Facile and green fabrication of cation exchange membrane adsorber with unprecedented adsorption capacity for protein purification

Accepted Manuscript Title: Facile and green fabrication of cation exchange membrane adsorber with unprecedented adsorption capacity for protein purification Authors: M. Kamran Khan, Jianquan Luo, Rashid Khan, Jinxin Fan, Yinhua Wan PII: DOI: Reference:

S0021-9673(17)31367-5 http://dx.doi.org/10.1016/j.chroma.2017.09.031 CHROMA 358860

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

28-6-2017 12-9-2017 13-9-2017

Please cite this article as: M.Kamran Khan, Jianquan Luo, Rashid Khan, Jinxin Fan, Yinhua Wan, Facile and green fabrication of cation exchange membrane adsorber with unprecedented adsorption capacity for protein purification, Journal of Chromatography Ahttp://dx.doi.org/10.1016/j.chroma.2017.09.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Facile and green fabrication of cation exchange membrane adsorber with unprecedented adsorption capacity for protein purification M. Kamran Khan, a, b Jianquan Luo, a, b* Rashid Khan, a, b Jinxin Fan,a, b Yinhua Wan a, b* a

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China b

University of Chinese Academy of Sciences, Beijing 100049, PR China

*

Corresponding authors: E-mail: [email protected] (J. Luo); [email protected] (Y. Wan) Tel/Fax: +86-10-62650673

Highlights 

A novel membrane adsorber is prepared by grafting alginate dialdehyde on nylon membrane.

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Alginate dialdehyde (ADA) reacts with sodium bisulfite to generate sulphonic groups on ligands.

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Sulphonic and carboxylic groups (both on membrane and ADA) endow a high binding capacity.

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The novel membrane adsorber captures lysozyme from chicken egg white solution.

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The purified lysozyme shows similar specific activity as commercial product.

ABSTRACT: Fabricating membrane adsorbers with high adsorption capacity and appreciable throughput for the separation and purification of protein products is challenging in biomedical and pharmaceutical industries. Herein, we report the synthesis of a novel membrane adsorber by functionalizing a nylon microfiltration membrane with alginate dialdehyde (ADA) followed by sulphonic addition, without any solvent usage, and its successful application in the purification of lysozyme. Taking advantage of abundant dual cation exchange (CEX) groups on sulphonic-ADA (S-ADA) ligands, this novel S-ADA-nylon membrane adsorber showed an unprecedented static binding capicity of 286 mg/mL for lysozyme adsorption. Meanwhile, the prepared membrane adsorber could be easily regenerated (complete protein elution) under mild conditions and be reused at least for five times. Featured with a unique selectivity, the S-ADA-nylon membrane also captured lysozyme from chicken egg white solution with a high purity (100%) and a high recovery 1

of 98%. The purified lysozyme showed similar specific activity as commercial product. The present work provides a facile, green and low-cost approach for the preparation of highperformance membrane adsorbers, which has a great potential in protein production.

KEYWORDS: Membrane chromatography; alginate dialdehyde; lysozyme; protein purification; microfiltration

1. Introduction Due to the vast significance of proteins in human life, its market industry has reached to almost 100 billion dollars per year and is still growing rapidly. Because of the advances in the upstream stage of bioprocesses the overall manufacturing costs have shifted towards downstream purification stage [1-3], therefor, high-throughput and high-recovery separation techniques are essential in therapeutic protein production [4]. Ion exchange (IEX) column chromatography is one of the most powerful approach for protein purification [5-7]. The separation mechanism in IEX purification depends on the charge difference between the desired molecule and the resin particles containing ionized or ionizable functional groups. For column chromatography, proteins go to the binding sites within the pores of beads via intra-particle diffusion transport, where the molecule adsorption and desorption efficiency is relatively low due to the long diffusion distances. As a result, protein degradation and denaturation are likely to occur during the column chromatography. Another main drawback of this method is the compression and compaction of chromatographic bed at high velocity, producing a higherpressure drop over the column and limited throughput. Furthermore, high consumption of chemicals and water usage in column chromatography at large scale also makes the process costly [8]. To circumvent these challenges, a variety of IEX membrane adsorbers were explored for the purification of a large number of useful proteins like antibodies, α1-Antitrypsin (AAT), BSA, γglobulin, lactoferrin, lysozyme and many others [4, 9-15]. Actually the microporous structure of membrane adsorbers and the fine membrane thickness allow high convective flow velocity for protein separation with a low pressure drop. Within intrinsic convective transport, IEX membranes present a high potential to bind proteins quickly due to highly accessible adsorption groups dispersed on/in the membrane matrix. Moreover, the elution process is mild, thus the protein 2

