Mixed-mode expanded-bed adsorption for human serum albumin

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Mixed-mode expanded-bed adsorption for human serum albumin separation Qi-Ci Wu, Qilei Zhang, Dong Gao, Lei Nie, Hai Bin Wang, Shan-Jing Yao, and Dong-Qiang Lin Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b03799 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Industrial & Engineering Chemistry Research

Mixed-mode expanded-bed adsorption for human serum albumin separation

Qi-Ci Wu1, Qi-Lei Zhang1, Dong Gao2, Lei Nie2, Hai-Bin Wang2, Shan-Jing Yao1, Dong-Qiang Lin1*

1. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China 2. Zhejiang Hisun Pharmaceutical Co., Ltd., 46 Waisha Rd., Jiaojiang, Taizhou 318000, China

*Corresponding author: Prof. Dong-Qiang Lin College of Chemical and Biological Engineering Zhejiang University Hangzhou 310027, China Fax: +86-571-87951982 E-mail: D.-Q. Lin ([email protected])

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ABSTRACT Mixed-mode chromatography (MMC) is a promising technology for protein separation with high adsorption capacity, good selectivity, salt-tolerance and facile elution. Expanded bed adsorption (EBA) can capture target proteins directly from bio-particle containing crude feedstock without solid-liquid separation. In this study, MMC and EBA were combined to develop a mixed-mode EBA technique for human serum

albumin

(HSA)

separation.

Two

mixed-mode

EBA resins

(using

quartz-densified agarose beads and tungsten carbide/agarose composite beads, respectively) with tryptamine as the ligand were prepared (TA-S and TA-T). Static and dynamic adsorption behaviors of HSA were determined under varying conditions and typical adsorption properties of pH dependence and salt-tolerance were found. The performance of the resins in expanded bed was studied, and the dynamic binding capacities at 10% breakthrough (Q10%) of TA-S and TA-T at bed expansion of 1.8 were 27.54 and 18.04 mg/ml settled resin, respectively. Moreover, these two resins were used to separate recombinant HSA (rHSA) from Pichia pastoris culture broth. TA-S showed better separation performance and the purity of rHSA monomer reached to 83.3% with the recovery of 91.6% through a two-step elution process. Total content of rHSA monomer and degraded fragment was 99.8%. The results demonstrate that the combination of MMC and EBA would be a potential platform for protein capture with less pretreatments and high process efficiency.


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Keywords: Expanded bed adsorption; Mixed-mode chromatography; Tryptamine ligand; Human serum albumin; Adsorption

1. INTRODUCTION Human serum albumin (HSA) is one of the most abundant proteins in human plasma (40-50 g/l),1 which is widely used in clinical practice2 and as drug excipient, stabilizer and supplement.3 Worldwide demands for HSA have increased to more than 500 tons per year.4 However, plasma-derived HSA (pHSA) is often in a shortage due to limited blood resources and risks of spreading blood-derived pathogens.5 Therefore, recombinant HSA (rHSA) expressed by microorganisms, transgenic animals or plants has become a potential pathogen-free and economic alternative, which could avoid virus infection and resolve blood shortage issues. However, high purity of rHSA is essential due to high dosage administration, which leads to a great challenge to purification processes.6 In addition, rHSA manufacturing processes should be economical and efficient for profitable commercial production. Cation ion-exchange chromatography is normally used for rHSA capture and some pretreatments of fermentation broth such as dilution and pH adjustment are needed to ensure high adsorption capacity, which results in a low process productivity and high operation cost.7 Therefore, alternative separation techniques with high selectivity and efficiency are expected to facilitate rHSA separation. Mixed-mode chromatography (MMC) and expanded-bed adsorption (EBA) are two innovative bioseparation technologies. Mixed-mode ligands combine multiple 3 ACS Paragon Plus Environment


