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Use of Polymeric Membranes for Purification of an E. Coli Expressed Biotherapeutic Protein a
a
S. Muthukumar & Anurag S. Rathore a
Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India Accepted author version posted online: 15 Jul 2015.
Click for updates To cite this article: S. Muthukumar & Anurag S. Rathore (2015): Use of Polymeric Membranes for Purification of an E. Coli Expressed Biotherapeutic Protein, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2015.1045609 To link to this article: http://dx.doi.org/10.1080/10826068.2015.1045609
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Use of Polymeric Membranes for Purification of an E. coli Expressed Biotherapeutic Protein S. Muthukumar1, Anurag S. Rathore1 1
Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India
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Corresponding author: Anurag Rathore Professor, Department of Chemical Engineering, Indian Institute of Technology, HauzKhas, New Delhi, 110016, India. Email: [email protected]
Abstract
Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed bed chromatography by eliminating the diffusion related limitations associated with the traditional resin beads. In this paper, we examine six membrane adsorbers for purification of Granulocyte Colony Stimulating Factor (GCSF), an E coli based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the paper. KEYWORDS: Membrane chromatography, Host cell protein, Granulocyte Colony Stimulating Factor (GCSF), Surface chemistry, Diffusion
1. INTRODUCTION Chromatography is by far the most widely used technique for purification of therapeutic proteins. Ion-exchange chromatography (IEC) forms the backbone of most biopharmaceutical drug purification processes as it offers high selectivity for clearance of 1
various process and product related impurities.[1, 2]Charge based differences between the native protein and other species that are being separated is the underlying mechanism behind the use of ion exchange chromatography, often used for achieving clearance of the various product related impurities such as oxidized forms, reduced forms and deamidated forms of the protein.[3]However, often these differences in the physicochemical properties
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are minor, making selective clearance a difficult task.
Designing an ion exchange chromatography step for selective clearance of such impurities requires optimization of numerous parameters, including mobile phase (pH, molarity, etc.), stationary phase (type of ion-exchange group, ion-exchange capacity, particle diameter, pore structure, pore size distribution, base matrix property, etc.), column parameters (length, diameter, etc.) and operating variables (flow rate, gradient slope, sample loading, etc.).[4, 5]In anion exchange chromatography, negatively charged amino acid side chains of the protein molecule interact with the positively charged ligands of the chromatography matrix. In cation exchange chromatography, positively charged amino acid side chains of the protein molecule interact with the negatively charged ligands of the chromatography matrix.[6, 7]
Ion exchange column chromatography has been widely used for purification of biotherapeutic proteins because of its high capacity and high efficiency. Most commercial available ion exchange beads are based on sephadex, agarose, silica and cross linked cellulose. However, high pressure drops, internal diffusion limitation, the compressibility of the soft beads and plugging are issues that are typically associated with them. In view
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of these limitations, use of membrane chromatography has been steadily increasing in the recent years.[8-10] A variety of anions (diethylaminoethyl, quaternary aminoethyl and quaternary ammonium) and cations (carboxymethyl, sulphopropyl and methyl sulphone) have been coupled to porous membranes based on nylon, regenerated cellulose, perfluoropolymer, poly (glycidyl methacrylate) and polyethylene. Three steps are usually involved in preparation of a typical ion exchange membrane chromatography process,
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namely preparation of the basic membrane, activation of the basic membrane, and coupling of ligands to the activated membrane.[11-15]
The effect of ligand density on cation exchange chromatography has been examined. The authors reported that ligand density affects the resolution of a basic charge variant as well as the host cell protein clearance.[16] Further, the performance of ion exchange chromatography resin has been found to depend not only on the ligand type and ligand density, but also on the pore accessibility of the target molecule. [17] The researchers demonstrated that ligand density directly influences the porosity of the materials as well as the pore diffusivity and the dynamic binding capacity.
The effect of ligand density, apparent pore size and protein charge on the dynamic binding capacity has been extensively investigated. [18] It has been found that the resin pore size is a factor in the occurrence of exclusion, with exclusion being largely absent in the larger pore size resins but appearing consistently in the smaller pore size based resins. The resin ligand density had very little effect on the maximum dynamic binding capacity. In another publication, the adsorption capacities of the resin were found to
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increase with an increase in ligand density and a decrease in pore size, and an integrative parameter was proposed to describe the combined effects of ligand density and pore size. [19]
It was also found that the effective pore diffusion coefficient of the adsorption kinetics
was influenced by pore sizes of resins, but was relatively independent on the ligand densities of the resins.
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A new type of ion exchanger has been reported in which the ionic groups are exclusively located on linear polymer chains grafted on the support surface. [20] This reduces the contact between the analyte and the matrix. Researchers have studied the influence of ligand density on antibody binding capacity of cation-exchange adsorbents and found that at high positive protein net charges (low pH, low ionic strength), low adsorption capacity values were obtained for cation-exchange materials with a grafted polymer layer and a high ligand density.[21] The impact of different pore sizes, dextran surface extender concentration and ligand density of cation exchange resins on the dynamic binding capacity of a therapeutic antibody has been explored.[22] The researchers identified that increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content resulted in higher dynamic binding capacity mainly at the optimal pore size and lower conductivities.
Granulocyte Colony Stimulating Factor (GCSF) is a 175 amino acid protein chain with five cysteine amino acids, four of which are involved in forming two disulphide linkages (Cys36-Cys42 and Cys64-Cys74) and the fifth (Cys17) is free. These two disulphide bonds form two small loops, which are separated by 21 amino acids. The GCSF molecule
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has a molecular weight of 18.8 kDa.[23, 24]It is known to stimulate the production of White Blood Cells (WBCs).[25]One of the major applications of this product is in ameliorating neutropenia, a condition where neutrophil count falls below 1.5 x 109/L.
The physical characteristics of the resin such as particle size, pore size, ligand density and base matrix chemistry also play a significant role in ion exchange chromatography. In
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particular, the effect of base matrix on impurity clearance has not been studied in a systematic manner for large scale purification processes. This paper explores the role of interactions between the protein and the chromatographic membrane surface in purification of GCSF (Figures 1A to 1F). Membranes that have been explored include those based on cation exchange, anion exchange, hydrophobic interaction and salt tolerance exchange interactions. Performance of the various membrane adsorbers has been characterized with respect to step recovery, product quality as well as non-specific binding of the product to the membrane.
2. EXPERIMENTAL 2.1. Materials Mustang S and Q membrane adsorbers were purchased from Pall Life Sciences, Bangalore. Sartobind®S, Q, HIC Phenyl and STIC AEXwerepurchased from Sartorius Stedim India Pvt Ltd, Bangalore. Glacial acetic acid, sodium acetate (anhydrous),sodium chloride, sodium hydroxide,glycerol,acetonitrile (HPLC grade), and trifluoroacetic acid (TFA) were purchased from Merck Chemicals, Bangalore, India.Acryl amide, ammonium persulfate, bisacrylamide, beta-mercaptoethanol, glycine, sodium dodecyl
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sulfate, sodium salt of ethylenediaminetetra acetic acid (EDTA), sodium thiosulphate and N, N, N', and N'-tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich Co, Bangalore,India.
2.2. Buffers Acetate buffers of different pH and buffer molarity were prepared for cation and anion
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exchange membrane chromatography. All buffers were degassed by ultrasonication prior to use.
2.3. Refolding and Sample Preparation Refolded GCSF was used in this investigation. Inclusion bodies from E. coli were initially solubilized using urea as a denaturant followed by the refolding using the dilution method at 7 ± 2°C.[26]The refolded product was analyzed by RP-HPLC for measuring the unfolded and refolded product forms and was thereafter concentrated by ultrafiltration using a 3 kDa MinimateTM tangential flow filtration capsule from Pall life sciences, Bangalore. The pH of the concentrated protein sample was then adjusted to pH 4.0. The protein sample was then centrifuged at 8000 rpm at 4ºC. Supernatant was collected and buffer exchanged in the respective buffers. The buffer exchanged samples were used as an input for the cation and anion exchange membrane chromatography. Approximately 90% of the total protein in the feed material was GCSF.