conformation could be well preserved. Therefore, IEX membrane adsorber was regarded as an ideal tool for efficient protein purification [13, 16-25]. For instance, Fan et al. captured AAT proteins from human plasma by anion exchange membrane with a high recovery of 86 % [4]. Knudsen et al. evaluated IEX membrane for process-scale antibody purification [26]. Arica et al. reported poly(2 hydroxy- ethylmethacrylate) (pHEMA) based membrane adsorbers, compopsed of carboxylic and sulphonic functiuonalities, with a binding capicity of 80 mg/mL for lysozyme purification form chicken egg white (CEW) [27]. Chen et al. used a carboxy functionalized chitosan membrane for lysozyme adsorption with a binding capacity of 170 mg/mL [28]. Fu et al. grafted citric acid on an electrospun nanofiber membrane to fabricate cation exchange (CEX) membrane adsorber, and due to a higher surface area, the lysozyme binding capacity could reach up to 284 mg/g [12]. IEX membrane adsorbers were extensively studied for protein purification and many of those have been also commercialized like Sartobind Q, Sartobind S, Mustang Q and ChromaSorb, but the potential for increasing the binding capacities of the membrane adsorbers still remains. For some of the membrane adsorbers, higher binding capacities have been achieved at the expense of using toxic materials for its functionalization[29]. Apart from the high price, these toxic functionalization materials may leach into the product during purification process. Therefore, fabricating a green and high-performance IEX membrane adsorber (i.e. high protein recovery, resolution and binding capacity) is still desired for protein purification. Alginate dialdehyde (ADA) is an intriguing bioadhesive polymer, possessing both carboxyl and aldehyde groups for functionalization. Due to the biocompatible, non-immunogenic, and biodegradable nature, it has been widely used in pharmaceutical, tissue engineering, cosmetic, and food industries [30]. Chen et al. reported that a liver tissue engineered scaffold was prepared by cross-linking ADA with galactosylated chitosan [31]. Recently ADA was also used as a linker for enzyme immobilization [30]. However, to the best of our knowledge, there is no relevant report regarding the application of ADA for protein purification. Theoretically, ADA with carboxylic (COOH) groups could be used as a CEX ligand. In this regard, we demonstrate a facile and green fabrication of CEX membrane adsorber by coating (also crosslinking) ADA on a highly porous, chemically stable nylon microfiltration membrane via a Schiff’s base reaction of aldehyde and amine. Nylon membrane also has carboxylic groups at the end of its polyamide chains, which make it more favorable for the CEX capacity. After ADA coating/crosslinking on the nylon membrane, the remaining aldehyde groups of ADA could further react with sodium bisulfite to 3

generate sulphonic groups on the ligands [32] (Fig. 1). The newly made sulphonic-ADA-nylon (SADA-nylon) CEX membrane adsorber with dual functionalities, has a larger surface area and more CEX sites, which is supposed to show much higher binding capacity for protein purification than the traditional membrane adsorbers. Lysozyme was employed as a model protein to explore the efficacy of the newly constructed ADA-nylon and S-ADA-nylon membrane adsorbers. Moreover, the influence of ADA coating time, pH, ionic concentration and initial lysozyme concentration on maximum lysozyme adsorption were examined. Furthermore, to determine the selectivity of the newly made membrane adsorbers, purification of lysozyme from CEW solution was conducted. Finally, the reusability of the obtained adsorber was also evaluated. To the best of our knowledge, this is the first attempt to fabricate CEX membrane adsorbers based on ADA platform for protein separation. [Fig. 1] 2. Experimental section 2.1. Materials Fresh chicken eggs were purchased from a local market. Lysozyme was bought from SigmaAldrich (Steinheim, Germany). A Mustang coin units (0.35 mL) was purchased from Pall Corporation, USA. Nylon membranes with a pore size of 0.45 µm were bought from Millipore USA. Sodium alginate was obtained from Zhejiang Jingyan Biotechnology Co. Ltd (China), Sodium periodate (NaIO4) was bought from Chengdu Kelong Chemical Reagent Company (China). Amicon Ultra-15 centrifugal filters with a molecular weight cut-off (MWCO) of 5,000 Da were bought from Millipore, USA. Chemicals used for buffer preparation, sample analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were of analytical grade. 2.2. Preparation of alginate dialdehyde (ADA) Na-alginate solution (6 grams in 200 mL of deionized water and 50 mL of ethanol) was stirred on a magnetic stirrer followed by adding 5 g of NaIO4 in the dark at room temperature to obtain ADA. The reaction was stopped after 24 h by adding ethylene glycol (20 mL) to reduce the excess periodate for 2 h with stirring. The product was purified by precipitation with the addition of 5 g sodium chloride and 800 ml pure ethanol. Afterward, the solution was dialyzed using dialysis tube (MWCO, 1000) against ultrapure water with several changes of water until the dialyzate was periodate free. The dialyzate was then lyophilized to obtain the product [31]. 4