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binding modes including hydrophobic, electrostatic interactions and/or hydrogen bonds, which lead to multimodal protein-ligand interactions to improve adsorption selectivity.8 Therefore, MMC has advantages of high adsorption capacity, good selectivity,9 salt-tolerance10 and facile elution.11 Meanwhile, EBA is a unique chromatographic technology which can capture target proteins directly from crude feedstock, e.g., culture suspensions or cell homogenates.12 EBA combines solid-liquid separation, concentration and primary purification into a single-unit operation,13, 14 which can reduce separation procedures and improve process efficiency with less requirements

of

capital

investment

and

consumables.15,

16

Generally,

specially-designed resin beads with functional ligands are crucial for EBA operation. During the past two decades, several commercialized resins have been developed for EBA, such as ion exchange,17-19 hydrophobic interaction and affinity resins.20 Kazumasa et al.21 used Streamline SP (a strong cationic adsorbent with quartz core) to substitute traditional adsorbents for rHSA capture and found that the total productivity could be improved by approximately 50% in terms of processing time and 45% in terms of yield.7 Hansson et al. reported that Streamline DEAE expanded bed could capture 90% recombinant fusion protein ZZ-M5 from a crude fermentation broth with high efficient removal of cells, host proteins, contaminating DNA and endotoxins.22 Normally, feedstock dilution has to be performed to ensure efficient protein capture with ion-exchange adsorption,23 which restrains the application of EBA technology. A new resin with a multi-modal functional ligand (Streamline Direct CST) was designed by GE Healthcare (Uppsala, Sweden) and the results indicated that BSA recovery 4 ACS Paragon Plus Environment

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could reach 95% with DBC5% of 28.5 mg/ml.24 Charoenrat et al. also used Streamline Direct HST to separate beta-glucosidase from P. pastoris high-cell-density culture broth with a higher recovery (74%) than that of ion-exchange EBA resin Streamline SP (about 48%).25 Kelly et al. applied Fastline HSA and Fastline MabDirect MM (UpFront Chromatography A/S, Denmark) to capture a recombinant protein from high-cell density yeast broth, and high binding capacities (54.7 and 72 mg/ml settled adsorbent) were obtained with the recovery of 96%.26 In general, EBA shows outstanding performance for protein capture.27, 28 Combining the advantages of EBA for high production and MMC for effective protein separation, new separation technology of MMC-EBA has an inspiring potential for protein separation directly from crude broth. In our previous study,29 a mixed-mode resin with tryptamine as the functional ligand showed good performance for rHSA separation with high selectivity, salt-tolerance and relatively wide working pH. In this work, tryptamine was further explored for EBA using tungsten carbide-densified agarose beads and Streamline quartz base matrix to prepare new MMC-based EBA resins. Static and dynamic adsorption behaviors of pHSA and rHSA were determined under varying conditions. Effects of pH and salt on binding capacity were focused. The new resins were used to separate rHSA directly from P. pastoris culture broth. The potential of MMC-based EBA processes for rHSA separation was discussed and the results were compared with that of Streamline SP which is a traditional commercial cation exchanger for preliminary capture. 5 ACS Paragon Plus Environment


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2. EXPERIMENTAL SECTION 2.1 Materials Streamline quartz base matrix with cross-linked 6% agarose was obtained from GE Healthcare (Uppsala, Sweden). Cross-linked 3% agarose beads containing tungsten carbide were prepared with methods similar to those published in our previous work.30 Tryptamine (purity > 98%) was purchased from Aladdin Industrial Inc. (Shanghai, China). Plasma-derived human serum albumin (pHSA, Mw=66.7 kDa) was purchased from Sigma (Milwaukee, WI, USA). P. pastoris culture broth (pH 6.0, conductivity of about 20 mS/cm, biomass wet weight of 40~50%) with rHSA concentration of 6.9 mg/ml was provided by a local biotechnology company. The protein marker for SDS-PAGE was purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). Other reagents were of analytical reagent grade and purchased locally.

