period with 100% mobile phase A, a linear gradient started from 0% B to 100% B in 1 min, continued with 5 min 100% eluent B and finished last 8 min 100% buffer A. All buffers and protein solutions were filtered before use. 2 ml of protein mixture was applied to the column. Absorbance was monitored at 280 nm. The separation was performed at room temperature. KBr was used as the void marker. Capacity factor (k ) and separation factor (˛) were calculated as k = (tR − to )/to , ˛ = k2 /k1 , where tR is the retention time of the protein and to is the retention time of the void marker (KBr), k2 is the capacity factor for IgG and k1 is the capacity factor for competitive protein (i.e., human serum albumin). The resolution (Rs ) and theoretical plate numbers (N) were calculated using the following equations:
t 2 R
N = 5.54
w0.5
(2)
RS =
2(tR,2 − tR,1 ) (w2 + w1 )
203
(3)
where w0.5 is the peak width at the corresponding peak height fraction, tR,1 and tR,2 are the retention times of two adjacent peaks, w1 and w2 are the widths of the two adjacent peaks at the baseline. 2.9. Desorption and repeated use Adsorbed IgG was desorbed using glycine–HCl elution buffer at pH 3.5. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the cryogel column at a flow-rate of 1.0 ml/min for 1 h. IgG concentration in the desorption medium was determined spectroscopically at 280 nm. After desorption, the cryogel was cleaned with 50 mM sodium hydroxide and then reequilibrated with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). The desorption ratio was calculated from the amount of IgG adsorbed on the cryogel and the final IgG concentration in the desorption medium. In order to test reuse of the PHEMA cryogel, IgG adsorption–desorption cycle was repeated for 10 times using the same cryogel. In order to regenerate and sterilize, after desorption, the PHEMA cryogel was washed with 50 mM NaOH solution. 3. Results and discussion A supermacroporous PHEMA cryogel was produced by copolymerization in the frozen state of HEMA with MBAAm in the presence of APS/TEMED. The hydroxyl groups on the PHEMA cryogel allowed modification with protein A. The SEM images of the pore structure of the cryogel are shown in Fig. 1. PHEMA cryogel have non-porous and thin polymer walls, large continuous interconnected pores (10–200 m in diameter) that provide channels for the mobile phase to flow through. Pore size of the matrix is much larger than the size of the protein molecules, allowing them to pass easily. As a result of the convective flow of the mobile phase through the pores, the mass transfer resistance is practically negligible. The equilibrium swelling degree of the PHEMA cryogel was 8.56 g H2 O/g cryogel. PHEMA cryogel is opaque, sponge like and elastic. This cryogel can be easily compressed by hand to remove water accumulated inside the pores. When the compressed piece of cryogel was submerged in water, it soaked in water and within 1–2 s restored its original size and shape. The pressure drop needed to drive the liquid through any system should be as low as possible. Pressure drop experiments through PHEMA cryogel column were performed in water as equilibration medium, and at linear flow-rates from 40 to 382 cm/h. The water was passed through the column for 1 min at each flow-rate. Due to the presence of large and highly interconnected macropores, the PHEMA cryogel column has very low liquid flow resistance. PHEMA cryogel column had low back pressure indicating the porous structure with large size of the interconnected macropores (Fig. 2). 3.1. Adsorption of IgG from aqueous solutions 3.1.1. Effect of flow-rate Fig. 3 shows that the adsorbed amount of IgG onto the PHEMA–protein A cryogel decreased when the flow-rate through the column increased. The adsorption capacity decreased from 83.2 to 58.9 mg/g polymer with the increase of the flow-rate from 0.5 to 4.0 ml/min. An increase in the flow-rate reduces the protein solution volume treated efficiently until breakthrough point and therefore decreases the retention time of the cryogel. When the flow-rate decreases the contact time in the column is longer. Thus, IgG molecules have more time to diffuse to the pore walls of the
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Fig. 3. Effect of flow-rate on IgG adsorption: protein A loading: 56 mg/g; IgG concentration: 2.0 mg/ml; adsorbing buffer PBS (pH 7.4); T: 25 ◦ C.
tively constant. In addition, the macropores in the cryogel structure are open and highly interconnected, forming a network of large channel. The mobile IgG phase is forced to flow through them, transporting the IgG molecules to the active attachment sites (i.e., protein A molecules) by convection. This result is an extremely fast mass exchange between the mobile IgG phase and the stationary PHEMA cryogel phase than the more traditional packed bed columns [26,27].
Fig. 1. SEM images of the PHEMA cryogel.
cryogel and to attach to the protein A, hence a better IgG adsorption capacity is obtained. In addition, for column operation the cryogel is continuously in contact with a fresh IgG solution. Consequently the IgG concentration in the solution in contact with a given layer of PHEMA–protein A cryogel in a column is rela-
3.1.2. Effect of protein A density Table 1 shows the effect of protein A density on IgG adsorption onto PHEMA cryogel. The decrease in surface area due to the supermacropores in cryogel structure was compensated by an increase in ligand density. When the surface protein A density (i.e., the number of protein A molecules per unit mass) increased the amount of IgG adsorbed onto PHEMA cryogel, first, increased and then reached almost a highest value. This maximum IgG adsorption capacity was 83.2 mg/g. With the increasing protein A density, the higher IgG adsorption is expected. But this may not be advantageous in all cases due to possible geometric (i.e., steric) effects. Protein A contains a tandem of five similar domains, each capable of binding the Fc region of immunoglobulin G (IgG) from various mammals. Each molecule of soluble protein A is able to bind two molecules of IgG. Steric hindrance prevents the binding of more than one or two IgG molecules to the immobilized protein A molecule. When higher protein A density is achieved, these receptors may not be accessible by IgG molecules because of the steric hindrances. Table 1 gives also the weight ratio of IgG/protein A adsorbed onto PHEMA cryogel containing different protein A density. This ratio is around 2.0 for all studied protein A surface density. This range is an agreement with values cited in the literature. Protein A–IgG binding stoichiometries of 1:2 were obtained using sedimentation data Table 1 Effect of protein A density on IgG adsorption: IgG concentration: 2.0 mg/ml; adsorbing buffer PBS (pH 7.4); T: 25 ◦ C.
Fig. 2. Pressure drop at different flow-rates.