2.3. Fabrication of ADA-nylon and sulphonyl-ADA-nylon membrane adsorbers Nylon membranes were activated with ADA according to the procedures described by Beeskow et al. with a little modification [33]. Nylon membrane with an area of 2.26 cm2 was soaked in an aqueous solution of 3 mL ADA (5% W/V) and 5 µL phosphoric acid (85% V/V) in a vial (shaken at 25˚c and 120 rpm) to bind ADA to the nylon membrane by one of its aldehyde via a Schiff’s base reaction. A time range of 4-16 h was given for different sets of membrane discs to study the effect of ADA coating time on protein binding. After ADA coating, a set of membranes was treated with glycine (0.1 M) and another one was reacted with sodium bisulfite solution (0.1 M) for 3 h (shaken at 25˚C and 120 rpm). The membranes reacted with glycine got only carboxylate groups while the membranes reacted with sodium bisulfite owned carboxylate as well as sulphonate groups. The membranes with dual functionality were further treated with HCl (0.1 M) for 30 min to wash the sodium ion from carboxylate and sulphonate groups [34]. These two kinds of membrane adsorbers were compared for lysozyme adsorption. The best one was selected for the process optimization to obtain maximum lysozyme adsorption and further used for the lysozyme purification from CEW solution. 2.4. Characterization methods The surface chemical composition of the pristine and modified nylon membranes were characterized by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATRFTIR) and X-ray photoelectron spectroscopy (XPS). ATR-FTIR spectra of the membrane adsorbers was recorded as absorbance using Nicolet IS50 spectrometer (Thermo Nicolet Instrument Corporation, WI, USA) equipped with an IS50 ATR multi range diamond sampling station. ESCA Lab 220i-XL electron spectrometer of VG scientific, using a monochromatized Al Kα X-ray source (1486.6 eV photons), was used for XPS characterization. The surface zeta potential of the membranes was measured by an electrokinetic analyzer (SurPASS Anton Parr, Austria) at a pH range of 3.0 to 10.0. The titration solutions used were 0.05 M KOH and 0.05 M HCl, while the electrolyte was 1 mM KCL solution. Contact angle (CA) measurement was conducted by an optical contact angle goniometer (OCA20, Data Physics Instruments Co., Germany). Trans membrane pressure (TMP) was measured using a chromatography system AKTA prime plus (GE healthcare). The morphology of the membrane was imaged using a scanning electron microscopy (SEM) (XL 30-SFEG, FEI/Philips, USA) at 5 kV accelerating voltage on gold sputter-coated samples. 5

2.5. Lysozyme static adsorption The static adsorption capacity of lysozyme with ADA-nylon and S-ADA-nylon membranes was determined at different adsorption time, pH, ionic strengths, and initial lysozyme concentrations. Phosphate buffer (PB 20 mM pH 7.5) was used as a binding buffer. Binding buffer with 1.5 M KSCN (pH 7.5) was used as elution buffer. For a typical static adsorption test, a 2.26 cm2 membrane (thickness=0.02 cm) was immersed in 8 mL of lysozyme solution (in binding buffer) and shaken at 25˚C and 110 rpm for 10 hours. The lysozyme adsorption capacity was obtained by calculating the difference between the initial and final amount of lysozyme in the feed solution. After the adsorption test the membrane was washed with binding buffer and the protein in the washing fraction was subtracted from the adsorption value. To elute the adsorbed lysozyme, membrane samples were immersed in elution buffer for 5 h. After each cycle, the membranes were washed three times with ultrapure water and finally with binding buffer to remove KSCN. The reversible binding capacity was calculated as the ratio of the eluted lysozyme in each cycle to the amount of lysozyme eluted in the first cycle. The lysozyme concentration was analyzed by highperformance liquid chromatography (HPLC). 2.6. Separation of lysozyme from chicken egg white (CEW) solution CEW solution from fresh chicken eggs was diluted three times by volume with a phosphate buffer (20 mM, pH 7.5) and homogenized in an ice bath for 6 h. The resulting solution was further centrifuged (15 000 rpm) at 4°C for 30 min, and the supernatant was diluted again three times by volume with phosphate buffer (20 mM, pH 7.5), and was used as lysozyme source [35]. For evaluating the binding efficiency of the membranes for capturing lysozyme from CEW solution, pure lysozyme was added to CEW solution with a final concentration of 2.22 mg/mL. Four pieces of membrane adsorbers with a 16 mm diameter were housed in the filter holder (Mustang coin units, 0.35 mL) and then integrated with chromatography system AKTA prime plus (GE Healthcare) to perform the separation. To study the effect of ADA coating time on the separation of lysozyme from CEW solution, four sets of membranes with 4, 8, 12 and 16 h ADA coating time were investigated. For CEX membrane chromatography, a phosphate buffer (20 mM, pH 7.5) was used as a binding buffer, and the same buffer with KSCN (1.5 M) was used to elute the adsorbed proteins. The operating procedure was described as follows: the membranes were first equilibrated with binding buffer followed by the injection of 5 mL feed (CEW solution) at 0.5 mL/min; the unbound proteins were removed by washing with binding buffer, and then the bound proteins were 6

