- Email: [email protected]
G for IgG purification
Journal of Membrane Science 319 (2008) 23–28
Contents lists available at ScienceDirect
Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Electrospun regenerated cellulose nanofiber affinity membrane functionalized with protein A/G for IgG purification Zuwei Ma ∗ , Seeram Ramakrishna Nanoscience and Nanotechnology Initiative, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore
a r t i c l e
i n f o
Article history: Received 1 May 2007 Accepted 7 March 2008 Available online 30 March 2008 Keywords: Electrospinning Cellulose nanofiber Affinity membrane IgG purification
a b s t r a c t The aim of this work is to prepare protein A/G functionalized electrospun regenerated cellulose (RC) nanofiber mesh as affinity membrane for immunoglobulin G (IgG) purification. Cellulose acetate (CA) nonwoven nanofiber membrane was prepared by electrospinning, followed by heat treatment and alkaline treatment to obtain RC nonwoven nanofiber membrane. After oxidization of the RC membrane by NaIO4 , protein A/G was covalently immobilized on the membrane, giving an affinity membrane capable of specifically capturing IgG molecules. Physical and chemical properties of the affinity membrane were characterized. Surface chemistry of the membranes during the membrane modification process was monitored with XPS and ATR-FTIR. Oxidization degree of the RC membrane was optimized to get a high ligand binding capacity without too much loss of mechanical strength for handling of the material. Ligand (protein A/G) amount immobilized on the RC membrane oxidized for 9 h was measured as 30 g/mg and the membrane’s IgG binding capacity was measured as 18 g/mg. The IgG purification ability of the affinity membrane was evaluated with BSA as a model impurity. Five layers of the cellulose affinity membrane were packed into a spin column to separate IgG from an IgG/BSA mixture solution. SDS-PAGE analysis showed that the BSA was completely removed after the purification. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Today’s biotechnological and biopharmaceutical industry devotes huge efforts in production of highly purified antibodies due to their wide applications in scientific research, immunodiagnostics and immunotherapy [1,2]. Particularly, recombinant monoclonal antibodies have continued to increase in importance as therapeutics for the treatment of cancer and other diseases thus dominate today’s biopharmaceutical pipeline [3,4]. Large-scale production of these therapeutic molecules requires not only a manufacturing process that delivers a high yield and reliability, but also an effective purification process to give extremely pure product. Affinity chromatography is a traditional technique employed in the later stages of protein purification. Traditional affinity chromatography purification 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. Affinity membranes, with binding domains (ligands) attached onto the membrane surface, provides a potential solution due to their low pressure drop and lack of diffusion resistances compared with traditional column-
∗ Corresponding author. E-mail address: [email protected] (Z. Ma). 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.03.045
based chromatography [5]. Affinity membrane chromatography provides an efficient fast protein purification method which will be important in antibody’s diagnostic application. The aim of this work is to develop an IgG purification affinity membrane using electrospun nonwoven nanofiber membrane. Electrospun polymer nonwoven nanofiber mesh has been recently studied as a novel filter media for both size exclusion filtration (for both gas and liquid) [6,7] and for affinity filtration [8]. We have reported that an electrospun PVDF nanofibrous membrane could be used for separation of microscale particles [7]. Application as affinity membrane was also reported for electrospun cellulose nanofiber having large surface area to volume ratio [8]. In this study, the material chosen for membrane preparation was cellulose acetate (CA). Previous studies [8–11] have shown that CA can be easily electrospun into nanofiber membrane and can be further deacetylized to obtain regenerated cellulose (RC) membrane, which is a hydrophilic material with low nonspecific protein adsorption thus widely used as chromatography packing materials. Another reason for choosing this material is that the polysaccharide can be partially oxidized by NaIO4 to generate aldehyde groups [12–14], upon which ligand molecules containing primary amino groups like most of protein molecules can be attached covalently. Popular ligands for IgG purification include protein A and protein G. Protein A, a cell wall protein component produced by several strains of Staphylococcus aureus, is a single polypeptide chain con-
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taining four high-affinity binding sites capable of interacting with the Fc region from IgG. Protein G is also a bacterial cell wall protein isolated from group G Streptococci containing two IgG-binding domains with higher affinity than protein A. Natural protein G also contains binding sites for albumin. The ligand used in this work is a genetically engineered gene fusion product called protein A/G (piece). Protein A/G is a 50,449 molecular weight protein that combines the four Fc-binding domains from protein A and the two from protein G with the albumin-binding site of protein G eliminated, making it a more universal tool in the IgG purification. In this work, the protein A/G attached RC nanofiber membrane was characterized in terms of physical structure, surface chemistry and IgG purification capability. 2. Experiments 2.1. Membrane preparation Regenerated cellulose nanofiber membrane was first prepared as described in [8]. Briefly, cellulose acetate (CA, Mr = 29,000, 40% acetyl groups, Fluka) solution (0.16 g/ml) in acetone/DMF/ trifluoroethanol (3:1:1) was electrospun to obtain CA nanofiber nonwoven membranes. After thermal treatment under 210 ◦ C for 1 h, the CA membrane was deacetylized by treating in 0.1 M NaOH (in H2 O/ethanol, 2:1) for 24 h to get RC nanofiber membrane. The RC membrane has a thickness of ∼100 m, an apparent density of ∼0.3 g/cm3 and a porosity of ∼80%, with fiber diameters ranging from 200 nm to 1 m, as shown in Fig. 1. The RC membrane was then oxidized in 5% sodium periodate (NaIO4 ) in order to produce dialdehyde groups. NaIO4 0.2 g was dissolved in 40 ml PBS, to which ∼10 mg cellulose membranes were added. The oxidization reaction was run under room temperature in darkness with gentle shaking, and was stopped by the addition of ethylene glycol after 3, 6, 9 or 12 h. Membranes obtained were designated as OXI-3, OXI-6, OXI-9 and OXI-12, respectively. The oxidized membranes were then washed with DI water thoroughly. To characterize the protein binding capability of the oxidized cellulose membrane, BSA (Sigma) was used as a model protein and was reacted with the oxidized RC membranes. OXI3, OXI6, OXI-9 and OXI-12 membranes were immersed in BSA solution (1 mg/ml in PBS) for 6 h under room temperature with gentle shaking. The membranes were shaken in 1% Tween 20 (Sigma) solution overnight in a shaking incubator under 37 ◦ C to minimize nonspecifically bond BSA then washed in DI water for 2 h. The amount
Fig. 2. Schematic representation of the spin column structure and the membrane packing process.
