Application of the Tunable Aqueous Polymer-Phase Impregnated Resins-Technology for protein purification

Separation and Purification Technology 136 (2014) 123–129

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Application of the Tunable Aqueous Polymer-Phase Impregnated Resins-Technology for protein purification F.A. van Winssen, J. Merz ⇑, L.-M. Czerwonka, G. Schembecker, TU Dortmund Laboratory for Plant and Process Design, Department of Biochemical and Chemical Engineering, Technische Universität Dortmund, D-44227 Dortmund, Germany

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Article history: Received 2 May 2014 Received in revised form 11 August 2014 Accepted 15 August 2014 Available online 3 September 2014 Keywords: Tunable Aqueous Polymer-Phase Impregnated Resins (TAPPIRÒ) Aqueous two-phase extraction Downstream processing Phase immobilization Protein separation

a b s t r a c t Aqueous Two-Phase Extraction (ATPE) represents a promising unit operation for downstream processing of biological products. It is known to provide gentle extraction conditions due to the aqueous phases’ high biocompatibility. However, some aqueous phases’ physicochemical properties can lead to long phase separation times that might be limiting for classical ATPE setups. The Tunable Aqueous Polymer-Phase Impregnated Resins (TAPPIRÒ)-Technology has been presented as novel approach to eliminate phase separation. One aqueous phase of the system is immobilized inside porous solids whereas the second aqueous phase represents the surrounding bulk phase creating the interphase for mass transfer between the two phases. In the present work, the application of the TAPPIRÒ-Technology for the separation of proteins using a polyethylene glycol 4000/sodium citrate aqueous two-phase system is shown. Lysozyme and myoglobin are separated due to their different partitioning behavior dependent on NaCl content. By variation of the porous solids’ properties like solid material, particle and pore size the influence on protein partitioning is investigated. As result, the same partitioning levels can be reached for the TAPPIRÒ-Technology as for classical ATPE mixer/settler experiments offering new alternative concepts for ATPE. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Biochemical industry represents one of the fastest growing markets. Apart from rising demands in pharmaceutical industry, biochemical and sustainable products are gaining more and more importance in food and cosmetic industry [1]. Novel findings in research areas related to cellular and molecular biology, biochemistry and biophysics lead to remarkable and fast developments in the upstream processing of biochemical products [2]. Due to the field of later product application, the subsequent downstream processing needs to cope with strict quality requirements by regulatory agencies concerning e.g. purity and the presence of critical contaminants. Additionally, separation tasks are characterized by high product sensitivities related to temperature, pH value and organic solvents, low product concentrations and complex and partly undefined mixtures [3]. Due to these facts, bioseparations are highly cost intensive and reach up to 80% of the total production costs [2,4]. Thus, alternative unit operations for downstream processing of proteins and enzymes need to be investigated. In this context, Aqueous Two-Phase Extraction (ATPE) has been intensively studied for more than 50 years [5]. It is known to be a gentle and efficient unit operation for sensitive biochemical products [6]

⇑ Corresponding author. http://dx.doi.org/10.1016/j.seppur.2014.08.030 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.

providing several advantages such as high biocompatibility due to high water contents. The phase forming compounds are mostly inert to biological molecules [7] and polyethylene glycol (PEG), for example, can have stabilizing effects on the tertiary structure of proteins [8,9]. Further advantages of ATPE are a negligible mass transfer resistance, a high extraction capacity and the possibility to adjust protein partitioning by the addition of neutral salts such as NaCl to the ATPS [10–13]. Apart from these advantages, the aqueous phases’ physicochemical properties namely low interfacial tensions, high viscosities and small density differences might lead to long phase separation times causing an increased footprint for the required mixer/settler devices in the production plant or the need for additional expensive equipment such as centrifuges [14]. Numerous attempts to speed up phase separation and enhance process throughput have been studied. Research includes classical apparatuses known from solvent extraction, specially designed mechanical devices, or techniques using additional energy or material input [15–22]. Another approach to overcome this ATPE limitation is the so-called ‘Tunable Aqueous Polymer-Phase Impregnated Resins’ (TAPPIRÒ)-Technology. The need for phase emulsification and separation is eliminated by immobilizing one aqueous phase inside porous solids prior to extraction [23]. Thus, the technology combines phase emulsification and separation of ATPE in a single step. In previous investigations, the

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TAPPIRÒ-Technology has been proven applicable as a technique for dye purification using the biodegradable PEG4000/sodium citrate ATPS [24]. In view of future applications in downstream processing, the application of the single-stage batch mode TAPPIRÒ-Technology for the extractive separation of lysozyme from myoglobin with a PEG4000/sodium citrate ATPS is presented. In order to get more insight into the mechanism of the TAPPIRÒ-Technology, the influence of the solids’ properties is investigated and discussed. Subsequently, the back-extraction of lysozyme is investigated in order to implement a holistic process concept of extractive protein purification by TAPPIRÒ.