Amount of protein A immobilized (mg/g)
Amount of IgG adsorbed (mg/g)
Weight ratio of IgG/protein A
28.7 29.8 56.0 26.9 25.2
49.3 52.9 83.2 38.9 33.5
1.72 1.77 1.49 1.44 1.32
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Table 2 Adsorption constants of Langmuir and Freundlich isotherms. Langmuir adsorption isotherm
Freundlich adsorption isotherm
Qmax = 102.1 mg/g b = 1.58 ml/mg R2 = 0.989
KF = 56.1 n = 1.98 R2 = 0.894
as structure, reactive functional groups, ligand loading amount, porosity, pore size, pore size distribution and accessible surface area.
Fig. 4. Effect of IgG concentration on IgG adsorption: protein A loading: 56 mg/g; flow-rate: 0.5 ml/min; pH: 7.4; T: 25 ◦ C.
and surface tension measurements [28,29]. In addition the binding ratio for a human IgG to protein A has been reported to be 3.3:1 [30]. 3.1.3. Adsorption isotherm Fig. 4 shows the adsorption data on PHEMA and PHEMA–protein A cryogel. The non-specific IgG adsorption on PHEMA cryogel was very low (about 0.38 mg/g). Since PHEMA cryogel retained water at the ratio of 8.56 g of water per gram of dry PHEMA, wet PHEMA cryogel is highly hydrophilic. Thus, PHEMA cryogel has to the advantage that IgG will not adsorb on it non-selectively. While specific adsorption (i.e., adsorption of IgG molecules onto the PHEMA cryogel through protein A molecules) was significant (up to 83.2 mg IgG/g), and increased with increasing IgG concentration. The adsorbed amount of IgG on the PHEMA cryogel via attached protein A molecules reached almost a plateau value around 1.5 mg/ml, due to the saturation of active attachment sites. Two important physico-chemical aspects for evaluation of the adsorption process as a unit operation are the kinetics and the equilibrium of adsorption. Equilibrium modelling data has been done using the Langmuir and Freundlich isotherms. The Langmuir and Freundlich isotherms are represented as follows Eqs. (4) and (5), respectively:
1 1 1 = + qe qmax qmax b ln qe =
1
1 (ln Ce ) + ln KF n
Ce
(4) (5)
where b is the Langmuir isotherm constant, KF is the Freundlich constant, and n is the Freundlich exponent. 1/n is a measure of the surface heterogeneity ranging between 0 and 1, becoming more heterogeneous as its value gets closer to zero. The ratio of qe gives the theoretical monolayer saturation capacity of PHEMA cryogel. Some model parameters are determined by nonlinear regression with commercially available software. The equilibrium data have been fitted with the Langmuir model and the relevant values are given in Table 2. A comparison of the maximum adsorption capacity, qmax , of the PHEMA cryogel with those of some other affinity adsorbents including commercially available adsorbents reported in literature is given in Table 3. The adsorption capacity of PHEMA–protein A was comparable with other adsorbents. Differences of IgG adsorption capacities are due to the properties of each adsorbent such
3.1.4. Effect of temperature The effect of temperature on IgG adsorption capacity of the PHEMA–protein A affinity cryogel was shown in Fig. 5. Non-specific adsorption of IgG (0.38 mg/g) due to van der Waals force was very low at all temperatures. No significant effect of temperature was observed on the physical adsorption of IgG onto the PHEMA cryogel. However, the equilibrium adsorption of IgG on the PHEMA–protein A cryogel significantly increased with increasing temperature and the maximum IgG adsorption was observed at 37 ◦ C (98.8 mg/g). From 4 to 37 ◦ C, the adsorption capacity increased about 125% for the PHEMA/protein A cryogel. This shows that IgG binding interaction of the PHEMA/protein A cryogel is not entropy driven as would be expected with hydrophobic interactions and is consistent with previous results [23]. Hydrophobic forces are dominant in protein A–IgG interactions. Hence, increasing the temperature enhances IgG adsorption and lowering the temperature generally promotes the protein elution. Van der waals attraction forces, which operate in hydrophobic interactions increase with increasing temperature. 3.2. IgG adsorption from human plasma Table 4 shows the IgG adsorption values from human plasma. As seen here, lower adsorption capacity of IgG was obtained for human Table 3 Comparison of the adsorption capacities for IgG of various adsorbents. Adsorbent
Ligand
Qmax (mg/g)
Reference
Cellulose Cellulose nanofiber Polymethylmethacrylate PHEMA PHEMA Eupergit, affigel PHEMA PHEMA Poly(caprolactam) Poly(ethylene) Sepharose 4B Poly(ethylene vinyl alcohol) Polysulfone Sartobind Polymethylmethacrylate Poly(vinyl alcohol) Sepharose 6B
Protein A Protein A Cu2+ l-Histidine Methacryloylamidohistidine Protein A Cu2+ , Ni2+ , Zn2+ , Co2+ Protein A Protein A Phenylalanine l-Histidine l-Histidine Protein A Protein A Protein A/G Protein A 3-Aminophenol 4-amino-1-naphthol Concanavalin A Biomimetic ligand Biomimetic ligand 4-Mercapto ethyl pyridine Mercapto methyl imidazole 2-Mercapto-5benzimidazole sulfonic acid Concanavalin A Bicyclic heteroatomic ligand Protein A
11.7 18.0 54.3 44.8 73.8 20.1 79.6 24.0 28.3 50.0 0.23 77.7 8.8 0.51 6.6 13.2 52.0
[1] [11] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
69.4 7.0 25.0 30 16 30
[43] [44] [45] [46] [47] [48]
25.6 29.2 88.1
[49] [50] In this study
PHEMA Sepharose CL 6B Sepharose 4B Cellulose Poly(ethylene vinyl alcohol) Cellulose
Poly(AAm-AGE) Agarose PHEMA
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Fig. 5. Effect of temperature on adsorption capacity: protein A loading: 56 mg/g; IgG concentration: 2.0 mg/ml; flow-rate: 0.5 ml/min; adsorbing buffer PBS.