2.4 Lab Scale Cation and Anion Exchange Membrane Chromatography
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The membrane chromatography disc was connected to ÄKTA Purifier chromatography system (GE Healthcare Bio-Sciences, Uppsala, Sweden). The disc was equilibrated using the selected equilibration buffer (5-10 CV). Pretreated GCSF protein solution was injected into the disc using a sample loop of capacities from 10-2000 µl (GE Healthcare Bio-Sciences).After sample loading, unbound protein sample was removed using a wash step with the equilibration buffer (5 CV).Elution was performed using a salt or pH based
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elution gradient. The output from the disc was monitored using pH, conductivity and UV detection at 280, 260 and 215 nm detectors.
2.5 Reversed Phase High Performance Liquid Chromatography (RP HPLC) Protein concentration for the refolded protein as well as membrane chromatography pools was determined by performing RP–HPLC using a 4.6 mm × 150 mm Zorbax Eclipse XDB C4 column from Agilent Technologies, Palo Alto, CA on theUltimate 3000 LC system from Dionex. The mobile phase consisted of solvents A and B. Solvent A was 0.1% (v/v) TFA in water and Solvent B was 0.1% (v/v) TFA in 98 % of acetonitrile. Flow rate was maintained at1 ml/min using a linear gradient of A to B at a wavelength of 214 nm.
2.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) SDS PAGE was used for identification of the impurities associated with GCSF. A 1 mm thick resolving polyacrylamide gel (13 %) was used under non-reducing condition at constant voltage conditions. Each sample was boiled for 5 min in the starting buffer
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before being loaded into the gel. Silver staining was used to detect proteins after electrophoretic separation.
2.7 Enzyme-Linked Immunosorbent Assay (ELISA) Concentration of host cell proteins (HCP) in the anion exchange chromatography flowthrough was analyzed using an ELISA (E. Coli. HCP analysis kit F 410 from Cygnus
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Technologies, USA). Samples containing E. coli HCP were reacted with a horseradish peroxidase (HRP) enzyme labeled anti-E. Coli antibody simultaneously in microtiter strips coated with an affinity purified capture anti-E. Coli antibody. The immunological reactions resulted in formation of a sandwich complex of solid phase antibody-HCPenzyme labeled antibody. The microtiter strips were washed to remove any unbound reactants. The substrate, tetramethylbenzidine (TMB) was then reacted. The amount of hydrolyzed substrate was read on a microtiter plate reader at 450 nm and was directly proportional to the concentration of E. coli HCP present.[27]
2.8. Circular Dichroism (CD) Spectroscopy Secondary structure of purified protein was determined by CD spectroscopy. Sample with 0.2 mg/ml of resolublized protein was taken into the respective buffer in a 2 mm path length cuvette and far UV CD spectrum was measured from 250 to 200 nm in a JASCO J-815 Spectropolarimeter (Jasco, Inc. Mary's Court, Easton, MD 21601, USA) with the spectral band width of 5 nm.[28] An average of three scans was plotted against the wavelength.
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2.9 Scanning Electron Microscopy (SEM) Membrane samples were removed from the Mustang S membranes after experimentation and gold was sputtered followed by SEM imaging. The sputtered samples were kept in the vacuum chamber and images were obtained using a ZEISS scanning electron microscope (Germany) with a secondary electrons detector at a voltage of 20 kV.
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2.10 Contact Angle Measurements Contact angles (θ) of deionized water, on different membrane surfaces were measured using a Kruss contact angle goniometer (KRÜSS GmbH, Borsteler Chaussee, Hamburg, Germany) at 25° C. [29]A drop of the liquid was placed on the test surface. After 5 min, the contact angle between the plane substrate (S) and the liquid meniscus (L) reached an equilibrium value corresponding to a constant drop volume. This equilibrium angle was reported and recorded as the static contact angle of the liquid on the substrate.
2.11 Statistical Analysis Design of experiments (DOE) were created as well as the statistical analysis of the resulting data was performed using JMP® 8.0 (SAS Institute Inc., Cary NC). For analysis of DOE data, the procedure outlined in a previous publication was followed.[30]
3. RESULTS AND DISCUSSION The objective of this article is to highlight the important role of interactions between the proteins and the polymer surface in purification of proteins. In this respect, we have examined performance of two different polymeric surfaces with four different ligands
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(Figure 1). Further, some key characteristics of these different polymeric membranes are also listed in Table 1. In the following subsections, we discuss the performance of the different adsorbers for purification of GCSF. Performance of the various membrane adsorbers has been characterized with respect to step recovery, product quality as well as non-specific binding of the product to the membrane.
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3.1. Purification Using Membrane Chromatography 3.1.1 Cation Exchange Membrane Chromatography Instability of GCSF at higher pH makes cation exchange membrane chromatography as the first choice for the separation of various product related impurities associated with GCSF31. Full-factorial design of experiments (DOE) was used to investigate the effect of pH and buffer molarity on product recovery both for the Sartobind S and Mustang S membranes (Figures 2 and 3). Based on the pH range under consideration, acetate buffer system was selected and buffers of varying molarity were prepared and used for equilibration in cation exchange membrane chromatography. Salt based elution strategy was selected in this study due to the reported instability of GCSF at higher pH values. Maximum salt concentration selected for the elution studies was limited to 1 M NaCl considering the precipitation effect of salt on GCSF.
As seen in Figures 2 and 3, product recovery is higher when the base matrix is PES (Mustang S) when compared to the base matrix of RC (Sartobind S) (average of 45% vs. 41%, maximum recovery of 78% vs. 72%). This is likely because the GCSF molecule is known to be very hydrophobic in nature and PES is known to be hydrophilic (more than
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RC). The lower recoveries are likely due to the presence of non-specific protein adsorption on the membrane surface [31]. The performance of the two matrices was quite similar with respect to purity of GCSF (average of 98.3% vs. 98.6% and maximum of 99.5% vs. 99.7%).
Figures 2 and 3also present detailed statistical analysis of the data for Mustang S and
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Sartobind S, respectively. It is seen in Figures 2B, 2D, 3B and 3D that in both cases the resulting models are statistically significant (R2=0.99). Further, both pH and buffer molarity have a significant impact on GCSF recovery and purity (Figures 2C, 2E, 3C and 3E). Optimization was performed for both the cases using the prediction profiler function of the JMP software. For the case of Mustang S, optimal conditions were identified as pH of 5.7 and buffer molarity of 33 mM (Figure 2C and 2E). Under these conditions, GSCF recovery of 80% and purity of 99.4% can be achieved. Similarly, in the case of Sartobind S, GCSF recovery of 73% and purity of 99.1% can be achieved at a pH of 5.7 and buffer molarity of 34 mM (Figures 3C and 3E).
Overall, we can conclude that the effects of pH and buffer molarity are quite similar for the two membranes, indicating the dominance of the surface chemistry (same for the two membranes) over the base matrix (different for the two membranes). Based on these results and our earlier experience with purification of GCSF, we think that the binding between the protein and the membrane is very strong at pH 4.0, and not all the protein leaves the membrane during elution. This results in lower recoveries at lower pH.
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3.1.2. Anion Exchange (Aex) Membrane Chromatography Usefulness of anion exchange chromatography as the first purification step is limited by the instability of GCSF at high pH.[31] In view of this, we evaluated use of AEX in the flow-through mode for clearance of host cell proteins, endotoxins and nucleic acids. The focus of optimization was thus on maximizing GCSF recovery.
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As seen in Figures 4 and 5, unlike the case of cation exchange chromatography, product recovery in anion-exchange chromatography is quite similar for the case when the base matrix is PES as in Mustang Q when compared to the base matrix of RC as in Sartobind Q (average of 94% and maximum recovery of 100% in both cases). This is likely due to absence of non-specific protein adsorption on the membrane surface under these conditions. The performance of the two matrices was also similar with respect to HCP clearance (average of 1.5 log clearance and maximum of 2 log clearance in both cases).
Figures 4 and 5 also present detailed statistical analysis of the data for Mustang Q and Sartobind Q, respectively. It is seen in Figures 4B, 4D, 5B and 5D that in both cases the resulting models are statistically significant (R2> 0.95). Further, both pH and buffer molarity have a significant impact on GCSF recovery and HCP clearance (Figures 4C, 4E, 5C, and 5E). Optimization was performed for both the cases using the prediction profiler function of the JMP software. For the case of Mustang Q, optimal conditions were identified as pH of 5.7 and buffer molarity of 42 mM (Figures4C and 4E). Under these conditions, GSCF recovery of 100% and HCP clearance of 1.8 logs can be achieved. Similarly, in the case of Sartobind Q, GCSF recovery of 99.3% and HCP
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clearance of 1.9 logs can be achieved at a pH of 5.7 and buffer molarity of 40mM (Figures 5C and 5E).
