Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

Journal of Membrane Science 260 (2005) 112–118

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography Raja Ghosh ∗ Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ont., Canada L8S 4L7 Received 21 January 2005; received in revised form 22 March 2005; accepted 23 March 2005 Available online 25 April 2005

Abstract This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 ␮m pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%. © 2005 Elsevier B.V. All rights reserved. Keywords: Membrane chromatography; Protein; Module; Hydrophobic interaction; Plasma protein; Immunoglobulin; Albumin

1. Introduction Membrane chromatography, which involves the use of a membrane or a stack of membranes as chromatographic media, is a fast growing protein bioseparation technique [1–12]. Membranes have been shown to possess some key advantages over packed beds. However, the use of membranes as chromatographic media faces some major challenges and hurdles, which need to be overcome in order for membrane chromatography to be more widely accepted and used. Some of these challenges include the generally lower protein binding capacities with most membranes, inadequacies in current membrane module designs and the limited range of separation chemistries with commercially available membranes [11]. These challenges have largely restricted the use of membrane chromatography to certain niche applications such as the capture of proteins from dilute solutions and the removal of endotoxins. However, the increase in research activities ∗

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in the area of membrane chromatography and the increasing visibility and availability of commercial products seem to indicate that membrane chromatography will be more widely used in the future. The main separation chemistry utilized in membrane chromatography is ion-exchange followed by affinity interactions [11]. There are relatively fewer reports on reverse phase and hydrophobic interaction-based membrane chromatographic separations. Column based hydrophobic interaction chromatography is widely used for the purification plasma proteins, monoclonal antibodies and membrane bound proteins [13]. The interactions take place between the hydrophobic residues on proteins and those on the chromatographic media in high anti-chaotropic salt concentration environment. Such interactions are frequently more discriminating than ion-exchange, particularly in the applications mentioned above. Hence, highly selective separation is frequently possible. Also, the high anti-chaotropic salt concentration environment is known to exert a stabilizing influence on the proteins being processed and hence the bioseparation is more benign [13].

R. Ghosh / Journal of Membrane Science 260 (2005) 112–118

As mentioned earlier, there are relatively fewer papers dealing with hydrophobic interaction-based membrane chromatography of proteins. Tennikova et al. [4] discussed the use of membranes modified with sulpho-, C4 and C8 groups for hydrophobic interaction membrane chromatography. Kubota et al. [14] have discussed the binding of bovine serum albumin on a phenyl-group modified polymeric membrane. In a recent paper, Coffinier and Vijayalakshmi [15] have discussed the use of mercaptoheterocyclic ligands for the thiophilic membrane chromatography of immunoglobulin G. The bioseparation of CAMPATH-1H monoclonal antibody by hydrophobic interaction membrane chromatography using hydrophilic polyvinylidine fluoride (PVDF) membrane having 0.2 ␮m pore size [16] and supported multi-liquid membrane [17] has been reported. More recently the use of Durapore PVDF membrane having 0.65 ␮m pore size for secondary capture of immunoglobulin G as part of an integrated or hybrid bioseparation technique has been discussed [18]. The binding capacity of the Durapore membrane was too low for it to be used for stand-alone membrane chromatography. This paper discusses the hydrophobic interaction membrane chromatographic separation of plasma proteins: human serum albumin (HSA) and human immunoglobulin G (HIgG) using 0.1 ␮m polyvinylidine fluoride (PVDF) microfiltration membrane. HSA and HIgG are the main constituents of human plasma, HSA comprising 70% of total plasma proteins while HIgG comprising another 17–20% [19]. Hence, these are the two main plasma proteins that are fractionated in bulk quantities for commercial use. HSA and HIgG are used as pharmaceutical products and are required in highly pure forms (as specified in the various Pharmacopoeias). The PVDF membrane used in this study was found to specifically and reversibly bind HIgG in a high ammonium sulphate concentration environment. At this operating condition i.e. 1.5 M ammonium sulphate concentration, HSA did not bind to the membrane. Hence, using this membrane, the separation of simulated mixtures of these plasma proteins could be carried out. The extent of HIgG binding on the membrane and the factors affecting it were first examined. HIgG/HSA separation was carried out in the pulse and step-input modes and the purity of individual proteins thus obtained were determined.