blood diluted with PBS buffer. But, there was a significant adsorption of IgG (up to 88.1 mg/g) on the PHEMA–protein A cryogel for non-diluted blood. The composition of bound and eluted protein was assayed by SDS-PAGE. About 85% of the protein was IgG indicating high specificity of the macroporous adsorbent even in the presence of large amount of other proteins like albumin. The purity of IgG eluted from PHEMA–protein A cryogel was assayed by SDS-PAGE as given in Section 2. As clearly seen in Fig. 6, IgG in serum (Lane 2) was almost disappeared in Lane 3 after adsorption onto PHEMA–protein A cryogel. Furthermore, the presence of only band at Lane 4 indicates the purity of IgG after desorption of PHEMA–protein A cryogel. The purity of IgG obtained was found to be in the range of 95.3–96.7%. The recovery of IgG from human plasma was 80.7%. In order to show the superiority of cryogel column for direct affinity purification, an experiment on whole blood flowing through the PHEMA–protein A cryogel column was performed. The supermacroporous structure of cryogels allows for direct processing of whole blood containing blood cells [19]. Blood was collected in EDTA-anticoagulation tubes. Non-coagulated blood (2 ml) was run over the column at 1.0 ml/min and washed with 50 ml isotonic buffer. The flow of the blood plug on the PHEMA–protein A cryogel column was monitored visually and photographed at intermediate times during the process. Blood is a complex mixture of cells with different sizes from 2 to 20 m. The pulse of blood applied on a PHEMA–protein A cryogel column under isotonic conditions passes through the column as a homogeneous plug without substantial tailing (Fig. 7).
Fig. 6. SDS-PAGE of serum fractions. The fractions were assayed by SDS-PAGE using 10% separating gel (9 cm × 7.5 cm), and 5% stacking gels were stained with 0.25% (w/v) Coomassie Brillant R 250 in acetic acid–methanol–water (1:5:5, v/v/v) and destained in ethanol–acetic acid–water (1:4:6, v/v/v). Lane 1, biomarker (Sigma); Lane 2, 1/10 diluted serum; Lane 3, 1/10 diluted serum after adsorption; Lane 4, eluted sample. Equal amounts of samples were applied to each line.
3.3. FPLC studies As shown in Fig. 8, HSA and IgG separation from protein mixture was observed at 1.94 and 9.58 min, respectively. As seen in Fig. 9, HSA and IgG separation from human plasma was observed at 1.89 and 9.54 min, respectively. The tR , N, k , a, Rs values are given in Table 4 IgG adsorption from human plasma: IgG concentration before dilution:14.8 mg/ml. Dilution agent
Adsorption capacity (mg/g)
Non-diluted plasma Diluted plasma (dilution ratio: 1/2, PBS pH: 7.4) Diluted plasma (dilution ratio: 1/10, PBS pH: 7.4)
88.1 ± 0.97 63.9 ± 1.20 9.4 ± 0.45
Fig. 7. Flow pattern of whole blood through a cryogel column. 2 ml of blood was applied to the cryogel column at a flow-rate of 1.0 ml/min in isotonic buffer solution. Column 1: column before application; column 2–6: column during processing; column 7: column after the flow of blood sample.
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Fig. 8. FPLC separation of HSA and IgG from protein mixture on a column with PHEMA–protein A cryogel: flow-rate: 3.0 ml/min; protein concentration: 0.5 mg/ml; detection was performed at 280 nm.
Fig. 10. Reusability of the PHEMA–protein A cryogel: protein A loading: 56 mg/g; flow-rate: 0.5 ml/min; IgG concentration: 2.0 mg/ml; adsorbing buffer PBS (pH 7.4); T: 25 ◦ C.
tain their adsorption capacity at almost constant value. Repeated separations did not show a decrease of separation performance. The PHEMA–protein A cryogel presented a good performance regarding reusability and stability. 4. Conclusion
Fig. 9. FPLC separation of HSA and IgG from human serum on a column with PHEMA–protein A cryogel: flow-rate: 3.0 ml/min; detection was performed at 280 nm.
Table 5. Rs values were calculated as 2.28 and 2.63 for binary protein mixture and human plasma, respectively. Because the Rs value should be higher than 1.0 for a good resolution of two peaks in such a chromatography system, the results for the resolution of IgG and HSA can be accepted as good resolution values. 3.4. Reusability of the affinity cryogel Reusability is very important for affinity adsorbent. This means that in designing a test system, attention must be paid to the chemical stability of the affinity adsorbent [51–53]. In order to determine the stability and reusability of the PHEMA–protein A cryogel, the cryogel column has been used up to 10 cycles. For sterilization after one adsorption–desorption cycle, the PHEMA cryogel was washed with 50 mM NaOH solution for 30 min. After this procedure, cryogel was washed with distilled water for 30 min, then equilibrated with the phosphate buffer at pH 7.4 for the next adsorption–desorption cycle. The results showed that about 95% of the adsorbed IgG were desorbed. As seen in Fig. 10 that the cryogel is very stable and mainTable 5 Chromatographic separation data. tR From protein mixture IgG 9.58 HSA 1.94 From human serum IgG 9.54 HSA 1.89
N
k
˛
Rs
130.35 10.95
6.85 0.59
– 11.61
– 2.28
164.64 14.71
6.82 0.55
– 12.4
– 2.63
Recently, a growing interest has been shown in using cryogels as adsorbents for diverse applications including protein purification and biomedical therapy [14,27,54–56]. In this article, the performance of a new affinity poly(hydroxyethyl methacrylate) cryogel carrying protein A for the purification of IgG has been investigated in detail. PHEMA cryogel present potentially attractive chromatographic medium, as it has low flow resistance. This cryogel are endowed with a very good selectivity for IgG and an interesting adsorption capacity, significantly improved with respect to the adsorbents including commercially available benchmark. The results obtained are very encouraging and show that PHEMA cryogel can be considered as a potential candidate for IgG purification. Acknowledgement We are greatful to DUBAP-07-01-31 for their financial support. References [1] C. Boi, S. Dimartino, G.C. Sarti, Biotechnol. Prog. 24 (2008) 640. [2] M.B. Ribeiro, M. Vijayalakshmi, D. Todorova-Balvay, S.M.A. Bueno, J. Chromatogr. B 861 (2008) 64. [3] S. Özkara, S. Akgöl, Y. Canak, A. Denizli, Biotechnol. Prog. 20 (2004) 1169. [4] G. Tishchenko, J. Dybal, K. Meszarosova, Z. Sedlakova, M. Bleha, J. Chromatogr. A 954 (2002) 115. [5] S. Özkara, B. Garipcan, E. Pis¸kin, A. Denizli, J. Biomater. Sci. Polym. Ed. 14 (2003) 761. [6] Y. Canak, S. Özkara, S. Akgöl, A. Denizli, React. Funct. Polym. 61 (2004) 369. [7] D. Türkmen, N. Öztürk, S. Akgöl, A. Elkak, A. Denizli, Biotechnol. Prog. 24 (2008) 1297. [8] H. Yavuz, A. Denizli, Macromol. Biosci. 5 (2005) 38. [9] V.I. Muronetz, T. Korpela, J. Chromatogr. B 790 (2003) 53. [10] J.F. Stotz, C. Rivat, C. Geschier, P. Colosetti, F. Streiff, Swiss Biotech. 8 (1990) 8. [11] Z. Ma, S. Ramakrishna, J. Membr. Sci. 319 (2008) 23. [12] K. Thommes, M.R. Kula, Biotechnol. Prog. 11 (1995) 357. [13] T.B. Tennikova, B. Reusch, J. Chromatogr. A 1065 (2005) 13. [14] N. Bereli, M. Andac¸, G. Baydemir, R. Say, I.Y. Galaev, A. Denizli, J. Chromatogr. A 1190 (2008) 18. [15] L. Uzun, R. Say, A. Denizli, React. Funct. Polym. 64 (2005) 93.