Overall, we can conclude that the effects of pH and buffer molarity are quite similar for the two membranes. Based on these results, we can conclude that anion exchange membrane chromatography can be successfully used in the flow-through mode for
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reducing host cell impurities.
3.1.3 Hydrophobic Interaction Phenyl Membrane Chromatography (Hic) Separation in hydrophobic interaction chromatography occurs due to a combination of hydrophobic and ionic interactions. Typically, feed is loaded under conditions of high salt and hence ionic interactions are minimal and hydrophobic interactions dominate the binding of the species to the chromatographic support. Salt ions in this solution reduce the solvation of solutes in the sample. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the hydrophobic membrane matrix. As the salt concentration is lowered during wash or elution, the ionic interactions come into play and impact the separation of the various species in the feed. The motivation for exploring HIC mode was the possibility of achieving enhanced separation due to duality of underlying interactions.
In view of the above, the possibility of using HIC membrane chromatography was explored as a possible purification step in the bind and elute mode as a possible substitute or an add-on to CEX chromatography. Table 2A shows the GCSF recovery and purity
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obtained. It is seen that while the purity obtained is acceptable (99.5%), the recovery is lesser (maximum of 70%) as compared to CEX membrane chromatography. It is seen that as the salt concentration increases the protein binding to the membrane matrix also improves up to a certain limit beyond which it start to decrease. Based on the results, it can be concluded that CEX membrane chromatography would be the preferred mode
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over HIC membrane chromatography for purification of GCSF.
3.1.4 Salt Tolerant Interaction Membrane Chromatography (Stic) Very recently a new kind of membrane adsorber has been introduced that uses the primary amine ligand for binding negatively charged impurities such as DNA, host cell proteins, endotoxins and viruses at much higher salt concentrations than in the case of conventional Q matrices. The motivation behind examining this membrane was the ease of operation STIC would offer as the elute from CEX chromatography could be directly loaded on the STIC column. Due to the high salt that this elute contains, the same is not possible with AEX chromatography discussed above and a diafiltration step is required in the intermediate to change the feed conditions.
In view of the above, the possibility of using STIC membrane chromatography was explored as a possible purification step in the flow-through mode as a substitute to AEX chromatography. Table 2B shows the GCSF recovery and purity obtained. It is seen that while the GCSF recovery obtained is high (98.5%), the HCP clearance is not as significant as in AEX chromatography (maximum of 1 log). It can be concluded that STIC offers a possible alternative to AEX membrane chromatography.
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3.1.5 Final Process Scheme Based on the results presented above, we can recommend two processing options. First would be using a CEX membrane chromatography followed by diafiltration and AEX membrane chromatography (CEX-DF-AEX). Second would be using CEX membrane chromatography followed by STIC membrane chromatography (CEX-STIC). Both
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options seem feasible and have their own strengths and weaknesses.
3.2 Product Characterization 3.2.1 Analysis Of Product Quality Non reducing SDS PAGE and RP-HPLC analysis were performed as identity and purity tests of GCSF, respectively. Figure 6 illustrates the data for CEX membrane chromatography. The SDS PAGE gel in Figure 6A clearly shows the significant purification that both CEX membrane chromatography (Lane 1) and HIC membrane chromatography (Lane 2) accomplish. Bulk of the impurities that are present in the feed material (Lanes 4-6) are removed during these steps resulting in almost pure product (Lane 3).Similar observation can be made from comparison of the RP-HPLC chromatogram for the feed material (Figure 6B) and the CEX membrane chromatography pool (Figure 6C). The latter shows a nearly complete removal of all product related impurities.
3.2.2 Circular Dichroism (CD) Spectroscopy For Secondary Structure Analysis
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CD spectroscopy was performed for the GCSF sample from CEX membrane chromatography and compared to that for the GCSF reference standard (Figure 6D). It is seen that the two spectra are near-identical and this confirms that the protein produced has the desired secondary structure.
3.3 Non-Specific Protein Adsorption
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Non-specific protein adsorption has been seen as a concern with GCSF due to the extreme hydrophobic nature of the protein [31]. This results in low product recovery during processing. Hence, it was investigated via two analytical tools namely contact angle measurements and scanning electron microscopy.
3.3.1 Scanning Electron Microscopy Scanning electron microscopy was used as an analytical tool to study reveals the images of the blank membrane surface (Figure 7A) and after the protein is adsorbed on to the membrane surface (Figure 7B). It is evident that there is considerable non-specific adsorption on the membrane surface. This is the reason for the lower product recovery in CEX and HIC membrane chromatography and will also be a concern for membrane reuse. Appropriate cleaning and sanitization protocols will need to be put in place to be able to recycle the membranes.
3.3.2 Contact Angle Measurements Contact angle measurements can be used for quantitating the hydrophobicity of a surface. The results of these measurements for the PES base matrix (Mustang S) and RC
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base matrix (Sartobind S) are shown in Figure 7C. The contact angle value for the new membrane is lower for PES (86±2 deg) than for RC (93±2 deg) and this agrees with our existing understanding that PES is known to be more hydrophilic than RC. It is also seen that in both cases, the contact angle decreases after the membrane has been used, thereby indicating presence of non-specific protein adsorption. Hence, for obtaining maximal recovery, it is necessary to identify conditions where non-specific protein adsorption is
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minimal (as shown above).
4. CONCLUSIONS This paper explores the use of a variety of membrane adsorbers for purification of Granulocyte Colony Stimulating Factor (GCSF), an E coli based biotherapeutic. Two different base matrices (polyethersulfone and regenerated cellulose) and four different ligands (sulfonic acid, quarternary ammonium, phenyl, and primary amine) were examined. Four different modes of chromatography, namely cation exchange, anion exchange, hydrophobic interaction and salt tolerant interaction, were experimentally investigated. It can be concluded that the base matrix have more significant impact on the separation than the ligands, particularly so in applications where the protein binds to the membrane and then elutes. Based on the results obtained, we can design an improved stationary phase for protein purification which has high pore accessibility for the particular protein. Many commercial vendors are likely to use existing base matrices for such new products, so the novelty of the design is likely to lie specifically in the polymer attachments. There are several broad classes of these, but a more complete understanding could allow incorporation of more nuanced design features towards exploiting such
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parameters as the polymer stiffness or ligand distributions. The paper clearly highlights the important role that polymers continue to play in the field of bioprocessing.
REFERENCES
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[1] Ishihara, T.; Yamamoto, S. Optimization of monoclonal antibody purification by ionexchange chromatography. Application of simple methods with linear gradient elution experimental data. J. Chromatogr. A. 2005, 1069, 99-106. [2] Srivastava,P.; Bhattacharaya, P.; Pandey, G.; Mukherjee, K. J. Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli. Protein Express Purif. 2005, 41, 313-322. [3] Bhambure, R.; Gupta, D.; Rathore, A. S. A Novel Multimodal Chromatography based Single Step Purification Process for Efficient Manufacturing of an E. coli based Biotherapeutic Protein Product. J. Chromatogr. A. 2013, 1314, 188-198. [4] Klatt, K. U.; Hanisch, F.; Dünnebier, G.; Engell, S.Model-based optimization and control of chromatographic processes. Comput. Chem. Eng. 2000, 24, 1119-1126. [5] Gallant, S. R.;Vunnum, S.; Cramer, S. M. Optimization of preparative ion-exchange chromatography of proteins: linear gradient separations. J. Chromatogr. A. 1996, 725, 295-314. [6] Petsch, D.; Beeskow, T. C.; Anspach, F. B.; Deckwer, W. D. Membrane adsorbers for selective removal of bacterial endotoxin. J. Chromatogr. B.1997, 693, 79-91.
18
[7] Stein, A.; Kiesewetter, A. Cation exchange chromatography in antibody purification: pH screening for optimised binding and HCP removal. J. Chromatogr. B.2007, 848, 151158. [8] Rathore, A. S.; Shirke, A.Recent developments in membrane-based separations in biotechnology processes. Prep. Biochem. Biotechnol. 2011, 41, 398-421. [9] Charcosset,C. Purification of proteins by membrane chromatography.
