Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

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Process Biochemistry 45 (2010) 1082–1087

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept Marcos Garza-Madrid 1 , Marco Rito-Palomares, Sergio O. Serna-Saldívar, Jorge Benavides ∗,1 Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico

a r t i c l e

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Article history: Received 18 November 2009 Received in revised form 16 March 2010 Accepted 19 March 2010 Keywords: Aqueous Two-Phase Systems Flexible devices Blood bag Human serum albumin Recovery and purification Downstream processing

a b s t r a c t In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Nowadays, the design and development of efficient processes for the production, recovery and purification of biological products is an area of major importance for the biotechnology industry. Such processes must meet strict validation requirements, which are particularly stern in the pharmaceutical industry. In order to assure the absence of pathogens and pyrogens in the system, process equipment must be carefully sterilized each time a new production batch is started. Although effective, this approach incurs in considerable energy and maintenance expenses. The development of bag/wave reactor technology aimed for the production of high value biotechnology compounds has allowed the application of sterile ready-to-use flexible disposable devices [1,2], avoiding sterilization related costs. Aside the economical advantages the application of this technology presents, the use of sterile disposable devices may also facilitate the validation process. Although this approach has been characterized in the case of bag/wave reactors, no information regarding the use of flexible disposable devices on downstream

∗ Corresponding author. Tel.: +52 81 8328 4132; fax: +52 81 8328 4136. E-mail address: [email protected] (J. Benavides). 1 These authors contributed equally to this work. 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.03.026

processing has been reported. In this context, the application of such devices for the fractionation, recovery and primary purification of biological products using Aqueous Two-Phase Systems (ATPS) is an interesting case of study. ATPS is a liquid–liquid fractionation technique used for recovery and primary purification of biological products, including proteins, genetic material, low molecular weight compounds, and even whole cells and organelles [3–7]. ATPS form when two hydrophilic compounds such as polymers (polyethylene glycol, dextran, among others) and salts (phosphates, sulfates, citrates, etc.) are combined over certain critical concentrations resulting in two immiscible phases which major constituent is water. When compared with other techniques, ATPS has demonstrated several advantages including biocompatibility, process integration and intensification capability, and scaling up feasibility [3]. The objective of the present study is to demonstrate the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using ATPS polymer–salt. Partition behavior of a model protein (human serum albumin, HSA) on conventional rigid cylindrical containers (tubes) and flexible disposable devices (blood bags) was compared. Additionally, selective fractionation of human serum proteins was studied. Phase formation kinetics of ATPS formed on flexible devices was also determined.

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Table 1 Composition of Aqueous Two-Phase Systems polyethylene glycol (PEG)–potassium phosphate (PO4 ). Volume ratio (VR ) and system pH was kept constant at 1 and 7 respectively. System

PEG MW (g/mol)

TLL (% w/w)

PEG (% w/w)

PO4 (% w/w)

