Highly Hydrophilic Ultraltration Membranes Synthesized From Acrylic Acid Grafted Polyethersulfone For Downstream Processing Of Therapeutic Insulin And Cobalamin

The signicance of the present study is to purify small therapeutic biomolecules such as urea, cobalamin, and insulin of molecular weights below 10 kDa through surface grafted polyethersulfone (PES) ultraltration (UF) membranes. The membranes were synthesized by adding an additive of 6 kDa polyethylene glycol (PEG) and grafted with acrylic acid (AA) by varying the concentrations from 2 to 6 wt. % under UV-induce phtocataltic reaction. These membranes were characterized by various tools such as Scanning electron microscope ((SEM), Fourier transforms infrared spectra (FTIR), thermogravimetric analysis (TGA), and contact angle for membrane morphological, structural, thermal stability, and hydrophilicity, respectively. The degree of grafting and their MWCOs of the indigenous membranes were analyzed using various molecular weight solutions of PEG. After PEG doping, the PWF of the membrane was enhanced to 41.50 L m − 2 h − 1 for PES [6+] [0], and a similar trend was also observed for the PEG doped PES grafted membranes. From the experimental results, the synthesized membranes of additive loaded with 5 and 6 wt. % AA grafted PES reject 90 % of the insulin and cobalamin. The results are found to be in correlation with the MWCO values of these membranes ranging from 1 to 10 kDa. From the overall characterization and experimental observations, it can be conrmed that these indigenously synthesized at sheet grafted membranes showed excellent permeabilities and higher % rejection towards the therapeutic biomolecules.


Introduction
Biomolecule puri cation is an important research frontier today, as the world market of proteins and their production is growing tremendously. For effective puri cation of the biomolecules, the most e cient separation techniques have to be focused. An ideal biomolecule puri cation strategy is achieved in few steps through easy and low-cost separation techniques, which have a high demand for mass production [1] [2]. The membranes used in the separation process can conduct without chemical treatment at an ambient condition where the membrane act as a selective sieving barrier. Hence, membrane ltration is used to separate biological components such as amino and organic acids, salts, proteins, sugars, peptides, and vitamins, growing in the biotechnological industries [3]. Ultra ltration (UF) is a separation process extensively used as a preferred technique and an alternative to size exclusion chromatography for protein concentration and buffer exchange [4]. UF membranes have a pore size in the range of 10-100 nm, especially for high retention of macromolecules and protein [5].
UF membranes can be prepared from various synthetic polymers, have high thermal stability, chemical resistivity, and restrict somewhat harsh cleaning chemicals [6]. Polyethersulfone (PES) is a widely used UF membrane material because of its high rigidity, creep resistance, moderate hydrophilicity, good thermal and dimensional stabilities [7]. The choice of the membrane is usually guided by its molecular weight cut-off (MWCO), which de nes the capability of the membrane where it could reject 90 % of the equivalent molecular size of the protein. PES-UF membranes, either hollow ber or at sheet, were extensively studied to separate proteins [8-10].
Various researchers have been synthesizing the polymeric membranes by adding additives to enhance the process e ciency of UF membranes. Li et al., 2014 [11] prepared blend membranes of PPTA and poly(vinylidene uoride) (PVDF) by in situ polycondensation using various additives such as PEG, inorganic salt of lithium chloride (LiCl), and Tween-80 surfactant were additives for the adequate performance of the membrane. Otitoju et al. [12] reviewed the blending of PES with different additives such as polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), PEG, and inorganic nanoparticles to improve the hydrophilicity and performance in terms of ux, solute rejection, and reduction of fouling. As per Rata et al., [13] polysulfone (PSf) and cellulose acetate UF membranes (UF1-UF36) were prepared by phase inversion method using 1000, 2000, and 4000 Da PEG additives for separation of whey protein. Lin et al. [14] synthesized a novel UF membrane by blending brominated poly(phenylene oxide) (BPPO) with its additive of quaternary phosphonium derivative (TPPOQP-Br) through the phase inversion method for suitable rejection property with high water ux.
On the other hand, the surface modi cation of the membranes plays a vital role in the separation process, especially for developing the selective separation of the solute molecules through molecular sieving [13]. Akbari et al. [15] developed NF membranes from PSf UF membrane surface modi cation with sodium pstyrene sulfonate (NaSS) and [2-(acryloyloxy)ethyl]trimethyl ammonium chloride (AC) by sensitive UVphoto induced grafting technique. These membranes were successfully removed six different dyes according to their charge and produced reused water in the process house. Shi et al.
The present study mainly focuses on the puri cation of semi-synthetic small therapeutic biomolecules of size below 10 kDa by UF membranes. There are several chromatographic methods in literature to purify such molecules. However, these methods have two limitations, i.e., expensive nature and di culty in scale-up the process. Therefore, this paper addresses the preparation of PEG additive added and AA grafted synthetic PES-UF membranes to purify small biomolecules below 10kDa. These porous UF membranes were synthesized using PES polymer with desired modi cation by adding PEG 6 kDa additive and grafted with various concentrations of AA (2 to 6%) on the membrane surface. These indigenous membranes were characterized by FTIR, SEM, TGA, and contact angle before and after membrane modi cation. The degree of grafting and MWCOs of various concentrations of AA grafted PES membranes were determined in terms of pure water ux (PWF). These membranes were applied to the separation of various biomolecules such as Urea, cobalamin, and Insulin, based on their size exclusion.
The separation speci city and improved biocompatibility of these membranes are discussed in this manuscript.