Downloaded by [Georgetown University] at 04:08 17 August 2015
J.Chem.Technol.Biotechnol.1998, 71, 95-110. [10] Knudsen, H. L.; Fahrner, R. L.; Xu, Y.; Norling, L. A.; Blank, G. S. Membrane ionexchange chromatography for process-scale antibody purification. J. Chromatogr. A.2001, 907, 145-154. [11] Chang, C. S.;Suen, S. Y. Modification of porous alumina membranes with nalkanoic acids and their application in protein adsorption.J. Membr. Sci. 2006, 275, 7081. [12] Ulbricht, M.Membrane separations using molecularly imprinted polymers. J. Chromatogr. B.2004, 804, 113-125. [13] Zydney, A.L. Membrane technology for purification of therapeutic proteins. Biotechnol. Bioeng.103, 227-230. [14] Xu, T. Ion exchange membranes: state of their development and perspective. J. Membr. Sci. 2005, 263, 1-29. [15] Wu, C.; Xu, T.; Gong, M.; Yang, W.Synthesis and characterizations of new negatively charged organic–inorganic hybrid materials: Part II. Membrane preparation and characterizations. J. Membr. Sci. 2005, 247, 111-118.
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[16] Fogle, J.; Mohan, N.; Cheung, E.; Persson, J. Effects of resin ligand density on yield and impurity clearance in preparative cation exchange chromatography. I. Mechanistic evaluation. J. Chromatogr. A. 2012, 1225, 62-69. [17] Franke, A.; Forrer, N.; Butté, A.; Cvijetić, B.; Morbidelli, M.; Jöhnck, M.; Schulte, M. Role of the ligand density in cation exchange materials for the purification of proteins. J. Chromatogr. A. 2010, 1217(15), 2216-2225.
Downloaded by [Georgetown University] at 04:08 17 August 2015
[18] Hardin, A. M.; Harinarayan, C.; Malmquist, G.; Axén, A.; Vanreis, R. Ion exchange chromatography of monoclonal antibodies: Effect of resin ligand density on dynamic binding capacity. J. Chromatogr. A. 2009, 1216(20), 4366-4371. [19] Lu, H. L.; Lin, D. Q.; Zhu, M. M.; Yao, S. J. Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion‐ exchange resins. J. Sep. Sci. 2012, 35(16), 2131-2137. [20] Müller, W. New ion exchangers for the chromatography of biopolymers. J. Chromatogr. A. 1990, 510, 133-140. [21] Wrzosek, K.; Gramblička, M.; Polakovič, M. Influence of ligand density on antibody binding capacity of cation-exchange adsorbents. J. Chromatogr. A. 2009, 1216(25), 5039-5044. [22] Hart, D. S.; Harinarayan, C.; Malmquist, G.; Axén, A.; Sharma, M.; Vanreis, R. Surface extenders and an optimal pore size promote high dynamic binding capacities of antibodies on cation exchange resins. J. Chromatogr. A. 2009, 1216(20), 4372-4376. [23] Herman, A. C.; Boone, T. C.; Lu, H. S. In Formulation, Characterization, and Stability of Protein Drugs: Case Histories;Pearlman, R.;Wang, Y.J, Eds.;Springer: New York, 2002;Vol 9,pp. 303.
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[24] Bartkowski, R.; Kitchel, R.; Peckham, N.; Margulis, L. Aggregation of recombinant bovine granulocyte colony stimulating factor in solution. J. Protein Chem. 2002, 21, 138. [25] Snee, R. D. Think strategically for Design of Experiments success. Bioprocess Int. 2011, 9, 18-25. [26] Bade, P. D.; Kotu, S. P.; Rathore, A. S. Optimization of a refolding step for a therapeutic fusion protein in the quality by design (QbD) paradigm. J. Sep. Sci. 2012, 35,
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3160-3169. [27] Cygnus application notes (http://cygnustechnologies.com/product_detail/bacterial/ecoli461 hcp-elisa-kit.html [28] Joshi, V.; Shivach, T.; Kumar,V.; Yadav,N.; Rathore, A.S. Avoiding antibody aggregation during processing: Establishing hold times. Biotechnol. J. 2014, 9, 11951205. [29] Bhambure, R.; Sharma, I.; Pattanayek, S. K.; Rathore, A.S. Qualitative and quantitative examination of non-specific protein adsorption on filter membrane disks of a commercially available high throughput chromatography device. J. Membr. Sci. 2014, 451, 312-318. [30] Kumar, V.;Bhalla, A.;Rathore, A. S. Design of experiments applications in bioprocessing: Concepts and approach. Biotechnol. Prog. 2014, 30, 86-99. [31] Muthukumar, S.; Rathore, A. S. High throughput process development (HTPD) platform for membrane chromatography. J. Membr. Sci.2013, 442, 245-253.
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Table 1. Key properties for the six membranes that were examined in this investigation for purification of GCSF.
S.
Property
Sartobind S
No
Mustang
Mustang Q
Sartobind Q
S
Sartobind
Sartobind
HIC Phenyl
STIC
.
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1
2
Type of
Ion
Ion
Ion
Ion
Hydrophobic
Ion
exchanger
exchange
exchange
exchange
exchange
interaction
exchange
Base
Regenerated Modified
Modified
Regenerated
Regenerated
Regenerated
Matrix
cellulose
Supor® PES
cellulose
cellulose
cellulose
Supor® PES
3
Pressure
4 bars
5.5 bars
5.5 bars
4 bars
4 bars
4 bars
Sulfonic
Sulfonic
Quarternary
Quarternary
Phenyl
Primary
acid
acid
ammonium
ammonium
2-14
2-14
2-14
2-14
tolerance 4
5.
Ligand
pH Stability
22
amine 2-14
2-14
Table 2. Effect of salt addition on GCSF recovery and product purity in Sartobind Phenyl and Sartobind STIC AEX membranes. A. Sartobind Phenyl Membrane pH
Buffer molarity
NaCl(mM)
Recovery (%)
Purity (%)
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(mM) 5.5
35
0
10.23
98.71
5.5
35
0.2
40.83
99.15
5.5
35
0.4
54.32
99.25
5.5
35
0.6
70.65
99.54
5.5
35
0.8
50.08
99.20
NaCl (mM)
Recovery (%)
Log HCP
B. Sartobind STIC AEX Membrane pH
Buffer molarity (mM)
reduction
5.5
35
0.2
94.50
0.59
5.5
35
0.4
97.89
0.82
5.5
35
0.6
98.45
0.93
5.5
35
0.8
92.45
0.96
23
Figure 1. Illustration of the various base matrices and surface chemistry of the different ion exchange polymeric supports used in this investigation. A. Regenerated cellulose base matrix as in Sartobind™ membrane adsorbers from Sartorius Stedim. B. Polyethersulfone base matrix as in Mustang™ membrane adsorbers from Pall Corporation. C. Quaternary ammonium surface chemistry. D. Amine surface chemistry.
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E. Sulfonyl surface chemistry. F. Phenyl surface chemistry.
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Figure 2. GCSF recovery and purity with cation exchange membrane chromatography
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(Mustang S) operated in bind and elute mode.
25
Figure 3. GCSF recovery and purity with cation exchange membrane chromatography
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(Sartobind S) operated in bind and elute mode.
26
Figure 4. GCSF recovery and purity with anion exchange membrane chromatography
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(Mustang Q) operated in flow-through mode.
27
Figure 5. GCSF recovery and purity with anion exchange membrane chromatography
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(Sartobind Q) operated in flow-through mode.
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Figure 6. A. SDS PAGE purity profile for the GCSF purified using cation exchange and HIC Phenyl membrane chromatography. Lane 1: CEX Output, Lane 2: HIC Phenyl output, Lane 3: GCSF Standard (0.2 mg/ml) with low molecular weight impurities, Lanes 4-6: Refolded material for experimentation. B. RP-HPLC chromatogram of the feed material to CEX membrane chromatography. C. RP-HPLC chromatogram of the CEX membrane chromatography pool. D. Far UV CD spectra for purified pool from CEX
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membrane chromatography and for the GCSF standard.
29
Figure 7. Scanning Electron Microscope images of the membrane chromatography samples. A. Scanning Electron microscope of the blank membrane. B. Scanning Electron microscope image of the membrane after use and cleaning. C. Contact angle measurements for both new and used membrane surfaces with base matrix as RC and
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PES.