I II III IV V VI

400 400 1000 1000 3350 3350

25 45 25 45 25 45

18.0 23.0 13.0 19.0 11.0 17.5

16.5 19.0 15.0 17.0 11.4 14.5

2. Materials and methods 2.1. Chemicals and reagents Polyethylene glycol (PEG) 400, 1000 and 3350 g/mol, and potassium phosphate monobasic (KH2 PO4 ) and dibasic (K2 HPO4 ) of ACS grade were purchased from Sigma (Sigma Chemicals, MO, USA). Human serum albumin stock solution (20%, w/v, 50 ml, CSL Behring, product number 4552) was purchased from a local drugstore. Bovine serum albumin stock solution (2 mg/ml) was purchased from Sigma (Sigma Chemicals, MO, USA). The ReadyPrep® rehydration buffer, 11 cm ReadyStrip® IPG strips (pH 3–10), Ready Gel® Precast Gels and Precision Plus® protein standard were purchased from Bio-Rad (Hercules, CA). Blue colorant used to facilitate following phase formation kinetics was purchased from Mane (Mane Mexico, Mexico). Bi-distilled water was used for all experiments. 2.2. Effect of polymer molecular weight, tie-line length and construction device upon human serum albumin partition behavior All ATPS used to establish the influence of PEG molecular weight (PEG MW), tie-line length (TLL) and construction device (either conical tubes or blood bags) upon HSA were prepared for convenience on a fixed mass basis. ATPS compositions were established from reported binodal curves [8]. Table 1 shows the composition of the ATPS used. The volume ratio (VR , defined as top phase volume/bottom phase volume) and the system pH were kept constant at 1 and 7, respectively. Conical centrifuge tubes with maximum capacity of 50 ml (21008-939, VWR International, PA, USA), and blood bags with maximum capacity of 450 ml (CPD-A 450, Grifols, Mexico) were used to construct in the ATPS in order to determine the influence of system geometry. In the case of bags system, before introducing PEG and phosphate stock solution the citrate phosphate dextrose adenine (CPD-A) contained in the bag was removed and the bag thoughtfully rinsed with water. Predetermined quantities of stock solutions of PEG and potassium phosphate (KH2 PO4 :K2 HPO4 ; 7:18) were mixed with a HSA solution with a concentration of 30 mg/ml, concentration that is usually found in human serum. The HSA solution represented 10% (w/w) from the system. Total weight for the ATPS was established in 50 and 400 g, for tubes and bags systems, respectively. Phases were dispersed by gentle mixing for 10 min at room temperature (25 ◦ C). Adjustment of pH was made by addition of orthophosphoric acid or potassium hydroxide. Systems were allowed to settle overnight (12 h) at 4 ◦ C in order to assure both, complete phase separation and thermodynamic equilibrium. Samples were carefully extracted from the phases and diluted for the determination of HSA concentration and subsequent estimation of its partition coefficient (Kp , defined as concentration of solute in the top phase/concentration of solute in the bottom phase). Results reported are the average of three independent experiments and standard errors were calculated dividing the standard deviation by the square root of the number of replicas. 2.3. Fractionation of human serum proteins on ATPS formed on flexible devices In order to demonstrate the potential application of ATPS formed on flexible devices for the fractionation of complex biological mixtures, selected systems (III and IV; Table 1) were loaded with human serum as sample. Human blood serum (∼50 ml) was obtained from a healthy volunteer. Blood was incubated in 50 ml conical centrifuge tubes at 37 ◦ C for 1 h. Afterwards, blood was kept under refrigeration (4 ◦ C) for 6 h to promote blood clot compaction. Tubes were then centrifuged at 5000 × g for 10 min at 4 ◦ C and the supernatant (human serum) was transferred to clean centrifuge tubes and stored at 4 ◦ C until used (no more than 2 days). Total protein concentration on serum was calculated to be 78.5 mg/ml. ATPS formed on flexible devices (blood bags) were conducted as previously mentioned, using 1% (w/w) sample loading. Samples were carefully extracted from the phases and diluted for the determination of total protein concentration and subsequent estimation of its partition coefficient (Kp ). Additionally, samples were used to analyze the protein fractionation pattern using two-dimensional electrophoresis (2DE). 300 ␮l of each phase was used for 2DE analysis. To eliminate interferences from phase-forming compounds and increase protein concentration, trichloroacetic acid (TCA) precipitation was performed to the top and bottom samples before isoelectric focusing according to the protocol reported by Gu and Glatz [9]. After precipita-

Fig. 1. Aqueous Two-Phase System polyethylene glycol (PEG)–potassium phosphate constructed on blood bags. A blue synthetic colorant was added to the system in order to facilitate the differentiation between both phases. 13% (w/w) PEG 1000 g/mol, 15% (w/w) potassium phosphate, TLL 25% (w/w), pH 7 and VR 1. Ruler shows scale in mm next to the bag system. Blood bag dimensions: 18 cm height, 12 cm width.