2. Materials and methods 2.1. Materials ATPS are prepared with aqueous solutions of tri-sodium citrate dihydrate (Na3C6H5O7 ⁄ 2H2O, P99 wt.%), citric acid (C6H8O7, P99 wt.%) from Carl Roth GmbH & Co. KG (Germany) and PEG with an average molecular weight of 4000 g/mol from Merck KGaA (Germany) resulting in a PEG and citrate mass fraction of 12.5 wt.% and a pH of the aqueous salt phase between 5.6 and 5.7. Water is received from a Milli-Q Synthesis apparatus with 0.22 lm Millipak express filters from EMD Millipore Corporation (USA). Sodium chloride (NaCl, P99.9 wt.%) is purchased from VWR International (Germany). The porous VitraPor glass pellets are purchased from ROBU Glasfilter-Geräte GmbH (Germany), Stuttgarter Masse solids with particle sizes 3–5 mm and 1.6– 3.15 mm are purchased from Pall GmbH (Germany). A dry mass of 5.00 g VitraPor glass pellets, 2.30 g Stuttgarter Masse 3–5 and 2.70 g Stuttgarter Masse 1.6–3.15 is used for each TAPPIRÒ and adsorption experiment. Lysozyme, from chicken egg white and myoglobin, from equine skeletal muscle (P95%) are purchased from Sigma Aldrich (Germany).

2.2. Methods 2.2.1. Analytics For all experiments, samples from the aqueous citrate phase are taken, filtered with 0.2 lm pre-syringe filters and analyzed for protein content. The protein concentration in the aqueous PEG phase was calculated according to the mass balance. For single protein experiments the protein concentration is determined via fluorescence measurements using the wellplate reader infinite M200 Pro, TECAN group (Switzerland). For lysozyme the wavelength used is 280 nm for excitation and 336 nm for emission and for myoglobin it is 283 nm for excitation and 329 nm for emission based on preceding wavelength scans. For protein mixture experiments the concentrations of lysozyme and myoglobin are determined via HPLC using a Nucleodur C18EC column with a pore size of 100 lm and a particle size of 3 lm from Macherey–Nagel (Germany). The method described by Sutherland et al. [25] was modified according to Table 1.

Table 1 HPLC method for lysozyme and myoglobin determination on Nucleodur C18EC column. Time (min)

Vol.% acetonitrile

Vol.% water

0 2 7 13 14 22

20 30 42.5 55 30 30

80 70 57.5 45 70 70

2.2.2. Experimental procedure of the TAPPIRÒ-Technology The procedure of the TAPPIRÒ-Technology for protein separation in single-stage batch mode consists of the steps schematically shown in Fig. 1. Steps (1)–(6) concern the preparation of the impregnated solids and the extractive separation of lysozyme and myoglobin. They are comparable with the procedure described for dye separation [24]. Different is, that two different ATPS are prepared. The ATPS for the extraction contains 4 wt.% NaCl and is further referred to as ATPS with NaCl (Fig. 1(1)). The ATPS for the back-extraction contains 0 wt.% NaCl and is further referred to as ATPS without NaCl. Lysozyme as target product and myoglobin as contaminant are added to the aqueous citrate phase (Fig. 1(4)). Lysozyme is extracted from the bulk phase into the immobilized phase while myoglobin stays in the aqueous citrate phase with NaCl due to their partitioning behavior (Fig. 1(6)). Steps (7) and (8) describe the back-extraction of lysozyme out of the immobilized aqueous PEG phase. The myoglobin that remains in the aqueous citrate phase with NaCl during extraction is removed by exchanging the aqueous citrate phase with NaCl with a preequilibrated aqueous citrate phase without NaCl (Fig. 1(7)). Due to the changed NaCl composition, lysozyme is back-extracted out of the immobilized PEG phase into the aqueous citrate phase without NaCl (Fig. 1(8)). Due to the phase immobilization in the porous solids, the phase separation step is eliminated. All experiments are performed on the rocking platform Rocky RTMG/R (LTF Labortechnik, Germany) at 10 cycles/min to enhance the mass transport and to ensure a constant mixing for all experiments. Furthermore, all experiments are performed as triplicates in a climate chamber at 21–23 °C to increase reproducibility. 2.2.3. Impregnation stability For the investigation of the impregnation stability, the same procedure is carried out for all porous solids and all experiments are performed in triplicates in a climate chamber at 21–23 °C to increase reproducibility. The first steps are comparable to the procedure described in Fig. 1. Different is, that no product or contaminant is added. Instead, the reactive dye CibacronÒ black F-2B (200 mg/L aqueous PEG phase) is added to the ATPS. Its partitioning behavior leads to the selective dyeing of the aqueous PEG phase and allows the visualization of impregnation and leaching processes as described in [24]. In order to investigate the impact of a change in NaCl content on the impregnation stability during extraction and back-extraction experiments, a second ATPS without NaCl is prepared. CibacronÒ black F-2B (200 mg/L aqueous PEG phase) is added to the ATPS and its phases are separated. The impregnated solids from the first experiment with ATPS containing NaCl are transferred and suspended in the second aqueous citrate phase without NaCl and mixed. To achieve equilibrium all suspensions are mixed for 16 h on the rocking platform Rocky RT-MG/R (LTF Labortechnik, Germany) at 10 cycles/min. Samples from the bulk phases are taken for all experiments and intensively mixed to achieve a fine emulsion of bulk phase and eventually leached impregnated phase. Samples are diluted with water to guarantee one-phasic samples. The concentration of CibacronÒ black F-2B in these samples is determined via absorption measurements and used to determine the volume of leached aqueous PEG phase with NaCl. The leaching is evaluated via the leaching factor (LV) defined as relation of leached aqueous PEG phase volume (Vleach) to initially immobilized aqueous PEG phase volume (Vimp) according to Eq. (1).

