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Cellulose-graft-Polyethyleneamidoamine Anion-Exchange Nanofiber Membranes for Simultaneous Protein Adsorption and Virus Filtration Rajesh Sahadevan, Caitlin Crandall, Steven Schneiderman, and Todd J. Menkhaus ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00519 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Applied Nano Materials

Cellulose-graft-Polyethyleneamidoamine Anion-Exchange Nanofiber Membranes for Simultaneous Protein Adsorption and Virus Filtration Sahadevan Rajesh, Caitlin Crandall, Steven Schneiderman, and Todd J. Menkhaus1*

Department of Chemical and Biological Engineering South Dakota School of Mines and Technology Rapid City, SD 57701, USA

Corresponding Author * Todd J. Menkhaus, Email: [email protected] Phone: (605) 394-2422: Fax: (605) 394-1232

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Abstract Development of bioseparation media that can perform separations based on both ion-exchange and size exclusion provide a transformative technology to purify therapeutic proteins with fewer operations in downstream processing. Thus, in this study we have developed cellulose-graftpolyethyleneamidoamine (CL-g-PEAA) anion-exchange nanofiber membrane adsorbers with a narrow effective pore size distribution using redox polymerization. Structural characterization illustrated the functionalization of each nanofibers of CL-g-PEAA membrane adsorbers with three dimensional nanolayers of amidoamine functionality. The ion-exchange separation efficiency of the CL-g-PEAA anionexchange membranes, evaluated using bovine serum albumin (BSA), exhibited an excellent static adsorption of 239 mg/g of nanofibers. The BSA dynamic binding capacity in flow through mode at 10% breakthrough of these membranes was 69 mg/g (corresponding to a volume capacity of 31 g/L) with an approximate residence time of 8s. Meanwhile, CL-g-PEAA membranes have shown 80-100% rejection for model bead particles of various sizes (200, 100 and 40 nm), which illustrated the potential in removing viruses, microbial agents, and cellular debris, from the harvested cell culture fluid (HCCF) during downstream processing. Overall performance of the CL-g-PEAA membranes illustrated that undesired impurities (viruses, host cell proteins, and DNA) in HCCF could be removed in a single step by an anionexchange membranes with ion-exchange and size exclusion based separation mechanisms. Keywords: Nanofiber, anion-exchange, redox polymerization, membrane chromatography, downstream processing 1. Introduction With the ever increasing prominence of monoclonal antibodies (mAbs) as therapeutic agents, the antibody titers in mammalian cell culture (MCC) have increased over ten-fold in last two decades

1-3

. Thus

it is important to develop highly efficient downstream purification processes that can handle higher volumes of mAbs in the harvested cell culture fluid (HCCF)

2-5

. Downstream processing of protein

purification involves the removal of non-essential components such as host cell protein, DNA, viruses, endotoxin and protein aggregates from the MCC

4,5

. The first step in the recovery of mAbs from MCC is

the removal of these unwanted cell components either by centrifugation or filtration to yield HCCF suitable

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5

for further purification by chromatography . The high affinity protein-A chromatography is then used to selectively capture mAb products directly from HCCF

4-5

. Two or more additional chromatographic steps

(ion-exchange, hydrophobic, mixed mode, etc.) and viral filtration are used as polishing steps to remove any surplus of non-essential cellular components mentioned above

5-9

. As a final step, diafiltration is used

to concentrate the final product. The need for these multiple steps in downstream processing accounts for almost 80% of the total manufacturing cost of biopharmaceutical production

10-11

. However, design of a

purification medium that is capable of both size exclusion (especially in the 40-100 nm range that is typical for virus filtration) and adsorptive separation simultaneously may help to achieve the targeted purity with lesser chromatographic polishing steps

5-11

.

Packed-bed chromatography (PBC) has been widely employed for the purification of therapeutic proteins because of its ability to perform ionic-site specific adsorption and desorption mechanism

12-13

.

Though PBC has high binding efficiency, it suffers from slow adsorption kinetics and pressure build-up during operation, and the highly expensive column packing and regeneration operations

6,14-15

. However,

membrane chromatography based on macro porous membrane is an attractive alternative to PBC for the recovery of mAbs

15-17

. The majority of the drawbacks associated with PBC can be overcome using a

membrane adsorber, as flow rate does not have any significant effect on the binding capacity. Nevertheless, one of the main disadvantages of membrane adsorbers, which is affecting its broad implementation, is the fewer number of ligand sites per unit volume, and thus lower binding capacity. However, compared to macroporous membranes, polymer eletrospun nanofiber membranes have high active surface area per unit volume and an open porous structure, which facilitates the fast adsorption kinetics

15,18-19

. Exploring these unique structural features, many previous attempts have been reported for

the effective utilization of nanofiber membranes for ionic contaminants removal 23

24

desalting , hemodialysis , enzyme immobilization

25

, bio-scaffolds

26

20-21

22

, metal ions removal ,

, therapeutic protein purification

15,27

etc. The nanofiber membranes modified with chemical functional groups have tremendous potential to address the drawbacks of traditional membranes due to their ability to transport biomolecules to the targeted binding sites by convection with low pressure.

14-15, 18, 27-29

. Previous research efforts from our

group and several attempts in the literature have explored these unique structural characteristics of the nanofiber membranes and reported the fabrication of cation-exchange nanofiber membranes with

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excellent binding capacity for positively charged proteins

Page 4 of 25

6, 14, 15, 18, 27-29

. However, the preparation of

anion-exchange (AEX) nanofiber membrane adsorbers remains a challenge due to its low adsorption capacity, rapid breakthrough resulting from the unfavorable interaction of large protein molecules with the binding sites, poor permeability and inability to be used as a self-supported structure. In the biopharmaceutical industry, AEX membranes are traditionally employed in polishing steps for the removal of impurities (aggregates, DNA, etc.) and virus particles, all of which are typically present at lower concentrations than the protein product

5, 14,30

. At the operation pH of 7.0, viruses, host cell proteins,

and DNA bind to the AEX membranes by electrostatic interactions, while allowing monoclonal antibodies to flow through the membranes due to its same charge (high isoelectric point, pI). Thus, efficiency of an AEX membrane depends on the nature of the ionic functional groups and physical properties of the membrane

17,30-32

. Many studies in the literature have proven that binding capacity of membrane

adsorbers can be improved by developing a multiple protein adsorption sites within the grafted layers in the pore walls of support structure membranes

17,32-34

. Several grafting methods, such as reversible

addition-fragmentation chain transfer polymerization (RAFT) (ATRP)

17, 37

, redox initiated

38

, radiation induced

39-40

35-36

, atom transfer radical polymerization

and photo-initiated polymerization

41

have been

successfully applied for the functionalization of macroporous and nanofiber membranes with three dimensional nanolayers. But, it is generally accepted that RAFT and ATRP require organic solvents and well controlled reaction conditions (preferably in a glove box with stringent control over ambient conditions and oxygen levels), making these difficult to implement in a commercial production unit

10,15, 42

. Thus,

redox polymerization was used in our development of anion-exchange membrane adsorbers because of its low activation energy requirements, quick initiation, feasibility in aqueous reaction medium and easy scale up

15, 38, 43

.