of the BSA on the oxidized cellulose membrane was characterized by BCA protein assay using a BCA Protein Assay Kit (Pierce). For the BCA assay, one piece of membrane (5 mm × 5 mm × 100 m) was added into one well of a 24-well plate where 2 ml BCA assay working solution was added. Optical absorption of the solution was measured at 562 nm after 4 h. Standard calibration curve was made using BSA solutions with known concentrations provided by the Kit. For immobilization of protein A/G (ImmunoPure® , Pierce, USA) onto the RC membrane, OXI-9 was used. Five pieces of OXI-9 (8 mm × 8 mm × 100 m for every piece, total weight ∼10 mg) was immersed into 5 ml protein A/G solution in PBS (1 mg/ml) and shaken gently over night under room temperature. The membranes were then washed in 1% Tween 20 solution under 37 ◦ C overnight, followed by washing in DI water for 2 h. The amount of protein A/G attached on the OXI-6 membrane was measured using the BCA test as described above. Calibration curve was obtained using protein A/G solutions with known concentrations. 2.2. Spin column packing The protein A/G immobilized cellulose membranes were cut into round shape pieces with diameter of 7 mm using a homemade cutter. Five such pieces with total thickness of ∼0.5 mm were packed into a spin column as illustrated in Fig. 2. The O-ring, the affinity membranes, the supporter and the column tube were fitted together tightly so that no side leaking could happen during the following IgG purification process. The active filtration area of the spin column is × (0.5 cm/2)2 = 0.20 cm2 . 2.3. IgG purification
Fig. 1. SEM micrograph of the RC membrane.
The spin column purification process is schematically shown in Fig. 3. Binding buffer was prepared by mixing ImmunoPure® (A/G) IgG binding buffer with PBS at a 1:1 volume ratio. The original solution was prepared by dissolving human IgG (Technical Grade, Sigma) in the binding buffer with a concentration of 1.12 mg/ml, to which BSA (400 g/ml) was added as a model impurity. The IgG/BSA mixture (400 l) was then filtered through the spin column by centrifuging at 300 rpm using an Eppendorf Centrifuge 5417C. The time for the original solution to flow through the membranes under this rotation speed was 4 min (this centrifuging condition was also employed in the following rinsing and elution process). The spin column was rinsed with 400 l PBS for two times to remove free proteins, and then was eluted by 400 l Pierce ImmunoPure® IgG elution buffer. The eluted IgG solution is collected for UV detection and SDS-PAGE analysis. The amount of the IgG solution bonded on the spin column was determined by measuring the eluted IgG solution’s concentration using an UV spectrometer at absorbance of 280 nm. Standard
Z. Ma, S. Ramakrishna / Journal of Membrane Science 319 (2008) 23–28
25
Fig. 3. Schematic representation of the spin column filtration process.
calibration curve was obtained using IgG solutions of known concentrations. Capturing capacity (A) of the affinity membrane is calculated with the following equation: A=
C × 400 l m × (5/7)
2
(1)
where the C stands for the concentration of the eluted IgG solution, and m stands for the total dry mass of the packed membranes. The factor (5/7)2 is due to the spin column’s active filtration area is smaller than the column’s cross-area, as shown in Fig. 2. 2.4. SDS-PAGE The purity of the eluted IgG solution was evaluated by SDS-PAGE analysis carried out on a Mini-PROTEAN® 3 Cell System (BIORAD). Detailed experimental conditions are described in the SDS-PAGE (Laemmli) Buffer System in the instruction manual (Mini-PROTEAN® 3 Cell System), with 4% stacking and 12% resolving gel formula adopted. Kaleidoscope prestained standards (broad range, BIORAD) were used as standards. After gel running, the gel was silver stained with a silver stain kit (BIORAD). 2.5. Material characterization FE-SEM (FEIQUANTA 200F) was used for microstructure observation of the electrospun nanofiber membrane. FTIR spectrum of the CA, RC and modified RC membrane was obtained on a Nicolet IR100 spectrometer (Thermo Electron Corporation, Madison, WI). The membranes (1–2 mg) were ground with 0.1 g KBr and pressed into a pellet, of which transmission FTIR spectrum were measured. XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu) analysis was carried out to verify the presence of the protein A/G on the modified RC membrane. For mechanical strength test, the membranes were cut into strips with a dimension of 1 cm × 4 cm, and then mounted on an Instron Microtester 5848 to test the tensile stress curve at a stretching speed of 10 mm/min. Precise mesh thickness measured by a micrometer and the sample width (1 cm) was input into the computer to calculate the tensile stress of the samples.