eluted using elution buffer at 3 mL/min; finally the “flow-through peak” and “elution peak” samples in flow-through and elution fractions respectively were collected and analyzed by HPLC. The recovery of lysozyme (%) was calculated as the mass ratio of the collected lysozyme in elution fraction and the total lysozyme in the injected CEW feed solution. The activity of lysozyme was measured by Shugar method, using Micrococcus lysodeikticus as a substrate (0.01% w/v in 0.1 M potassium phosphate buffer, pH 6.2). For the activity reaction, a 2.9 mL substrate was reacted with 0.1 mL solution of purified lysozyme and CEW solution (with similar concentration of lysozyme) and the decrease in absorbance was recorded for 5 mins at 450 nm using a UV spectrophotometer. The specific activity of the purified lysozyme as well as the lysozyme in CEW solution was compared. 2.7. Sample assay Protein samples were concentrated by centrifugal filters with an MWCO of 3,000 Da (Amicon Ultra-15 Millipore Ireland ) using a Sigma 4-15 K Germany centrifuge at 4000 rpm, before analysis if necessary. Protein concentrations were examined by HPLC using Agilent TC C18 column. Buffer A was 0.1% (v/v) trifluoroacetic acid (TFA) in water and Buffer B was water/acetonitrile solution (2:8) with 0.05 mL TFA. A 20 µL sample was injected by the autosampler, and peak detection was performed at 215 nm. A temperature of 30˚C and a flow rate of 0.5 mL/min was used. The samples collected from membrane chromatography were also analyzed by reducing SDS-PAGE performed with 12.5% gels. Coomassie blue staining protocol was employed to visualize the protein bands. The activity of lysozyme was monitored by UV spectrophotometer at 450 nm and 25 ºC using a 9000S spectrophotometer from Metash Shanghai. 2.8. Reusability test To evaluate the reusability of the prepared membrane adsorbers, the adsorption-desorption experiments were conducted for 5 cycles for the same membranes. Lysozyme adsorption experiment was performed as explained in static lysozyme adsorption then the membrane adsorbers were washed with binding buffer and finally treated with elution buffer until no lysozyme could be detected in the eluent. After each cycle, the membranes were washed three times with ultrapure water and finally with binding buffer to remove KSCN.

3. Results and discussion 3.1 Membrane characterization 7

The schematic diagram of surface functionalization of microporous nylon membranes with ADA and sodium bisulfite is shown in Fig. 1. ADA molecules were grafted on the nylon membranes by the reaction between aldehyde groups of ADA and amine groups on the membrane. Successful surface functionalization was confirmed by FTIR and XPS scan for the pristine, ADAnylon and S-ADA-nylon membranes. The FTIR spectrum of pristine nylon, ADA-nylon and SADA-nylon were shown in Fig. 2. The peak at 1529 cm-1 corresponds to amides while the peak at 1625 cm-1 shows carboxyl and carbonyl groups. The peak due to amine groups appeared around 3300 cm-1 for all the nylon membranes. Successful ADA coating was confirmed by the appearance of a new aldehyde peak around 1725 cm-1 and an increase in the carboxyl peak for ADA-nylon membrane. Furthermore, the decrease in amine peak would be attributed to the Schiff’s base formation. In the scan for S-ADA-nylon membrane, the disappearance of aldehyde peak and the two new peaks for sulphonic groups at 1150 cm-1 and 1346 cm-1 confirmed the successful fabrication of S-ADA-nylon adsorber. XPS scan for the pristine, ADA-nylon and S-ADA-nylon membranes also verified this conclusion (See Fig S1-S3 in Supplementary Information). In order to further clarify the variation of functional groups on the nylon membrane, zeta potential with varying pH for the pristine nylon, ADA coated nylon and S-ADA-nylon membranes was measured. As shown in the Fig. 3, the pristine nylon membrane was positively charged from pH 3 to 5.5 thanks to the amide/amine ionization, while at pH higher than 5.5, it became negatively charged due to the presence of –COOH functional groups at the ends of the polyamide chains. However, because of the abundant carboxyl groups on ADA, the zeta potential maintained negative for both ADA coated nylon and S-ADA-nylon adsorbers. Moreover, the zeta potential of S-ADA-nylon membrane was always more negative than that of ADA-coated nylon membrane at tested pH, which was probably due to the incorporation of sulphonic functionality. Overall the zeta potential for both ADA and S-ADA-nylon membranes was quite stable, demonstrating the stability of the newly fabricated membrane adsorbers at a broad pH range. S-ADA-nylon membrane adsorbers with a high zeta potential of -83 mV at pH 7.0 could strongly bind positively charged lysozyme around neutral pH. Moreover, the decrease in water contact angle and transmembrane pressure (TMP) with increasing ADA coating time indicated the improvement in hydrophilicity and permeability for SADA-nylon membrane adsorber by ADA modification (See Fig. S4 ). SEM data of surface and cross-section analysis for the pristine nylon and S-ADA-nylon adsorbers were given in Fig. S5, 8