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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Use of Polymeric Membranes for Purification of an E. Coli Expressed Biotherapeutic Protein a
a
S. Muthukumar & Anurag S. Rathore a
Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India Accepted author version posted online: 15 Jul 2015.
Click for updates To cite this article: S. Muthukumar & Anurag S. Rathore (2015): Use of Polymeric Membranes for Purification of an E. Coli Expressed Biotherapeutic Protein, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2015.1045609 To link to this article: http://dx.doi.org/10.1080/10826068.2015.1045609
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Use of Polymeric Membranes for Purification of an E. coli Expressed Biotherapeutic Protein S. Muthukumar1, Anurag S. Rathore1 1
Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India
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Corresponding author: Anurag Rathore Professor, Department of Chemical Engineering, Indian Institute of Technology, HauzKhas, New Delhi, 110016, India. Email: [email protected]
Abstract
Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed bed chromatography by eliminating the diffusion related limitations associated with the traditional resin beads. In this paper, we examine six membrane adsorbers for purification of Granulocyte Colony Stimulating Factor (GCSF), an E coli based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the paper. KEYWORDS: Membrane chromatography, Host cell protein, Granulocyte Colony Stimulating Factor (GCSF), Surface chemistry, Diffusion
1. INTRODUCTION Chromatography is by far the most widely used technique for purification of therapeutic proteins. Ion-exchange chromatography (IEC) forms the backbone of most biopharmaceutical drug purification processes as it offers high selectivity for clearance of 1
various process and product related impurities.[1, 2]Charge based differences between the native protein and other species that are being separated is the underlying mechanism behind the use of ion exchange chromatography, often used for achieving clearance of the various product related impurities such as oxidized forms, reduced forms and deamidated forms of the protein.[3]However, often these differences in the physicochemical properties
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are minor, making selective clearance a difficult task.
Designing an ion exchange chromatography step for selective clearance of such impurities requires optimization of numerous parameters, including mobile phase (pH, molarity, etc.), stationary phase (type of ion-exchange group, ion-exchange capacity, particle diameter, pore structure, pore size distribution, base matrix property, etc.), column parameters (length, diameter, etc.) and operating variables (flow rate, gradient slope, sample loading, etc.).[4, 5]In anion exchange chromatography, negatively charged amino acid side chains of the protein molecule interact with the positively charged ligands of the chromatography matrix. In cation exchange chromatography, positively charged amino acid side chains of the protein molecule interact with the negatively charged ligands of the chromatography matrix.[6, 7]
Ion exchange column chromatography has been widely used for purification of biotherapeutic proteins because of its high capacity and high efficiency. Most commercial available ion exchange beads are based on sephadex, agarose, silica and cross linked cellulose. However, high pressure drops, internal diffusion limitation, the compressibility of the soft beads and plugging are issues that are typically associated with them. In view
2
of these limitations, use of membrane chromatography has been steadily increasing in the recent years.[8-10] A variety of anions (diethylaminoethyl, quaternary aminoethyl and quaternary ammonium) and cations (carboxymethyl, sulphopropyl and methyl sulphone) have been coupled to porous membranes based on nylon, regenerated cellulose, perfluoropolymer, poly (glycidyl methacrylate) and polyethylene. Three steps are usually involved in preparation of a typical ion exchange membrane chromatography process,
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namely preparation of the basic membrane, activation of the basic membrane, and coupling of ligands to the activated membrane.[11-15]
The effect of ligand density on cation exchange chromatography has been examined. The authors reported that ligand density affects the resolution of a basic charge variant as well as the host cell protein clearance.[16] Further, the performance of ion exchange chromatography resin has been found to depend not only on the ligand type and ligand density, but also on the pore accessibility of the target molecule. [17] The researchers demonstrated that ligand density directly influences the porosity of the materials as well as the pore diffusivity and the dynamic binding capacity.
The effect of ligand density, apparent pore size and protein charge on the dynamic binding capacity has been extensively investigated. [18] It has been found that the resin pore size is a factor in the occurrence of exclusion, with exclusion being largely absent in the larger pore size resins but appearing consistently in the smaller pore size based resins. The resin ligand density had very little effect on the maximum dynamic binding capacity. In another publication, the adsorption capacities of the resin were found to
3
increase with an increase in ligand density and a decrease in pore size, and an integrative parameter was proposed to describe the combined effects of ligand density and pore size. [19]
It was also found that the effective pore diffusion coefficient of the adsorption kinetics
was influenced by pore sizes of resins, but was relatively independent on the ligand densities of the resins.
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A new type of ion exchanger has been reported in which the ionic groups are exclusively located on linear polymer chains grafted on the support surface. [20] This reduces the contact between the analyte and the matrix. Researchers have studied the influence of ligand density on antibody binding capacity of cation-exchange adsorbents and found that at high positive protein net charges (low pH, low ionic strength), low adsorption capacity values were obtained for cation-exchange materials with a grafted polymer layer and a high ligand density.[21] The impact of different pore sizes, dextran surface extender concentration and ligand density of cation exchange resins on the dynamic binding capacity of a therapeutic antibody has been explored.[22] The researchers identified that increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content resulted in higher dynamic binding capacity mainly at the optimal pore size and lower conductivities.
Granulocyte Colony Stimulating Factor (GCSF) is a 175 amino acid protein chain with five cysteine amino acids, four of which are involved in forming two disulphide linkages (Cys36-Cys42 and Cys64-Cys74) and the fifth (Cys17) is free. These two disulphide bonds form two small loops, which are separated by 21 amino acids. The GCSF molecule
4
has a molecular weight of 18.8 kDa.[23, 24]It is known to stimulate the production of White Blood Cells (WBCs).[25]One of the major applications of this product is in ameliorating neutropenia, a condition where neutrophil count falls below 1.5 x 109/L.
The physical characteristics of the resin such as particle size, pore size, ligand density and base matrix chemistry also play a significant role in ion exchange chromatography. In
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particular, the effect of base matrix on impurity clearance has not been studied in a systematic manner for large scale purification processes. This paper explores the role of interactions between the protein and the chromatographic membrane surface in purification of GCSF (Figures 1A to 1F). Membranes that have been explored include those based on cation exchange, anion exchange, hydrophobic interaction and salt tolerance exchange interactions. Performance of the various membrane adsorbers has been characterized with respect to step recovery, product quality as well as non-specific binding of the product to the membrane.
2. EXPERIMENTAL 2.1. Materials Mustang S and Q membrane adsorbers were purchased from Pall Life Sciences, Bangalore. Sartobind®S, Q, HIC Phenyl and STIC AEXwerepurchased from Sartorius Stedim India Pvt Ltd, Bangalore. Glacial acetic acid, sodium acetate (anhydrous),sodium chloride, sodium hydroxide,glycerol,acetonitrile (HPLC grade), and trifluoroacetic acid (TFA) were purchased from Merck Chemicals, Bangalore, India.Acryl amide, ammonium persulfate, bisacrylamide, beta-mercaptoethanol, glycine, sodium dodecyl
5
sulfate, sodium salt of ethylenediaminetetra acetic acid (EDTA), sodium thiosulphate and N, N, N', and N'-tetramethylethylenediamine (TEMED) were purchased from Sigma Aldrich Co, Bangalore,India.
2.2. Buffers Acetate buffers of different pH and buffer molarity were prepared for cation and anion
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exchange membrane chromatography. All buffers were degassed by ultrasonication prior to use.
2.3. Refolding and Sample Preparation Refolded GCSF was used in this investigation. Inclusion bodies from E. coli were initially solubilized using urea as a denaturant followed by the refolding using the dilution method at 7 ± 2°C.[26]The refolded product was analyzed by RP-HPLC for measuring the unfolded and refolded product forms and was thereafter concentrated by ultrafiltration using a 3 kDa MinimateTM tangential flow filtration capsule from Pall life sciences, Bangalore. The pH of the concentrated protein sample was then adjusted to pH 4.0. The protein sample was then centrifuged at 8000 rpm at 4ºC. Supernatant was collected and buffer exchanged in the respective buffers. The buffer exchanged samples were used as an input for the cation and anion exchange membrane chromatography. Approximately 90% of the total protein in the feed material was GCSF.