excellent material for affinity membrane fabrication. The RC membrane used in this work has an apparent density of ∼0.3 g/cm3 and a porosity of ∼80%, and a membrane thickness of ∼100 m. Micro-morphology of the original CA nanofiber membrane is presented in Fig. 1. The fiber diameters were between 200 and 800 nm as measured by image analysis software (ImageJ, National Institutes of Health, USA) and a mean diameter of 400 nm was calculated. 3.2. Oxidization of cellulose membrane by NaIO4 The main steps involved in the chemical modification of the RC membrane were the membrane’s derivatization to generate aldehyde groups and subsequent protein A/G functionalization. Oxidization by NaIO4 is a popular approach to attach ligands on cellulose-based chromatographic materials [12–14]. The moderate oxidization by NaIO4 generates cellulose aldehyde groups that can be further employed as binding site for covalent ligand immobilization. The chemical structure of oxidized RC membrane was monitored by FTIR spectroscopy (Fig. 4), which showed expected appearance of characteristic absorption peak for the aldehyde group at ∼1720 cm−1 , although the shoulder peaks were not strong. As expected, longer oxidization time gave stronger absorption peak. As shown in Fig. 4, FTIR spectrum of the CA membrane, in which the characteristic adsorption peaks [11] attributed to the vibrations
3. Results and discussion 3.1. Electrospun cellulose membrane Previous works [8–11] have demonstrated that regenerated cellulose nanofiber can be formed by deacetylization of electrospun cellulose acetate nanofiber in sodium hydroxyl solution. The RC nanofiber membrane has strong hydrophilicity and available reactivity for further chemical functionalization, thus provides an
Fig. 4. FTIR transmission spectra of the CA, RC and oxidized RC membranes with different oxidization time.
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Table 1 Tensile stress test results of the RC and oxidized RC membranes Samples
Elongation at break (%)
Tensile strength (MPa)
RC membrane OXI-3 OXI-6 OXI-9 OXI-12
4.6 9.5 10.1 5.8 5.8
7.6 6.0 4.4 3.1 1.9
of the acetate group at 1745 cm−1 (C O ), 1375 cm−1 (C–CH3 ), and 1235 cm−1 (C–O–C ) can be found. Disappearance of these peaks in the RC membrane and significant increase of the peak at 3500 cm−1 (O–H ) in the RC membrane indicated a total removal of the acetyl groups by the NaOH treatment. 3.3. Optimization of the oxidization degree A long enough oxidization time for the NaIO4 oxidization reaction should be applied to yield as much aldehyde groups as possible to maximize the RC membrane’s ligand binding capacity. On the other hand, however, an adverse side reaction in the oxidization reaction by NaIO4 is that the oxidization also causes the gradual breaking of the polymer chain [14], leading to gradual degradation of the cellulose molecules and loss of mechanical strength. This problem may be more serious for the RC nanofiber membrane due to the fiber’s small diameter and large surface. It was found that oxidization of the RC nanofiber membrane for 15 h will give a material with very poor mechanical strength which is actually impractical for use. Therefore, the oxidization time of the RC nanofiber membrane need to be optimized to obtain a high ligand binding capacity while at the same time make the material strong enough. Table 1 shows mechanical strength of the RC nanofiber membranes after oxidization for different times. Oxidization process obviously decreased the tensile strength of the cellulose membrane. To evaluate the ligand binding ability of the oxidized RC nanofiber membrane with different oxidization degree, BSA was chosen as a model to be attached on the oxidized RC membrane. Protein A/G was not used for this purpose because of its high cost. BSA here served as a good model protein due to its similar molecular weight (66,000) with that of protein A/G (50,449). More over, a BSA molecule contains about 60 lysines providing primary amino groups for reaction with the cellulose aldehyde groups, while a protein A/G molecule contains a comparable number of 36 lysine residues. Fig. 5 shows the relationship between the attached BSA content on the oxidized RC membrane and the oxidization time. Although the membrane was rinsed with Tween® 20 thoroughly, nonspecific adsorbed BSA on the membrane could not be completely removed, indicated by the presence of BSA on the non-oxidized RC membrane (oxidization time is zero). However,
Fig. 5. Relation ship between protein ligand binding ability on the NaIO4 oxidized RC membrane and the oxidization time.
the covalently attached BSA amount increased with the oxidization time and when the oxidization time is 9 h the nonspecifically adsorbed BSA should be less than 4% of the covalently attached BSA. Considering both the protein binding ability (Fig. 5) and the mechanical strength of the oxidized RC membrane, optimal oxidization time is determined as 9 h in this work. The membrane with this oxidization degree is still strong enough to be handled easily, although the tensile strength is only half of the original material. SEM picture of the original RC and OXI-9 membrane is shown in Fig. 6a and b. The micro-fractures developed on the oxidized nanofiber surface were due to the degradation effect, directly leading to decreased mechanical strength. 3.4. Membrane functionalziation with protein A/G Protein A/G, a genetically engineered, 50,449 molecular weight protein that combines the IgG binding profiles of both Protein A and Protein G was covalently attached on the OXI-9 membrane using the same method for BSA attachment described above. The covalently attached protein A/G amount, or ligand capacity, was determined as 30 g/mg using the BCA protein assay method. Fig. 6c shows the morphology of the protein A/G modified RC nanofiber, where the micro-fractures on the fiber surface were covered by the attached protein A/G. Presence of protein A/G was also indicated by the appearance of N1s peak in the XPS spectrum of the RC membranes (Fig. 7).
Fig. 6. SEM graphs of the (a) RC membrane, (b) OXI-9 membrane and (c) protein A/G attached OXI-9 membrane.
Z. Ma, S. Ramakrishna / Journal of Membrane Science 319 (2008) 23–28
Fig. 7. XPS scan spectra of the (upper) OXI-9 membrane and (lower) protein A/G functionalized OXI-9 membrane.