showing that some pore narrowing and coverage occurred due to the ADA coating layer on the nylon membrane. [Fig. 2] [Fig. 3] 3.2. Comparison of ADA-nylon and S-ADA-nylon membrane adsorbers The ADA-nylon membrane with only carboxyl groups and the S-ADA-nylon membrane with both carboxyl and sulphonic groups were compared in terms of lysozyme maximum adsorption. The ADA-nylon membrane adsorber with carboxyl functionality showed a maximum static binding of 180 mg/mL for lysozyme adsorption, which is higher than the lysozyme adsorption (170 mg/mL & 105 mg/g) reported by Chen and Chiu et al. respectively for carboxy cation exchange membranes [28, 34]. The S-ADA-nylon adsorber with dual CEX groups (carboxyl & Sulphonic) bound lysozyme with a maximum adsorption capacity of 286 mg/mL, which is even higher than the total sum of the static adsorption capacities of carboxyl (170 mg/mL) and sulphonic (84 mg/mL) [36] CEX membranes as shown in Table 1. S-ADA-nylon membrane adsorber showed a 10% DBC of 95 mg/mL which was quite higher than the commercial Sartobind S CEX membrane (29 mg/mL) (Fig. S6). Therefore, it was revealed that a CEX membrane adsorber with dual functional groups could synergistically adsorb proteins with an unprecedented capacity. Accordingly, the S-ADA-nylon membrane adsorber was selected for further separation of lysozyme from CEW solution. [Table 1] 3.3. Process optimization of membrane adsorber preparation and application 3.3.1. Effect of ADA coating time on lysozyme adsorption and purification The density of ADA ligands in/on the membrane could directly govern the protein binding capacity. Thus, the S-ADA-nylon membranes with different ADA coating time (4-16 h) was investigated for static lysozyme binding. There was a great increase in the static binding capacity from 120 to 280 mg/mL as ADA coating time increased from 4 to 12 h (Fig. 4). The membrane with a coating time of 16 h had only a little increase in binding capacity with a total value of 286 mg/mL. Moreover, the effect of ADA coating time on the purification of lysozyme from CEW solution was also investigated in flow-through mode. As shown in Table 2, the recovery of lysozyme improved from 36 % to 98 % by increasing the ADA coating time from 4 to 16 h because of an increase in the CEX groups on the membrane surface. Comparing the results in Fig. 4 and 9