2.4 Lab Scale Cation and Anion Exchange Membrane Chromatography
6
The membrane chromatography disc was connected to ÄKTA Purifier chromatography system (GE Healthcare Bio-Sciences, Uppsala, Sweden). The disc was equilibrated using the selected equilibration buffer (5-10 CV). Pretreated GCSF protein solution was injected into the disc using a sample loop of capacities from 10-2000 µl (GE Healthcare Bio-Sciences).After sample loading, unbound protein sample was removed using a wash step with the equilibration buffer (5 CV).Elution was performed using a salt or pH based
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elution gradient. The output from the disc was monitored using pH, conductivity and UV detection at 280, 260 and 215 nm detectors.
2.5 Reversed Phase High Performance Liquid Chromatography (RP HPLC) Protein concentration for the refolded protein as well as membrane chromatography pools was determined by performing RP–HPLC using a 4.6 mm × 150 mm Zorbax Eclipse XDB C4 column from Agilent Technologies, Palo Alto, CA on theUltimate 3000 LC system from Dionex. The mobile phase consisted of solvents A and B. Solvent A was 0.1% (v/v) TFA in water and Solvent B was 0.1% (v/v) TFA in 98 % of acetonitrile. Flow rate was maintained at1 ml/min using a linear gradient of A to B at a wavelength of 214 nm.
2.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) SDS PAGE was used for identification of the impurities associated with GCSF. A 1 mm thick resolving polyacrylamide gel (13 %) was used under non-reducing condition at constant voltage conditions. Each sample was boiled for 5 min in the starting buffer
7
before being loaded into the gel. Silver staining was used to detect proteins after electrophoretic separation.
2.7 Enzyme-Linked Immunosorbent Assay (ELISA) Concentration of host cell proteins (HCP) in the anion exchange chromatography flowthrough was analyzed using an ELISA (E. Coli. HCP analysis kit F 410 from Cygnus
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Technologies, USA). Samples containing E. coli HCP were reacted with a horseradish peroxidase (HRP) enzyme labeled anti-E. Coli antibody simultaneously in microtiter strips coated with an affinity purified capture anti-E. Coli antibody. The immunological reactions resulted in formation of a sandwich complex of solid phase antibody-HCPenzyme labeled antibody. The microtiter strips were washed to remove any unbound reactants. The substrate, tetramethylbenzidine (TMB) was then reacted. The amount of hydrolyzed substrate was read on a microtiter plate reader at 450 nm and was directly proportional to the concentration of E. coli HCP present.[27]
2.8. Circular Dichroism (CD) Spectroscopy Secondary structure of purified protein was determined by CD spectroscopy. Sample with 0.2 mg/ml of resolublized protein was taken into the respective buffer in a 2 mm path length cuvette and far UV CD spectrum was measured from 250 to 200 nm in a JASCO J-815 Spectropolarimeter (Jasco, Inc. Mary's Court, Easton, MD 21601, USA) with the spectral band width of 5 nm.[28] An average of three scans was plotted against the wavelength.
8
2.9 Scanning Electron Microscopy (SEM) Membrane samples were removed from the Mustang S membranes after experimentation and gold was sputtered followed by SEM imaging. The sputtered samples were kept in the vacuum chamber and images were obtained using a ZEISS scanning electron microscope (Germany) with a secondary electrons detector at a voltage of 20 kV.
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2.10 Contact Angle Measurements Contact angles (θ) of deionized water, on different membrane surfaces were measured using a Kruss contact angle goniometer (KRÜSS GmbH, Borsteler Chaussee, Hamburg, Germany) at 25° C. [29]A drop of the liquid was placed on the test surface. After 5 min, the contact angle between the plane substrate (S) and the liquid meniscus (L) reached an equilibrium value corresponding to a constant drop volume. This equilibrium angle was reported and recorded as the static contact angle of the liquid on the substrate.
2.11 Statistical Analysis Design of experiments (DOE) were created as well as the statistical analysis of the resulting data was performed using JMP® 8.0 (SAS Institute Inc., Cary NC). For analysis of DOE data, the procedure outlined in a previous publication was followed.[30]
3. RESULTS AND DISCUSSION The objective of this article is to highlight the important role of interactions between the proteins and the polymer surface in purification of proteins. In this respect, we have examined performance of two different polymeric surfaces with four different ligands
9
(Figure 1). Further, some key characteristics of these different polymeric membranes are also listed in Table 1. In the following subsections, we discuss the performance of the different adsorbers for purification of GCSF. Performance of the various membrane adsorbers has been characterized with respect to step recovery, product quality as well as non-specific binding of the product to the membrane.
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3.1. Purification Using Membrane Chromatography 3.1.1 Cation Exchange Membrane Chromatography Instability of GCSF at higher pH makes cation exchange membrane chromatography as the first choice for the separation of various product related impurities associated with GCSF31. Full-factorial design of experiments (DOE) was used to investigate the effect of pH and buffer molarity on product recovery both for the Sartobind S and Mustang S membranes (Figures 2 and 3). Based on the pH range under consideration, acetate buffer system was selected and buffers of varying molarity were prepared and used for equilibration in cation exchange membrane chromatography. Salt based elution strategy was selected in this study due to the reported instability of GCSF at higher pH values. Maximum salt concentration selected for the elution studies was limited to 1 M NaCl considering the precipitation effect of salt on GCSF.
As seen in Figures 2 and 3, product recovery is higher when the base matrix is PES (Mustang S) when compared to the base matrix of RC (Sartobind S) (average of 45% vs. 41%, maximum recovery of 78% vs. 72%). This is likely because the GCSF molecule is known to be very hydrophobic in nature and PES is known to be hydrophilic (more than
10
RC). The lower recoveries are likely due to the presence of non-specific protein adsorption on the membrane surface [31]. The performance of the two matrices was quite similar with respect to purity of GCSF (average of 98.3% vs. 98.6% and maximum of 99.5% vs. 99.7%).
Figures 2 and 3also present detailed statistical analysis of the data for Mustang S and
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Sartobind S, respectively. It is seen in Figures 2B, 2D, 3B and 3D that in both cases the resulting models are statistically significant (R2=0.99). Further, both pH and buffer molarity have a significant impact on GCSF recovery and purity (Figures 2C, 2E, 3C and 3E). Optimization was performed for both the cases using the prediction profiler function of the JMP software. For the case of Mustang S, optimal conditions were identified as pH of 5.7 and buffer molarity of 33 mM (Figure 2C and 2E). Under these conditions, GSCF recovery of 80% and purity of 99.4% can be achieved. Similarly, in the case of Sartobind S, GCSF recovery of 73% and purity of 99.1% can be achieved at a pH of 5.7 and buffer molarity of 34 mM (Figures 3C and 3E).
Overall, we can conclude that the effects of pH and buffer molarity are quite similar for the two membranes, indicating the dominance of the surface chemistry (same for the two membranes) over the base matrix (different for the two membranes). Based on these results and our earlier experience with purification of GCSF, we think that the binding between the protein and the membrane is very strong at pH 4.0, and not all the protein leaves the membrane during elution. This results in lower recoveries at lower pH.
11
3.1.2. Anion Exchange (Aex) Membrane Chromatography Usefulness of anion exchange chromatography as the first purification step is limited by the instability of GCSF at high pH.[31] In view of this, we evaluated use of AEX in the flow-through mode for clearance of host cell proteins, endotoxins and nucleic acids. The focus of optimization was thus on maximizing GCSF recovery.
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As seen in Figures 4 and 5, unlike the case of cation exchange chromatography, product recovery in anion-exchange chromatography is quite similar for the case when the base matrix is PES as in Mustang Q when compared to the base matrix of RC as in Sartobind Q (average of 94% and maximum recovery of 100% in both cases). This is likely due to absence of non-specific protein adsorption on the membrane surface under these conditions. The performance of the two matrices was also similar with respect to HCP clearance (average of 1.5 log clearance and maximum of 2 log clearance in both cases).
Figures 4 and 5 also present detailed statistical analysis of the data for Mustang Q and Sartobind Q, respectively. It is seen in Figures 4B, 4D, 5B and 5D that in both cases the resulting models are statistically significant (R2> 0.95). Further, both pH and buffer molarity have a significant impact on GCSF recovery and HCP clearance (Figures 4C, 4E, 5C, and 5E). Optimization was performed for both the cases using the prediction profiler function of the JMP software. For the case of Mustang Q, optimal conditions were identified as pH of 5.7 and buffer molarity of 42 mM (Figures4C and 4E). Under these conditions, GSCF recovery of 100% and HCP clearance of 1.8 logs can be achieved. Similarly, in the case of Sartobind Q, GCSF recovery of 99.3% and HCP
12
clearance of 1.9 logs can be achieved at a pH of 5.7 and buffer molarity of 40mM (Figures 5C and 5E).