Materials
The polymer PES was procured from Solvay, Vadodara, India, to synthesize at sheet membranes. The solvent N-methyl-2-pyrrolidone (NMP), a crystalline compound of bismuth subnitrate (BiONO 3 ) and hydrochloric acid (HCl) with 35-38 % concentration, were purchased from Sd Fine Chemicals, Mumbai, India. The molecular weight cut-off of the synthesized membrane was determined using PEG with different molecular weights, i.e., 600, 1000, 2000, 4000, 6000, 10,000, and 20,000, along with AA were supplied from Sigma-Aldrich Chemical Private Limited, USA. Urea, a therapeutic biomolecule, was purchased from LOBA Chemie Pvt Ltd., Mumbai, India. Analytical grade purity of potassium iodide (KI) and sodium hydroxide (NaOH) purchased from Molychem, Mumbai, India provided. Human mixtard and Cobalamin insulin injections were purchased from NOVO Nordisk India Pvt Ltd., Bangalore, and Mankind Pharma Ltd., New Delhi, India. The deionized water with TDS < 2 ppm was used for sample preparation and experimental studies in the laboratory using the RO membrane cascade system.

Synthesis of at sheet membranes
The UF at sheet membranes were synthesized and analyzed for their pore size and MWCOs. The method used in this study for the fabrication of the membrane was by phase inversion. Initially, the honey-like viscous polymer solution was prepared by dissolving 17 % of PES in 83 wt % of the NMP solvent to synthesize the pristine membrane. On the other hand, 8 wt % 6,000 Da molecular weight of PEG solution was prepared using NMP solvent and stirred continuously for 30 mins at 900 rounds per minute (rpm) under ambient conditions until it dissolves completely. Then the desired amount of polymer was added into that PEG solution was provide in Table 1. The obtained solution was stirred for another 2 h at 45 ± 2 ºC using a magnetic stirrer to get the homogeneous solution, further degassed to make it bubble-free. The obtained polymer solution was cast on polyester (PE) non-woven fabric support using a doctor's blade to the desired thickness. While casting the solution, the air gap between the blade and the support fabric was maintained at such a distance to get the resultant membrane of 60 µm polymeric coating, measured using a micron gauge. After casting the membrane on the support, it was transferred immediately into a non-solvent bath, where type-II water was used as non-solvent and kept for 30 min to obtain desired porous membrane. Initially, 6 wt % of AA, a week acid considered a monomer, was selected to grafting the membrane. After the synthesis of membranes through phase inversion, washed thoroughly with type-II water to remove excess residual solvents from the matrix. These membranes were subjected to grafting by immersing them into an AA solution under 265 nm UV light at 50 cm distance for about 40 min as the monomer gets attached to the PES polymer and forms polyacrylic acid around the pores, which leads to a decrease in the membrane pore size. The reaction mechanism of UV-induced AA graft polymerization at the pores of the PES support was provided in Scheme 1. The prepared membranes were denoted in the order of PES and used additive PEG to prepare the membrane represented as the molecular weight (kDa), followed by the percentage of AA used for grafting. For example, the membrane synthesized by PES polymer solution with additive PEG 2 kDa and grafted with 6 % AA represented as PES [2+] [6]. The combinations of PES membrane with PEG additive and AA grafting polymers in terms of weight percentage used in this present study were provided in Table 1.