Table 2, the much lower lysozyme binding from the CEW solution by membrane chromatography than that from model solution by static binding is caused by the lower lysozyme amount in the real solution (11.1 vs. 48 mg) and larger membrane volume in the chromatography system (0.16 vs. 0.045 mL) . [Table 2] [Fig. 4] 3.3.2. Effect of pH on lysozyme adsorption and purification For the performance of CEX membrane adsorber, pH plays a crucial role both in adsorption capacity and protein selectivity. Lysozyme has an isoelectric point (pI) of 10.7 which is especially higher than that of the other CEW proteins (the highest one is 6.5). Therefore, a suitable operating pH could be found between 6.5 and 10.7 since the lysozyme was positively charged while the others were negatively charged. As shown in Fig. 5, the optimal pH for lysozyme adsorption by the S-ADA-nylon membrane adsorber was 7.5. The decline of lysozyme adsorption at pH higher than 7.5 could be explained by the reduction in positive charge of lysozyme [37], but the increasing lysozyme adsorption at pH from 6.5 to 7.5 was unexpected (this membrane adsorber was stable at pH 4-9 at least for 8 hours, see Fig. S7). Arica et al. claimed that the ion-exchange property of a protein was primarily governed by the distribution of charges on the proteins rather than their “net charge”[27]. Therefore, a pH of 7.5 was selected for the purification of lysozyme from the CEW solution. [Fig. 5] 3.3.3. Effect of ionic strength on lysozyme static adsorption It is clear from Fig. 6 that the lysozyme adsorption greatly depends on ionic concentration. It was noticed that the lysozyme adsorption dramatically decreased with the increase in KSCN concentration. The decrease was from 286 mg/mL for no salt to only 10 mg/mL at 1.0 M salt concentration. The trend could be expected and was also reported in the literature [38]. It can be explained by the electrostatic shielding and salting-out effect at higher ionic strength. That is, at higher salt concentration, the charge on the ligands and proteins would be shielded, and the proteins tend to aggregate due to salting-out effect, resulting in less surface-exposed binding groups on the proteins. Hence, increasing the ionic strength plays a negative role in lysozyme adsorption. But it offers a mild approach to elute the lysozyme from the S-ADA-nylon membranes. 10

[Fig. 6] 3.3.4. Effect of lysozyme concentration on lysozyme static adsorption As shown in Fig. 7, the lysozyme binding was directly proportional to the lysozyme concentration in the feed to a certain extent, and then it became constant because of the saturation of all the available active sites on the membrane. However, regarding the lysozyme binding efficiency (the ratio of bound lysozyme to the total lysozyme amount in the feed), it decreased from 79% to 54% with an increase of lysozyme concentration from 0.5 to 3.0 mg/mL. This implies that the bound lysozyme in/on the membrane would produce a negative effect on the further protein adsorption due to the charge repulsion and steric hindrance. Furthermore, to quantitatively analyze the adsorption process of lysozyme on S-ADA-nylon adsorber, Langmuir model was employed to fit the experimental data. The form of Langmuir isotherms is described by the following equation. 1 1 1 = + q e qmax K d q max Ce Where qmax is the maximum adsorption capacity, qe is the lysozyme adsorption amount at different initial concentrations, Ce is the equilibrium lysozyme concentration, Kd is the Langmuir isotherm constant. The Langmuir fitted curve is displayed in Fig. 7, with a qmax = 555 mg/mL and Kd = 3.1 x 10-5 M. [Fig. 7] 3.5. Purification of lysozyme from CEW solution CEW is composed of different proteins, among these lysozyme have the highest pI) of 10.7 (for other proteins the highest pI is 6.5). At a pH of 7.5 lysozyme being positively charged will be successfully captured by S-ADA-nylon membrane adsorber while the other proteins will be repelled since they are negatively charged. As seen in Table 2, the lysozyme recovery enhanced from 36.3% to 98.1% when the ADA coating time increased from 4 h to 16 h, indicating that more ligands were grafted on the membrane at higher coating time. It is worth mentioning that the lysozyme binding efficiency is 45.4% at a coating time of 4 h, being much higher than its recovery (36.3%), while at higher coating time, almost all the bound lysozyme can be eluted. This can be explained by a fact that when the ADA coating is not enough to fully cover the membrane surface, some lysozyme molecules may bind on the membrane via hydrophobic adsorption, and these bound lysozyme molecules cannot be eluted from the membrane adsorber by 1.5 M KSCN. 11

SDS-PAGE and HPLC were used to determine the purity of captured lysozyme from the CEW solution. As illustrated in Fig. 8, the chromatogram of CEW shows four major peaks for conalbumin, ovalbumin, ovomucin, and lysozyme with molecular weights of 78k, 45k, 28k, and 14k Dalton, respectively. For the chromatogram of the purified lysozyme, only one major peak for lysozyme is observed, which is equivalent to the chromatogram of the commercial lysozyme. The SDS-PAGE image in Fig. 9 confirmed the above results. In the first lane for the CEW solution, there are different bands representing a mixture of proteins. The bands in the second and fourth lanes corresponding to standard and eluted lysozyme samples respectively are similar, and the third lane expressing the flow-through fraction demonstrates that almost all the lysozyme in CEW is captured by the membrane adsorber, verifying that a high purity (100%) and a high recovery (98%) of lysozyme are obtained by the S-ADA-nylon membrane adsorber in treating the CEW solution. Moreover, the specific activity of the purified lysozyme was 65,000 U/mg, while the CEW solution showed a specific activity of 733.33 U/mg. The specific activity of the commercial lysozyme was 70000 U/mg. [Fig. 8] [Fig. 9] 3.6. Reusability and cost Efficient elution and easy regeneration are essential for an excellent CEX membrane adsorber, thus this newly made S-ADA-nylon membrane adsorber was used for the adsorption of lysozyme for five cycles. As seen in Fig. 10, the protein binding was similar in all the five cycles. The good reusability of the newly made membrane adsorbers can also be confirmed in Fig. S8, in which both of the chromatograms are similar for the two cycles of lysozyme capturing from the CEW solution. This indicates that the ligands attached to the nylon membrane are not washed away during the elution step and the bound lysozyme can be completely removed by the elution. Furthermore the total cost of the membrane adsorber was calculated which was around 0.20 USD/cm2 while the commercial Sartobind S CEX adsorber was about 6.0 USD/cm2 (including the cost of the packed materials). Although it was a rough comparison, the raw materials cost for fabrication of this new membrane adsorber was relatively low. Thanks to its reusability and cost effectiveness, the S-ADA-nylon membrane adsorber is promising in the downstream processing of protein production. [Fig. 10] 12