In this study we fabricated novel self-supported AEX nanofiber membranes by grafting diethylenetriamine on to electrospun cellulose (CL) nanofiber by redox polymerization for the recovery of mAbs from HCCF with fewer purifications steps. AEX membranes were prepared using CL as the base material because of its naturally low adsorptive surface as an unmodified fiber (e.g., the ability to interact specifically and uniquely with biological molecules through directed adsorption of added functional groups), abundant availability and the presence of flexible hydroxyl (-OH) groups, which can be

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transformed into desired functionality

14-18

. The CL-g-PEAA nanofiber adsorptive membrane in the present

study was prepared by following a multi-step procedure. Cellulose-graft-polyacrylonitrile (CL-g-PAN) was first prepared by the continuous grafting of acrylonitrile (AN) onto the surface of CL nanofibers. Subsequently, diethylenetriamine was grafted to the -C≡N group of CL-g-PAN to form CL-g-PEAA anion exchange membrane adsorber. The hybrid membrane material obtained by combining CL of biological nature and synthetic PEAA would surely imparts mechanical integrity to the AEX membranes during practical applications. For the fabricated CL-g-PEAA AEX membrane adsorbers, the role of synthesis conditions of CL-g-PEAA membrane adsorbers and its morphology on the purification of negatively charged proteins were evaluated. In addition, sized-based separation efficiency of hypothetical virus particles by the membranes was studied with beads as model particles. These AEX membranes were also subjected to the separation of negatively charged proteins and have shown excellent binding capacity with fast kinetic equilibrium. 1 M NaCl solutions could effectively desorbed the bound proteins from the

CL-g-PEAA nanofiber membrane adsorbers, which illustrated the efficiency of

new AEX

membranes in the fast and effective removal of negatively charged biomolecules and protein aggregates from HCCF. 2. Experimental 2.1. Materials The specifications of polymers, chemicals and all other reagents used for the current study are provided in the supporting information. 2.2. Fabrication of cellulose-graft-polyethyleneamidoamine membranes The cellulose nanofiber used as the base material for the fabrication of CL-g-PEAA membrane adsorber was prepared by electrospinning process and detailed procedure was described in our earlier publications

14, 15, 27

. CL-g-PEAA nanofiber AEX membrane adsorber was prepared by a multi step

method. The initial step of the CL-g-PEAA nanofiber AEX membrane preparation in this study was common to that of cationic membrane adsorber reported in our previous publication

15

. Briefly, the

cellulose-graft-polyacrylonitrile (CL-g-PAN) was first prepared by the continuous grafting of acrylonitrile (AN) onto the surface of CL nanofibers. CL-g-PAN was then reacted with diethylenetriamine, DETA (33.0

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mL) at 100°C for 2 h in presence of Na2CO3 (1.0g) and water (67.0 mL) to complete the preparation of CL-g-PEAA membrane adsorber. The CL-g-PEAA AEX membrane adsorber thus prepared was washed several times with DI water and then immersed in pH adjusted DI water (pH-2.0, adjusted with HCl) for 12 h. The CL-g-PEAA membranes prepared were then dried at 80°C thoroughly prior to use for BSA purification and bead particle separation. 2.3. CL-g-PEAA nanofiber membrane characterization The morphological changes of CL nanofibers during CL-g-PEAA preparation were recorded at 3 kV using ZEISS Supra40 field-emission scanning electron microscope (FESEM). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR, Agilent Technologies, Cary 660) and Xray photoelectron spectroscopy (XPS, PHI Quantera II, ULVAC-PHI Inc., USA) were used to study the chemical structural changes of the CL nanofiber at various stages of CL-g-PEAA preparation. 2.4. Static and dynamic protein adsorption experiments Static protein adsorption capacity of the AEX membrane adsorbers was studied using BSA at a concentration of 2g/L @ pH-8.0. The BSA adsorption capacity (qe) was calculated based on the equation 15, 29

,  =

  

(1)



Where Ci and Cf respectively represent BSA concentrations before and after adsorption, V0 and m are the volume of protein solution and dry weight of the nanofiber used respectively. AKTA 0070ure chromatography system (GE Healthcare Life Sciences) was employed to study the dynamic BSA adsorption capacity of the AEX adsorptive membranes as described in our earlier studies

14, 15

. Three layers of CL-g-PEAA adsorptive membranes with an approximate total thickness of 3

0.03-0.04 cm, dry volume of 0.09-0.14 cm , and dry mass of 43-62 mg was used. Dynamic adsorption capacity based on membrane mass and volume at 10% breakthrough were estimated from the AKTA chromatogram. The detailed procedure for the static and dynamic adsorption studies are provided in the supporting information.

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2.5. Filtration performance of bead particles The feasibility of the removal of small microbial agents, cellular debris and viruses from HCCF by size-based separation efficiency of AEX membranes was studied by filtering model bead particles of various sizes under gravitational feed pressure. An aqueous solution of 200 nm blue dyed polystyrene microspheres, 100 nm yellow dyed polystyrene microspheres and 40 nm nanobead NIST traceable particle size standard were used for the filtration experiments (each with an initial concentration of 0.5 g/L).