2. Experimental 2.1. Materials Human serum albumin (HSA) and human immunoglobulin G (HIgG) were kindly donated by the Scottish Blood Transfusion Services, UK. HSA has a molecular weight of 69 kDa and an isoelectric point of 4.9 while HIgG has a molecular weight of 155 kDa and an isoelectric point of 7.0. The human serum albumin was used as available while the human immunoglobulin G was purified to 99% purity before

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being used in the membrane chromatographic experiments. PVDF micro-filtration membrane sheets (20 cm × 20 cm, 0.1 ␮m pore size, type VVPP) were kindly donated by Millipore. In the membrane chromatographic experiments, 42 mm discs were cut out from these flat sheets and were either used singly or in the from of membrane stacks. All buffers and feed solutions were prepared using ultra-pure water (18.2 M
cm) obtained from a Barnstead NANOPURE Diamond water purification unit. All chemical used in the experiments e.g. sodium phosphate (di-basic and mono-basic) and ammonium sulphate were purchased from Sigma. 2.2. Chromatographic equipment An AKTAprime system (Amersham Biosciences) was used for carrying out the membrane chromatographic studies. The chromatographic column normally used in such a system was replaced by a custom designed membrane module which was of the same type as disclosed in a recent patent application [20]. Within this module stacks of flat sheet membrane discs could be housed. The effective diameter of the membrane discs within the module was 42 mm. This membrane module was integrated with the AKTAprime system using PEEK tubing and tubing connectors and the dead volume was kept at a minimum. A 50 ml Superloop was used for injecting protein solutions in breakthrough binding and step-input separation experiments while appropriate smaller sized loops were used in the pulse input binding and separation experiments. In all these experiments the UV absorbance (at 280 nm) of the effluent stream from the module, the system pressure, pH and conductivity data was continuously recorded and logged into a computer using Prime View (Amersham Biosciences) software. 2.3. Buffers and protein samples Preliminary experiments showed that HIgG bound to the microporous PVDF membranes only above 1.4 M ammonium sulphate concentration in the binding buffer. This threshold ammonium sulphate concentration was significantly lower than that required for CAMPATH-1H monoclonal antibody binding on 0.2 ␮m PVDF membrane [16] and supported multi-liquid membrane [17] and for immunoglobulin G binding on 0.65 ␮m Durapore membrane [18]. In these papers, threshold concentrations around 1.8 M have been reported. In the current study, a solution containing 1.5 M ammonium sulphate was chosen as the binding buffer which was prepared using 20 mM sodium phosphate buffer (pH 6.5) as the base buffer. Henceforth in this paper the binding buffer is referred to as buffer A. The base buffer was also used as the eluting buffer and is referred to in this paper as buffer B. All buffers were micro-filtered and degassed prior to use. Protein feed solutions were prepared in buffer A. Prior to use these feed solutions were centrifuged at 10,000 rpm for 20 min to remove precipitates and aggregates and the concentration of protein remaining in solution was measured.

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Fig. 1. Breakthrough of HIgG at 0.45 mg/ml feed concentration. Number of discs in stack = 1; membrane volume = 0.173 ml; flow rate =10 ml/min.

Fig. 3. Analytical separation of HIgG and HSA in the pulse mode using a 500 ␮l sample. Number of discs in stack = 1; membrane volume = 0.173 ml; flow rate = 10 ml/min.

2.4. Experimental methods Two types of chromatographic experiments were carried out in this study i.e. step input and pulse input. The step input experiments were carried out to obtain HIgG breakthrough curves and for step input HSA/HIgG separations. In these experiments buffer A was passed through the membrane until a stable UV absorbance baseline was obtained. Appropriate amount of feed solution prepared in buffer A was then injected into the membrane module using a Super-

loop. The unbound material within the membrane module was then removed using buffer A and the bound HIgG was finally recovered from the membrane using buffer B which provided a low salt concentration environment that favoured desorption. The concentration of HIgG in the effluent was obtained from the UV absorbance data using appropriate absorbance–concentration calibration for this protein. While presenting breakthrough-binding data, an accurate hydraulic correction was done using an unbound tracer in order to ac-

Fig. 2. Breakthrough curves at different flow rates. Number of discs in stack = 1; membrane volume = 0.173 ml; HIgG concentration in feed = 0.45 mg/ml.

Fig. 4. Preparative separation of HIgG and HSA in the pulse mode using a 5 ml sample. Number of discs in stack = 1; membrane volume = 0.173 ml; flow rate = 10 ml/min.

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Fig. 5. Protein-A affinity chromatograms of feed and purified samples from preparative separation of HIgG and HSA.

count for the dead volume i.e. due to the tubing and the module. The pulse chromatographic experiments were carried by injecting pulses of feed solutions prepared in buffer A. The bound HIgG was subsequently eluted using buffer B. In the step and pulse input separation experiments, effluent samples were collected at various stages of separation and these samples were analysed by affinity chromatography and SDS-PAGE. A HiTrapTM rProtein-A FF affinity column (Amersham Biosciences) was used for the affinity chromatographic analysis. HIgG bound to this column while HSA did not. The binding buffer used in this assay was 20 mM sodium phosphate (pH 7.0), the eluting buffer was 100 mM sodium

citrate buffer (pH 3.0) and the flow rate was 1 ml/min. Samples were appropriately diluted in binding buffer before the affinity assays. The protein purity was determined from the affinity chromatograms using calibrations prepared with pure HSA and HIgG. This was necessary due to the high difference in specific absorbance of these two proteins. The SDS-PAGE was obtained using a 10% gel [21].