3.5. IgG purification ability and binding capacity of the RC affinity membrane The next step is to evaluate the protein A/G functionalized RC membrane’s purification ability for IgG. The key point for the affinity membrane is that it should be able to specifically bind IgG molecule while having no or very low nonspecific adsorption for other proteins. The affinity membranes were packed into a commercial spin column as described in Fig. 2. The original solution to be purified was a mixture of IgG and BSA, with BSA as a model impurity. The original solution was then filtered through the spin column and the bonded IgG was then eluted out, with detailed steps described in Fig. 3 and Section 2. The eluted solution was analyzed by SDS-PAGE. Fig. 8 shows the SDS-PAGE result containing the bands of both the unpurified original solution and the purified solution. There are two heavy chains (2× 50,000) and two light chains (2× 25,000) in an IgG molecule (150,000) combined together covalently by three pairs of disulfide bond. By treatment in heated buffer solution containing SDS and thiol reducing agent prior to the electrophoresis, one IgG molecule is disassembled into two heavy chains and two light chains, leading to two bands with corresponding molecular weight of 50,000 and 25,000, respectively. Thus, the feeding solution contains three bands corresponding to the BSA (66,000), heavy chain and light chain, respectively. While in the
27
band of the purified solution, the band for BSA disappeared, indicating that the BSA was removed during the purification process, and indicating the protein A/G spin column has strong binding specificity towards IgG molecules. Another important parameter for the affinity membrane is its binding capacity. Thus, the IgG binding capacity of the affinity membrane was evaluated. The concentration of the eluted IgG solution was measured by UV adsorption at 280 nm to calculate the amount of the IgG captured by the spin column, and the capturing capacity of the affinity membrane was calculated according to Eq. (1) as 18 g/mg. The ligand (protein A/G) capacity (30 g/mg) and the IgG binding capacity (18 g/mg) of the affinity membrane can be further analyzed to obtain information on the utilization efficiency of the binding domains in the attached protein A/G on the affinity membrane. The molecular weight of the protein A/G and the IgG is 50,000 and 150,000, respectively, thus the molar ratio of the protein A/G and the bonded IgG can be calculated as 5:1. Considering every protein A/G has 6 binding domains, it can be known that every 30binding domain captured only one IgG molecule. This low binding efficiency may be attributed to three reasons. First, it is not possible to preserve all the 6 binding domains in a protein A/G molecule since some domain will be destroyed when their lysine groups were reacted with the aldehyde groups. Second, not all the binding domains can be exposed on the membrane surface. Finally, IgG has a molecular weight of 150,000 so that steric effect between the huge IgG molecules determines not all the binding domain exposed on the membrane surface can bind a IgG molecule. Finally, the novel affinity membrane developed in this work should be compared with current commercial products in the market. Using protein A as ligand, Vivapure® Protein A Mini spin column is a commercially available spin column for IgG purification with claimed IgG capturing capacity of 200–300 g IgG per spin column. Since our affinity membrane has an IgG capturing capacity of 18 g/mg, the spin column containing 5 pieces of membrane used in this work (total thickness ∼0.5 mm, total active dry mass ∼3 mg) could capture 54 g IgG. Thus, a spin column stacked with twenty layers of the affinity membranes (total thickness ∼2 mm, total active dry mass ∼12 mg) would have the similar IgG capture ability as the Vivapure® Protein A Mini spin column. Another commercially affinity membrane for IgG purification is Satorbind® Protein A 75 membrane with protein A as ligand and with a IgG binding capacity of 9.3 g/mg. Our membrane’s binding capacity (18 g/mg) is almost as twice as that of this commercial membrane. 4. Conclusions Regenerated electrospun nanofiber membrane derived from electrospun cellulose acetate nanofiber membrane was oxidized with NaIO4 to generate aldehyde groups, upon which protein A/G ligand containing six IgG binding domains was covalently attached. The NaIO4 oxidization time for the RC membrane was optimized as 9 h to obtain a high protein A/G capacity (30 g/mg) at the same time preserving the membrane’s mechanical strength for easy handling. The affinity membrane has a strong binding specificity towards IgG molecules, and has a capturing capacity of 18 g/mg which is higher than current commercial product. This work provides a good tool for fast antibody purification at a small scale. References
Fig. 8. SDS-PAGE gel running result. (a and b) Purified IgG and (c) original solution containing IgG and BSA. Band (a) corresponds to a purified IgG solution using the spin column for the first time, and band (b) the second time. This result indicated a good reusability of the spin column.
[1] M.C. Glassy, R.O. Dillman, Molecular biotherapy with human monoclonal antibodies, Mol. Biother. 1 (1988) 7–13. [2] D.L. Siegel, Recombinant monoclonal antibody technology, Transfus. Clin. Biol. 9 (2002) 15–22. [3] R.L. Fahrner, H.L. Knudsen, Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography processes, Biotechnol. Genet. Eng. Rev. 18 (2001) 301–327.
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[4] J.X. Zhou, T. Tressel, Basic concepts in Q membrane chromatography for largescale antibody production, Biotechnol. Prog. 22 (2006) 341–349. [5] H. Zou, Q. Luo, Affinity membrane chromatography for the analysis and purification of proteins, J. Biochem. Biophys. Methods 49 (2001) 199–240. [6] P. Gibson, H. Schreuder-Gibson, D. Rivin, Transport properties of porous membranes based on electrospun nanofibers, Colloids Surf. A: Physicochem. Eng. Aspects 187 (2001) 469–481. [7] R. Gopal, S. Kaur, Z.W. Ma, C. Chan, S. Ramakrishna, T. Matsuura, Electrospun nanofibrous filtration membrane, J. Membr. Sci. 281 (2006) 581–586. [8] Z.W. Ma, M. Kotaki, S. Ramakrishna, Electrospun cellulose nanofiber as affinity membrane, J. Membr. Sci. 265 (2005) 115–123. [9] H.Q. Liu, Y.L. Hsieh, Ultrafine fibrous cellulose membranes from electrospinning of cellulose acetate, J. Polym. Sci. B: Polym. Phys. 40 (2002) 2119–2129.