Bead solution fluxes and percentage rejections were then estimated using equation, 2 and 3,

respectively

23, 44

,

 =

∆

(2)

 × 

Where ∆V represents the volume of water passed through the AEX membranes of area ‘A,’ in time ‘t’. %R = 1 −

 



(3)

Here cf and cp respectively represents the feed and permeate bead solutions concentrations. 3. Results and Discussion 3.1. Morphology of CL-g-PEAA membranes Anion-exchange membrane chromatography purification has been commonly employed in the polishing stage of protein purification to remove host cell proteins, endotoxins, DNA, viruses, leached protein-A and other non-essential cellular components

5,15

. Since most of these components are

negatively charged at neutral pH, the amount of functional polymer nanolayer grafted on to the base polymer thus have a significant effect on the separation efficiency of these anion-exchange membranes. In the current study we developed a positively charged CL-g-PEAA membrane adsorber by grafting amidoamine groups on to the surface of CL nanofibers by a multistep process. The schematic representation of the packing of CL-g-PEAA membrane adsorber in to a bed and its use in the removal of BSA by a binding-elution cycle are given in scheme 1.

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Scheme 1. Schematic representation of the dynamic BSA adsorption by CL-g-PEAA anion-exchange membrane adsorber using an AKTA pure chromatographic system in the current study. (a) the packing of CL-g-PEAA nanofiber discs in to a cartridge (bed volume: 0.13 mL), BSA solution at a desired flow rate is then flow through the membrane column which selectively bind BSA molecules because of its positive charge at pH-8.0. (b) Illustration of a single CL-g-PEAA nanofiber capturing BSA molecules and (c) adsorbed BSA molecules are eluted by 1 M NaCl to regenerate the CL-g-PEAA membrane for next cycles. The chemical route and reaction conditions used to prepare CL-g-PEAA membrane adsorbers are given in Fig. 1 (a-c) and Scheme S1 (supporting Information). Preparation of CL-g-PEAA membranes involved a multistep process. CL-g-PAN intermediate was first prepared by surface grafting AN monomer on to CL nanofibers using cerium ion as redox initiator. The various stages of reaction mechanism involved in the synthesis of CL-g-PAN intermediate is provided in Scheme S2. As illustrated in S2 Ce(IV) induce the formation of CL free radicals in the reaction medium, which initiated the polymerization of AN monomer on to CL. The reaction was propagated by chemically binding more AN monomer to the growing PAN chains. The reaction was then terminated by coupling and disproportionation with transfer of protons between the macroradicals to form CL-g-PAN intermediate. As formed CL-g-PAN intermediate was reacted with diethylenetriamine in the second step to form CL-g-PEAA membrane adsorber. As illustrated in the reaction mechanism in scheme S3, -C≡N groups of CL-g-PAN formed initially are hydrolyzed to from acid groups. These acid groups then condense and rearrange with amine groups of diethylenetriamine to form amidoamine functionalized CL-g-PEAA anion-exchange membrane adsorber. Similar types of reaction mechanisms are reported in the literature too

43, 45-47

.

The change in microstructure take place at each step of the synthesis of CL-g-PEAA membranes -3

from 28.2x10 M of AN concentration in the initial step and 3.1 M DETA concentration in the final step are

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Fig. 1. A schematic of the reaction pathway involved and corresponding morphological changes in the fabrication of CL-g-PEAA anion-exchange membrane adsorber by redox polymerization. (a) representative image of a single CL nanofiber used as the base material, (b) intermediate step in which CL reacted with acylonitrile monomer at 60 °C for 6h to form CL-g-PAN, and (c), CL-g-PAN was finally converted in to CL-g-PEAA by reaction with diethylenetriamine at 100 °C for 1.5 h. (a1), (b1) and (c1) respectively are the SEM images of nanofibers at synthesis stages (a), (b) and (c). (a2), (b2) and (c2) are the histograms of average nanofiber diameters calculated for images (a1), (b1) and (c1) respectively using -3 ImageJ software. AN and DETA concentrations of 28.2x10 and 3.1 M were respectively used for the intermediate and final step of the CL-g-PEAA preparation. presented in Fig.1 (a1-c1). The morphological changes at different stages of the synthesis were monitored by measuring change in thickness of the nanofibers using ImageJ software and are shown Fig. 1 (a2-c2). The CL electrospun nanofibers used as the base material had an initial fiber thickness of 160 nm (Fig. 1a1&a2). After the grafting of CL mat with PAN, the thickness of individual nanofibers was increased significantly as a function of AN concentration indicating higher degree of grafting -3

nanofibers modified with AN concentration of 28.2x10

45

. The CL-g-PAN

M have an approximate thickness of 350nm

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(Fig.1b1&b2), which increased to 550 nm after reaction with DETA (Fig.1c1&c2). Fig. S1 and Fig. S2 represent the SEM micrographs of CL-g-PAN intermediate synthesized from various AN concentration and reaction time respectively. It is important to note that lower AN concentrations produced uniform PAN -3

coating along the individual nanofibers, but beyond 28.2x10 M AN concentrations, nanofibers started to aggregate. The effect of AN grafting time on the CL-g-PAN intermediate was the same as that of concentration with a dense structure for longer than 6h of reaction time (Fig. S2c&d). Such a conclusion was corroborated by the similar graft yield obtained for CL-g-PAN intermediate prepared from higher AN -3

-3

concentration of 37.7x10 M for 6h reaction and lower AN concentration of 28.2x10 M for 10h (Fig.S3). In membrane chromatographic operations, an open pore structure means that desired biomolecules in -3

the HCCF reaches the membrane binding sites by convection; thus, AN concentration of 28.2x10 M with a functionalization time of 6 h could be considered as ideal conditions for the intermediate step (Fig. 1b1). CL-g-PAN synthesized in the intermediate step was finally treated with DETA to form CL-g-PEAA anion exchange membrane adsorber. SEM images of the CL-g-PEAA membrane adsorbers fabricated with various DETA functionalization time are provided in Fig. S4. As could be seen, reaction time had a significant effect on the CL-g-PEAA nanofiber morphology, with the formation of broken fibers beyond 1.5 h of reaction (Fig. S4c). Also, it is important to mention that during the amination reaction with DETA, the initial white color of cellulose nanofibers mat changed to pale yellow (≥ 10min) to pale orange (≥1 h) and finally a fragile white fiber mat beyond 2.5h of amination reaction. From the morphological changes in Fig. S4 and visual examination of the membranes, it could be considered that 1.5 h of amination reaction time is optimum for CL-g-PEAA membrane adsorber preparation (Fig. 1c1). For comparative evaluation, PAN electrospun nanofiber prepared in our laboratory (with a fiber thickness of 60-70 nm) was also functionalized by direct treatment with DETA

48

. The morphological changes of amidoamine functionalized

PAN nanofiber are given in Fig. S5 and it follows the same trend as that of CL-g-PEAA membranes with a fragile structure with 2h of reaction. Comparing the CL-g-PEAA and PAN membranes, it could be noticed that membrane integrity directly related to the amount of -C≡N groups present and its subsequent conversion in amination step. A fewer number of -C≡N groups and its uniform distribution in a given volume of CL-g-PEAA membranes relative to pure PAN membranes help to maintain the mechanical integrity after treatment with DETA. These DETA nanolayers coated along the individual nanofibers

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imparts anion-exchange functionality to CL nanofibers, which can be used for the separation of negatively charged impurities in protein purification and that will discussed in the subsequent sections

46,48

.