3. Results and discussion 3.1. Binding of HIgG on PVDF membrane Fig. 1 shows the breakthrough and elution curve obtained with a feed solution consisting of 0.45 mg/ml HIgG prepared

Fig. 6. SDS-PAGE of feed and purified samples from HIgG/HSA separation experiments. Gel = 10%, lane 1 = empty; lane 2 = molecular weight markers; lane 3 = pure HSA standard; lane 4 = pure HIgG standard; lane 5 = HSA/HIgG mixture (feed); lane 6 = HSA purified by pulse input separation; lane 7 = HIgG purified by pulse input separation; lane 8 = HSA purified by step input separation using stack of five discs, lane 9 = HIgG purified by step input separation using stack of five discs; lane 10 = empty.

Fig. 7. Step-input separation of HIgG and HSA using one membrane disc at 10 ml/min flow rate.

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in buffer A. This experiment was carried out at a feed flow rate of 10 ml/min using one PVDF membrane disc having an effective diameter of 42 mm and a bed volume of 0.173 ml. The HIgG binding capacity of the membrane was determined from the breakthrough curve after taking into consideration the dead volume of the system. The incipient breakthrough HIgG binding capacity of the membrane which is defined as the binding capacity at the point at which the HIgG first appeared in the effluent determined to be 37.62 mg/ml. The 10% breakthrough binding capacity, which is defined as the binding capacity at the point when the effluent concentration reached 10% of the feed concentration was determined as 42.83 mg/ml. Another experiment was carried with one membrane disc using a 0.2 mg/ml HIgG feed solution at a flow rate of 10 ml/min. The incipient breakthrough binding

capacity at this experimental condition was determined as 31.61 mg/ml while the 10% breakthrough binding capacity of the membrane was determined as 35.91 mg/ml. Quite clearly the lower HIgG concentration in the feed solution was responsible for the lower binding capacity. The HIgG binding capacity of the 0.1 ␮m PVDF membrane used in this study was an order of magnitude higher than that reported for the 0.65 ␮m Durapore membrane used for the integrated bioseparation of immunoglobulin G [18] and significantly higher than those for various membranes reported by Castilho et al. [22]. Hence, this membrane could be used for efficient stand-alone membrane chromatographic separation of plasma proteins. Fig. 2 shows the breakthrough curves obtained at four different flow rates, i.e. 2.5, 5.0, 7.5 and 10 ml/min, respectively. All these experiments were carried out with one membrane

Fig. 8. Protein-A affinity chromatograms of purified samples from step-input separation of HIgG and HSA. (A) HSA purified using one membrane disc, (B) HIgG purified using one membrane disc, (C) HSA purified using stack of five membrane discs and (D) HIgG purified using stack of five membrane discs.

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disc within the membrane module using 0.45 mg/ml HIgG feed solutions. These breakthrough curves clearly indicate that in the experimental range examined the flow rate had no effect on the binding, this being a characteristic feature of membrane chromatography. This de-coupling of binding capacity from flow rate is one of the major advantages of membrane chromatography since this leads to significant operational flexibility. 3.2. Separation of HSA and HIgG Pulse chromatography is frequently used for analytical as well as small and medium scale preparative protein separations. Fig. 3 shows the separation of HIgG and HSA in the pulse chromatographic mode at what could be described as analytical scale. This separation was carried out using one PVDF membrane disc at a flow rate of 10 ml/min. The feed solution consisted of 0.2 mg/ml HIgG and 0.8 mg/ml HSA, respectively prepared in buffer A. This concentration ratio, i.e. 1:4 is representative of the ratio in which these proteins are present in human plasma [19]. In this experiment 500 ␮l of HSA/HIgG mixture was injected in the pulse mode. The amount of HIgG injected in the pulse was significantly lower than the binging capacity of the membrane. Hence the first peak obtained around 9.49 ml effluent volume was that of HSA while the second peak, which was obtained by eluting the bound protein using buffer B was that of HIgG. Fig. 4 shows the preparative separation of HSA and HIgG in the pulse chromatographic mode using one PVDF membrane disc at a flow rate of 10 ml/min. The feed solution consisted of 0.2 mg/ml HIgG and 0.8 mg/ml HSA, 5 ml of this being injected in the pulse. Once again, the amount of HIgG injected in the pulse was significantly lower than the binding capacity of the membrane. Hence, the first peak was that of unbound HSA while the second peak was that of initially bound and subsequently eluted HIgG. Effluent samples corresponding to these peaks were collected and analysed. Fig. 5 shows the protein-A affinity chromatograms for the feed, and the purified HSA and HIgG samples. Based on area under the curve calculations, the HSA purity was found to be in excess of 98% while the HIgG purity was above 97%. These calculations took into account the difference in specific absorbance of HSA and HIgG. Fig. 6 shows the SDSPAGE of pure HSA and HIgG standards, the HSA/HIgG feed and the purified samples. HSA having a molecular weight of 69 kDa showed up on the stained gel as a single band between the 50 and 75 kDa markers. HIgG showed up on the gel as two bands, one corresponding to the heavy chains (approximately 50 kDa) and other corresponding to the light chain (approximately 25 kDa). Fig. 6 clearly shows that the HSA and HIgG samples obtained by pulse input separation were pure. Fig. 7 shows the separation of HIgG and HSA in the step input mode using one membrane disc at a flow rate of 10 ml/min. The average back-pressure during this experiment was 80 kPa. This pressure includes the transmembrane pres-