[10] H.Q. Liu, Y.L. Hsieh, Surface methacrylation and graft copolymerization of ultrafine cellulose fibers, J. Polym. Sci. B: Polym. Phys. 41 (2003) 953–964. [11] W.K. Son, J.H. Youk, T.S. Lee, W.H. Park, Electrospinning of ultrafine cellulose acetate fibers: studies of a new solvent system and deacetylation of ultrafine cellulose acetate fibers, J. Polym. Sci. B: Polym. Phys. 42 (2003) 5–11. [12] D. Bhattacharyya, J.A. Hestekin, P. Brushaber, L. Cullen, L.G. Bachas, S.K. Sikdar, Novel poly-glutamic acid functionalized microfiltration membranes for sorption of heavy metals at high capacity, J. Membr. Sci. 141 (1998) 121– 135. [13] U.J. Kim, S. Kuga, Polyallylamine-grafted cellulose gel as high-capacity anionexchanger, J. Chromatogr. A 946 (2002) 283–289. [14] L.A. Fransson, Periodate oxidation of l-iduronic acid residues in dermatan sulphate, Carbohydr. Res. 36 (1974) 339–348.
Contents lists available at ScienceDirect
Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Electrospun regenerated cellulose nanofiber affinity membrane functionalized with protein A/G for IgG purification Zuwei Ma ∗ , Seeram Ramakrishna Nanoscience and Nanotechnology Initiative, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore
a r t i c l e
i n f o
Article history: Received 1 May 2007 Accepted 7 March 2008 Available online 30 March 2008 Keywords: Electrospinning Cellulose nanofiber Affinity membrane IgG purification
a b s t r a c t The aim of this work is to prepare protein A/G functionalized electrospun regenerated cellulose (RC) nanofiber mesh as affinity membrane for immunoglobulin G (IgG) purification. Cellulose acetate (CA) nonwoven nanofiber membrane was prepared by electrospinning, followed by heat treatment and alkaline treatment to obtain RC nonwoven nanofiber membrane. After oxidization of the RC membrane by NaIO4 , protein A/G was covalently immobilized on the membrane, giving an affinity membrane capable of specifically capturing IgG molecules. Physical and chemical properties of the affinity membrane were characterized. Surface chemistry of the membranes during the membrane modification process was monitored with XPS and ATR-FTIR. Oxidization degree of the RC membrane was optimized to get a high ligand binding capacity without too much loss of mechanical strength for handling of the material. Ligand (protein A/G) amount immobilized on the RC membrane oxidized for 9 h was measured as 30 g/mg and the membrane’s IgG binding capacity was measured as 18 g/mg. The IgG purification ability of the affinity membrane was evaluated with BSA as a model impurity. Five layers of the cellulose affinity membrane were packed into a spin column to separate IgG from an IgG/BSA mixture solution. SDS-PAGE analysis showed that the BSA was completely removed after the purification. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Today’s biotechnological and biopharmaceutical industry devotes huge efforts in production of highly purified antibodies due to their wide applications in scientific research, immunodiagnostics and immunotherapy [1,2]. Particularly, recombinant monoclonal antibodies have continued to increase in importance as therapeutics for the treatment of cancer and other diseases thus dominate today’s biopharmaceutical pipeline [3,4]. Large-scale production of these therapeutic molecules requires not only a manufacturing process that delivers a high yield and reliability, but also an effective purification process to give extremely pure product. Affinity chromatography is a traditional technique employed in the later stages of protein purification. Traditional affinity chromatography purification 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. Affinity membranes, with binding domains (ligands) attached onto the membrane surface, provides a potential solution due to their low pressure drop and lack of diffusion resistances compared with traditional column-
∗ Corresponding author. E-mail address: [email protected] (Z. Ma). 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.03.045
based chromatography [5]. Affinity membrane chromatography provides an efficient fast protein purification method which will be important in antibody’s diagnostic application. The aim of this work is to develop an IgG purification affinity membrane using electrospun nonwoven nanofiber membrane. Electrospun polymer nonwoven nanofiber mesh has been recently studied as a novel filter media for both size exclusion filtration (for both gas and liquid) [6,7] and for affinity filtration [8]. We have reported that an electrospun PVDF nanofibrous membrane could be used for separation of microscale particles [7]. Application as affinity membrane was also reported for electrospun cellulose nanofiber having large surface area to volume ratio [8]. In this study, the material chosen for membrane preparation was cellulose acetate (CA). Previous studies [8–11] have shown that CA can be easily electrospun into nanofiber membrane and can be further deacetylized to obtain regenerated cellulose (RC) membrane, which is a hydrophilic material with low nonspecific protein adsorption thus widely used as chromatography packing materials. Another reason for choosing this material is that the polysaccharide can be partially oxidized by NaIO4 to generate aldehyde groups [12–14], upon which ligand molecules containing primary amino groups like most of protein molecules can be attached covalently. Popular ligands for IgG purification include protein A and protein G. Protein A, a cell wall protein component produced by several strains of Staphylococcus aureus, is a single polypeptide chain con-