3.2. Structural characterization of CL-g-PEAA membranes The chemical structural variations takes place in two different stages of the CL-g-PEAA anionexchange membrane adsorber synthesis process were recorded using ATR-FTIR spectroscopy (Fig. 2). Pure CL nanofibers displayed an adsorption peak at 1065 cm

-1

due to the –C-O linkage between

glycosidic carbon and –OH groups. After the redox polymerization step, CL-g-PAN intermediate -1

nanofibers exhibited a new stretching vibration at 2240 cm illustrating the successful grafting of -C≡N groups. The CL-g-PEAA anionic membrane adsorber, the nitrile group stretching vibration at 2240 cm

-1

-1

completely vanished with the emergence of a fresh peak at 1560 cm due to the bending vibration of – -1

NH2 groups. This new peak at 1560 cm illustrated effective grating of DETA on to CL nanofibers through the grafted -C≡N groups in the intermediate step. Similarly, amidoamine functionalized PAN membranes spectra were provided in Fig. S6. From the spectra, it was observed that with 2 h of amination reaction the -C≡N groups present in PAN membranes were completely converted to amidoamine functionality. In the comparison of the spectra of pure PAN and CL-g-PAN nanofibers, it could be seen that intensity of C≡N groups grafted on to CL nanofibers with 6h reaction is much lower than pure PAN membranes. Thus it can be elucidated that such a uniform spatial distribution of -C≡N groups grafted along the CL nanofibers with fewer number per unit area have significant effect on the hydrophilic/hydrophobic balance, mechanical robustness and ion-exchange functionality of the resultant CL-g-PEAA membranes.

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Fig. 2. ATR-FTIR spectra of CL-g-PEAA anion-exchange membrane adsorbers developed in this study. The upper spectra represents pure CL, middle and lower spectra correspond CL-g-PAN intermediate and AEX anion-exchange membranes respectively. Chemical structural features takes place in two different stages of CL-g-PEAA anion-exchange membranes preparation were further evaluated by monitoring the surface composition using XPS. The XPS survey scan and deconvoluted C 1s regions of these membranes are provided in Fig. 3 (a-c) and (a1-c1), respectively. The presence of C 1s in these structures was confirmed by binding energy peaks at 285.0 eV. Similarly, peaks at 399.8 eV and 532.2 eV confirmed the presence of N 1s and O1 s respectively. However, these peak intensities varied significantly among these structures indicating the structural evolution in each stage of synthesis process. The pure CL nanofibers displayed a tiny N 1s peak and this can be justified considering the small percentage of PAN used in the preparation of CL nanofibers (small amount of PAN was used during the preparation of CL via electrospinning, Fig. 3a). The deconvoluted C 1s peaks of pure CL nanofibers thus corresponds to; 284.7 eV for -C-C/-C-H linkage, 286.1 eV for -C-O linkage and finally 287.8 eV for -C≡N linkage (Fig. 3a1). This characteristic N 1s peak of -C≡N group at 287.8 eV is in accordance with the value reported for pure PAN membranes in the literature

15, 49

.

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Fig. 3. XPS spectra of the CL-g-PEAA anion-exchange membrane adsorbers; (a), pure the CL (b) CL-gPAN prepared in the redox polymerization and (c) CL-g-PEAA membrane adsorbers. (a1), (b1) and (c1) respectively are the high resolution C 1s region scan of these membranes. The CL-g-PEAA anionexchange membrane adsorber synthesis conditions were same as that provided in Fig. 1. In the CL-g-PAN nanofibers survey scan, intensity of N 1s peak is much higher than pure CL nanofibers. The increase in intensity of N 1s peak demonstrated successful grafting of PAN onto CL in the redox polymerization step (Fig. 3b). In addition, in the deconvoluted C 1s scan, the peak positions for C-C/-C-H, -C-O and -C≡N linkages are unchanged (Fig. 3 b1). In the CL-g-PEAA anion-exchange membrane adsorber survey scan, the intensity of peaks of N 1s and O 1s were further increased, illustrating the grafting of -NH2 groups onto the CL nanofibers through an amide bond (Fig. 3c). Also, in the deconvoluted C 1s scan, the peak position for -C-C/C-H linkage was shifted to a higher binding energy of 285.4 eV because of the presence of aliphatic ethylene in the side chain. Two additional peaks at binding energy 286.7 eV and 287.4 eV, respectively, correspond to the –C-N and –N-C=O linkage of

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amidoamine group (Fig. 3 c1)

49

Page 14 of 25

. This structural rearrangement observed in the CL-g-PEAA membranes

confirmed effective grafting of amidoamine groups to CL nanofibers. The disappearance of –C-O and C≡N peaks in the C 1s curve fitting of CL-g-PEAA indicated uniform covering of newly formed amidoamine group along the nanofibers