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sure and any additional system pressure for the AKTAprime liquid chromatography system. The feed solution consisted of 0.1 mg/ml HIgG and 0.4 mg/ml HSA prepared in buffer A. In this experiment 29.8 ml of feed solution was injected using a Superloop. The breakthrough observed around 6–7 ml was due to the HSA which did not bind to the membrane. The absorbance increased rapidly and reached a plateau value corresponding to the feed concentration of HSA i.e. 0.4 mg/ml. Around 32–33 ml effluent volume a secondary breakthrough was observed, this being due to the HIgG in excess of the binding capacity of the membrane. The peak obtained at 132 ml effluent volume was that of HIgG. Effluent samples corresponding to the HSA breakthrough (collected around 15 ml effluent volume) and the HIgG peak were collected and analysed. Fig. 8 shows the protein-A affinity chromatograms of the purified HSA and HIgG samples (chromatograms A and B). Using area under the curve calculations, the HSA and HIgG purities were both determined to be above 97%. Fig. 9 shows the separation of HIgG and HSA in step input mode using a stack of five membrane discs (bed volume = 0.865 ml) at a flow rate of 10 ml/min. The average back-pressure during this experiment was 140 kPa. The feed solution consisted of 0.4 mg/ml HIgG and 1.6 mg/ml HSA prepared in buffer A. In this experiment 29.6 ml of feed solution was injected using a Superloop. Effluent samples corresponding to the HSA breakthrough and the HIgG peak were collected and analysed. Fig. 8 shows the protein-A affinity chromatograms of the purified HSA and HIgG samples (chromatograms C and D). Based on the area under the curve calculations, the HSA and HIgG purities were both determined to be above 98%. Fig. 6 shows the SDS-PAGE of the purified samples. Quite clearly, the HSA and HIgG samples obtained by the step input separation were pure.

Fig. 9. Step-input separation of HIgG and HSA using a stack of five membrane discs at 10 ml/min flow rate.

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4. Conclusion The 0.1 ␮m PVDF membrane used in this study was able to specifically and reversibly bind HIgG by hydrophobic interaction mechanism. The membrane bound 42.8 mg of HIgG/ml of membrane volume, this being significantly higher than most membrane binding capacities for HIgG reported in literature. At conditions which promoted HIgG binding, HSA did not bind to the membrane. Hence, this membrane could be used for efficient stand-alone separation of plasma proteins HSA and HIgG. The amount of protein bound depended on the feed concentration, the higher binding capacity being observed at the higher feed concentration. Separation of simulated HSA/HIgG mixtures could be carried out in the pulse and step-input modes and using these, pure HSA and HIgG samples could be obtained.

Acknowledgements Canada Foundation for Innovation and Ontario Innovation Trust are acknowledged for funding in the form of a CFI New Opportunities Grant which enabled the purchase of equipment used in this study. Paul Gatt of the Department of Chemical Engineering, McMaster University is acknowledged for fabricating the membrane modules used in this study based on design provided by the author. Special thanks go out to Dr Q.Y. Li of the Scottish Blood Transfusion Services, UK for donation of the HSA and HIgG used in the experiments. Millipore is acknowledged for donating the VVPP 0.1 ␮m PVDF membranes. D. Kanani and Xin Ge are thanked for help with protein analysis.

Nomenclature C C0

concentration of protein in effluent (kg/m3 ) concentration of protein in feed (kg/m3 )

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