2.2. Preparation of MMC-EBA Resins Tryptamine-based EBA resins were prepared according to the method published previously.31, 32 Cross-linked agarose beads containing tungsten carbide or quartz were activated with allyl bromide (AB). The allyl-activated matrices were then brominated by N-bromosuccinimide (NBS). Finally, tryptamine was coupled onto the brominated matrices. Typically, 10 g drained agarose beads were mixed with 4 ~ 5 ml AB and 4 ~ 5 g sodium hydroxide in 10 ml 20% dimethyl sulfoxide (DMSO) solution in a 100 ml 6 ACS Paragon Plus Environment

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conical flask. The mixture was continuously agitated at 180 rpm under 30 oC for 24 h. The allyl-activated agarose beads were washed with ethanol and deionized water, and then mixed with 3.0 molar excess of NBS in 50% acetone at 180 rpm and 30 oC for 3 h. The brominated beads were then washed with deionized water. Finally, 3.0 molar excess of tryptamine was coupled onto the brominated beads in 1 M carbonate buffer (pH 12) at 180 rpm and 30 oC for 12 h. The beads were washed with 0.1 M HCl, 0.1 M NaOH and deionized water, and the resins were stored in 20% (v/v) ethanol. Two MMC-EBA resins with tryptamine as the functional ligand were prepared with quartz base matrix and tungsten carbide-densified beads and named TA-S and TA-T, respectively. The double bond and ligand density were determined via titration as reported previously.30

2.3. Adsorption Equilibrium Experiments The adsorption isotherms of pHSA and rHSA on TA-S and TA-T resins were determined by batch adsorption experiments. 20 mM acetate buffer (pH 4.0 and 5.0) and 20 mM sodium phosphate buffer (pH 6.0, 7.0 and 8.0) were used as the liquid phases for different pH conditions. Sodium chloride and ammonium sulfate were added into the buffer with different concentrations (0 ~ 1 M) to investigate the influence of salt concentration. pHSA concentrations in the supernatant were determined at 280 nm with One Drop Spectrophotometer (Nanjing Wins Technology Co., Ltd., Nanjing, China). rHSA concentration was analyzed by SEC-HPLC. Langmuir equation was used to describe the adsorption isotherms, and the saturated 7 ACS Paragon Plus Environment


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adsorption capacity Qm (mg/ml resin) and the association constant Ka (ml/mg) were obtained.

2.4. Frontal Adsorption Experiments Dynamic binding capacity of HSA was measured through frontal adsorption experiments in packed and expanded beds. 20 mM acetate buffer (pH 5.0) was used as the equilibrium buffer. For packed bed adsorption (PBA) mode, 5 mm i.d column (Tricorn 5/50, GE Healthcare, Uppsala, Sweden) was packed with 1.0 ml of TA-S or TA-T resins (about 5 cm bed height). For EBA mode, a 1 cm diameter homemade column was used, and ~15 ml of TA-S or TA-T resins were packed with a settled-bed height of 20 cm. pHSA solution (2 mg/ml) was loaded into the column at different linear velocities. The protein concentration in the effluent was monitored at 280 nm. The column was then eluted with acetate buffer (pH 4.0), regenerated with 0.1 M NaOH and re-equilibrated with 20 mM acetate buffer (pH 5.0) in sequence. The dynamic binding capacity (DBC) at 10% breakthrough (Q10%) was calculated as follows:33

C0 × Q10% =

∫

V 0

 C  1 − dV10% C 0   Vs

(1)

where C and C0 (mg/ml) are the protein concentration of outlet and loading fluid, respectively. V10% (ml) is the loading volume at 10% breakthrough and Vs is the packing volume of the resin.


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2.5. rHSA Separation from Yeast Broth with Expanded Bed The same expanded bed with ~15 ml of TA-S or TA-T resins was used for rHSA separation from yeast culture broth, which contained 6.9 mg/ml rHSA. Added 10 mM sodium caprylate as a stabilizing agent, the broth was heated at 68 oC for 30 mins to inactivate the proteases produced by P. pastoris, and then used as the crude sample. The bed was expanded to 1.8 bed expansion (H/H0) for 20 mins with 20 mM acetate buffer (pH 5.0). The sample adjusted to pH 5.0 was then loaded. After washing by the equilibrium buffer (15 CV), the column was washed with 20 mM sodium phosphate buffer (pH 7.0, 10 CV) and then eluted with 20 mM acetate buffer (pH 3.6 or 4.0, 10 CV). Finally, 0.1 M NaOH was used for the regeneration and clean in place (CIP). The fractions were collected during the chromatographic process and analyzed with SDS-PAGE and SEC-HPLC to determine the recovery and purity of rHSA.