tion, the protein pellet was completely re-dissolved using 200 ␮L of ReadyPrep® rehydration buffer (Bio-Rad) and used for first-dimension isoelectric focusing. The first-dimension isoelectric focusing (IEF) was performed using 11.0 cm pH 3–10 linear immobilized pH gradient strips (IPG ReadyStrip® , Bio-Rad) in an Ettan IPGphor3® apparatus (GE Healthcare). Strips were rehydrated using 185 ␮L of sample to a maximum of 200 ␮g of protein per strip during 16 h at room temperature. IEF was carried out for a total of 50250 Vh. For the second dimension, the focused IPG strips were equilibrated with 6 M urea, pH 8.8, 75 mM Tris–HCl, 2% (w/v) SDS, 29.3% (v/v) glycerol, 0.002% (w/v) bromophenol blue and 2% (w/v) DTT for 15 min, and then acetylated for another 15 min using the same solution except replacing DTT with 2.5% (w/v) iodoacetamide. Strips were placed onto Ready Gel® Precast Gels (12.5%, w/v linear polyacrylamide gels; Bio-Rad) and electrophoresis was performed using a Criterion® Dodeca Cell (Bio-Rad). Precision Plus® protein standard was used during second dimension for molecular weight determination. The gels were visualized by staining with Coomassie Blue G-250, and scanned at 600-dpi resolution using a flat bed scanner ImageScanner III (GE Healthcare). Experiments were done by triplicate.

2.4. Phase formation kinetics in bag systems Phase formation kinetics in bag systems were determined using the systems described in Table 1, keeping constant the VR and system pH at 1 and 7, respectively. A solution of blue colorant (0.5 mg/ml, Mane Mexico, Mexico) with complete affinity towards the top phase of the system was used as marker during this experimental stage in order to facilitate the visual observation of phase formation through time. Once complete mixing of the forming phases was achieved, the systems were allowed to settle and phases separated under gravity. The changing distance from the interface to the bottom of the bag was recorded every 10 s on a piece of paper. Distance was determined using a graduated millimeter ruler after the system reached equilibrium. Distance ratio (d/D, defined as the distance from the interface to the bottom of the bag at t time/the distance from the interface to the bottom of the bag once system reached equilibrium) was calculated, and the time needed for each system to reach complete phase formation estimated. Fig. 1 shows an ATPS at equilibrium in a blood bag, where top phase can be easily visualized due to the presence of the colorant used in the phase formation kinetics experiments.

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Table 2 Human serum albumin (HSA) partition behavior in Aqueous Two-Phase Systems polyethylene glycol (PEG)–potassium phosphate (PO4 ) constructed on rigid and flexible devices. System

PEG MW (g/mol)

TLL (% w/w)

Device

Kp

I

400

25

Tube Bag

32.77 ± 6.94 35.29 ± 4.28

II

400

45

Tube Bag

6.43 ± 0.84 8.21 ± 0.13

III

1000

25

Tube Bag

0.41 ± 0.02 0.46 ± 0.01

IV

1000

45

Tube Bag

32.93 ± 4.72 38.91 ± 15.66

V

3350

25

Tube Bag

0.04 ± 0.01 0.05 ± 0.00

VI

3350

45

Tube Bag

0.22 ± 0.02 0.21 ± 0.01

2.5. Analytical procedures HSA concentration of each phase was determined using the Bradford method [10], adapted for 96-well plates (BD353261, VWR International, PA, USA). Briefly, 5 and 250 ␮l of sample and Bradford reagent, respectively, were added to each well. Standard curve was constructed using bovine serum albumin ranging from 0.1 to 1.4 mg/ml on water. Assay was incubated at room temperature (25 ◦ C) for 10–15 min. Absorbance was measured at 595 nm using a Synergy HT multi-mode microplate reader (Bio-Tek Instruments, VT, USA).