49

. Table S1 represents the C, O and N chemical compositions of

CL-g-PEAA anion-exchange membranes estimated from XPS analysis. 3.3. Static protein binding capacity (SPBC) The performance efficiency of CL-g-PEAA membranes in the adsorptive removal of proteins was evaluated using BSA as the model protein in static mode at pH-8.0. Isoelectric point (pI) of BSA is 4.7 and pKa value of primary amine groups in CL-g-PEAA membranes is about 9.3-10.5. At our operational pH8.0, BSA existed with a negative charge and CL-g-PEAA existed as positively charged anion-exchange membranes. The total magnitude of this positive charge of CL-g-PEAA membranes depended on the density of amine groups formed on the CL nanofibers. Thus the role of AN concentration, AN reaction +

time and amination reaction time on the growth of the cationic group (-NH3 ) was studied by evaluating the BSA separation performance. The BSA separation behavior of the CL-g-PEAA anion-exchange membrane adsorber prepared with three different AN concentrations and various reaction times were shown in Fig. 4. The adsorption performance measured under the same experimental conditions revealed that pure CL substrate and intermediate CL-g-PAN respectively have a BSA binding of 9 mg/g and 14 mg/g. As could be seen, the BSA adsorption of CL-g-PEAA anion-exchange membranes increased up to 6h of AN reaction time for a particular concentration. Among the three CL-g-PEAA anion-exchange -3

membranes in Fig.4, membrane adsorber prepared from AN concentration of 9.4x10 M showed a peak adsorption performance (170 mg/g) with 6h of reaction. This increase in adsorption performance indicated +

the uniform growth of cationic tentacles/anion-exchange sites (-NH3 ) along the CL nanofibers with increase in AN grafting. However, a further increase in AN concentration and reaction time did not contribute to any increase in SPBC, indicating the non-uniform growth and overlapping of active sites resulted in poor electrostatic interaction with BSA.

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Fig. 4. Static BSA adsorption performance with the respect to CL-g-PAN intermediate step reaction time for CL-g-PEAA anion-exchange membranes fabricated from three different AN concentrations. In the CLg-PEAA preparation step, a DETA concentration of 3.1 M with reaction time of 2 h was used. BSA in Tris buffer (2g/L, pH-8.0) was used for evaluating the adsorption efficiency. The BSA concentrations before and after binding experiments were recorded to estimate the adsorption efficiency. The SPBC of CL-g-PEAA anion-exchange membranes were then studied by changing the amination reaction time for four different AN concentrations and the results were shown in Fig. 5. The CL-3

g-PEAA anion-exchange membranes synthesized with AN concentration of 9.4x10

-3

and 18.8x10

M,

respectively, have a SPBC of 239 and 210 mg/g of nanofibers for 1.5 h of amination reaction. It is important to note that contrary to our expectation, doubling the AN concentration did not provide a corresponding increase in SPBC. The SPBC of anion exchange membrane adsorbers prepared with AN -3

-3

concentration of 28.2x10 M is even lower than membranes prepared from 4.7x10 M AN concentration. Also, when we proceeded with amination reactions beyond 1.5 h, a decrease in SPBC was observed. This decrease in binding efficiency as a function of an increase in AN concentration and amination time was due to the large number of amine groups grafted on to CL nanofibers, which caused considerable steric hindrance to the approaching protein molecules. Also, due to the non-uniform distribution and overlapping of adjacent –NH2 groups, all binding sites formed on the CL nanofibers were not accessible -3

-3

to BSA molecules. Thus, it is important to note that an AN concentrations of 9.44x10 /18.8x10 M with a reaction time of 6h and subsequent amination reaction of 1.5 h in the second step resulted in the

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Fig. 5. Static BSA binding as a function of DETA functionalization time for CL-g-PEAA anion-exchange membrane adsorbers fabricated with various AN concentrations. AN reaction time of 6h was maintained constant in the CL-g-PAN intermediate step. An DETA concentrations of 3.1 M was used for the synthesis of CL-g-PEAA preparation step. BSA in Tris buffer (2g/L, pH-8.0) was used for evaluating the adsorption efficiency. The BSA concentrations before and after binding experiments were recorded to estimate the adsorption efficiency. formation of CL-g-PEAA membranes with optimum binding capacity. Fig. S7 and Fig. S8 represent the effect of time of adsorption experiments and initial BSA concentration on the adsorption respectively. Kinetic parameters and adsorption isotherms parameters are provided in Table S2 and S3 respectively. From these evaluations, it could be conclude that physical interactions and monolayer adsorption are the mechanisms facilitating highly efficient BSA adsorption in the CL-g-PEAA anion-exchange membranes. As a comparative study, we measured the SPBC of pure electrospun PAN nanofibers functionalized with 3.3 M DETA for a reaction time of 1 h and it showed a SPBC of 83 mg/g of nanofibers (Fig. S9a). The BSA adsorption capacity of CL-g-PEAA membranes was thus three times higher than the amidoamine functionalized PAN nanofiber membranes. In addition, SPBC’s values obtained for the CL-gPEAA anion-exchange membrane adsorbers in this study are six times higher than the cellulose nanofibers modified with diethylaminoethyl (DEAE)

14

. This higher BSA binding capacity of CL-g-PEAA

anion-exchange membranes may be due to the more efficient functionalization, uniform spatial +

distribution of –NH3 groups and lower overall density, higher resulting hydrophilic/hydrophobic balance and thus more specific interaction with biological molecules.

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3.4. Dynamic protein binding capacity (DPBC) Dynamic protein adsorption performance of CL-g-PEAA anion-exchange membranes were then carried out to evaluate the adsorption performance and anion-exchange site utilization efficiency with relatively short residence times within flowing operations. The schematic representation of the packing of nanofiber membranes as a bed for the DPBC studies is shown in scheme 1. Dynamic BSA capacities (on a membrane mass basis) at 10% breakthrough of the CL-g-PEAA anion-exchange membranes synthesized with four different AN concentrations for three different flow rates are shown in Fig. 6a.The corresponding binding capacities based on volume of membrane are given in Fig. S10. The 10% DPBC -3

capacity increased linearly with an increase in AN concentration up to 8.8x10 M, and then it decreased significantly. At a flow rate of 1.0 mL/min (which is equivalent to an approximate residence time of less than 8s), these CL-g-PEAA anion-exchange membrane adsorber synthesized from intermediate step AN -3

concentration of 18.8x10 M had a maximum DPBC value of 69 mg/g which is almost 35% of the SPBC reported in Fig. 5. This adsorption behavior illustrated that interaction between the active sites in the CL-3

g-PEAA membrane adsorber and BSA molecules was hindered beyond 18.8x10 M AN concentration. The slight difference in DPBC values calculated at different flow rates substantiated the minimal mass transfer resistance for BSA in reaching the functional regions of the CL-g-PEAA anion-exchange membranes. The significant difference in SPBC and DPBC of CL-g-PEAA anion-exchange membranes could be explained by the small membrane column used in our DPBC studies (membrane diameter-25 mm, column volume of 0.09-0.14 mL and column height of 0.3-0.4 mm). Considering the short residence time of less than 4 s at 2.0 mL/min flow rate for CL-g-PEAA anion-exchange membranes, BSA molecules passes through membranes column rapidly during flow through operation leading to a minimal contact time between BSA molecules and -NH3