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Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel Hüseyin Alkan a , Nilay Bereli b , Zübeyde Baysal a , Adil Denizli b,∗ a b
Department of Chemistry, Dicle University, Diyarbakir, Turkey Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 15 January 2009 Received in revised form 10 March 2009 Accepted 27 March 2009 Keywords: Protein A Cryogels PHEMA Antibody purification IgG
a b s t r a c t Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 m size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Human immunoglobulin G (IgG) is an important plasma protein with many applications in therapeutics, immunodiagnostics and immunochromatography [1]. These applications generally require highly pure IgG [2]. For medical applications, immunoglobulins (IgGs) have been thoroughly purified using a combination of various methods, mainly precipitation and chromatographic techniques including ion exchange, hydrophobic interaction, histidine affinity, metal-chelate affinity and dye-affinity [3–8]. Fractionation by ethanol precipitation is the most used process to purify IgG from human plasma on industrial scale (27 ton/year) [9]. But this technique is not specific and can give partially denatured proteins [10]. Conventional chromatography technique uses gel beads column chromatography, which have certain limitations such as a high pressure drop, low flow-rates and hence low productivities and difficulties in efficient scale up [11]. It has also a major limitation; incapability of highly viscous fluids, for example human
∗ Corresponding author at: P.K. 1, Samanpazarı, 06242, Ankara, Turkey. Tel.: +90 312 2992163; fax: +90 312 2992163. E-mail address: [email protected] (A. Denizli). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.03.013
blood. The most obvious progress has been made in the development of alternative chromatography formats such as membrane adsorbents, monoliths and cryogels [12–15]. Cryogels provides a potential solution due to their low pressure drop and lack of diffusion resistances due to the macropores compared to traditional column chromatography [16–18]. Cryogels allow high flow-rates enabling the processing of large volumes within short process times. Whole blood can be applied to cryogels without any pretreatment [19]. Cryogels are also cheap materials and they can be used as disposable avoiding cross-contamination between batches [20]. Protein A is a cell wall associated protein domain exposed on the surface of the Gram-positive bacterium Staphylococcus aureus [21]. Immunoglobulin G (IgG) binds protein A via its Fc region [22]. The interaction appears to be characterized by hydrophobic interaction together with some hydrogen bonds and salt bridges [23,24]. Protein A consists of five homologous IgG binding domains [25]. Each of five domains in protein A is arranged in an anti-parallel there ␣-helical bundle and the three-dimensional structure is stabilized via a hydrophobic core [21]. In this study, we used poly(hydroxyethyl methacrylate) [PHEMA] cryogel carrying protein A as a model supermacroporous monolithic matrix capable of purification of IgG from human plasma.
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2. Experimental
2.4. CNBr activation
2.1. Materials
PHEMA cryogel was activated with cyanogen bromide in order to prepare active attachment sites for protein A. Prior to activation process, PHEMA cryogel was kept in distilled water for about 24 h and washed with 0.5 M NaCl solution and water. 2 ml of 0.5 M sodium carbonate buffer (pH 10.5) was added and stirred slowly. The mixture was placed in a fume hood and the glass pH electrode was immersed into this solution. CNBr was weighed carefully and added to the solution. The pH of this solution was quickly adjusted to 11.5 with 4 M NaOH and the pH was maintained between 10.5 and 11.5 during the activation reaction. The CNBr solution was recirculated through the column at 1.0 ml/min at room temperature (25 ◦ C) and activation procedure was continued for 60 min. After the activation reaction, in order to remove the excess activation agent, the PHEMA cryogel was washed with 0.1 M NaHCO3 and any remaining active groups (e.g., isourea) on the cryogel surface were blocked by the treatment with ethanol amine (pH 9.1) and FeCl3 solution for 1 h. Then, the CNBr-activated PHEMA cryogel was washed four times with distilled water containing 0.5 M NaCl. In the last stage the cryogel was washed with cold sodium citrate buffer (0.1 M; pH 6.5).