LV ¼ V leach =V imp  100%

ð1Þ

2.2.4. Lysozyme extraction and back-extraction To evaluate the performance of the TAPPIRÒ-Technology for protein purification, the protein lysozyme is used as exemplary

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Fig. 1. Concept of the TAPPIRÒ-Technology for protein purification by extraction and back-extraction. (1) Preequilibration of ATPS, (2) phase separation, (3) impregnation of porous solids, (4) addition of target product lysozyme and contaminant myoglobin to aqueous citrate phase with NaCl, (5) suspension of impregnated porous solids in aqueous citrate phase with NaCl, (6) extraction of lysozyme into immobilized aqueous PEG phase with NaCl, (7) removal of myoglobin remaining in aqueous citrate phase with NaCl by exchange of phase and (8) back-extraction of lysozyme into aqueous citrate phase without NaCl.

target product and myoglobin serves as exemplary contaminant. The TAPPIRÒ-Technology is compared to classical ATPE and adsorption setups. The respective experiments are performed simultaneously and in triplicates for each single experimental run. All experiments considering the extraction are performed using the ATPS with NaCl to shift the partitioning behavior of lysozyme to the aqueous PEG phase whereas myoglobin stays in the aqueous citrate phase. The TAPPIRÒ setup is prepared as described before. The volume of aqueous citrate phase with NaCl and an initial lysozyme and myoglobin concentration of 0.117 mg/mL is 7 mL for all experimental setups. For the TAPPIRÒ setups the volume of the aqueous PEG phase with NaCl results from the impregnation volume of the porous solids. For ATPE setups, preequilibrated aqueous PEG phase with NaCl and aqueous citrate phase with NaCl containing dissolved lysozyme and myoglobin are mixed. To guarantee comparability, the same volume ratio of aqueous PEG phase to aqueous salt phase as for the TAPPIRÒ setups is used. The adsorption setups represent a negative control to proof that the depletion of lysozyme in TAPPIRÒ is due to ATPE and not due to the interaction of the molecules with the solids’ surface. The same dry mass of porous solid as used in TAPPIRÒ setups is mixed with the aqueous citrate phase containing NaCl, lysozyme and myoglobin. For back-extraction of lysozyme experiments are performed with aqueous citrate phase containing 0 wt.% NaCl to shift the lysozyme partitioning back to the aqueous citrate phase. For the TAPPIRÒ-Technology the impregnated solids used before and7 mL fresh aqueous citrate phase without NaCl are mixed. The ATPE setup for back-extraction consists of the aqueous PEG phase used in the ATPE setup before and aqueous citrate phase without NaCl is added. The experiments are performed for 16 h when partitioning equilibrium is reached. For all setups, samples from the aqueous citrate phase are taken and filtered with 0.2 lm pre-syringe filters. ATPE setups are centrifuged for 3 min at 1500 rpm to separate the phases. To describe the separation performance for extraction, the depletion of the proteins in the aqueous citrate phase is determined. The depletion is defined as the relation of the protein concentration in the aqueous citrate phase after a distinct time (ct) to the initial protein concentration in the aqueous citrate phase (c0).