2.6. SDS-PAGE Analysis The feedstock and collected fractions from the chromatographic separation of rHSA were analyzed by 10% SDS-PAGE gel under non-reducing conditions. The protein solution was diluted to the concentration of 0.5 ~ 2.0 mg/ml. The amount of loading sample was 5 µl of protein marker and 10 µl of protein sample. The protein migration was performed under 200 V for 45 mins. The gel was stained with Coomassie Blue R-250 and then destained. The stained protein gel was scanned using the Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA, USA), and analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA). 9 ACS Paragon Plus Environment


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2.7. SEC-HPLC Analysis The analytical SEC-HPLC was performed with LC3000 HPLC system (Beijing ChuangXinTongHeng Science and Technology Co., Ltd., Beijing, China) using the TSK G3000SWXL column (7.8 mm × 30.0 cm, TOSOH, Japan). The mobile phase of 0.2 M sodium phosphate buffer (pH 7.0, containing 1% isopropanol) was used after 0.22 µm membrane filtration and degassing. The operation flow rate was 0.6 ml/min. The HPLC purity of rHSA was defined as the percentage of the peak area of rHSA monomer to the total integrated peak areas. The recovery of rHSA was calculated as the percentage of rHSA monomer amount in the elution fraction to that in the feedstock.

2.8. HCPs Determination HCPs in the feedstock and fractions collected were determined using ELISA by P. pastoris HCP analysis kit (Catalogue No. F140, Cygnus Technologies, Southport, NC, USA). All operations were performed according to the product protocol.

3. RESULTS AND DISCUSSION 3.1. Preparation of TA-S and TA-T Resins Two MMC-EBA resins (TA-S and TA-T) with tryptamine as the functional ligand

were

prepared

with

quartz-densified

agarose

beads

and

tungsten

carbide/agarose composite beads as matrices, respectively. The preparation process 10 ACS Paragon Plus Environment

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includes three procedures, i.e. 1) activation with AB, 2) bromination with NBS and 3) tryptamine ligand coupling. The activation and bromination steps were the same as our previous work and the optimized conditions were used.29,

32

The activation

densities were about 300 µmol/ml gel and 120 µmol/ml gel for quartz-densified agarose beads and tungsten carbide/agarose composite beads, respectively. After brominated by NBS, the ligand coupling reaction was conducted under alkali condition as published previously.30, 32,

34

The ligand densities of TA-S and TA-T

resins were ~200 µmol/ml gel and 105 µmol/ml gel, respectively, which was suitable for mixed-mode protein adsorption and separation studies. The results indicate that the preparation process is feasible with high ligand coupling efficiency.

3.2. Adsorption Equilibrium of pHSA The pH of liquid phase is the first important factor for protein adsorption with MMC resins, which could affect the net charge of proteins and alter the electrostatic interactions between proteins and resins. The adsorption behaviors of pHSA on the two resins at the pH range of 4.0 ~ 8.0 were investigated. The correlated saturated adsorption capacity (Qm) and association constant (Ka) were obtained by Langmuir equation and are compared in Figure 1 and Table S1 (in the Supporting information). The adsorption profiles on the two MMC-EBA resins are similar to that of TA-B-6FF resin with same ligand for packed bed in our previous work.29 The highest Qm was found at pH 5.0. Compared to TA-B-6FF, the adsorption capacity of TA-S had advantage but TA-T showed less Qm. Qm and Ka of TA-S were 164.18 11 ACS Paragon Plus Environment


mg/ml and 14.93 ml/mg at pH 5.0, which were 127.8% and 183.6% higher than that of TA-T, respectively. When pH < 5.0 or pH > 5.0, the adsorption capacity decreased significantly, which showed a typical pH-dependent adsorption behavior.

Figure 1. Adsorption equilibrium of pHSA adsorption with TA-S (a) and TA-T (b) resins at different pHs.