Protein A (from S. aureus, Cowan Strain I; Product No.: P6031), immunoglobulin G (IgG), N,N -methylene-bis(acrylamide) (MBAAm) and ammonium persulfate (APS) were supplied by Sigma (St. Louis, MO, USA). N,N,N ,N -tetramethylene diamine (TEMED) was obtained from Fluka A.G. (Buchs, Switzerland). All other chemicals were of reagent grade and were purchased from Merck AG (Darmstadt, Germany). All water used in the adsorption experiments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure® organic/colloid removal and ion exchange packed-bed system. Laboratory glassware was kept overnight in a 5% nitric acid solution. Before use all the glassware was rinsed with deionised water and dried in a dust-free environment. 2.2. Preparation of PHEMA cryogel Preparation of the PHEMA cryogel is described below. Briefly, monomers (1.6 ml HEMA and 0.3 g N,N -methylene-bis(acrylamide) (MBAAm) were dissolved in deionised water (5 ml) and the mixture was degassed under vacuum for 5 min to eliminate soluble oxygen. Total concentration of monomers was 12% (w/v). The cryogel was produced by free radical polymerization initiated by TEMED and APS. After adding APS (25 mg, 1% (w/v) of the total monomers) the solution was cooled in an ice bath for 2–3 min. TEMED (20 L, 1% (w/v) of the total monomers) was added and the reaction mixture was stirred for 1 min. Then, the reaction mixture was poured into a plastic syringe (5 ml, id. 0.8 cm) with closed outlet at the bottom. The polymerization solution in the syringe was frozen at −16 ◦ C for 24 h and then thawed at room temperature. After washing with 200 ml of water, the cryogel was stored in buffer containing 0.02% sodium azide (NaN3 ) at 4 ◦ C until use. 2.3. Characterization of PHEMA cryogel The swelling degree of the cryogel (S) was determined as follows: cryogel was washed with water until washing water was clear. Then it was sucked dry and then transferred to pre-weighed vial and weighed (mwet gel ). After drying to constant mass in the oven at 60 ◦ C, the mass of dried sample was determined (mdry gel ). The swelling degree was calculated as: S=
mwet gel − mdry gel mdry gel
(1)
The morphology of a cross-section of the cryogel was investigated by scanning electron microscope (SEM). The sample was fixed in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer overnight, post-fixed in 1% osmium tetroxide for 1 h. Then the sample was dehydrated stepwise in ethanol and transferred to a critical point drier temperated to 10 ◦ C where the ethanol was changed for liquid carbon dioxide as transitional fluid. The temperature was then raised to 40 ◦ C and the pressure to ca. 100 bar. Liquid CO2 was transformed directly to gas uniformly throughout the whole sample without heat of vaporization or surface tension forces causing damage. Release of the pressure at a constant temperature of 40 ◦ C resulted in dried cryogel. Finally, it was coated with gold–palladium (40:60) and examined using a JEOL JSM 5600 scanning electron microscope.
2.5. Protein A attachment Protein A attachment was carried out in a recirculating system equipped with a water jacket glass column for temperature control. The cryogel was washed with 100 ml of water. After washing procedure, the cryogel was treated with 50 ml of carbonate buffer (pH 10) at 1.0 ml/min for 1 h. Then, 50 ml of protein A solution (2.0 mg/ml, pH 6.5) was pumped through the column under recirculation at 1.0 ml/min for 2 h. The amount of protein A attachment on the cryogel was determined by measuring the decrease of protein A concentration and also by considering the protein A adsorbed nonspecifically (amount of protein A adsorbed on the PHEMA cryogel), by Lowry method. The protein sample (200–400 l) was diluted to 1.0 ml with phosphate buffered saline (PBS, 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4 , 8.1 mM Na2 HPO4 ). Thereafter, 1.0 ml of a freshly prepared Lowry reagent was added. After 30 min incubation at room temperature, 500 l of freshly prepared Folin-Ciocalteu’s reagent was added and the solution was mixed using a vortex. The blank solution was prepared analogously as the protein sample using 200–400 l of a 3.0% sodium dodecyl sulfate solution in PBS instead of the protein solution. After 30 min incubation at room temperature, the absorbance of the protein sample was measured at 730 nm against the blank solution. A calibration curve was established using protein A with known concentration to relate the protein concentration in the solution with the absorbance of the sample. The leakage of protein A from the cryogel was followed by treating the cryogel with PBS for 24 h at room temperature. Protein A released after this treatment was measured in the liquid phase spectrophotometrically at 280 nm. 2.6. IgG adsorption from aqueous solutions IgG adsorption studies were also performed in a glass column equipped with a water jacket for temperature control. The cryogel was washed with 50 ml of water and then equilibrated with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). Then, IgG solution was pumped through the column. In a typical adsorption system, IgG solution was passed through the cryogel, by a peristaltic pump. Dynamic adsorption capacity was calculated from IgG breakthrough curves. The adsorption was followed by monitoring the decrease in UV absorbance at 280 nm. Effects of flow-rate, initial concentration of IgG and temperature of the medium on the adsorption capacity were studied. The flow-rate of the solution was in the
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range of 0.5–4.0 ml/min. To observe the effects of the IgG concentration on adsorption, it was changed between 0.1 and 2.0 mg/ml. To determine the effect of temperature on the adsorption, temperature of the solution was changed between 4 and 37 ◦ C. 2.7. IgG purification from human plasma Human blood is collected from thoroughly controlled voluntary blood donors. Each unit separately controlled and found negative for hepatitis B surface antigen and HIV I, II and hepatitis C antibodies. No preservatives are added to the blood samples. Human blood was collected into EDTA-containing vacutainers and frozen at −20 ◦ C. Before application, the blood sample was diluted with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). Dilution ratios were 1/2 and 1/10. Human blood (50 ml) with a IgG content of 14.8 mg/ml was pumped through the cryogel at a flow-rate of 1.0 ml/min for 1 h. The amount of IgG adsorbed on the PHEMA cryogel was determined by ELISA. Human anti-IgG (Sigma, I-9384) diluted 1/1000 in 50 mM NaHCO3 , pH 9.6, was adsorbed to PVC microtitre plates at 4 ◦ C for 12 h. The plates were washed with PBS containing 0.05% Tween 20 (wash buffer) and blocked with PBS containing 0.05% Tween 20, 1.5% bovine serum albumin, and 0.1% sodium azide (blocking buffer). Samples (2.5 ml, neutralized with 0.5 ml of 1.0 M trisodium citrate) or controls containing known amounts of IgG were added and incubated at 37 ◦ C for 1 h. Attached IgG was detected with the anti-IgG labeled with biotin followed by peroxidase-conjugated streptavidin and o-phenylenediamine. The absorbance was measured at 492 nm. SDS-PAGE analysis of the serum samples was performed on 10% separating mini gels (9 cm × 7.5 cm) for 120 min at 200 V. Stacking gels (5%) were stained with 0.25% (w/v). Coomassie Brillant R 250 in acetic acid–methanol–water mixture (1:5:5, v/v/v) and destained in ethanol–acetic acid–water mixture (1:4:6, v/v/v). The SDS-PAGE gels were scanned using a Shimadzu dual-wavelength flying spot scanning densitometer (Shimadzu, Tokyo, Japan). 