as well as undefined (Stuttgarter Masse particles) solids. In previous investigations, these solids have been proven applicable for a stable impregnation with a PEG4000/sodium citrate ATPS without NaCl [24]. In this study, the impregnation stability for protein extraction and back-extraction conditions were investigated. 3.1. Impregnation stability A stable impregnation of the porous solids with the immobilized phase during extraction and back-extraction is essential for the process concept of the TAPPIRÒ-Technology for protein purification. VitraPor glass pellets represented defined solids, being monodisperse and regularly shaped and showing an average particle size comparable to Stuttgarter Masse 3–5. Hence, the influence of particle size distribution and particle uniformity could be investigated. Additionally, the pore size distribution from 40 to 100 lm was narrower than for Stuttgarter Masse particles. Stuttgarter Masse represented a solid with undefined properties such as irregularly shaped particles, a larger particle size distribution, and a larger pore size distribution (0.1–100 lm) than VitraPor glass pellets. This offered the possibility to investigate the influence of small pore sizes on the impregnation stability. Consisting of ceramic, Stuttgarter Masse particles showed influences of the solid material. The ceramic surface was rougher than the smooth glass surface of VitraPor glass pellets. The influence of the solids particle size was examined using two different particle size fractions of Stuttgarter Masse. It was shown, that the impregnation was stable for all solids using the ATPS with 4 wt.% NaCl. The leaching factor was below 1% for all solids and no differences related to the solids properties were detected. For changing the NaCl content of the aqueous citrate phase to 0 wt.% the impregnation of the aqueous PEG phase with NaCl was also stable, showing leaching factors below 4% for all solids tested. It has to be mentioned, that the aqueous salt phase without NaCl was not pre-equilibrated with the immobilized aqueous PEG phase and thus, the higher leaching factor could be explained. Nevertheless, the impregnation of VitraPor glass pellets and Stuttgarter Masse has been proven to be stable for changing ATPS containing 4 wt.% NaCl to 0 wt.% NaCl. This offered the possibility to use the TAPPIRÒ setup for selective protein extraction and back-extraction induced by a change in NaCl concentration in the aqueous citrate bulk phase.

3. Results

3.2. Single protein extraction in TAPPIRÒ

The influence of the porous solids’ properties on the TAPPIRÒTechnology was examined using defined (VitraPor glass pellets)

In order to investigate the applicability of the TAPPIRÒTechnology for protein separation, the extraction of single proteins

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was investigated. The partitioning behavior of lysozyme strongly depends on the NaCl content of the ATPS used. The partition coefficients of lysozyme were determined to 0.1 at 0 wt.% NaCl and to 15.1 at 4 wt.% NaCl. To compare the lysozyme behavior in TAPPIRÒ, ATPE and adsorption, experiments of all setups were performed simultaneously. Single protein experiments were performed with VitraPor glass pellets. A dry mass of 5.00 ± 0.02 g (average of 3  12 experimental setups) VitraPor glass pellets resulted in 0.778 ± 0.057 mL (average of 3  12 experimental setups) immobilized aqueous PEG phase. The initial lysozyme concentration was 0.117 mg/mL aqueous citrate phase. Fig. 2 shows the depletion of lysozyme and Fig. 3 the depletion for myoglobin respectively in the aqueous citrate phase for TAPPIRÒ, ATPE and adsorption setups at 0 wt.% NaCl (a) and 4 wt.% NaCl (b). In setups without NaCl (Fig. 2a) lysozyme did not show significant depletions. The lysozyme depletion for ATPE and TAPPIRÒ was below 7.4%. For the adsorption setup, depletions were measured below 3.3% indicating no significant adsorption of lysozyme on the solid material. At 4 wt.% NaCl (Fig. 2b), the extractive depletion of lysozyme was high due to NaCl induced salting out effects. In equilibrium, lysozyme was extracted in the same amount into the impregnated solid of the TAPPIRÒ setup as it was extracted by classical ATPE. For ATPE the equilibrium depletion of 91.1% was reached after approximately 60 min. The partitioning in TAPPIRÒ took longer because the transition of the lysozyme molecules from the aqueous salt phase into the aqueous polymer phase was possible only at the phase contact area of the pores outlet. Hence, the kinetic was limited by transport process via intra-particle pore diffusion. The equilibrium depletion of 88.8% was reached after 360–480 min. Nevertheless, the results gained show that the equilibrium depletions for APTE and TAPPIRÒ reached comparable