The maximum values of Qm of the two resins were both found around the isoelectric point (pI) of HSA (pH 4.7)

35

as reported in the literatures.36, 37 Generally, 12

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the interactions between proteins and mixed-mode ligands are complicated which combine hydrophobic, electrostatic interactions and hydrogen bonds. At pH 5.0, HSA shows high total hydrophobic surface area, thin hydrated layer and small protein hydrodynamic diameters,29, 36, 37 which are favorable for the adsorption of HSA on the hydrophobic interaction-based resins. In addition, tryptamine has an indole ring to provide hydrophobic interaction with the hydrophobic area of protein surface. The results indicate that the hydrophobic interaction might dominate the adsorption of HSA on the tryptamine-based resin. When pH increases from 5.0 to 8.0, the adsorption capacities gradually decline due to the decrease of hydrophobicity of HSA.38 When the pH is below the pI of HSA and the pKa of the tryptamine ligand (pH 10.2),39 the electrostatic repulsion between positively-charged HSA and the tryptamine ligand reduces the binding strength of HSA on resins, which certainly cause the desorption of HSA from the resin. Salt-tolerance is another key factor of MMC resins. The effect of NaCl addition on the two resins was determined at pH 5.0. The adsorption isotherm curves are showed in Figure S1 (in the Supporting information), and the correlated Qm and Ka are compared in Table S2 (in the Supporting information). The results showed that some adsorption isotherms could not reach plateaus under the protein concentration tested. Therefore, the fitted Qm values would be not suitable to describe the adsorption ability. As suggested by Sun and coworkers,40-43 the adsorbed protein density (Qc) at an equilibrium liquid-phase concentration (5 mg/ml) calculated by Langmuir equation



was used as a substitute to describe the adsorption capacity. The results are compared in Figure 2.

Figure 2. Qc of pHSA with TA-S and TA-T at the equilibrium liquid-phase concentration of 5 mg/ml (pH 5.0) as the function of different sodium chloride concentrations.

NaCl addition greatly reduced HSA adsorption. Qc decreased sharply when salt concentration increased to 0.125 M, and the adsorption capacity declined slightly with the increase of salt concentration from 0.125 to 1 M. Qcs on TA-S and TA-T dropped by ~26% when 0.125 M NaCl was added. When NaCl concentration increased to 0.5 ~ 1.0 M, Qcs kept in the ranges of 87 ~ 97 mg/ml and 36 ~ 43 mg/ml, respectively. As mentioned above, the surface topology/hydrophobicity play the dominant role on HSA adsorption at pH 5.0. The net charge of HSA is low around the pI, but the distribution of surface charges is non-homogenous.29, 37 Therefore, there might be some electrostatic attractions between the positively-charged ligands and the locally 14 ACS Paragon Plus Environment

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negatively-charged areas on HSA. With the increase of salt concentration, the electrostatic interaction can be shielded and the adsorption capacity thereby decreases. The electrostatic interaction might be restrained at high salt concentrations, but the hydrophobic interaction could be enhanced. The balance between electrostatic and hydrophobic interactions can induce a typical salt-tolerant property of MMC resins which has the special ligands with the charged and hydrophobic groups together. Thus, TA-S and TA-T showed a relative stable adsorption ability under wide range of NaCl concentrations (0.25 ~1.0 M), which are more suitable to capture HSA from crude feedstock with medium-to-high conductivity.

3.3. Dynamic Binding Capacity of HSA on TA-S and TA-T Resins Dynamic binding capacities of pHSA with two resins at different flow velocities were determined by the frontal adsorption experiments in both packed bed and expanded bed. The breakthrough curves are shown in Figure 3 and the dynamic binding capacities at 10% breakthrough (Q10%) are listed in Table 1. For PBA mode, with the increase of flow rate from 100 to 300 cm/h, Q10% decreased dramatically by 72.0% for TA-S, but that of TA-T decreased slightly. For higher linear velocities (400 ~ 800 cm/h), TA-T still kept a relative high capacity Q10% (12.1 ~ 15.0 mg/ml). Compared with TA-T, flow rate had less effects on HSA adsorption on TA-S. Generally, pore diffusion resistance is the main restraining factor for mass transfer of proteins from bulk solution into the resin, especially at high operation velocity.44, 45 TA-T resin prepared with lower agarose concentration has 15 ACS Paragon Plus Environment


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larger pore size, which means lower pore diffusion resistance. This can facilitate mass transport and binding efficiency of target protein.