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Z. Ma, S. Ramakrishna / Journal of Membrane Science 319 (2008) 23–28
taining four high-affinity binding sites capable of interacting with the Fc region from IgG. Protein G is also a bacterial cell wall protein isolated from group G Streptococci containing two IgG-binding domains with higher affinity than protein A. Natural protein G also contains binding sites for albumin. The ligand used in this work is a genetically engineered gene fusion product called protein A/G (piece). Protein A/G is a 50,449 molecular weight protein that combines the four Fc-binding domains from protein A and the two from protein G with the albumin-binding site of protein G eliminated, making it a more universal tool in the IgG purification. In this work, the protein A/G attached RC nanofiber membrane was characterized in terms of physical structure, surface chemistry and IgG purification capability. 2. Experiments 2.1. Membrane preparation Regenerated cellulose nanofiber membrane was first prepared as described in [8]. Briefly, cellulose acetate (CA, Mr = 29,000, 40% acetyl groups, Fluka) solution (0.16 g/ml) in acetone/DMF/ trifluoroethanol (3:1:1) was electrospun to obtain CA nanofiber nonwoven membranes. After thermal treatment under 210 ◦ C for 1 h, the CA membrane was deacetylized by treating in 0.1 M NaOH (in H2 O/ethanol, 2:1) for 24 h to get RC nanofiber membrane. The RC membrane has a thickness of ∼100 m, an apparent density of ∼0.3 g/cm3 and a porosity of ∼80%, with fiber diameters ranging from 200 nm to 1 m, as shown in Fig. 1. The RC membrane was then oxidized in 5% sodium periodate (NaIO4 ) in order to produce dialdehyde groups. NaIO4 0.2 g was dissolved in 40 ml PBS, to which ∼10 mg cellulose membranes were added. The oxidization reaction was run under room temperature in darkness with gentle shaking, and was stopped by the addition of ethylene glycol after 3, 6, 9 or 12 h. Membranes obtained were designated as OXI-3, OXI-6, OXI-9 and OXI-12, respectively. The oxidized membranes were then washed with DI water thoroughly. To characterize the protein binding capability of the oxidized cellulose membrane, BSA (Sigma) was used as a model protein and was reacted with the oxidized RC membranes. OXI3, OXI6, OXI-9 and OXI-12 membranes were immersed in BSA solution (1 mg/ml in PBS) for 6 h under room temperature with gentle shaking. The membranes were shaken in 1% Tween 20 (Sigma) solution overnight in a shaking incubator under 37 ◦ C to minimize nonspecifically bond BSA then washed in DI water for 2 h. The amount
Fig. 2. Schematic representation of the spin column structure and the membrane packing process.
of the BSA on the oxidized cellulose membrane was characterized by BCA protein assay using a BCA Protein Assay Kit (Pierce). For the BCA assay, one piece of membrane (5 mm × 5 mm × 100 m) was added into one well of a 24-well plate where 2 ml BCA assay working solution was added. Optical absorption of the solution was measured at 562 nm after 4 h. Standard calibration curve was made using BSA solutions with known concentrations provided by the Kit. For immobilization of protein A/G (ImmunoPure® , Pierce, USA) onto the RC membrane, OXI-9 was used. Five pieces of OXI-9 (8 mm × 8 mm × 100 m for every piece, total weight ∼10 mg) was immersed into 5 ml protein A/G solution in PBS (1 mg/ml) and shaken gently over night under room temperature. The membranes were then washed in 1% Tween 20 solution under 37 ◦ C overnight, followed by washing in DI water for 2 h. The amount of protein A/G attached on the OXI-6 membrane was measured using the BCA test as described above. Calibration curve was obtained using protein A/G solutions with known concentrations. 2.2. Spin column packing The protein A/G immobilized cellulose membranes were cut into round shape pieces with diameter of 7 mm using a homemade cutter. Five such pieces with total thickness of ∼0.5 mm were packed into a spin column as illustrated in Fig. 2. The O-ring, the affinity membranes, the supporter and the column tube were fitted together tightly so that no side leaking could happen during the following IgG purification process. The active filtration area of the spin column is × (0.5 cm/2)2 = 0.20 cm2 . 2.3. IgG purification
Fig. 1. SEM micrograph of the RC membrane.
The spin column purification process is schematically shown in Fig. 3. Binding buffer was prepared by mixing ImmunoPure® (A/G) IgG binding buffer with PBS at a 1:1 volume ratio. The original solution was prepared by dissolving human IgG (Technical Grade, Sigma) in the binding buffer with a concentration of 1.12 mg/ml, to which BSA (400 g/ml) was added as a model impurity. The IgG/BSA mixture (400 l) was then filtered through the spin column by centrifuging at 300 rpm using an Eppendorf Centrifuge 5417C. The time for the original solution to flow through the membranes under this rotation speed was 4 min (this centrifuging condition was also employed in the following rinsing and elution process). The spin column was rinsed with 400 l PBS for two times to remove free proteins, and then was eluted by 400 l Pierce ImmunoPure® IgG elution buffer. The eluted IgG solution is collected for UV detection and SDS-PAGE analysis. The amount of the IgG solution bonded on the spin column was determined by measuring the eluted IgG solution’s concentration using an UV spectrometer at absorbance of 280 nm. Standard
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Fig. 3. Schematic representation of the spin column filtration process.