2.8. FPLC studies The selectivity of PHEMA–protein A cryogel (diameter: 1.0 cm, length: 5.0 cm) with respect to the competitive proteins was also applied by utilizing Fast Protein Liquid Chromatography (FPLC). FPLC separation was performed using an AKTA-FPLC (Amersham Bioscience, Uppsala, Sweden) system equipped with a UV detection system. The system includes M-925 mixer, P-920 pump, UPC-900 monitor, INV-907 injection valve and Frac920 fraction collector. Separation was carried out at GE Healthcare column (10/10, 195001-01) with PHEMA–protein A cryogel. FPLC mobile phases A and B were prepared using 25 mM phosphate buffer (pH 7.4) and 0.1 M glycine–HCl buffer (pH 3.5), respectively. The chromatographic separation was performed using a linear gradient at 3 ml/min flow-rate. After a 7-min starting period with 100% mobile phase A, a linear gradient started from 0% B to 100% B in 1 min, continued with 5 min 100% eluent B and finished last 8 min 100% buffer A. All buffers and protein solutions were filtered before use. 2 ml of protein mixture was applied to the column. Absorbance was monitored at 280 nm. The separation was performed at room temperature. KBr was used as the void marker. Capacity factor (k ) and separation factor (˛) were calculated as k = (tR − to )/to , ˛ = k2 /k1 , where tR is the retention time of the protein and to is the retention time of the void marker (KBr), k2 is the capacity factor for IgG and k1 is the capacity factor for competitive protein (i.e., human serum albumin). The resolution (Rs ) and theoretical plate numbers (N) were calculated using the following equations:
t 2 R
N = 5.54
w0.5
(2)
RS =
2(tR,2 − tR,1 ) (w2 + w1 )
203
(3)
where w0.5 is the peak width at the corresponding peak height fraction, tR,1 and tR,2 are the retention times of two adjacent peaks, w1 and w2 are the widths of the two adjacent peaks at the baseline. 2.9. Desorption and repeated use Adsorbed IgG was desorbed using glycine–HCl elution buffer at pH 3.5. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the cryogel column at a flow-rate of 1.0 ml/min for 1 h. IgG concentration in the desorption medium was determined spectroscopically at 280 nm. After desorption, the cryogel was cleaned with 50 mM sodium hydroxide and then reequilibrated with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). The desorption ratio was calculated from the amount of IgG adsorbed on the cryogel and the final IgG concentration in the desorption medium. In order to test reuse of the PHEMA cryogel, IgG adsorption–desorption cycle was repeated for 10 times using the same cryogel. In order to regenerate and sterilize, after desorption, the PHEMA cryogel was washed with 50 mM NaOH solution. 3. Results and discussion A supermacroporous PHEMA cryogel was produced by copolymerization in the frozen state of HEMA with MBAAm in the presence of APS/TEMED. The hydroxyl groups on the PHEMA cryogel allowed modification with protein A. The SEM images of the pore structure of the cryogel are shown in Fig. 1. PHEMA cryogel have non-porous and thin polymer walls, large continuous interconnected pores (10–200 m in diameter) that provide channels for the mobile phase to flow through. Pore size of the matrix is much larger than the size of the protein molecules, allowing them to pass easily. As a result of the convective flow of the mobile phase through the pores, the mass transfer resistance is practically negligible. The equilibrium swelling degree of the PHEMA cryogel was 8.56 g H2 O/g cryogel. PHEMA cryogel is opaque, sponge like and elastic. This cryogel can be easily compressed by hand to remove water accumulated inside the pores. When the compressed piece of cryogel was submerged in water, it soaked in water and within 1–2 s restored its original size and shape. The pressure drop needed to drive the liquid through any system should be as low as possible. Pressure drop experiments through PHEMA cryogel column were performed in water as equilibration medium, and at linear flow-rates from 40 to 382 cm/h. The water was passed through the column for 1 min at each flow-rate. Due to the presence of large and highly interconnected macropores, the PHEMA cryogel column has very low liquid flow resistance. PHEMA cryogel column had low back pressure indicating the porous structure with large size of the interconnected macropores (Fig. 2). 3.1. Adsorption of IgG from aqueous solutions 3.1.1. Effect of flow-rate Fig. 3 shows that the adsorbed amount of IgG onto the PHEMA–protein A cryogel decreased when the flow-rate through the column increased. The adsorption capacity decreased from 83.2 to 58.9 mg/g polymer with the increase of the flow-rate from 0.5 to 4.0 ml/min. An increase in the flow-rate reduces the protein solution volume treated efficiently until breakthrough point and therefore decreases the retention time of the cryogel. When the flow-rate decreases the contact time in the column is longer. Thus, IgG molecules have more time to diffuse to the pore walls of the
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Fig. 3. Effect of flow-rate on IgG adsorption: protein A loading: 56 mg/g; IgG concentration: 2.0 mg/ml; adsorbing buffer PBS (pH 7.4); T: 25 ◦ C.
tively constant. In addition, the macropores in the cryogel structure are open and highly interconnected, forming a network of large channel. The mobile IgG phase is forced to flow through them, transporting the IgG molecules to the active attachment sites (i.e., protein A molecules) by convection. This result is an extremely fast mass exchange between the mobile IgG phase and the stationary PHEMA cryogel phase than the more traditional packed bed columns [26,27].