+

functional groups. Under similar experimental conditions,

amidoamine functionalized PAN nanofiber membrane adsorbers with a 1h treatment showed a DPBC of 27 mg/g (Fig. S8b). Though the mass based DPBCs of CL-g-PEAA membranes are two times higher than the amidoamine functionalized PAN membranes, the volume based capacities were almost similar (Fig. S9b and S10). The lower density (mass per volume) of the CL-g-PEAA membrane adsorber thus offers a significant advantage when packed as a bed for the chromatographic separations. Since the DPBC of volume based CL-g-PEAA membranes was two times higher than mass based DPBC, same amount of

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membrane material can be packed either as much larger column or in to multiple columns. Thus the CLg-PEAA membranes enhance the economic efficiency of the chromatographic purification process by minimizing the cost involved in the material preparation and subsequent ion-exchange membrane modification.

Fig. 6. (a) Mass based BSA binding capacity in dynamic mode at three different flow rates for CL-g-PEAA anion-exchange adsorbers fabricated with different AN concentrations in the intermediate step. CL-g-PAN intermediate was prepared with AN reaction time of 6h. For the final step of CL-g-PEAA preparation DETA concentration of 3.1 M with a treatment time of 1.5 h was used. b) A distinctive AKTA pure chromatogram obtained at a flow rate of 0.5mL/min for the CL-g-PEAA anion-exchange membranes -3 synthesized with AN concentration of 18.8x10 M in the intermediate step (only variations in absorbance and conductivity values are displayed). In this chromatogram, step (1) was used to wash the system, step (2) for BSA adsorption, step (3) to remove the loosely bound BSA molecules by buffer washing, step (4) for the recovery of bound BSA using elution buffer and finally step (5) was used for the final washing. For the binding and washing experiments 1g/L BSA and 1M NaCl solutions in Tris buffer were used respectively (both maintained at pH-8.0). The AKTA pure chromatogram generated at a flow rate of 0.5 mL/min for CL-g-PEAA anion-3

exchange membranes synthesized from AN concentration of 18.8x10

M are shown in Fig. 6b (only

variations in absorbance and conductivity values are displayed). As seen in the chromatogram, approximately 4 mL of BSA solution adsorbed reversibly to the CL-g-PEAA membrane in step 2. Elution of this bound protein in step 4 resulted in a sharp peak and more than 85% of these bound proteins could be collected in a volume fraction of less than 3 mL. The five steps showed in the chromatogram clearly illustrate the reversible nature of the binding of BSA to the CL-g-PEAA and its reusability in protein purification applications. The adsorptive binding performance of CL-g-PEAA anion-exchange adsorbers was compared with commercial membranes and other macroporous/nanofiber membranes recently reported in the literature (Table S4). According to the manufacturer data, Sartobind D and Q membranes

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have DPBC of 22 mg/mL and 29 mg/mL respectively (static binding capacities of 22 mg/mL). Comparison of these data revealed that CL-g-PEAA membrane adsorption performance is higher than the commercial Sartobind D and Q membranes. Comparing the performance of the ATRP modified macroporous membranes, our membranes performance may not appear to be as good. However, ATRP procedures are hard to use in a large scale production unit and does not have any scope beyond lab scale production. Also, considering the significant variation in static (240 mg/g) and dynamic (69mg/g) adsorption capacities of the anion-exchange membranes in the present study, in the future it is likely to improve the dynamic adsorption capacity by efficient process design studies such as use of large column, high packing density and creation of tortuous permeation path for protein molecules within the chromatography column. 3.5. Size-based separation efficiency of CL-g-PEAA membranes In order evaluate the feasibility of CL-g-PEAA membrane adsorbers for the removal of viruses, cellular debris and other larger molecules by size exclusion, the separation efficiency of model bead particles of various sizes (200, 100 and 40 nm) was studied. Under gravitational feed pressure, CL-g-3

PEAA anion-exchange membranes synthesized from a AN concentration of 18.8x10

M with DETA

-2 -1

functionalization of 1.5 h showed a pure water flux of 460 Lm h . The solution flux and percentage rejection behavior of the AEX membrane adsorbers for 100 mL of bead solutions fed through the membranes are provided in Fig. 7 (a&b). From the Fig. 7a, it was evident that solution flux decreased for all bead particles upon increasing the volume of solution fed/increasing the filtration time. The 200 nm bead particles maintained 99.5% rejection throughout the filtration of 100 mL of feed solution. However, for 100 and 40 nm beads particles, percentage rejections decreased from 99.5 to 94.5%, and 88.2 to 78.8%, respectively, at the end of 100 mL of solution fed. The CL-g-PEAA membranes prepared with other AN concentrations followed the same trend (Fig. S11). It is important to note that at the beginning of the filtration of all bead particles the flux of the solutions decreased rapidly, and then the decrease in solution flux observed was comparatively slower. It is important to note that in the initial stage of filtration solution flux for all bead solutions decreased significantly, and during the later stages a steady decrease was observed. Whereas, the rejection behavior followed an opposite trend with a steady values initially

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and a substantial decrease after 75 mL of feed volume. This separation behavior of CL-g-PEAA membranes could be explained by the pore-plugging and cake-formation theory. The insignificant flux reduction after a certain filtration time is due to the stabilization of the additional filtration resistance contributed by the cake layer

44

.