4. Conclusion This work demonstrated a novel, facile and green approach for fabricating CEX membrane adsorber with unprecedented adsorption capacity for protein purification by grafting ADA and sodium bisulfite on the nylon microfiltration membranes. The inherent enormous surface area and hydrophilicity enables S-ADA-nylon membrane adsorber to exhibit high binding capacity toward protein molecules, as well as excellent resistance towards nonspecific biomolecule adsorption. This novel membrane adsorber showed a 10% DBC of 95 mg/mL which was quite higher than the commercial Sartobind S CEX membrane (29 mg/mL). Furthermore, this CEX membrane adsorber not only provided a high selectivity for purifying lysozyme from the CEW solution but also maintained a high recovery of 98% in binding-eluting mode. It also showed an excellent reusability for static lysozyme adsorption. Thanks to the simplicity and cost effectiveness of the synthesis process, as well as the high performance of the resultant S-ADA-nylon membrane adsorber, we anticipate that such a CEX adsorber will provide a new kind of functional materials for the fast, efficient and low-cost production of various protein products. Acknowledgments The authors thank the National Science Foundation of China (No. 21506229) and Youth Innovation Promotion Association (2017069) of Chinese Academy of Sciences. This work is supported by ‘100 Talents Program’ of Chinese Academy of Sciences. References [1] M. Mayani, C.D.M. Filipe, M.D. McLean, J.C. Hall, R. Ghosh, Purification of transgenic tobaccoderived recombinant human monoclonal antibody, Biochem. Eng. J. 72 (2013) 33-41. [2] H.F. Tong, D.Q. Lin, D. Gao, X.M. Yuan, S.J. Yao, Caprylate as the albumin-selective modifier to improve IgG purification with hydrophobic charge-induction chromatography, J. Chromatogr. A, 1285 (2013) 88-96. [3] J. Weaver, S.M. Husson, L. Murphy, S.R. Wickramasinghe, Anion exchange membrane adsorbers for flow-through polishing steps: Part I. Clearance of minute virus of mice, Biotechnol. Bioeng. 110 (2013) 491-499. [4] J. Fan, J. Luo, W. Song, X. Chen, Y. Wan, Directing membrane chromatography to manufacture α1antitrypsin from human plasma fraction IV, J. Chromatogr. A, 1423 (2015) 63-70. [5] S.S. Yan Luding, Yun Junxian, Yao Kejian, Isolation of lysozyme from chicken egg white using polyacrylamide-based cation-exchange cryogel, Chin. J. Chem. Eng. 19 (2011) 876-880. [6] S. Fekete, A. Beck, J.L. Veuthey, D. Guillarme, Ion-exchange chromatography for the characterization of biopharmaceuticals, J. Pharm. Biomed. Anal. 113 (2015) 43-55.

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Figures

15

Figure 1. Schematic diagram for fabrication of S-ADA-nylon membrane adsorber and its surface chemistry. N-H

0.6

Pristine Nylon

C=O

N-H

0.3

Absorbance

0.0 ADA-Nylon

0.6 Aldehyde 0.3 0.0 0.6

Sulphonic Peaks

S-ADA-Nylon

0.3 0.0 1000

1500

2000

2500

3000

Wavenumbers (cm-1)

3500

Figure 2. ATR-FTIR spectrum for pristine nylon, ADA-nylon and S-ADA-nylon membrane adsorbers.

16

90 Pristine nylon ADA coated nylon S-ADA-nylon membrane

60

Zeta Potential (mV)

30 0 -30 -60 -90 -120 4

6

8

10

pH

Figure 3. Zeta potential of pristine nylon, ADA-nylon and S-ADA-nylon membrane adsorbers.