3. Results and discussion 3.1. Effect of polymer molecular weight, tie-line length and construction device upon human serum albumin partition behavior The effect of PEG molecular weight (PEG MW) and tie-line length (TLL) on the partition coefficient (Kp ) of HSA was established on both, conical tubes and blood bags. Table 2 summarizes the obtained results. The construction device has not significant influence on the Kp . The partition behavior of biologicals on ATPS is determined by the thermodynamic interaction between such products and the constituents of the system [11]. This interaction is related to the physicochemical and biochemical characteristics of the molecules, as well as the energy net balance in the system. However, is not related with macroscopic characteristics, such as the geometry of the system and the total weight. Therefore, independently of the device (geometry) used to construct the system, the thermodynamic equilibrium, and therefore the partition coefficient, remained constant in both cases. Regarding the effect of PEG MW and TLL both parameters are significant. When PEG 400 and 3350 g/mol are used, HSA partitions preferently to the top (Kp > 6) and bottom (Kp < 0.25) phase of the system, respectively. This behavior may be explained in terms of phase free volume and hydrophobicity [11–13]. As PEG molecular weight increases the free volume of the top phase of the system decreases, causing the migration of HSA towards the bottom phase. Additionally, as the length of the PEG molecule increases the hydrophobicity of the top phase also increases due to the reduction in the number of PEG terminal hydroxyl groups. Since HSA is a hydrophilic protein this increment in hydrophobicity may also drive its partition towards the opposite phase. When PEG 1000 g/mol is used, the affinity of HSA shifts from bottom to top phase as the TLL increases. This behavior may be explained in terms of free volume at both phases and salting-out effects. As TLL increases, the free volume at both system phases decreases. However, since PEG 1000 g/mol is a rather small polymer molecule, the

reduction of free volume at the top phase seems to be easily overcome by the increment on salt concentration at the bottom phase. Additionally, as a result of this increment in salt concentration at the bottom phase of the systems salting-out effects may also influence the partition behavior of HSA. The tendency of a particular protein to partition towards either top or bottom phase is highly dependent upon surface properties [14]. The effect of salting-out forces becomes particularly significant on the partition behavior at high TLL values due to the extremely high salt concentration at the bottom phase. These high salt concentrations may induce changes on the structure and surface properties of macromolecular products such as proteins, therefore influencing their partition behavior [14]. In the case of ATPS constructed with PEG 1000 g/mol (III and IV, Table 1) the TLL increase from 25 to 45% (w/w) represented an increase of salt concentration at the bottom phase from ∼21.6 to ∼31.0% (w/w) [8]. Therefore, this increment in salt concentration may have been influenced the partition of HSA towards the top phase (IV, Table 2). These results demonstrate that the effect of system parameters such PEG MW and TLL on HSA partition are independent of the geometry of the system. The partition behavior of numerous biological products on ATPS constructed in rigid geometry containers is well characterized. Such knowledge may be extrapolated to establish the partition behavior of biological in ATPS constructed in flexible devices, thus facilitating the integration of this technology to existing purification protocols. HSA recovery at top and bottom phase, based on the original amount of HSA feed to the system, was determined (Table 3). HSA recovery at the top phase is favored in systems constructed using PEG 400 g/mol (regardless TLL) and PEG 1000 g/mol (45%, w/w). The highest HSA recovery (85%) at the top phase was achieved when PEG 1000 g/mol and TLL 45% (w/w) were used. On the other hand, the use of PEG 3350 favored the partition of HSA towards the bottom phase. The highest HSA recovery (92%) at the bottom phase was achieved when PEG 3350 g/mol and TLL 25% (w/w) were used. The obtained results may be explained once again in terms of phase excluded volume and hydrophobicity [11–13]. ATPS are well known for their capability to achieve high recovery yields of biologicals [6]. 3.2. Fractionation of human serum proteins on ATPS formed on flexible devices The fractionation of human serum proteins was carried out using selected ATPS PEG–potassium phosphate (III and IV, Table 1) formed on blood bags in order to evaluate the potential application of flexible devices as containers for liquid–liquid separation technologies. Table 4 shows the obtained results concerning partition behavior and recovery yield of human serum total protein. As seen, in the case of system III (PEG 1000 g/mol, TLL 25%, w/w) similar protein concentrations were found on both phases (Kp 0.92), while for system IV (PEG 1000 g/mol, TLL 45%, w/w) most of the protein partitioned principally towards the top phase. Differences in Kp and total protein accumulated on the interface (0.84 and 24.4% for III and IV, respectively) are attributed to the increment on excluded volume and salting-out effects at the bottom phase of the system as the TLL increases. Since water available for solute solvatation is reduced as TLL increases, a fraction of the total protein is unable to stay in solution, accumulating at the interface. The effect of excluded volume and salting-out forces on the partition behavior is remarkably significant for solutes of high molecular weight [13]. Although total protein concentration on both phases of system III was similar, the protein profile differed as shown in Fig. 2. Fig. 2A and B shows the protein profile of the top and bottom phase, respectively. HSA may be located at pI 5.8–6.0 [15] and ∼67 kDa molecular weight on both phases, in accordance with the Kp estimated on model systems (III, Table 2). However, the