Fig. 1. SEM images of the PHEMA cryogel.
cryogel and to attach to the protein A, hence a better IgG adsorption capacity is obtained. In addition, for column operation the cryogel is continuously in contact with a fresh IgG solution. Consequently the IgG concentration in the solution in contact with a given layer of PHEMA–protein A cryogel in a column is rela-
3.1.2. Effect of protein A density Table 1 shows the effect of protein A density on IgG adsorption onto PHEMA cryogel. The decrease in surface area due to the supermacropores in cryogel structure was compensated by an increase in ligand density. When the surface protein A density (i.e., the number of protein A molecules per unit mass) increased the amount of IgG adsorbed onto PHEMA cryogel, first, increased and then reached almost a highest value. This maximum IgG adsorption capacity was 83.2 mg/g. With the increasing protein A density, the higher IgG adsorption is expected. But this may not be advantageous in all cases due to possible geometric (i.e., steric) effects. Protein A contains a tandem of five similar domains, each capable of binding the Fc region of immunoglobulin G (IgG) from various mammals. Each molecule of soluble protein A is able to bind two molecules of IgG. Steric hindrance prevents the binding of more than one or two IgG molecules to the immobilized protein A molecule. When higher protein A density is achieved, these receptors may not be accessible by IgG molecules because of the steric hindrances. Table 1 gives also the weight ratio of IgG/protein A adsorbed onto PHEMA cryogel containing different protein A density. This ratio is around 2.0 for all studied protein A surface density. This range is an agreement with values cited in the literature. Protein A–IgG binding stoichiometries of 1:2 were obtained using sedimentation data Table 1 Effect of protein A density on IgG adsorption: IgG concentration: 2.0 mg/ml; adsorbing buffer PBS (pH 7.4); T: 25 ◦ C.
Fig. 2. Pressure drop at different flow-rates.
Amount of protein A immobilized (mg/g)
Amount of IgG adsorbed (mg/g)
Weight ratio of IgG/protein A
28.7 29.8 56.0 26.9 25.2
49.3 52.9 83.2 38.9 33.5
1.72 1.77 1.49 1.44 1.32
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Table 2 Adsorption constants of Langmuir and Freundlich isotherms. Langmuir adsorption isotherm
Freundlich adsorption isotherm
Qmax = 102.1 mg/g b = 1.58 ml/mg R2 = 0.989
KF = 56.1 n = 1.98 R2 = 0.894
as structure, reactive functional groups, ligand loading amount, porosity, pore size, pore size distribution and accessible surface area.
Fig. 4. Effect of IgG concentration on IgG adsorption: protein A loading: 56 mg/g; flow-rate: 0.5 ml/min; pH: 7.4; T: 25 ◦ C.
and surface tension measurements [28,29]. In addition the binding ratio for a human IgG to protein A has been reported to be 3.3:1 [30]. 3.1.3. Adsorption isotherm Fig. 4 shows the adsorption data on PHEMA and PHEMA–protein A cryogel. The non-specific IgG adsorption on PHEMA cryogel was very low (about 0.38 mg/g). Since PHEMA cryogel retained water at the ratio of 8.56 g of water per gram of dry PHEMA, wet PHEMA cryogel is highly hydrophilic. Thus, PHEMA cryogel has to the advantage that IgG will not adsorb on it non-selectively. While specific adsorption (i.e., adsorption of IgG molecules onto the PHEMA cryogel through protein A molecules) was significant (up to 83.2 mg IgG/g), and increased with increasing IgG concentration. The adsorbed amount of IgG on the PHEMA cryogel via attached protein A molecules reached almost a plateau value around 1.5 mg/ml, due to the saturation of active attachment sites. Two important physico-chemical aspects for evaluation of the adsorption process as a unit operation are the kinetics and the equilibrium of adsorption. Equilibrium modelling data has been done using the Langmuir and Freundlich isotherms. The Langmuir and Freundlich isotherms are represented as follows Eqs. (4) and (5), respectively:
1 1 1 = + qe qmax qmax b ln qe =
1
1 (ln Ce ) + ln KF n
Ce
(4) (5)
where b is the Langmuir isotherm constant, KF is the Freundlich constant, and n is the Freundlich exponent. 1/n is a measure of the surface heterogeneity ranging between 0 and 1, becoming more heterogeneous as its value gets closer to zero. The ratio of qe gives the theoretical monolayer saturation capacity of PHEMA cryogel. Some model parameters are determined by nonlinear regression with commercially available software. The equilibrium data have been fitted with the Langmuir model and the relevant values are given in Table 2. A comparison of the maximum adsorption capacity, qmax , of the PHEMA cryogel with those of some other affinity adsorbents including commercially available adsorbents reported in literature is given in Table 3. The adsorption capacity of PHEMA–protein A was comparable with other adsorbents. Differences of IgG adsorption capacities are due to the properties of each adsorbent such
3.1.4. Effect of temperature The effect of temperature on IgG adsorption capacity of the PHEMA–protein A affinity cryogel was shown in Fig. 5. Non-specific adsorption of IgG (0.38 mg/g) due to van der Waals force was very low at all temperatures. No significant effect of temperature was observed on the physical adsorption of IgG onto the PHEMA cryogel. However, the equilibrium adsorption of IgG on the PHEMA–protein A cryogel significantly increased with increasing temperature and the maximum IgG adsorption was observed at 37 ◦ C (98.8 mg/g). From 4 to 37 ◦ C, the adsorption capacity increased about 125% for the PHEMA/protein A cryogel. This shows that IgG binding interaction of the PHEMA/protein A cryogel is not entropy driven as would be expected with hydrophobic interactions and is consistent with previous results [23]. Hydrophobic forces are dominant in protein A–IgG interactions. Hence, increasing the temperature enhances IgG adsorption and lowering the temperature generally promotes the protein elution. Van der waals attraction forces, which operate in hydrophobic interactions increase with increasing temperature. 3.2. IgG adsorption from human plasma Table 4 shows the IgG adsorption values from human plasma. As seen here, lower adsorption capacity of IgG was obtained for human Table 3 Comparison of the adsorption capacities for IgG of various adsorbents. Adsorbent
Ligand
Qmax (mg/g)
Reference
Cellulose Cellulose nanofiber Polymethylmethacrylate PHEMA PHEMA Eupergit, affigel PHEMA PHEMA Poly(caprolactam) Poly(ethylene) Sepharose 4B Poly(ethylene vinyl alcohol) Polysulfone Sartobind Polymethylmethacrylate Poly(vinyl alcohol) Sepharose 6B
Protein A Protein A Cu2+ l-Histidine Methacryloylamidohistidine Protein A Cu2+ , Ni2+ , Zn2+ , Co2+ Protein A Protein A Phenylalanine l-Histidine l-Histidine Protein A Protein A Protein A/G Protein A 3-Aminophenol 4-amino-1-naphthol Concanavalin A Biomimetic ligand Biomimetic ligand 4-Mercapto ethyl pyridine Mercapto methyl imidazole 2-Mercapto-5benzimidazole sulfonic acid Concanavalin A Bicyclic heteroatomic ligand Protein A
11.7 18.0 54.3 44.8 73.8 20.1 79.6 24.0 28.3 50.0 0.23 77.7 8.8 0.51 6.6 13.2 52.0
[1] [11] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
69.4 7.0 25.0 30 16 30
[43] [44] [45] [46] [47] [48]
25.6 29.2 88.1
[49] [50] In this study
PHEMA Sepharose CL 6B Sepharose 4B Cellulose Poly(ethylene vinyl alcohol) Cellulose
Poly(AAm-AGE) Agarose PHEMA
206
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Fig. 5. Effect of temperature on adsorption capacity: protein A loading: 56 mg/g; IgG concentration: 2.0 mg/ml; flow-rate: 0.5 ml/min; adsorbing buffer PBS.