calibration curve was obtained using IgG solutions of known concentrations. Capturing capacity (A) of the affinity membrane is calculated with the following equation: A=
C × 400 l m × (5/7)
2
(1)
where the C stands for the concentration of the eluted IgG solution, and m stands for the total dry mass of the packed membranes. The factor (5/7)2 is due to the spin column’s active filtration area is smaller than the column’s cross-area, as shown in Fig. 2. 2.4. SDS-PAGE The purity of the eluted IgG solution was evaluated by SDS-PAGE analysis carried out on a Mini-PROTEAN® 3 Cell System (BIORAD). Detailed experimental conditions are described in the SDS-PAGE (Laemmli) Buffer System in the instruction manual (Mini-PROTEAN® 3 Cell System), with 4% stacking and 12% resolving gel formula adopted. Kaleidoscope prestained standards (broad range, BIORAD) were used as standards. After gel running, the gel was silver stained with a silver stain kit (BIORAD). 2.5. Material characterization FE-SEM (FEIQUANTA 200F) was used for microstructure observation of the electrospun nanofiber membrane. FTIR spectrum of the CA, RC and modified RC membrane was obtained on a Nicolet IR100 spectrometer (Thermo Electron Corporation, Madison, WI). The membranes (1–2 mg) were ground with 0.1 g KBr and pressed into a pellet, of which transmission FTIR spectrum were measured. XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu) analysis was carried out to verify the presence of the protein A/G on the modified RC membrane. For mechanical strength test, the membranes were cut into strips with a dimension of 1 cm × 4 cm, and then mounted on an Instron Microtester 5848 to test the tensile stress curve at a stretching speed of 10 mm/min. Precise mesh thickness measured by a micrometer and the sample width (1 cm) was input into the computer to calculate the tensile stress of the samples.
excellent material for affinity membrane fabrication. The RC membrane used in this work has an apparent density of ∼0.3 g/cm3 and a porosity of ∼80%, and a membrane thickness of ∼100 m. Micro-morphology of the original CA nanofiber membrane is presented in Fig. 1. The fiber diameters were between 200 and 800 nm as measured by image analysis software (ImageJ, National Institutes of Health, USA) and a mean diameter of 400 nm was calculated. 3.2. Oxidization of cellulose membrane by NaIO4 The main steps involved in the chemical modification of the RC membrane were the membrane’s derivatization to generate aldehyde groups and subsequent protein A/G functionalization. Oxidization by NaIO4 is a popular approach to attach ligands on cellulose-based chromatographic materials [12–14]. The moderate oxidization by NaIO4 generates cellulose aldehyde groups that can be further employed as binding site for covalent ligand immobilization. The chemical structure of oxidized RC membrane was monitored by FTIR spectroscopy (Fig. 4), which showed expected appearance of characteristic absorption peak for the aldehyde group at ∼1720 cm−1 , although the shoulder peaks were not strong. As expected, longer oxidization time gave stronger absorption peak. As shown in Fig. 4, FTIR spectrum of the CA membrane, in which the characteristic adsorption peaks [11] attributed to the vibrations
3. Results and discussion 3.1. Electrospun cellulose membrane Previous works [8–11] have demonstrated that regenerated cellulose nanofiber can be formed by deacetylization of electrospun cellulose acetate nanofiber in sodium hydroxyl solution. The RC nanofiber membrane has strong hydrophilicity and available reactivity for further chemical functionalization, thus provides an
Fig. 4. FTIR transmission spectra of the CA, RC and oxidized RC membranes with different oxidization time.
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Table 1 Tensile stress test results of the RC and oxidized RC membranes Samples
Elongation at break (%)
Tensile strength (MPa)
RC membrane OXI-3 OXI-6 OXI-9 OXI-12
4.6 9.5 10.1 5.8 5.8
7.6 6.0 4.4 3.1 1.9
of the acetate group at 1745 cm−1 (C O ), 1375 cm−1 (C–CH3 ), and 1235 cm−1 (C–O–C ) can be found. Disappearance of these peaks in the RC membrane and significant increase of the peak at 3500 cm−1 (O–H ) in the RC membrane indicated a total removal of the acetyl groups by the NaOH treatment. 3.3. Optimization of the oxidization degree A long enough oxidization time for the NaIO4 oxidization reaction should be applied to yield as much aldehyde groups as possible to maximize the RC membrane’s ligand binding capacity. On the other hand, however, an adverse side reaction in the oxidization reaction by NaIO4 is that the oxidization also causes the gradual breaking of the polymer chain [14], leading to gradual degradation of the cellulose molecules and loss of mechanical strength. This problem may be more serious for the RC nanofiber membrane due to the fiber’s small diameter and large surface. It was found that oxidization of the RC nanofiber membrane for 15 h will give a material with very poor mechanical strength which is actually impractical for use. Therefore, the oxidization time of the RC nanofiber membrane need to be optimized to obtain a high ligand binding capacity while at the same time make the material strong enough. Table 1 shows mechanical strength of the RC nanofiber membranes after oxidization for different times. Oxidization process obviously decreased the tensile strength of the cellulose membrane. To evaluate the ligand binding ability of the oxidized RC nanofiber membrane with different oxidization degree, BSA was chosen as a model to be attached on the oxidized RC membrane. Protein A/G was not used for this purpose because of its high cost. BSA here served as a good model protein due to its similar molecular weight (66,000) with that of protein A/G (50,449). More over, a BSA molecule contains about 60 lysines providing primary amino groups for reaction with the cellulose aldehyde groups, while a protein A/G molecule contains a comparable number of 36 lysine residues. Fig. 5 shows the relationship between the attached BSA content on the oxidized RC membrane and the oxidization time. Although the membrane was rinsed with Tween® 20 thoroughly, nonspecific adsorbed BSA on the membrane could not be completely removed, indicated by the presence of BSA on the non-oxidized RC membrane (oxidization time is zero). However,
Fig. 5. Relation ship between protein ligand binding ability on the NaIO4 oxidized RC membrane and the oxidization time.