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values differing only in the magnitude of experimental triplets’ errors. These findings indicate that the depletion in TAPPIRÒ was based on extraction of lysozyme from the aqueous salt phase into the immobilized aqueous polymer phase. As well as for the experiment without NaCl, the experiment with NaCl did not show significant adsorption. The depletions after 300 min for the adsorption setup are assumed to be due to salting out or namely precipitation effects resulting from the long time the molecules were mixed at high ionic strength. For ATPE and TAPPIRÒ setups, the lysozyme molecules were pushed into the second aqueous PEG phase and thus, were stabilized. In case of myoglobin (Fig. 3) no strong NaCl dependency on the partitioning behavior in the ATPS used was observed which is expressed by its partition coefficients of 0.2 for 0 wt.% NaCl and 0.5 for 4 wt.% NaCl. The TAPPIRÒ and ATPE experiments for 0 wt.% NaCl (Fig. 3a) showed depletions below 4.9%, for 4 wt.% NaCl (Fig. 3b) the depletion was below 8.9%. Thus, myoglobin stayed in the aqueous salt phase at both NaCl concentrations tested in this study. The results for the adsorption setup differed slightly from the ones obtained for lysozyme. An average adsorption of 16.0% for 0 wt.% NaCl and of 24.0% for 4 wt.% NaCl was determined. This showed that adsorption occured and increased with higher salt concentration by higher salting out effects. In the adsorption setup, the inner pore surface was available for the adsorption of myoglobin molecules. In contrast, in the TAPPIRÒ setup, however, the pores are filled with aqueous PEG phase. According to the partition coefficient myoglobin prefers the aqueous salt phase and does not enter the pores which are filled with PEG phase. Thus, the myoglobin molecules do not approach the inner pores of the impregnated solids and thus do not adsorb. Hence, the TAPPIRÒ-Technology works comparable to the ATPE approach and the adsorption of myoglobin can be neglected when using TAPPIRÒ as separation method.

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To show the applicability of extraction and back-extraction of lysozyme using TAPPIRÒ an independent experimental setup was investigated. The equilibrium enrichment of lysozyme for the back-extraction was determined to 84.8 ± 5.0% for TAPPIRÒ, and to 90.2 ± 13.4% for ATPE. The results show, that lysozyme was back-extracted out of the immobilized aqueous PEG phase from the extraction step by a shift in NaCl concentration of the aqueous citrate phase. Combined with depletions of 94.9 ± 0.3% for TAPPIRÒ and 90.5 ± 0.7% for ATPE during extraction, the overall lysozyme yields using the TAPPIRÒ-Technology (81%) and ATPE (82%) resulted in comparable values. 3.3. Protein mixture separation in TAPPIRÒ Based on the single protein experiments, the separation of the binary lysozyme/myoglobin mixture was performed. Additionally the influence of, pore and particle sizes of VitraPor glass pellets and Stuttgarter Masse particles was investigated. The initial lysozyme and myoglobin concentration was each 0.117 mg/mL aqueous citrate phase. First, the experiments with VitraPor glass pellets were performed. A dry mass of 5.00 ± 0.02 g (average of 3  5 experimental setups) VitraPor glass pellets was impregnated with 0.793 ± 0.063 mL (average of 3  5 experimental setups) immobilized aqueous PEG phase. In Fig. 4 the depletions of lysozyme (a)) and myoglobin (b)) for TAPPIRÒ, ATPE and adsorption setups using VitraPor glass pellets are displayed. For ATPE, the lysozyme equilibrium depletion of 94.5% was reached after 60 min, for TAPPIRÒ the equilibrium of 94.4% was reached after 300 min. The results show, that lysozyme was extracted into the immobilized PEG phase in the same amount as for ATPE. The average depletion measured in the adsorption setup was 16.6%. The depletion in the adsorption setup was slightly higher than in the single protein experiments. However, the same