Preparation of Standard solution of insulin
The insulin stock solution of 1000 ppm was prepared by dissolving The experimental laboratory setup for a UF system using at-sheet membranes is shown in Fig. 1. The system consists of a 1 L capacity feed connected to a at-sheet membrane module with a membrane surface area of 90 cm 2 through 300 gallons per day (GPD) pump. The module another has an exit of permeate and reject streams, where the reject line is directly connected to the feed tank, and the permeate was collected into permeate tank. Pressure gauge and control valves were xed at the reject line to measure the applied pressure on the membrane module, and the control valve was used to restrict the ow in the system.
Initially, the feed container was lled with 500ml of PEG solution of desired molecular weight and passed across the membrane under 30 psi feed pressure in a cross-ow manner. Before collecting the nal permeate, the system permeates and rejects streams recycled for about 30 min to attain a steady state. The feed and permeate samples were then collected every 5 min time intervals to analyze the samples.
The UV-VIS spectrophotometer was used to analyze the concentrations of the samples over the standard calibration curve. The same procedure was followed for all the membranes to nd MWCO. The degree of grafting is used calculated the amount of AA grafted on the membrane surface and is estimated as the following equation; where W 1 and W 2 are the weight of the samples before and after grating the PES membrane, respectively.

Pure Water Flux (PWF)
The volumetric ow rate of the permeate is measured as a function of time during the UF process. The permeate ux is estimated by accounting for the volumetric ow rate per unit effective area of the membrane, as shown in Eq. (2).
Where permeate volumetric owrate Q and Membrane effective area A

Percentage Rejection
Percentage rejection is one of the essential parameters that de ne the potentiality of the membrane in particle retention. The percentage of rejection is calculated from Eq. (3).
Where C P and C F the solute concentrations of permeate and feed, respectively.

SEM studies
Morphological studies of the PES membrane before and after adding additive and surface grafting were shown in Fig. 2. The pristine PES membrane without grafting and without any additive resulted in uneven distribution of pores throughout the surface (Fig. 2a). After adding the PEG 6000 additive to pristine PES, the pore density was increased from 0.86 (pristine) to 2.45 µm − 2 on the surface with uniform distribution throughout the membrane can be seen from Fig. 2b. Various percentages of AA, i.e., 2, 4, and 6 % grafted on (8 % PEG 6000) additive added PES substrates, and surface pores were cured by crosslinking of AA monomer at the pores, which can be clearly seen from Figs. 2c, d, and e. The crosslinking mechanism at the pores was provided in Scheme 1 and further con rmed from FTIR in the following section. This observation increases membrane performance in the separation process by increasing the pore density on the surface PEG and shrinking these pores by AA grafting, which helps develop different MWCO membranes.

FTIR studies
The FTIR spectra of the pristine PES and modi ed with acrylic acid surface functional groups can be observed from Fig. 3 (a and b). From Fig. 3a

Contact angle
The contact angle of the pristine, PEG additive loaded and various concentrations of AA grafted PEG loaded PES membranes were measure and provided in Table 2. From

Thermo Gravimetric Analysis (TGA)
The TGA analysis of the various compositions of at sheet PES membranes was illustrated in Fig. 4 Fig. 4., it can be clearly observed that as the degree of grafting increases the derivative, second weight loss increases, which is possibly due to the degradation of grafted polyacrylic acid (PAA) [29]. From TGA analysis, it can be concluded that the PAA deposition on the PES porous substrate increased with the grafting solution concentration and also supports the structural elucidation of FTIR.