Overall, we can conclude that the effects of pH and buffer molarity are quite similar for the two membranes. Based on these results, we can conclude that anion exchange membrane chromatography can be successfully used in the flow-through mode for
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reducing host cell impurities.
3.1.3 Hydrophobic Interaction Phenyl Membrane Chromatography (Hic) Separation in hydrophobic interaction chromatography occurs due to a combination of hydrophobic and ionic interactions. Typically, feed is loaded under conditions of high salt and hence ionic interactions are minimal and hydrophobic interactions dominate the binding of the species to the chromatographic support. Salt ions in this solution reduce the solvation of solutes in the sample. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the hydrophobic membrane matrix. As the salt concentration is lowered during wash or elution, the ionic interactions come into play and impact the separation of the various species in the feed. The motivation for exploring HIC mode was the possibility of achieving enhanced separation due to duality of underlying interactions.
In view of the above, the possibility of using HIC membrane chromatography was explored as a possible purification step in the bind and elute mode as a possible substitute or an add-on to CEX chromatography. Table 2A shows the GCSF recovery and purity
13
obtained. It is seen that while the purity obtained is acceptable (99.5%), the recovery is lesser (maximum of 70%) as compared to CEX membrane chromatography. It is seen that as the salt concentration increases the protein binding to the membrane matrix also improves up to a certain limit beyond which it start to decrease. Based on the results, it can be concluded that CEX membrane chromatography would be the preferred mode
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over HIC membrane chromatography for purification of GCSF.
3.1.4 Salt Tolerant Interaction Membrane Chromatography (Stic) Very recently a new kind of membrane adsorber has been introduced that uses the primary amine ligand for binding negatively charged impurities such as DNA, host cell proteins, endotoxins and viruses at much higher salt concentrations than in the case of conventional Q matrices. The motivation behind examining this membrane was the ease of operation STIC would offer as the elute from CEX chromatography could be directly loaded on the STIC column. Due to the high salt that this elute contains, the same is not possible with AEX chromatography discussed above and a diafiltration step is required in the intermediate to change the feed conditions.
In view of the above, the possibility of using STIC membrane chromatography was explored as a possible purification step in the flow-through mode as a substitute to AEX chromatography. Table 2B shows the GCSF recovery and purity obtained. It is seen that while the GCSF recovery obtained is high (98.5%), the HCP clearance is not as significant as in AEX chromatography (maximum of 1 log). It can be concluded that STIC offers a possible alternative to AEX membrane chromatography.
14
3.1.5 Final Process Scheme Based on the results presented above, we can recommend two processing options. First would be using a CEX membrane chromatography followed by diafiltration and AEX membrane chromatography (CEX-DF-AEX). Second would be using CEX membrane chromatography followed by STIC membrane chromatography (CEX-STIC). Both
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options seem feasible and have their own strengths and weaknesses.
3.2 Product Characterization 3.2.1 Analysis Of Product Quality Non reducing SDS PAGE and RP-HPLC analysis were performed as identity and purity tests of GCSF, respectively. Figure 6 illustrates the data for CEX membrane chromatography. The SDS PAGE gel in Figure 6A clearly shows the significant purification that both CEX membrane chromatography (Lane 1) and HIC membrane chromatography (Lane 2) accomplish. Bulk of the impurities that are present in the feed material (Lanes 4-6) are removed during these steps resulting in almost pure product (Lane 3).Similar observation can be made from comparison of the RP-HPLC chromatogram for the feed material (Figure 6B) and the CEX membrane chromatography pool (Figure 6C). The latter shows a nearly complete removal of all product related impurities.
3.2.2 Circular Dichroism (CD) Spectroscopy For Secondary Structure Analysis
15
CD spectroscopy was performed for the GCSF sample from CEX membrane chromatography and compared to that for the GCSF reference standard (Figure 6D). It is seen that the two spectra are near-identical and this confirms that the protein produced has the desired secondary structure.
3.3 Non-Specific Protein Adsorption
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Non-specific protein adsorption has been seen as a concern with GCSF due to the extreme hydrophobic nature of the protein [31]. This results in low product recovery during processing. Hence, it was investigated via two analytical tools namely contact angle measurements and scanning electron microscopy.
3.3.1 Scanning Electron Microscopy Scanning electron microscopy was used as an analytical tool to study reveals the images of the blank membrane surface (Figure 7A) and after the protein is adsorbed on to the membrane surface (Figure 7B). It is evident that there is considerable non-specific adsorption on the membrane surface. This is the reason for the lower product recovery in CEX and HIC membrane chromatography and will also be a concern for membrane reuse. Appropriate cleaning and sanitization protocols will need to be put in place to be able to recycle the membranes.
3.3.2 Contact Angle Measurements Contact angle measurements can be used for quantitating the hydrophobicity of a surface. The results of these measurements for the PES base matrix (Mustang S) and RC
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base matrix (Sartobind S) are shown in Figure 7C. The contact angle value for the new membrane is lower for PES (86±2 deg) than for RC (93±2 deg) and this agrees with our existing understanding that PES is known to be more hydrophilic than RC. It is also seen that in both cases, the contact angle decreases after the membrane has been used, thereby indicating presence of non-specific protein adsorption. Hence, for obtaining maximal recovery, it is necessary to identify conditions where non-specific protein adsorption is
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minimal (as shown above).
4. CONCLUSIONS This paper explores the use of a variety of membrane adsorbers for purification of Granulocyte Colony Stimulating Factor (GCSF), an E coli based biotherapeutic. Two different base matrices (polyethersulfone and regenerated cellulose) and four different ligands (sulfonic acid, quarternary ammonium, phenyl, and primary amine) were examined. Four different modes of chromatography, namely cation exchange, anion exchange, hydrophobic interaction and salt tolerant interaction, were experimentally investigated. It can be concluded that the base matrix have more significant impact on the separation than the ligands, particularly so in applications where the protein binds to the membrane and then elutes. Based on the results obtained, we can design an improved stationary phase for protein purification which has high pore accessibility for the particular protein. Many commercial vendors are likely to use existing base matrices for such new products, so the novelty of the design is likely to lie specifically in the polymer attachments. There are several broad classes of these, but a more complete understanding could allow incorporation of more nuanced design features towards exploiting such
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parameters as the polymer stiffness or ligand distributions. The paper clearly highlights the important role that polymers continue to play in the field of bioprocessing.
REFERENCES
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[1] Ishihara, T.; Yamamoto, S. Optimization of monoclonal antibody purification by ionexchange chromatography. Application of simple methods with linear gradient elution experimental data. J. Chromatogr. A. 2005, 1069, 99-106. [2] Srivastava,P.; Bhattacharaya, P.; Pandey, G.; Mukherjee, K. J. Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli. Protein Express Purif. 2005, 41, 313-322. [3] Bhambure, R.; Gupta, D.; Rathore, A. S. A Novel Multimodal Chromatography based Single Step Purification Process for Efficient Manufacturing of an E. coli based Biotherapeutic Protein Product. J. Chromatogr. A. 2013, 1314, 188-198. [4] Klatt, K. U.; Hanisch, F.; Dünnebier, G.; Engell, S.Model-based optimization and control of chromatographic processes. Comput. Chem. Eng. 2000, 24, 1119-1126. [5] Gallant, S. R.;Vunnum, S.; Cramer, S. M. Optimization of preparative ion-exchange chromatography of proteins: linear gradient separations. J. Chromatogr. A. 1996, 725, 295-314. [6] Petsch, D.; Beeskow, T. C.; Anspach, F. B.; Deckwer, W. D. Membrane adsorbers for selective removal of bacterial endotoxin. J. Chromatogr. B.1997, 693, 79-91.
18
[7] Stein, A.; Kiesewetter, A. Cation exchange chromatography in antibody purification: pH screening for optimised binding and HCP removal. J. Chromatogr. B.2007, 848, 151158. [8] Rathore, A. S.; Shirke, A.Recent developments in membrane-based separations in biotechnology processes. Prep. Biochem. Biotechnol. 2011, 41, 398-421. [9] Charcosset,C. Purification of proteins by membrane chromatography.