Fig. 7. (a) Permeate solution fluxes and (b) percentage rejection behavior of CL-g-PEAA anion-exchange membrane adsorbers throughout the filtration of 200, 100 and 40 nm polystyrene microspheres for 100 mL of solution. Aqueous bead solutions used as the feed have initial concentrations of 0.5 g/L. Solutions were flowed through the membranes under gravitational pressure effect. CL-g-PEAA anion-exchange -3 membrane adsorbers fabricated with AN concentration of 18.8x10 M (reaction time of 6h) and DETA concentration of 3.1 M (treatment time of 1.5 h) was used for filtration experiments. UV-Vis spectrophotometer was used to measure the bead solution concentration during the filtration experiments. During filtration, particles larger than the pore size of membrane deposited on the surface and form a cake layer. Particles with sizes comparable to the membrane pore size first infiltrate partially in to the pores and then form a cake layer. When we compare the morphologies of pure CL (Fig. 1a1) and CLg-PEAA membranes (Fig. S4a&b), it should be noted that separation performance was inconsistent with the pore size of the membranes. Pure CL nanofibers had pore sizes greater than 2 µm did not reject any of these bead particles. However, the CL-g-PEAA membranes, the effective pore sizes were significantly reduced by uniform coating of amidoamine groups along the nanofibers which could completely reject 200 and 100 nm particles and 80% of 40 nm particles. Since pore sizes of the CL-g-PEAA membranes were above 0.2 µm (Fig. S4a&b), rejection of smaller particles could be explained by the interconnected pore structure and depth filtration mechanism of CL-g-PEAA nanofiber membranes. It is likely that the smaller pores responsible for the majority of the rejection became clogged/constricted, which allowed more flow through the larger open pores that could not reject the smaller particles. The decrease in

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rejection of 40 nm bead particles after 75 mL of feed volume may be due to the defects in the nanofiber membranes, which could be mitigated by layering the membrane sheets during filtration. The rejection results indicated the potential use of CL-g-PEAA AEX nanofiber membrane adsobers in clearance of viruses (influenza>100 nm, retro>100 nm and parvovirus >20 nm) DNA (radius of gyration > 50 nm), and protein aggregates (> 50 nm) by size exclusion

11, 30, 51

.

4. Conclusions In this study, we have developed a novel CL-g-PEAA nanofiber based anion-exchange membrane adsorbers that could perform separation both by ion-exchange and size exclusion. CL-gPEAA membranes were fabricated by a multi-step process. Acrylonitrile was first polymerized and grafted on to CL nanofibers by redox polymerization and subsequently grafted nitrile group was converted to amine by reaction with DETA. Structural characterization illustrated the three dimensional coating of amidoamine functionally along the individual nanofibers. Ion-exchange separation efficiency of the CL-gPEAA anion-exchange membranes were evaluated in flow through mode using BSA as model protein. At 10% breakthrough these membranes displayed a DPBC of 69 mg/g (corresponding to a volume based binding capacity of 31 g/L), with an approximate residence time of 8 s. This BSA adsorption performance of non-optimized CL-g-PEAA anion-exchange adsorbers was higher than Sartobind D and comparable to Sartobind Q commercial membranes. Also, CL-g-PEAA membranes have showed 80-100% rejection for model bead particles of various sizes (200, 100 and 40 nm), which illustrated the efficiency in the separation of viruses, DNA, host cell proteins, and other higher molecular weight aggregates from the harvested cell culture fluid (HCCF) during downstream processing. Overall performance of the CL-gPEAA membranes illustrated that undesired impurities (viruses, host cell proteins, and DNA) in HCCF could be removed in a single step by an anion-exchange membranes with ion-exchange and size exclusion based separation mechanism. Considering the feasible synthesis process employed, remarkable separation efficiency and recyclability, CL-g-PEAA anion-exchange membranes developed in this study offered a unique platform for the ion-exchange separations of biomolecules by chromatography and other ion-exchange based separation processes. Promising results of this study should pave the

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way for broader implementation of anion-exchange membranes in the polishing stage of downstream processing. Acknowledgements The authors would like to thank the financial support provided by the US Air Force Civil Engineering Center under Contract Number FA4819-14-C-0004. The authors would also like to acknowledge Professor Hao Fong and his research group for their help with the electrospinning process. Supplementary Content Chemical route of synthesis of CL-g-PEAA anion-exchange membranes (Scheme S1), CL-g-PAN reaction mechanism (Scheme S2), CL-g-PEAA reaction mechanism (Scheme S3), SEM images of CL-gPAN adsorbers fabricated from various AN concentrations (Figure S1 a-f), SEM images of CL-g-PEAA adsorbers fabricated from various AN reaction time (Figure S2 a-d), Graft Yield in the grafting AN on to CL nanofibers (Figure S3 a&b), SEM images of CL-g-PEAA membranes prepared with various DETA reaction time (Figure S4 a-d), SEM images of PAN membranes prepared with various DETA reaction time (Figure S5 a-d), ATR FT-IR spectra of amidoamine functionalized PAN (Figure S6), XPS Chemical composition (Table S1), Static binding Vs Time (Figure S7), Kinetic parameters (Table S2), Static binding Vs initial BSA concentration (Figure S8), Isotherm constants (Table S3), Static and dynamic BSA adsorption capacity of amidoamine functionalized PAN (Figure S9), Dynamic volume based BSA adsorption capacity of CL-g-PEAA (Figure S10), solution flux and rejection of CL-g-PEAA membranes and comparative study of the performance AEX membrane adsorbers in this study with recently reported new anion-exchange membranes in the literature (Table S4). This material can be found in the online version. References 1. Jungbauer, A., Continuous Downstream Processing of Biopharmaceuticals. Trends Biotechnol. 2013, 31, 479-492. 2. Cramer, S. M.; Holstein, M. A., Downstream Bioprocessing: Recent Advances and Future Promise. Curr. Opin. Chem. Engg. 2011, 1, 27-37. 3. Goodman, M., Market watch: Sales of Biologics to Show Robust Growth Through to 2013. Nat. Rev. Drug Discovery 2009, 8, 837-837. 4. Spears, R., Overview of Downstream Processing. In Biotechnology, Wiley-VCH Verlag GmbH: 2008; pp 39-55. 5. Liu, H. F.; Ma, J.; Winter, C.; Bayer, R., Recovery and Purification Process Development for Monoclonal Antibody Production. mAbs 2010, 2, 480-499. 6. Schneiderman, S.; Zhang, L.; Fong, H.; Menkhaus, T. J., Surface-Functionalized Electrospun Carbon Nanofiber Mats as an Innovative Type of Protein Adsorption/Purification Medium with High Capacity and High Throughput. J. Chromatogr. A 2011, 1218, 8989-8995. 7. Yigzaw, Y.; Piper, R.; Tran, M.; Shukla, A. A., Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Monoclonal Antibody Purification. Biotechnol. Prog. 2006, 22, 288-296. 8. Follman, D. K.; Fahrner, R. L., Factorial Screening of Antibody Purification Processes Using Three Chromatography Steps Without Protein A. J. Chromatogr. A 2004, 1024, 79-85. 9. Shukla, A. A.; Thömmes, J., Recent Advances in Large-Scale Production of Monoclonal Antibodies and Related Proteins. Trends Biotechnol. 2010, 28, 253-261. 10. Liu, Z.; Wickramasinghe, S. R.; Qian, X., Membrane Chromatography for Protein Purifications from Ligand Design to Functionalization. Sep. Sci. Technol. 2017, 52, 299-319. 11. van Reis, R.; Zydney, A., Bioprocess Membrane Technology. J Membrane Sci 2007, 297, 16-50.