Effect of AA concentration on the degree of grafting
The AA concentration in the grafting bath was varied from 2 to 6 % to understand the trade-off. All the membranes were grafted under the same operational condition, i.e., UV light working distance and grafting period. From Fig. 5., it is observed that the percentage of AA monomer increased from 2 to 6 %, resulting in the grafting percentage increased from 12.4 % to 17.8 %. The reason is possibly due to the availability of monomer units increased on the membrane surface, which is further con rmed from TGA analysis, where the additional weight loss of the grafted lms, i.e., PES[6+] [2] to PES[6+][6] is corresponding to the grafted PAA.

Calibration curve for PEG concentration determination
The calibration curve was generated using various prepared known concentrations of 20, 40, 60, 80, and 100 ppm of PEG 1000 Da solution; spectra were recorded in the range of 300 to 700 nm wavelength. The maximum wavelength was observed at 465 nm for PEG 1000 Da, and the absorbance values are linearly increasing with PEG concentration, shown in Fig. 6a. The rst-order linear regression is tted to recorded absorbance values at the maximum wavelength of 465 nm to establish the co-relation between PEG concentration and its corresponding absorbance, as shown in Fig. 6b Various grafted membranes prepared in this study were tested for separation performance of small biomolecules of urea, cobalamin (vitamin B12), and insulin. Figure 9 represents the permeability data of urea, cobalamin, and insulin solutions of 50ppm concentration that was passed through PES[6+] [2]  Similarly, the permeability of cobalamin and insulin was decreased from 27 to 12 L h − 1 m − 2 bar − 1 and from 22 to 4 L h − 1 m − 2 bar − 1 , respectively (Fig. 9). The decreasing permeability trend is due to the size exclusion of biomolecules in urea < cobalamin < insulin.
From Fig. 10 (Fig. 10), the insulin and cobalamin were getting rejected, and the concentration of this molecule in permeate is minimal. Therefore, the insulin and cobalamin permeability and permeate concentrations were decreased with increasing the degree of grafting on the membranes. As per the size exclusion principle, the low molecular weight biomolecule of urea permeates faster through the membrane than other therapeutic molecules. Therefore, the size exclusion of biomolecules is evaluated in terms of the membrane transport phenomenon provided in Fig. 11. From the graphical and transport properties of the grafted membrane, it can be concluded that these membranes could pass urea molecules easily and concentrate the remaining biomolecules in the reject stream. Hence, from the overall characterization and experimental observations, it can be con rmed that these indigenously synthesized at sheet grafted membranes showed excellent permeability for all the biomolecules and applicable for the separation of therapeutic molecules with an MWCO range of 1 to 10 kDa.

Conclusions
In the present study, the PES -UF membranes were prepared by phase inversion method in 6 kDa PEG as a pore-forming agent to enhance the surface pore density. Surface modi cation was performed by AA grafting through UV-induced photo-catalytic reaction. The molecular weight cut-off of the indigenous membranes was controlled using various monomer grafting concentrations. The characterization studies were carried out with various SEM, FTIR, TGA, and contact angle tools, where SEM revealed the role of PEG additive in the dope solution and AA grafting on the membrane surface. Before and after grafting, the functional groups and new bonds formed on the membrane surface were clearly evident from the FTIR studies. The thermal analysis of the grafted membranes exhibited an additional weight loss of PAA, supporting the structural analysis. The trade-offs between the grafting solution concentration with a degree of grafting and corresponding MWCOs of the prepared membranes were established. From the experimental results, the PWF of the grafted membranes was signi cantly enhanced in the presence of the additive (PES [6+] [6]) than the pristine grafted membrane (PES [0] [6]). The urea permeability for all the membranes was found to be similar, whereas the retention of cobalamin and insulin was maximum in the case of PES[6+][6] and PES[6+] [5]. All the essential peptides and proteins present in human blood above 2kDa will be retained. Only uremic toxins, which are less than 1 kDa, are removed with these membranes. Therefore, from the results, it can be concluded that PES[6+][6] and PES[6+] [5] membranes are helpful to remove uremic toxins for renal patients instead of tedious dialysis procedure by surgically implanting this membrane cassette into the chronic renal patients so that the permeate is attached to the urinary bladder and reject back to the bloodstream, where the uremic toxins are removed continuously. The continuous removal of small uremic toxins will reduce these small toxins binding to signi cant proteins and peptides present in the blood.