blood diluted with PBS buffer. But, there was a significant adsorption of IgG (up to 88.1 mg/g) on the PHEMA–protein A cryogel for non-diluted blood. The composition of bound and eluted protein was assayed by SDS-PAGE. About 85% of the protein was IgG indicating high specificity of the macroporous adsorbent even in the presence of large amount of other proteins like albumin. The purity of IgG eluted from PHEMA–protein A cryogel was assayed by SDS-PAGE as given in Section 2. As clearly seen in Fig. 6, IgG in serum (Lane 2) was almost disappeared in Lane 3 after adsorption onto PHEMA–protein A cryogel. Furthermore, the presence of only band at Lane 4 indicates the purity of IgG after desorption of PHEMA–protein A cryogel. The purity of IgG obtained was found to be in the range of 95.3–96.7%. The recovery of IgG from human plasma was 80.7%. In order to show the superiority of cryogel column for direct affinity purification, an experiment on whole blood flowing through the PHEMA–protein A cryogel column was performed. The supermacroporous structure of cryogels allows for direct processing of whole blood containing blood cells [19]. Blood was collected in EDTA-anticoagulation tubes. Non-coagulated blood (2 ml) was run over the column at 1.0 ml/min and washed with 50 ml isotonic buffer. The flow of the blood plug on the PHEMA–protein A cryogel column was monitored visually and photographed at intermediate times during the process. Blood is a complex mixture of cells with different sizes from 2 to 20 m. The pulse of blood applied on a PHEMA–protein A cryogel column under isotonic conditions passes through the column as a homogeneous plug without substantial tailing (Fig. 7).
Fig. 6. SDS-PAGE of serum fractions. The fractions were assayed by SDS-PAGE using 10% separating gel (9 cm × 7.5 cm), and 5% stacking gels were stained with 0.25% (w/v) Coomassie Brillant R 250 in acetic acid–methanol–water (1:5:5, v/v/v) and destained in ethanol–acetic acid–water (1:4:6, v/v/v). Lane 1, biomarker (Sigma); Lane 2, 1/10 diluted serum; Lane 3, 1/10 diluted serum after adsorption; Lane 4, eluted sample. Equal amounts of samples were applied to each line.
3.3. FPLC studies As shown in Fig. 8, HSA and IgG separation from protein mixture was observed at 1.94 and 9.58 min, respectively. As seen in Fig. 9, HSA and IgG separation from human plasma was observed at 1.89 and 9.54 min, respectively. The tR , N, k , a, Rs values are given in Table 4 IgG adsorption from human plasma: IgG concentration before dilution:14.8 mg/ml. Dilution agent
Adsorption capacity (mg/g)
Non-diluted plasma Diluted plasma (dilution ratio: 1/2, PBS pH: 7.4) Diluted plasma (dilution ratio: 1/10, PBS pH: 7.4)
88.1 ± 0.97 63.9 ± 1.20 9.4 ± 0.45
Fig. 7. Flow pattern of whole blood through a cryogel column. 2 ml of blood was applied to the cryogel column at a flow-rate of 1.0 ml/min in isotonic buffer solution. Column 1: column before application; column 2–6: column during processing; column 7: column after the flow of blood sample.
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Fig. 8. FPLC separation of HSA and IgG from protein mixture on a column with PHEMA–protein A cryogel: flow-rate: 3.0 ml/min; protein concentration: 0.5 mg/ml; detection was performed at 280 nm.
Fig. 10. Reusability of the PHEMA–protein A cryogel: protein A loading: 56 mg/g; flow-rate: 0.5 ml/min; IgG concentration: 2.0 mg/ml; adsorbing buffer PBS (pH 7.4); T: 25 ◦ C.
tain their adsorption capacity at almost constant value. Repeated separations did not show a decrease of separation performance. The PHEMA–protein A cryogel presented a good performance regarding reusability and stability. 4. Conclusion
Fig. 9. FPLC separation of HSA and IgG from human serum on a column with PHEMA–protein A cryogel: flow-rate: 3.0 ml/min; detection was performed at 280 nm.
Table 5. Rs values were calculated as 2.28 and 2.63 for binary protein mixture and human plasma, respectively. Because the Rs value should be higher than 1.0 for a good resolution of two peaks in such a chromatography system, the results for the resolution of IgG and HSA can be accepted as good resolution values. 3.4. Reusability of the affinity cryogel Reusability is very important for affinity adsorbent. This means that in designing a test system, attention must be paid to the chemical stability of the affinity adsorbent [51–53]. In order to determine the stability and reusability of the PHEMA–protein A cryogel, the cryogel column has been used up to 10 cycles. For sterilization after one adsorption–desorption cycle, the PHEMA cryogel was washed with 50 mM NaOH solution for 30 min. After this procedure, cryogel was washed with distilled water for 30 min, then equilibrated with the phosphate buffer at pH 7.4 for the next adsorption–desorption cycle. The results showed that about 95% of the adsorbed IgG were desorbed. As seen in Fig. 10 that the cryogel is very stable and mainTable 5 Chromatographic separation data. tR From protein mixture IgG 9.58 HSA 1.94 From human serum IgG 9.54 HSA 1.89
N
k
˛
Rs
130.35 10.95
6.85 0.59
– 11.61
– 2.28
164.64 14.71
6.82 0.55
– 12.4
– 2.63
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