350

(mg/mL membrane)

Lysozyme adsorption

280

210

140

70

0 4

8

12

16

ADA Coating Time (h)

Figure 4. Effect of ADA coating time on lysozyme static adsorption by S-ADA-nylon membrane. Static binding was carried out for 10 hours at a shaking speed of 110 rpm in sodium phosphate buffer (20 mM, pH 7.5) with a lysozyme concentration of 6 mg/mL. 17

350

Lysozyme adsorption (mg/mL membrane)

280

210

140

70

0 6.5

7.0

7.5

8.0

8.5

pH

Figure 5. Effect of pH on lysozyme static adsorption by S-ADA-nylon membrane with an ADA coating time of 16 h. Static binding was carried out for 10 hours at a shaking speed of 110 rpm and 25 ºC in 20 mM sodium phosphate buffer with a lysozyme concentration of 6 mg/mL. 350

Lysozyme adsorption (mg/mL membrane)

280

210

140

70

0 0.0

0.2

0.4

0.6

0.8

1.0

KSCN (mol/L)

Figure 6. Effect of ionic strength on lysozyme static adsorption by S-ADA-nylon membrane with an ADA coating time of 16 h. The different salt concentrations were added in 20 mM sodium phosphate binding buffer with a final pH of 7.5. Static binding was carried out for 10 hours at a shaking speed of 110 rpm and 25 ºC with a lysozyme concentration of 6 mg/mL. 18

300

Lysozyme adsorption (mg/mL membrane)

250 200 150 100 50 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Lysozyme initial conc. (mg/mL)

Figure 7. Effect of initial lysozyme concentration on static adsorption by S-ADA-nylon membrane with 16 h ADA coating time (Langmuir fitting). Static binding was carried out for 10 hours in sodium phosphate buffer (20 mM, pH 7.5) at a shaking speed of 110 rpm.

12000 8000

Ovalbumin

CEW Ovomucin

Conalbumin

Lysozyme

4000 0

UV(mAu)

-4000 4000

Purified Lys Lysozyme

0 -4000 4000

Comercial Lys Lysozyme

0 -4000 10

20

30

40

Time(min)

Figure 8. HPLC chromatogram for CEW solution, purified lysozyme and commercial lysozyme. Buffer A was 0.1% (v/v) trifluoroacetic acid (TFA) in water and buffer B was water/acetonitrile solution (2:8) with 0.05 mL TFA. The flow rate was 0.5mL/min at 30 ºC.

19

Figure 9. SDS-PAGE analysis of CEW (lane 1), standard lysozyme (lane 2), flow-through fraction (lane 3) and elution fraction (lane 4).

500

Relative RBC (%)

100 80

400

Relative RBC (%) Lysozyme adsorption(mg/ml)

60 40

300

20 0

Lysozyme adsorption Capicity (mg/mL membrane)

120

200 1

2

3

4

5

Number of cycle

Figure 10. Relative RBC (reversible binding capacity) and lysozyme adsorption capacity during five lysozyme adsorption elution cycles conducted by S-ADA-nylon membrane adsorber. The RBC was the ratio of eluted lysozyme mass to the membrane volume. The relative RBC was calculated by comparing RBC in each cycle to the RBC in first cycle.

20

Tables Table. 1. Comparison of lysozyme maximum adsorption capacity by different cation exchange membrane adsorbents. Cation exchange adsorbents

Lysozyme maximum adsorption capacity

Reference

EVOH –CCA nano filtration membrane a

284 mg/g

12

170 mg/mL

28

Weak acidic polyacrylonitrile membrane

105 mg/g

34

Sulphonic cation exchange hollow fiber membrane

84 mg/mL

36

ADA-nylon membrane b

180 mg/mL

This study

S-ADA-nylon membrane c

286 mg/mL (322 mg/g)

This study

Carboxymethylcellulose chitosan membrane

a. EVOH-CCA= Ethylene vinyl alcohol- citric acid b. ADA= Alginate dialdehyde c. S-ADA= Sulphonic-alginate dialdehyde

Table. 2. Purification of lysozyme from real CEW solution by the S-ADA-nylon adsorbers with different ADA coating time. Recovery is the ratio of total lysozyme in elution fraction to the amount of total lysozyme in feed solution. For finding binding efficiency of the membrane adsorbers pure lysozyme was added to CEW solution with a final concentration of 2.22 mg/mL. ADA Coating Time (h)

Lysozyme binding (mg/mL membrane)

Recovery (%)

04

31.50

36.3

08

46.50

65.5

12

66.25

95.3

16

68.12

98.1

21