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trend of larger depletions with increasing time was obtained. This indicated the lower lysozyme stability in the aqueous salt phase with NaCl as before and it seemed that the presence of myoglobin increased the salting out effect of lysozyme. The higher total protein concentration led to the decrease of free water molecules due to the hydration layer formation around the protein. Thus, the protein partitioning and protein–protein interactions were increased. For this reason the depletion equilibrium in TAPPIRÒ was reached faster than for the single protein experiments. The myoglobin depletions for TAPPIRÒ and ATPE in the protein mixture experiments were higher than the ones obtained in the single protein experiments due to the higher overall protein concentration. The adsorption process of myoglobin seemed unaffected by the protein mixture. Initial depletions for all three setups of 17% were determined. Apart from an increased partitioning, these effects most probably referred to denaturation effects because myoglobin did not partition into the aqueous PEG phase due to its partition coefficient. Therefore, depletions measured were assumed to be due to precipitation as observed during the experiments before. In view of kinetics, the results showed, that the protein partitioning in TAPPIRÒ took longer than in ATPE. This was due to mass transfer hindrance by pore diffusion in the immobilized PEG phase. Nevertheless, the results proved that the same extractive depletion was achieved in TAPPIRÒ and in ATPE. In order to investigate the influence of the VitraPor glass pellets’ defined solid properties such as particle size distribution, particle uniformity and pore size distribution, the same experiments were performed using Stuttgarter Masse 3–5 particles. They had the same average particle size, a larger particle size distribution and a larger pore size distribution than VitraPor glass pellets and were characterized by irregular particle shapes. A dry mass of 2.30 ± 0.01 g (average of 3  5 experimental setups) Stuttgarter Masse 3–5 resulted in 0.775 ± 0.045 mL (average of 3  5 experimental setups) immobilized aqueous PEG phase. Fig. 5 shows the depletions of lysozyme (a) and myoglobin (b) for TAPPIRÒ, ATPE and adsorption setups using Stuttgarter Masse 3–5. For the ATPE setup, the equilibrium of lysozyme depletion of 95.7% was reached after 60 min. For TAPPIRÒ, the equilibrium of 94.1% was reached after 600 min. The results showed, that lysozyme was extracted into the immobilized PEG phase in the same amount as during ATPE within the experimental error. The lysozyme depletion in the adsorption setup was 30.1% in average which indicated a solid material dependent adsorption behavior. However, the driving forces governing protein partitioning seem to be the same in TAPPIRÒ as in ATPE, as the TAPPIRÒ and ATPE kinetics and partitioning behavior were comparable. The myoglobin depletions slightly varied from those for VitraPor glass pellets. An initial depletion for all three experiments of 10% was determined. Comparing the depletion profiles using VitraPor glass pellets and Stuttgarter Masse 3–5, an influence of the VitraPor glass pellets defined particle properties on the lysozyme transport processes can be stated. The initial depletion rate was faster using VitraPor glass pellets, indicated by a steeper slope of the depletion curve. This effect can be explained by the VitraPor glass pellets’ monodispersity and regular shape which lead to an equal relation of impregnated aqueous PEG phase volume for each solid particle. Additionally, VitraPor glass pellets showed a pore size distribution from 40 to 100 lm, which was narrower than for Stuttgarter Masse particles with a comparable average particle size. Hence, the diffusion length was more similar for all proteins that are extracted from the bulk aqueous salt phase to the immobilized aqueous PEG phase. Therefore, the initial depletion rate was faster using VitraPor glass pellets. Compared to the dye depletion kinetics published [24], the impact of impregnated particles’ uniformity and defined pore size distribution was bigger for protein than for dye

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Fig. 5. Depletion of (a) lysozyme and (b) myoglobin for TAPPIRÒ ( ), ATPE ( ) and adsorption ( ) setups using Stuttgarter Masse 3–5 at 4 wt.% NaCl, pH 5.7, 21.4 °C and 10 rpm.

Fig. 6. Depletion of lysozyme (a) and myoglobin (b) for TAPPIRÒ (), ATPE (j) and adsorption (N) setups using Stuttgarter Masse 1.6–3.15 at 4 wt.% NaCl, pH 5.68, 22.3 °C and 10 rpm.

kinetics. This was due to the proteins’ larger molecular size. Intraparticle transport processes played a more important role and kinetics were enhanced by more defined particle properties. In order to investigate the influence of particle size, Stuttgarter Masse 1.6–3.15 was used. A dry mass of 2.70 ± 0.00 g (average of 3  5 experimental setups) Stuttgarter Masse 1.6–3.15 resulted in 0.786 ± 0.065 mL (average of 3  5 experimental setups) immobilized aqueous PEG phase. Fig. 6 shows the depletions of lysozyme (a) and myoglobin (b) for TAPPIRÒ, ATPE and adsorption setups using Stuttgarter Masse 1.6–3.15. For ATPE the equilibrium of 95.2% was reached after 60 min, for TAPPIRÒ the time to reach the equilibrium of 95.2% was decreased to 100–200 min compared to 600 min. Experiments with the adsorption setup resulted in average depletions of 15.6% after 300 min. After a longer experimental time the depletion increased up to 29.05%. This trend was similar to the one in the previous experiments. The myoglobin depletion resulted in less than 13% for the TAPPIRÒ and ATPE setup and to 23.7% for the adsorption which was also in line with previous experiments. In terms of kinetics, the results showed, that the decrease of the average particle size to 40% can decrease the time needed for depletion equilibrium in TAPPIRÒ by 75%. In ATPE, the partitioning of molecules is fast due to the systems’ low interfacial tension and does not represent the rate-limiting step. In TAPPIRÒ, this fact may also be true for all used particle sizes because the same ATPS is used as in the ATPE approaches. The proteins’ partitioning from the bulk aqueous salt phase to the immobilized aqueous PEG phase takes place at the pore outlets on the outer particle surface and further diffusion is needed to transport the molecules into the complete immobilized phase volume inside the particle’s pores. Even though a decrease in particle size leads to a larger outer particle surface and more pore outlets per impregnated aqueous PEG phase volume are available for phase transition, the main reason for a faster depletion kinetic using smaller particles is the decrease in

