Modification of poly(vinylidene fluoride) microfiltration
membrane with surface-copolymerized poly(ethylene glycol)
methacrylate and its applications on protein purification
Kuo Lung Tung*, Yung Chang* , Chia Yu Chen* and Nien Jung Lin*
*R&D Center for Membrane Technology and Department of Chemical Engineering
Chung Yuan University, Chungli 320, Taiwan
(E-mail : kuolun@cycu.edu.tw )
Abstract
In this study, a poly (vinylidene fluoride) (PVDF) microfiltration membrane was chosen to modify and analyze
the protein purification. Two kinds of monomers (3-Sulfopropyl methacrylate potassium salt, SA and poly
(ethylene glycol) methacrylate, PEGMA) were grafted onto a PVDF membrane using three steps: (i)
immersing PVDF membranes in the monomer solutions at different molar ratio regarded as a physical
coating; (ii) dried membranes were modified using low pressure argon plasma treatment under various times;
(iii) using ultrasonic cleaning to wash out non-grafting monomers. Lysozyme and BSA were regarded as
model proteins to analyze adsorption efficiency. Moreover, the experiment operated under different molar
ratios of monomer solution, pH value and ion strength to analyze membrane efficiency. Finally, the results
show the cation-exchange membrane performed well in the separation of the two proteins.
Keywords. Cation-exchange – argon plasma treatment– membrane chromatography – protein adsorption
INTRODUCTION
In biotechnology, membrane separation has already been widely used for upstream and downstream
processes, for example ultrafiltration, microfiltration, virus filtration, depth filtration, membrane bioreactor,
membrane contactor and membrane chromatography. Membrane chromatography has been studied for
nearly two decades. Its benefits are a low operating pressure and the short operation time of adsorption due
to the convective flow taking place in the transport of protein to the binding site (Ghost 2002, Reis 2007). In
this work, we used a physical coating following an argon plasma grafting method to graft two monomers on
the PVDF membrane (Zou 2002) to produce a cation-exchange adsorptive membrane.
METHODS
The 0.22μm PVDF membranes which were chosen were purchased from Millipore. First, two kinds of
monomers (SA and PEGMA) were mixed into the solution containing water and isopropyl alcohol (1:1). The
molar ratio of PEGMA/SA was adjusted from 0 to 100 %. In addition, the SA monomer has SO3 group to
provide protein capture and the PEGMA monomer has an antifouling function (Chang, 2008) to prevent nonspecific binding. The PVDF membrane was immersed into the solution for 10 minutes and dried at room
temperature for 24 hours. Subsequently, the coating membrane was treated by a low pressure argon plasma
treatment under various times. Finally, ultrasonic cleaning was used to wash out non-grafting monomers.
The static adsorption experiments of lysozyme (LY) and bovine serum albumin (BSA) were operated on
batch type individually. The modified membranes were cut into a circle with 1.2 cm diameter and immersed
into phosphate buffer solution for 24 hours and placed in a protein solution (1mg/mL) for 2 hours. Later the
concentration of protein solutions was detected by a UV/VIS detector with wave length at 280 cm-1.
Moreover, these experiments were operated under different parameters including plasma treating times (3090s), molar ratios of monomer (0-100%), pH values (3-12) and ionic strengths (0-1M NaCl) to analyze
efficiency of the modified membranes.
RESULTS
The results were divided into two sections to discuss the protein adsorption amount and the LY ratio (defined
as the ratio of LY adsorption to the sum of LY and BSA adsorption amounts). In Figure 1, the modified
membrane with molar ratio of PEGMA:SA = 7:3 has a higher adsorption amount and LY ratio under longer
plasma treating time due to the higher grafting density. From this result, all of the analyzed experiments are
operated with modified membranes at a plasma treating time of 90s because of its higher efficiency. Figure 2
reveals that modified membranes under various molar ratios with plasma treatment at 90s have different
protein adsorption amounts. As the SA molar ratio was higher than 90%, the grating density was decreased.
In Figure 3, when the pH value was under 11 (pI. of LY), lysozyme’s positive charges allowed it to be
adsorbed by the SO3 groups (negative charges). The same results would be found at pH values below 4.7
(pI. of BSA). In Figure 4, the adsorption experiments were operated in different ionic strengths of phosphate
buffer solution, because of the screening of electrostatic attraction, the adsorption amount was decreased
under the higher salt concentration.
Figure 1. Protein adsorption at different plasma
treating time. (PEGMA:SA=7:3)
Figure 2. Protein adsorption at different SA molar
ratio. (90s-plasma treatment)
Figure 3. Protein adsorption at different pH value.
(90s-plasma treatment, PEGMA:SA=7:3)
Figure 4. Protein adsorption at different ionic
strength. (90s-plasma treatment, PEGMA:SA=7:3)
CONCLUSION
In this work, the cation-exchange membrane was successfully made by a physical coating following a low
pressure argon plasma modification method. We find there is better efficiency of protein purification (the LY
ratio was higher than 94%) with a molar ratio of PEGMA : SA = 7:3 and a treating time at 90 sec.
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