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12. Chen, J.; Tetrault, J.; Zhang, Y.; Wasserman, A.; Conley, G.; DiLeo, M.; Haimes, E.; Nixon, A. E.; Ley, A., The Distinctive Separation Attributes of Mixed-Mode Resins and Their Application in Monoclonal Antibody Downstream Purification Process. J. Chromatogr. A 2010, 1217, 216-224. 13. Johansson, B.-L.; Belew, M.; Eriksson, S.; Glad, G.; Lind, O.; Maloisel, J.-L.; Norrman, N., Preparation and Characterization of Prototypes for Multi-Modal Separation Media Aimed for Capture of Negatively Charged Biomolecules at High Salt Conditions. J. Chromatogr. A 2003, 1016, 21-33. 14. Zhang, L.; Menkhaus, T. J.; Fong, H., Fabrication and Bioseparation Studies of Adsorptive Membranes/Felts Made from Electrospun Cellulose Acetate Nanofibers. J. Membr. Sci. 2008, 319, 176184. 15. Rajesh, S.; Schneiderman, S.; Crandall, C.; Fong, H.; Menkhaus, T. J., Synthesis of Cellulosegraft-Polypropionic Acid Nanofiber Cation-Exchange Membrane Adsorbers for High-Efficiency Separations. Acs Appl. Mater. Interfaces 2017, 9, 41055-41065. 16. Bhut, B. V.; Wickramasinghe, S. R.; Husson, S. M., Preparation of High-Capacity, Weak AnionExchange Membranes for Protein Separations Using Surface-Initiated Atom Transfer Radical Polymerization. J. Membr. Sci. 2008, 325, 176-183. 17. Bhut, B. V.; Weaver, J.; Carter, A. R.; Wickramasinghe, S. R.; Husson, S. M., The Role of Polymer Nanolayer Architecture on the Separation Performance of Anion-Exchange Membrane Adsorbers: I. Protein Separations. Biotechnol. Bioeng. 2011, 108, 2645-2653. 18. Hardick, O.; Dods, S.; Stevens, B.; Bracewell, D. G., Nanofiber Adsorbents for High Productivity Downstream Processing. Biotechnol. Bioeng. 2013, 110, 1119-1128. 19. Dods, S. R.; Hardick, O.; Stevens, B.; Bracewell, D. G., Fabricating Electrospun Cellulose Nanofibre Adsorbents for Ion-Exchange Chromatography. J. Chromatogr. A 2015, 1376, 74-83. 20. Rajesh, S.; Zhao, Y.; Fong, H.; Menkhaus, T. J., Polyacrylonitrile Nanofiber Membranes Modified with Ionically Crosslinked Polyelectrolyte Multilayers for the Separation of Ionic Impurities. Nanoscale 2016, 8, 18376-18389. 21. Xiao, M.; Chery, J.; Frey, M. W., Functionalization of Electrospun Poly(vinyl alcohol) (PVA) Nanofiber Membranes for Selective Chemical Capture. Acs Appl. Nano Materials 2018, 1, 722-729. 22. Xu, G.; Zhao, Y.; Hou, L.; Cao, J.; Tao, M.; Zhang, W., A Recyclable Phosphinic Acid Functionalized Polyacrylonitrile Fiber for Selective and Efficient Removal of Hg2+. Chem. Eng. J. 2017, 325, 533-543. 23. Rajesh, S.; Zhao, Y.; Fong, H.; Menkhaus, T. J., Nanofiber Multilayer Membranes with Tailored Nanochannels Prepared by Molecular Layer-by-Layer Assembly for High Throughput Separation. J Mater Chem A 2017, 5, 4616-4628. 24. Yu, X.; Shen, L.; Zhu, Y.; Li, X.; Yang, Y.; Wang, X.; Zhu, M.; Hsiao, B. S., High Performance Thin-Film Nanofibrous Composite Hemodialysis Membranes with Efficient Middle-Molecule Uremic Toxin Removal. J. Membr. Sci. 2017, 523, 173-184. 25. Feng, Q.; Hou, D.; Zhao, Y.; Xu, T.; Menkhaus, T. J.; Fong, H., Electrospun Regenerated Cellulose Nanofibrous Membranes Surface-Grafted with Polymer Chains/Brushes via the Atom Transfer Radical Polymerization Method for Catalase Immobilization. Acs Appl. Mater. Interfaces 2014, 6, 2095820967. 26. Xu, T.; Miszuk, J. M.; Zhao, Y.; Sun, H.; Fong, H., Electrospun Polycaprolactone 3D Nanofibrous Scaffold with Interconnected and Hierarchically Structured Pores for Bone Tissue Engineering. Adv. Healthcare Mater. 2015, 4, 2238-2246. 27. Menkhaus, T. J.; Varadaraju, H.; Zhang, L.; Schneiderman, S.; Bjustrom, S.; Liu, L.; Fong, H., Electrospun Nanofiber Membranes Surface Functionalized with 3-Dimensional Nanolayers as an Innovative Adsorption Medium with Ultra-High Capacity and Throughput. Chem. Commun. 2010, 46, 3720-3722. 28. Hardick, O.; Dods, S.; Stevens, B.; Bracewell, D. G., Nanofiber Adsorbents for High Productivity Continuous Downstream Processing. J. Biotechnol. 2015, 213, 74-82. 29. Fu, Q.; Wang, X.; Si, Y.; Liu, L.; Yu, J.; Ding, B., Scalable Fabrication of Electrospun Nanofibrous Membranes Functionalized with Citric Acid for High-Performance Protein Adsorption. Acs Appl. Mater. Interfaces 2016, 8, 11819-11829. 30. Weaver, J.; Husson, S. M.; Murphy, L.; Wickramasinghe, S. R., Anion Exchange Membrane Adsorbers for Flow-Through Polishing Steps: Part II. Virus, Host Cell Protein, DNA Clearance, and Antibody Recovery. Biotechnol. Bioeng. 2013, 110 (2), 500-510.

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