Figure 3. Breakthrough curves of pHSA on TA-S and TA-T resin at different flow rates and pH 5.0 in packed beds and expanded beds. PBA mode: (a) TA-S; (b) TA-T. EBA mode: (c) TA-S; (d) TA-T.


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Table 1. Comparison of Q10% of pHSA with TA-S and TA-T resins at different flow rates in packed beds and expanded beds (pH 5.0).

Resins

Operation mode

Flow rate (U, cm/h)

Q10% (mg/ml)

TA-S

PBA

100

35.5

PBA

200

14.8

PBA

300

9.9

EBA

209

27.5

EBA

275

23.9

EBA

330

21.2

PBA

100

20.9

PBA

200

18.1

PBA

300

15.7

PBA

400

15.0

PBA

500

13.5

PBA

600

12.5

PBA

800

12.1

EBA

1037

18.0

EBA

1390

17.6

EBA

1711

16.4

TA-T

For EBA mode, bed expansions (H/H0) of the two resins were first determined and the results are shown in Figure S2 (in the Supporting information). The flow rates of TA-S and TA-T were at the range of 209 ~ 500 cm/h and 1037 ~ 2515 cm/h, corresponding to bed expansion of 1.8 ~ 2.8, respectively. The operation velocity of TA-T is higher than that of TA-S due to higher bead density with tungsten carbide addition in the matrix. The breakthrough curves and Q10% at bed expansion of 1.8, 2.0 17 ACS Paragon Plus Environment


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and 2.2 were determined (Figure 3 and Table 1). The results showed that Q10% declined with the increase of flow rate and bed expansion. When bed expansion increased from 1.8 to 2.2, Q10% of TA-S decreased slightly, while TA-T remained a stable adsorption performance even at higher operation velocity. The flow rate had little effects on the adsorption of HSA with TA-T expanded bed, which could be used for higher operation velocity to improve process productivity. It was interesting that Q10% values with expanded beds were significantly higher than that with packed beds when flow rates were above 200 cm/h, as reported by Shi et al.12 Due to the expansion of bed height in expanded bed, the residence time in column would increase obviously, which can enhance protein adsorption and result in higher capacity. Compared with different flow rates, the operations at 209 cm/h for TA-S and 1037 cm/h for TA-T (corresponding to bed expansion of 1.8) showed the highest dynamic binding capacities, which would be suitable for EBA separation of HSA.

3.4. Adsorption Equilibrium of rHSA from Culture Broth TA-S and TA-T showed high capacity and good salt-tolerance for HSA adsorption in a wide range of pH. In addition, dynamic binding in expanded beds was better than that in packed beds at similar conditions, which indicates that EBA is a promising potential for HSA capture from crude feedstock with medium-to-high conductivity. Therefore, two resins were further challenged to separate rHSA from P. pastoris culture broth. The adsorption isotherms of rHSA from P. pastoris broth are


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shown in Figure S3 (in the Supporting information), and Qcs at 5 mg/ml are compared in Figure 4.

Figure 4. Comparison of Qc for rHSA adsorption on TA-S (white) and TA-T (grey) resin at the equilibrium liquid-phase concentration of 5 mg/ml.

The maximum Qc of the two resins were both found at pH 5.0 (63.43 mg/ml for TA-S and 23.65 mg/ml for TA-T). Qc values decreased dramatically at pH 4.0 and reduced slowly with the increase of pH from 5.0 to 8.0. The conductivity of P. pastoris culture broth was about 20 mS/cm (corresponding to 0.25 M NaCl). It could be found that Qc of rHSA decreased by 40 ~ 50% compared with pHSA adsorption under similar conditions. The crude feedstock of culture broth was abundant in complex impurities, such as host cell proteins (HCPs), peptide and pigment, which might affect rHSA adsorption. Compared to TA-B-6FF resin published in our previous paper,29 the ligand density of TA-S was 33.3% higher but some pores in the beads might be occupied by quartz, which probably leaded to a 16.6% increase of Qc. 19 ACS Paragon Plus Environment


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Nevertheless, rHSA could be reasonably adsorbed at pH 5.0 ~ 8.0, and pH 5.0 was the most suitable pH for feedstock loading. In addition, buffer dilution at pH 5.0 and pH 6.0 was also investigated to evaluate the effects of feedstock conductivity. The adsorption capacity of rHSA increased by less than 15% for TA-S and TA-T when the broth was diluted with 1.0 or 2.0 equal volume of buffer with the conductivity of about 10 mS/cm. Thus, buffer dilution had limited effects on rHSA adsorption, which also verified the salt-tolerant property of the mixed-mode resins. The results indicated that rHSA could be capture directly from P. pastoris culture broth by TA-S and TA-T without dilution or even pH adjustments, which would certainly simplify the procedure and improve process efficiency.