Downloaded by [Georgetown University] at 04:08 17 August 2015
J.Chem.Technol.Biotechnol.1998, 71, 95-110. [10] Knudsen, H. L.; Fahrner, R. L.; Xu, Y.; Norling, L. A.; Blank, G. S. Membrane ionexchange chromatography for process-scale antibody purification. J. Chromatogr. A.2001, 907, 145-154. [11] Chang, C. S.;Suen, S. Y. Modification of porous alumina membranes with nalkanoic acids and their application in protein adsorption.J. Membr. Sci. 2006, 275, 7081. [12] Ulbricht, M.Membrane separations using molecularly imprinted polymers. J. Chromatogr. B.2004, 804, 113-125. [13] Zydney, A.L. Membrane technology for purification of therapeutic proteins. Biotechnol. Bioeng.103, 227-230. [14] Xu, T. Ion exchange membranes: state of their development and perspective. J. Membr. Sci. 2005, 263, 1-29. [15] Wu, C.; Xu, T.; Gong, M.; Yang, W.Synthesis and characterizations of new negatively charged organic–inorganic hybrid materials: Part II. Membrane preparation and characterizations. J. Membr. Sci. 2005, 247, 111-118.
19
[16] Fogle, J.; Mohan, N.; Cheung, E.; Persson, J. Effects of resin ligand density on yield and impurity clearance in preparative cation exchange chromatography. I. Mechanistic evaluation. J. Chromatogr. A. 2012, 1225, 62-69. [17] Franke, A.; Forrer, N.; Butté, A.; Cvijetić, B.; Morbidelli, M.; Jöhnck, M.; Schulte, M. Role of the ligand density in cation exchange materials for the purification of proteins. J. Chromatogr. A. 2010, 1217(15), 2216-2225.
Downloaded by [Georgetown University] at 04:08 17 August 2015
[18] Hardin, A. M.; Harinarayan, C.; Malmquist, G.; Axén, A.; Vanreis, R. Ion exchange chromatography of monoclonal antibodies: Effect of resin ligand density on dynamic binding capacity. J. Chromatogr. A. 2009, 1216(20), 4366-4371. [19] Lu, H. L.; Lin, D. Q.; Zhu, M. M.; Yao, S. J. Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion‐ exchange resins. J. Sep. Sci. 2012, 35(16), 2131-2137. [20] Müller, W. New ion exchangers for the chromatography of biopolymers. J. Chromatogr. A. 1990, 510, 133-140. [21] Wrzosek, K.; Gramblička, M.; Polakovič, M. Influence of ligand density on antibody binding capacity of cation-exchange adsorbents. J. Chromatogr. A. 2009, 1216(25), 5039-5044. [22] Hart, D. S.; Harinarayan, C.; Malmquist, G.; Axén, A.; Sharma, M.; Vanreis, R. Surface extenders and an optimal pore size promote high dynamic binding capacities of antibodies on cation exchange resins. J. Chromatogr. A. 2009, 1216(20), 4372-4376. [23] Herman, A. C.; Boone, T. C.; Lu, H. S. In Formulation, Characterization, and Stability of Protein Drugs: Case Histories;Pearlman, R.;Wang, Y.J, Eds.;Springer: New York, 2002;Vol 9,pp. 303.
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[24] Bartkowski, R.; Kitchel, R.; Peckham, N.; Margulis, L. Aggregation of recombinant bovine granulocyte colony stimulating factor in solution. J. Protein Chem. 2002, 21, 138. [25] Snee, R. D. Think strategically for Design of Experiments success. Bioprocess Int. 2011, 9, 18-25. [26] Bade, P. D.; Kotu, S. P.; Rathore, A. S. Optimization of a refolding step for a therapeutic fusion protein in the quality by design (QbD) paradigm. J. Sep. Sci. 2012, 35,
Downloaded by [Georgetown University] at 04:08 17 August 2015
3160-3169. [27] Cygnus application notes (http://cygnustechnologies.com/product_detail/bacterial/ecoli461 hcp-elisa-kit.html [28] Joshi, V.; Shivach, T.; Kumar,V.; Yadav,N.; Rathore, A.S. Avoiding antibody aggregation during processing: Establishing hold times. Biotechnol. J. 2014, 9, 11951205. [29] Bhambure, R.; Sharma, I.; Pattanayek, S. K.; Rathore, A.S. Qualitative and quantitative examination of non-specific protein adsorption on filter membrane disks of a commercially available high throughput chromatography device. J. Membr. Sci. 2014, 451, 312-318. [30] Kumar, V.;Bhalla, A.;Rathore, A. S. Design of experiments applications in bioprocessing: Concepts and approach. Biotechnol. Prog. 2014, 30, 86-99. [31] Muthukumar, S.; Rathore, A. S. High throughput process development (HTPD) platform for membrane chromatography. J. Membr. Sci.2013, 442, 245-253.
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Table 1. Key properties for the six membranes that were examined in this investigation for purification of GCSF.
S.
Property
Sartobind S
No
Mustang
Mustang Q
Sartobind Q
S
Sartobind
Sartobind
HIC Phenyl
STIC
.
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1
2
Type of
Ion
Ion
Ion
Ion
Hydrophobic
Ion
exchanger
exchange
exchange
exchange
exchange
interaction
exchange
Base
Regenerated Modified
Modified
Regenerated
Regenerated
Regenerated
Matrix
cellulose
Supor® PES
cellulose
cellulose
cellulose
Supor® PES
3
Pressure
4 bars
5.5 bars
5.5 bars
4 bars
4 bars
4 bars
Sulfonic
Sulfonic
Quarternary
Quarternary
Phenyl
Primary
acid
acid
ammonium
ammonium
2-14
2-14
2-14
2-14
tolerance 4
5.
Ligand
pH Stability
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amine 2-14
2-14
Table 2. Effect of salt addition on GCSF recovery and product purity in Sartobind Phenyl and Sartobind STIC AEX membranes. A. Sartobind Phenyl Membrane pH
Buffer molarity
NaCl(mM)
Recovery (%)
Purity (%)
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(mM) 5.5
35
0
10.23
98.71
5.5
35
0.2
40.83
99.15
5.5
35
0.4
54.32
99.25
5.5
35
0.6
70.65
99.54
5.5
35
0.8
50.08
99.20
NaCl (mM)
Recovery (%)
Log HCP
B. Sartobind STIC AEX Membrane pH
Buffer molarity (mM)
reduction
5.5
35
0.2
94.50
0.59
5.5
35
0.4
97.89
0.82
5.5
35
0.6
98.45
0.93
5.5
35
0.8
92.45
0.96
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Figure 1. Illustration of the various base matrices and surface chemistry of the different ion exchange polymeric supports used in this investigation. A. Regenerated cellulose base matrix as in Sartobind™ membrane adsorbers from Sartorius Stedim. B. Polyethersulfone base matrix as in Mustang™ membrane adsorbers from Pall Corporation. C. Quaternary ammonium surface chemistry. D. Amine surface chemistry.
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E. Sulfonyl surface chemistry. F. Phenyl surface chemistry.
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Figure 2. GCSF recovery and purity with cation exchange membrane chromatography
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(Mustang S) operated in bind and elute mode.
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Figure 3. GCSF recovery and purity with cation exchange membrane chromatography
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(Sartobind S) operated in bind and elute mode.
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Figure 4. GCSF recovery and purity with anion exchange membrane chromatography
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(Mustang Q) operated in flow-through mode.
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Figure 5. GCSF recovery and purity with anion exchange membrane chromatography
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(Sartobind Q) operated in flow-through mode.
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Figure 6. A. SDS PAGE purity profile for the GCSF purified using cation exchange and HIC Phenyl membrane chromatography. Lane 1: CEX Output, Lane 2: HIC Phenyl output, Lane 3: GCSF Standard (0.2 mg/ml) with low molecular weight impurities, Lanes 4-6: Refolded material for experimentation. B. RP-HPLC chromatogram of the feed material to CEX membrane chromatography. C. RP-HPLC chromatogram of the CEX membrane chromatography pool. D. Far UV CD spectra for purified pool from CEX
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membrane chromatography and for the GCSF standard.
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Figure 7. Scanning Electron Microscope images of the membrane chromatography samples. A. Scanning Electron microscope of the blank membrane. B. Scanning Electron microscope image of the membrane after use and cleaning. C. Contact angle measurements for both new and used membrane surfaces with base matrix as RC and
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PES.
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