inner-particle diffusion lengths. The length of pores and the diffusion distance in the particles decreases with the decrease in particle diameter. Thus, for protein separation, a decreased particle size can be used to significantly fasten the transport processes. To evaluate the back-extraction of lysozyme using VitraPor glass pellets and Stuttgarter Masse 3–5 and 1.6–3.15 the last set of impregnated solids from the kinetic studies of the extraction was further used. For TAPPIRÒ using VitraPor glass pellets, the extraction resulted in 94.4 ± 0.8% lysozyme depletion and in 94.5 ± 1.6% for ATPE. The subsequent back-extraction using the aqueous salt phase without NaCl resulted in an enrichment of 63.5 ± 2.4% for TAPPIRÒ and 77.5 ± 2.5% for ATPE respectively. Hence, lysozyme was back-extracted from the PEG phase. For ATPE the back-extraction was better. However, the back-extraction performed for the single protein experiments showed higher yields. The overall lysozyme mean recovery achieved for the TAPPIRÒTechnology resulted to 60% and to 73% for ATPE. Using Stuttgarter Masse 3–5, the extraction resulted to 94.1 ± 0.8% lysozyme depletion for TAPPIRÒ and to 95.7 ± 0.1% for ATPE, the back-extraction to 92.5 ± 0.1% enrichment of lysozyme for TAPPIRÒ and to 99.1 ± 0.8% for ATPE, respectively. The overall lysozyme mean recovery achieved for the TAPPIRÒ-Technology was 87%, the one with ATPE 95%. With Stuttgarter Masse 1.6–3.15 the depletion for the TAPPIRÒ extraction resulted to 95.2 ± 0.4%, for ATPE to 95.2 ± 0.4%. The enrichment during back-extraction resulted to 64.6 ± 3.1% for the TAPPIRÒ-Technology and to 79.0 ± 3.3% for ATPE. For TAPPIRÒ and ATPE, the overall mean yield resulted to 62% and 75%, respectively. According to all received results, lysozyme extraction and back-extraction using TAPPIRÒ and ATPE was possible also for the separation from a protein mixture. All experiments have been performed independently from each other using freshly prepared ATPS each time. The extraction equilibrium experiments lead to comparable results. However, the TAPPIRÒ and ATPE results for

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back-extraction enrichment from the lysozyme myoglobin mixture differed for all solids used over a range from 6% to 14%. This difference was potentially due to protein adsorption on the inner solids’ surface that remains during back-extraction which may have led to a protein amount missing in TAPPIRÒ enrichment. The additional difference for back-extraction enrichment for VitraPor and Stuttgarter Masse 1.6–3.15 may have resulted from the deviation of an external condition such as temperature or the pH value of the ATPS as previously seen within the single protein extraction experiments, where the depletions’ deviation was up to approximately 10%. Nevertheless, the presented results showed the applicability of the extraction/back-extraction strategy for the separation of a binary protein mixture using different solid materials for the TAPPIRÒ-Technology. The equilibrium results received using the solid particles VitraPor glass pellets and Stuttgarter Masse 3–5 and 1.6–3.15 did not give any insight into the back-extraction kinetics which were probably influenced by the solids’ material, solids’ form, particle size, particle size distribution, pore size and pore size distribution as discussed earlier for the extraction kinetics. These effects during back-extraction have to be investigated furthermore in order to get more insights into the TAPPIRÒ efficiency. 4. Conclusions In this manuscript, the Tunable Aqueous Polymer-Phase Impregnated Resins (TAPPIRÒ)-Technology has been presented as alternative to classical ATPE for protein purification. The application of the TAPPIRÒ process concept consisting of protein extraction and back-extraction induced by a shift in NaCl content was demonstrated for an exemplary protein mixture. The comparison to classical ATPE setups showed the comparability of the separation using the TAPPIRÒ-Technology. However, the protein partitioning in the TAPPIRÒ-Technology was slower than in classical ATPE because of increased mass transfer hindrance. Porous solids properties were identified to have large potential to fasten protein partitioning velocities. Defined particle properties such as monodispersity and smaller particle and narrow pore size distributions enhanced the initial partitioning velocities offering the possibility to close the gap between TAPPIRÒ and ATPE kinetics. The particle size has been identified as the most important property to significantly enhance protein depletion kinetics in the TAPPIRÒ-Technology. Back-extraction experiments showed the possibility to backextract proteins with NaCl dependent partitioning behavior out of the immobilized aqueous PEG phase using all different porous solids investigated. The investigations show, that for applications with biological material, the TAPPIRÒ-Technology is potentially advantageous due to the elimination of phase emulsification and separation by phase immobilization while maintaining ATPE advantages in terms of gentle extraction conditions. In the experimental setup presented, the time necessary for TAPPIRÒ is governed by product and contamination partitioning. This is