the covalently attached BSA amount increased with the oxidization time and when the oxidization time is 9 h the nonspecifically adsorbed BSA should be less than 4% of the covalently attached BSA. Considering both the protein binding ability (Fig. 5) and the mechanical strength of the oxidized RC membrane, optimal oxidization time is determined as 9 h in this work. The membrane with this oxidization degree is still strong enough to be handled easily, although the tensile strength is only half of the original material. SEM picture of the original RC and OXI-9 membrane is shown in Fig. 6a and b. The micro-fractures developed on the oxidized nanofiber surface were due to the degradation effect, directly leading to decreased mechanical strength. 3.4. Membrane functionalziation with protein A/G Protein A/G, a genetically engineered, 50,449 molecular weight protein that combines the IgG binding profiles of both Protein A and Protein G was covalently attached on the OXI-9 membrane using the same method for BSA attachment described above. The covalently attached protein A/G amount, or ligand capacity, was determined as 30 g/mg using the BCA protein assay method. Fig. 6c shows the morphology of the protein A/G modified RC nanofiber, where the micro-fractures on the fiber surface were covered by the attached protein A/G. Presence of protein A/G was also indicated by the appearance of N1s peak in the XPS spectrum of the RC membranes (Fig. 7).
Fig. 6. SEM graphs of the (a) RC membrane, (b) OXI-9 membrane and (c) protein A/G attached OXI-9 membrane.
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Fig. 7. XPS scan spectra of the (upper) OXI-9 membrane and (lower) protein A/G functionalized OXI-9 membrane.
3.5. IgG purification ability and binding capacity of the RC affinity membrane The next step is to evaluate the protein A/G functionalized RC membrane’s purification ability for IgG. The key point for the affinity membrane is that it should be able to specifically bind IgG molecule while having no or very low nonspecific adsorption for other proteins. The affinity membranes were packed into a commercial spin column as described in Fig. 2. The original solution to be purified was a mixture of IgG and BSA, with BSA as a model impurity. The original solution was then filtered through the spin column and the bonded IgG was then eluted out, with detailed steps described in Fig. 3 and Section 2. The eluted solution was analyzed by SDS-PAGE. Fig. 8 shows the SDS-PAGE result containing the bands of both the unpurified original solution and the purified solution. There are two heavy chains (2× 50,000) and two light chains (2× 25,000) in an IgG molecule (150,000) combined together covalently by three pairs of disulfide bond. By treatment in heated buffer solution containing SDS and thiol reducing agent prior to the electrophoresis, one IgG molecule is disassembled into two heavy chains and two light chains, leading to two bands with corresponding molecular weight of 50,000 and 25,000, respectively. Thus, the feeding solution contains three bands corresponding to the BSA (66,000), heavy chain and light chain, respectively. While in the
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band of the purified solution, the band for BSA disappeared, indicating that the BSA was removed during the purification process, and indicating the protein A/G spin column has strong binding specificity towards IgG molecules. Another important parameter for the affinity membrane is its binding capacity. Thus, the IgG binding capacity of the affinity membrane was evaluated. The concentration of the eluted IgG solution was measured by UV adsorption at 280 nm to calculate the amount of the IgG captured by the spin column, and the capturing capacity of the affinity membrane was calculated according to Eq. (1) as 18 g/mg. The ligand (protein A/G) capacity (30 g/mg) and the IgG binding capacity (18 g/mg) of the affinity membrane can be further analyzed to obtain information on the utilization efficiency of the binding domains in the attached protein A/G on the affinity membrane. The molecular weight of the protein A/G and the IgG is 50,000 and 150,000, respectively, thus the molar ratio of the protein A/G and the bonded IgG can be calculated as 5:1. Considering every protein A/G has 6 binding domains, it can be known that every 30binding domain captured only one IgG molecule. This low binding efficiency may be attributed to three reasons. First, it is not possible to preserve all the 6 binding domains in a protein A/G molecule since some domain will be destroyed when their lysine groups were reacted with the aldehyde groups. Second, not all the binding domains can be exposed on the membrane surface. Finally, IgG has a molecular weight of 150,000 so that steric effect between the huge IgG molecules determines not all the binding domain exposed on the membrane surface can bind a IgG molecule. Finally, the novel affinity membrane developed in this work should be compared with current commercial products in the market. Using protein A as ligand, Vivapure® Protein A Mini spin column is a commercially available spin column for IgG purification with claimed IgG capturing capacity of 200–300 g IgG per spin column. Since our affinity membrane has an IgG capturing capacity of 18 g/mg, the spin column containing 5 pieces of membrane used in this work (total thickness ∼0.5 mm, total active dry mass ∼3 mg) could capture 54 g IgG. Thus, a spin column stacked with twenty layers of the affinity membranes (total thickness ∼2 mm, total active dry mass ∼12 mg) would have the similar IgG capture ability as the Vivapure® Protein A Mini spin column. Another commercially affinity membrane for IgG purification is Satorbind® Protein A 75 membrane with protein A as ligand and with a IgG binding capacity of 9.3 g/mg. Our membrane’s binding capacity (18 g/mg) is almost as twice as that of this commercial membrane. 4. Conclusions Regenerated electrospun nanofiber membrane derived from electrospun cellulose acetate nanofiber membrane was oxidized with NaIO4 to generate aldehyde groups, upon which protein A/G ligand containing six IgG binding domains was covalently attached. The NaIO4 oxidization time for the RC membrane was optimized as 9 h to obtain a high protein A/G capacity (30 g/mg) at the same time preserving the membrane’s mechanical strength for easy handling. The affinity membrane has a strong binding specificity towards IgG molecules, and has a capturing capacity of 18 g/mg which is higher than current commercial product. This work provides a good tool for fast antibody purification at a small scale. References
Fig. 8. SDS-PAGE gel running result. (a and b) Purified IgG and (c) original solution containing IgG and BSA. Band (a) corresponds to a purified IgG solution using the spin column for the first time, and band (b) the second time. This result indicated a good reusability of the spin column.
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