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Table 3 Human serum albumin (HSA) recovery in Aqueous Two-Phase Systems polyethylene glycol (PEG)–potassium phosphate (PO4 ) constructed on rigid and flexible devices. System

PEG MW (g/mol)

TLL (% w/w)

Device

Recovery Top Phase (%)

Recovery Bottom Phase (%)

I

400

25

Tube Bag

76.81 ± 2.32 80.03 ± 4.99

2.51 ± 0.41 2.36 ± 0.41

II

400

45

Tube Bag

71.60 ± 1.66 77.27 ± 2.29

11.55 ± 1.55 9.41 ± 0.15

III

1000

25

Tube Bag

24.57 ± 0.93 31.93 ± 0.41

59.81 ± 5.08 69.92 ± 1.31

IV

1000

45

Tube Bag

89.07 ± 1.06 84.78 ± 3.05

2.82 ± 0.41 2.82 ± 0.81

V

3350

25

Tube Bag

3.43 ± 0.81 4.35 ± 0.41

VI

3350

45

Tube Bag

12.47 ± 0.81 16.61 ± 1.25

83.86 ± 12.74 91.82 ± 3.71 55.98 ± 1.48 80.03 ± 0.81

Table 4 Human serum protein partition behavior and recovery yield in Aqueous Two-Phase Systems polyethylene glycol (PEG)–potassium phosphate (PO4 ) constructed on flexible devices. System

PEG MW (g/mol)

TLL (% w/w)

Total protein Kp

Recovery top phase (%)

Recovery bottom phase (%)

III IV

1000 1000

25 45

0.92 ± 0.07 5.91 ± 0.13

47.24 ± 1.13 64.66 ± 2.06

51.92 ± 3.96 10.94 ± 0.40

Fig. 2. Two-dimensional electrophoresis (2DE) analysis of human serum proteins fractionated on Aqueous Two-Phase System polyethylene glycol (PEG)–potassium phosphate constructed on blood bags. A: top phase, B: bottom phase.13% (w/w) PEG 1000 g/mol, 15% (w/w) potassium phosphate, TLL 25% (w/w), pH 7 and VR 1.

selective fractionation of particular proteins towards either the top or bottom phase is observed. A visual tentative identification of major spots was conducted through comparison with the literature [16]. Bands corresponding to the light (∼25 kDa, pI 6.0–9.5)

and heavy (∼50 kDa, pI 6.0–9.5) chains of immunoglobulin (manly IgG) are observed just at the top phase of the system (Fig. 2A). On the other hand, spots tentatively corresponding to transferrin (∼80 kDa, pI 7.0–7.5) and prothrombin (∼80 kDa, pI 5.4–5.6) are just

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studied. Both system parameters have significant effect on phase formation kinetics as seen in Fig. 3. As the PEG MW increased, the time needed to reach phase equilibrium (d/D = 1) also increased. This behavior may be explained in terms of polymer viscosity. When low molecular weight polymer (PEG 400 g/mol) was used (Fig. 3A), the increment of TLL had no effect on the time needed to reach equilibrium. For both systems (I and II, Table 1) the time needed to reach phase equilibrium was exactly the same (2 min). On the other hand, when PEG 1000 and 3350 g/mol were used (Fig. 3B and C, respectively), TLL had significant effect upon phase formation kinetics. As the TLL increased, the time needed to reach the phase equilibrium decreased. The higher the TLL, the more different the phases of a particular system become, promoting a faster separation. As a direct result the time needed to reach phase equilibrium reduced. TLL effect became more evident when high PEG MW (3350 g/mol) was used. While on system V (Table 1) d/D = 1 was reached on 5.5 min, system VI just required 3 min. Phase formation kinetics are known to be related with several factors, including PEG MW, TLL, and system geometry [17]. This last parameter is important since the highest the interface area, the more favorable is the mass transfer between phases, and therefore phase formation is facilitated [17]. In the case of blood bag ATPS, regardless of PEG MW and TLL, all systems studied (I–VI, Table 1) required a short time (
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