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because the particle sizes used for the TAPPIRÒ experiments are comparably large leading to long transport processes inside the particles’ inner pores. In future applications, the particle size should be further decreased to improve TAPPIRÒ kinetics in order to compare total experimental time necessary for TAPPIRÒ and ATPE in larger scale. Further investigations may focus on the question if biological activity of enzymes can be maintained using the TAPPIRÒ-Technology as well as the application of multistage extractions with TAPPIRÒ. Acknowledgement The research leading to these results has received funding from the Ministry of Innovation, Science and Research of North Rhine-Westphalia in the frame of CLIB-Graduate Cluster Industrial Biotechnology, Contract No. 314-108 001 08. References [1] A.K. Pavlou, J.M. Reichert, Nat. Biotechnol. 22 (2004) 1513–1519. [2] R. Ghosh, Principles of Bioseparations Engineering, World Scientific, Singapore, Hackensack, N.J., 2006. [3] B.K. Nfor, T. Ahamed, G.W.K. van Dedem, L.A.M. van der Wielen, E.J.A.X. van de Sandt, M.H.M. Eppink, M. Ottens, J. Chem. Technol. Biotechnol. 83 (2008) 124– 132. [4] K. Bauer, Synthese von Downstream-Prozessen, Dissertation, first ed., Verl. Dr. Hut, Dortmund, 2011. [5] P.-A. Albertsson, Nature 182 (1958) 709–711. [6] O. Aguilar, V. Albiter, L. Serrano-Carreón, M. Rito-Palomares, J. Chromatogr. B 835 (2006) 77–83. [7] R. Gupta, S. Bradoo, R.K. Saxena, Curr. Sci. 77 (1999) 520–523. [8] P.-A. Albertsson, Biochem. Pharmacol. 5 (1961) 351–358. [9] R. Hatti-Kaul, Mol. Biotechnol. 19 (2001) 269–277. [10] H. Walter, G. Johansson, Methods in Enzymology: Aqueous Two-Phase Systems, Academic Press, San Diego, 1994. [11] A. Kaul, R. Pereira, J. Asenjo, J. Merchuk, Biotechnol. Bioeng. 48 (1995) 246– 256. [12] A.M. Azevedo, P.A.J. Rosa, I.F. Ferreira, M.R. Aires-Barros, J. Biotechnol. 132 (2007) 209–217. [13] B.A. Andrews, A.S. Schmidt, J.A. Asenjo, Biotechnol. Bioeng. 90 (2005) 380–390. [14] U. Gündüz, A. Tolga, J. Chromatogr. B 807 (2004) 13–16. [15] G. Blomquist, P.A. Albertsson, J. Chromatogr. A 73 (1972) 125–133. [16] N.D. Srinivas, A.V. Narayan, K.S.M.S. Raghavarao, Process Biochem. 38 (2002) 387–391. [17] A. Hamidi, M. van Berlo, K.C. Luyben, L. van der Wielen, J. Chem. Technol. Biotechnol. 74 (1999) 244–249. [18] L. Igarashi, T.G. Kieckbusch, T.T. Franco, J. Chromatogr. B 807 (2004) 75– 80. [19] P.A.J. Rosa, A.M. Azevedo, S. Sommerfeld, W. Bäcker, M.R. Aires-Barros, J. Chromatogr. B 880 (2012) 148–156. [20] T. Ban, M. Shibata, F. Kawaizumi, S. Nii, K. Takahashi, J. Chromatogr. B 760 (2001) 65–72. [21] R. Hu, X. Feng, P. Chen, M. Fu, H. Chen, L. Guo, B.-F. Liu, J. Chromatogr. A 1218 (2011) 171–177. [22] P. Vázquez-Villegas, O. Aguilar, M. Rito-Palomares, Sep. Purif. Technol. 78 (2011) 69–75. [23] G. Schembecker, B. Burghoff, F.A. van Winssen, Verfahren zur Trennung/ Reinigung von Biomolekülen, 01.04.2011, 10 2011 001 743. 7, 2011. [24] F.A. van Winssen, J. Merz, G. Schembecker, J. Chromatogr. A (2014) 38–44. [25] I.A. Sutherland, G. Audo, E. Bouton, F. Couillard, D. Fisher, I. Garrard, P. Hewitson, O. Intes, J. Chromatogr. A (2008) 57–62.