3.5. rHSA Separation from Culture Broth with TA-S and TA-T The adsorption of P. pastoris cells on TA-S and TA-T was determined by a biomass-pulse-response technique as reported in the literatures.46 The results showed that the resins could hardly adsorb yeast cells at pH 5.0 ~ 6.0 with 0.25 M NaCl addition. In addition, the stability of the expanded bed could be maintained during the load of crude feedstock containing P. pastoris cells. Therefore, TA-S and TA-T resins were used to separate rHSA directly from P. pastoris culture broth containing 6.9 mg/ml rHSA (pH 5.0). Acetate buffer (20 mM, pH 5.0) was used as the equilibrium buffer. The suitable operation conditions were bed expansion of 1.8, loading at pH 5.0 and elution at pH 3.6 or 4.0. An additional washing step with sodium phosphate buffer 20 ACS Paragon Plus Environment

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(20 mM, pH 7.0) was performed before the elution to improve the removal of impurities as our previous work.29 The feedstock and the fractions collected were analyzed by SEC-HPLC and SDS-PAGE.

Figure 5. Chromatographic separation of rHSA from yeast broth with EBA process. (a) Typical EBA process chromatogram with TA-T resin; (b) Non-reducing SDS-PAGE analysis. M, protein marker; F, feedstock; FT, flow-through; W, washing fraction; E, elution fraction; C, CIP fraction.



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Figure 5(a) is a typical EBA process chromatogram. Figure 5(b) shows the SDS-PAGE results of the fractions collected during rHSA separation. The results showed that there were observable low-molecular-weight (LMW) impurities in the feedstock and some protein impurities could be found in the flow-through, washing and CIP fractions. For the elution pool, the band was relatively pure, which meant high purity of rHSA separated. Except for rHSA monomer, the prominent impurities were LMW proteins which might be the degraded rHSA fragment (about 43 kDa) due to protease in the broth.47 SEC-HPLC analysis was used to identify the purity and recovery of rHSA, and the results are compared in Figure 6 and Table 2. After EBA separation process with TA-S and TA-T, most of the impurities in the feedstock could be removed. For TA-S resin with pH 4.0 elution, there were only rHSA monomer (83.3%) and some degraded rHSA fragments (16.5%) in the elution pool. The recovery and purity of rHSA monomer were 91.6% and 83.3% respectively, with the purification factor of 37.9. For TA-T resin with pH 4.0 elution, the purity of rHSA monomer was 78.6% and the amount of total rHSA could reach to 99.9%, but the recovery was 56.4%. Therefore, elution at pH 3.6 was further tested and the recovery of rHSA was improved to 74.3% with similar rHSA purity. In addition, HCPs content in the fractions was also measured and the results show that HCPs could be reduced by more than 80% for two resins. In general, TA-S would be more suitable to capture rHSA from P. pastoris culture broth with high purity of rHSA monomer and practicable recovery.


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Figure 6. SEC-HPLC analysis of the fractions collected during rHSA separation with EBA process. (a) Feedstock; (b) pH 4.0 elution fraction with TA-S; (c) pH 4.0 elution fraction with TA-T; (d) pH 3.6 elution fraction with TA-T; (e) Elution fraction with Streamline SP.


Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 24 of 35

Table 2. Separation performance of expanded bed adsorption processes with three resins. Resin

Component content (%)a

Fraction

Feedstock

Total rHSA (%)b

rHSA

(monomer+ fragment)

monomer

rHSA

rHSA

rHSA

LMW

Dimer

monomer

fragment

impurity

0.2

2.2

1.8

95.5

3.9

recovery (%)

TA-S

pH 4.0 elution

83.3

16.5

0.1

99.8

91.6

TA-T

pH 4.0 elution

78.6

21.3

0.05

99.9

56.4

pH 3.6 elution

76.2

23.5

0.2

99.7

74.3

48.3

16.5

26.2

64.8

90.1

Streamline SP

Elution

8.9

a: area percentage, SEC-HPLC analysis. b: total rHSA percentage (monomer+fragment), SEC-HPLC analysis.

24

ACS Paragon Plus Environment

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Mitsubishi Tanabe Pharma Co. reported an EBA process using Streamline SP resins to capture rHSA from yeast culture broth.48 Here Streamline SP was also tested to separate rHSA from the same culture broth for comparison. The separation conditions were based on the patent reported by Mitsubishi Tanabe Pharma Co..48 The culture broth was diluted by 2 folds with the buffer and adjusted to pH 4.0. 13 ml of the diluted sample was loaded onto 7 ml Streamline SP bed at pH 4.0, and the elution was done using 0.1 M acetate buffer (pH 6.5 with 0.3 M NaCl). SDS-PAGE results (Figure 5) reveal that the purity of rHSA was relatively low with obvious impurities. Based on the SEC-HPLC analysis, the recovery of rHSA monomer was about 90.1% but the purity was only 48.3%, which was much lower than TA-S or TA-T. As shown in Figure 6, there were some rHSA dimers and LWM impurities in the elution faction. Compared with Streamline SP, mixed-mode EBA resins developed in the present work showed some advantages on rHSA purity and the simplified feedstock pretreatments without dilution or even pH adjustment. The results demonstrate that the two EBA resins are suitable to capture rHSA directly from complicated feedstock. TA-S shows higher recovery and purity but a relative lower operation velocity, and TA-T is more suitable for high operation velocity. New EBA processes with tryptamine as the functional mixed-mode ligand could combine the advantages of MMC and EBA, which shows a promising potential for protein capture with high productivity and process efficiency.



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4. CONCLUSIONS EBA can capture target proteins directly from unclarified feedstock, and MMC combines multiple binding interactions to improve the adsorption selectivity. In this work, EBA and MMC were integrated into one new unit (mixed-mode EBA) for HSA separation, using tryptamine as the MMC ligand coupled onto two matrices for EBA. Effects of pH and salt addition on pHSA adsorption were investigated, and typical properties of high adsorption capacity, pH dependence and salt-tolerance were found. The results of dynamic adsorption showed that flow rate had less effects on HSA adsorption in expanded beds than that in packed beds. Q10%s of TA-S and TA-T reached 27.54 and 18.04 mg/ml settled resin, respectively. Moreover, TA-S and TA-T were used to capture rHSA directly from P. pastoris culture broth without dilution and pH adjustment. TA-S resin showed better separation performance, and the recovery and purity of rHSA monomer reached 91.6% and 83.3%, with purification factor of 37.9, which was obviously better than that of Streamline SP resin. The results demonstrated that MMC-based EBA resins with tryptamine as the functional ligand is a promising new technology for rHSA capture directly from complex feedstock. The combination of EBA and MMC is a favorable new platform for protein capture with reduced feedstock pretreatments and improved process efficiency, which can be expanded to other bioproduct separation.


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXX. Adsorption equilibrium of pHSA at different NaCl concentrations;

Bed

expansion in expanded bed; Adsorption equilibrium of rHSA at different pHs; Equilibrium parameters for pHSA adsorption at different pHs; Equilibrium parameters for pHSA adsorption at various NaCl concentrations.

ACKNOWLEDGEMENTS This work was financially supported by the International Science& Technology Cooperation Program of China and National Natural Science Foundation of China. The authors have declared no conflict of interests.

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Combination of EBA and MMC Matrix for expanded bed adsorption

rHSA Separation

EBA

Densified agarose beads

(1) Quartz-densified (2) Tungsten carbide densified

Ligand for mixed-mode chromatography

MMC Tryptamine

Yeast culture broth

MMC-EBA Process ACS Paragon Plus Environment

Mixed-mode expanded-bed adsorption for human serum albumin

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