SUPPORTING INFORMATION
A multi-dimensional approach for fractionating proteins using charged membranes
Mirco Sorci1, Minghao Gu1, Caryn L Heldt1,2, Elizabeth Grafeld1 and Georges Belfort1*
1
Howard P. Isermann Department of Chemical and Biological Engineering
and Center for Biotechnology and Interdisciplinary Studies
Rensselaer Polytechnic Institute, Troy, NY 12180
2
Present address: Department of Chemical Engineering, Michigan Tech, Houghton, MI 49931
Running Title: Charged membrane filtration
Keywords: Lysozyme, RNase A, BSA, Hemoglobulin, Membrane modification, Electrostatic
interactions
*To whom correspondence should be addressed: Georges Belfort, Howard P. Isermann
Department of Chemical and Biological Engineering and Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA, Tel.: (518)
276-6948; Fax: (518) 276-4030; Email: belfog@rpi.edu
1
Charged membrane filtration
EXPERIMENTAL
Material
Buffers - Lys (0.5 mg/ml) - RNase A (0.15 mg/ml) mixture was prepared in 20 mM Trisbase, 20 mM boric acid, pH 6.8 (Buffer A). The solution ionic strength was controlled by the
addition of appropriate amounts of NaCl: Buffer A + 300 mM NaCl (Buffer B). The solution
pH was controlled 20 mM Tris-base, 250 mM boric acid (Acid) and 20 mM Tris-base, 100
mM NaOH (Base). BSA (0.5 mg/ml) – Hb (0.15 mg/ml) mixture was prepared in 20 mM
Na2HPO4, 0.68 mM citric acid, pH 7.6 (Buffer A). The solution ionic strength was controlled
by the addition of appropriate amounts of NaCl: Buffer A + 200 mM NaCl (Buffer B). The
solution pH was controlled by Buffer A + 50 mM citric acid (Acid).
Methods
UV-induced graft polymerization - PES membranes were surface-modified using a dip
technique (Pieracci et al. 2000). For grafting and polymerization, irradiation at ~ 300 nm was
conducted in a UV chamber (F300S, Fusion UV Systems, Inc. Gaithersburg, MD) containing
an electrode-less microwave lamp (Pieracci et al. 2002). After UV irradiation, the
membranes were soaked with DI water for 24 h to remove homopolymer and unreacted
monomer residues (Pieracci et al. 2000).
Degree of grafting (DG) by ATR/FTIR - Attenuated Total Reflection/Fourier Transform
Infrared spectroscopy (ATR/FTIR, Magna-IR 550 Series II, Nicolet Instruments, Madison,
WI) was used to measure the DG of modified membranes. DG was defined as the ratio of the
absorbance peak height at ~ 1727 cm-1 (carbonyl stretching) for grafted SPMP and MPTA and
at ~ 1034 cm-1 (phosphate) for grafted SSAS to ~ 1578 cm-1, a reference peak of the benzene
2
Charged membrane filtration
carbon-carbon double bond of PES membrane. IR beam penetration was 0.1-1.0 μm.
Filtration under either pH or conductivity gradient – In the buffer mixer, each 8-liter
buffer tank was kept pressurized by compressed N2 and connected to the mixing loop.
Buffers were chosen taking into account the mixing effect in order to operate at constant
protein concentration in the feed solution (e.g. protein was added in Buffer B when operating
under conductivity gradient and constant pH). Once the set-points values were reached in the
mixing loop both for pH and conductivity (with a precision of 0.01), the control system
opened the feed line from the mixing loop to the cross-flow UF system. Two additional pH
and conductivity probes, located before the cross-flow UF system, monitored the pH and
conductivity values of the feed solution and compared them to the values in the mixing loop.
Protein analysis - The amounts of Lys and RNase A in the different streams were
measured by chromatography (Breeze 2 HPLC system, Waters, Milford, MA) using a
BioSuite SP, 10 μm CXC, 7.5 x 75 mm column. The mobile phase consisted of 20 mM TrisHCl, 10 mM sodium tetraborate, pH 8.0 and the separation of the two protein peaks was
realized using a salt gradient of NaCl at a flow rate of 1 ml/min. Details of the analysis are in
Fig. S2. The injection volume was kept constant and equal to 40 μl, protein concentration
was measured at 280 nm and the mass was calculated using commercial software (Breeze 2
software, Waters, Milford, MA). The amounts of BSA and Hb in the different streams were
estimated by spectroscopy (Hitachi U-2000, Hitachi High Technologies America, San Jose,
CA), measuring the absorbance at 280 and 416 nm, following the method by Shukla et al.
(2000). Hb absorbs strongly at 416 nm, while BSA does not. Thus, the Hb concentration in
the BSA-Hb mixture was estimated from the absorbance reading of the sample at 416 nm.
3
Charged membrane filtration
Then, the BSA concentration was estimated from absorbance readings at 280 nm, after
subtracting the computed Hb contribution at this wavelength. Details of protein spectra and
calibration curves are shown in Fig. S3.
REFERENCES
Pieracci J, Crivello JV, Belfort G. 2002. Increasing membrane permeability of UV-modified poly(ether sulfone)
ultrafiltration membranes. Journal of Membrane Science 202(1-2):1-16.
Pieracci J, Wood DW, Crivello JV, Belfort G. 2000. UV-assisted graft polymerization of N-vinyl-2-pyrrolidinone
onto poly(ether sulfone) ultrafiltration membranes: Comparison of dip versus immersion modification
techniques. Chemistry of Materials 12(8):2123-2133.
Shukla R, Balakrishnan M, Agarwal GP. 2000. Bovine serum albumin-hemoglobin fractionation: significance of
ultrafiltration system and feed solution characteristics. Bioseparation 9(1):7-19.
4
Charged membrane filtration
Figure S1
Digital display
Pressurized
buffer tanks
pH-1
Conductivity-1
pH-2
Conductivity-2
2
1
Conductivity &
pH probes
Mixing loop
Figure S1. Photograph of the buffer mixer, highlighting the key components of the system:
Mixing loop and probes before (1) and after (2) the mixing loop, to control pH and
conductivity.
5
Charged membrane filtration
Figure S2
A
Lys
t = 9.049 min
RNase A
t = 6.512 min
B
RNase A
Lys
Figure S2. HPLC analysis of Lys-RNase A mixture, using a BioSuite SP, 10 μm CXC, 7.5 x
75 mm column. (A) Chromatogram of Lys-RNase A mixture, showing the separation of the
characteristic peaks of the two proteins realized with a NaCl gradient (insert) between 20 mM
Tris-HCl, 10 mM sodium tetraborate, pH 8.0 (buffer A) and 20 mM Tris-HCl, 10 mM sodium
tetraborate, 500 mM NaCl, pH 8.0 (buffer B). (B) Calibration curves for RNase A (R2 =
0.9997) and Lys (R2 = 0.9999) obtained by injecting different known amounts of proteins and
reading absorbance values at 280 nm.
6
Charged membrane filtration
Figure S3
A
B
0.9
BSA
Hb
0.8
y = 4.0317x - 0.0192
R² = 0.9995
Absorbance (-)
0.7
0.6
0.5
y = 1.4642x - 0.0078
R² = 0.9998
0.4
0.3
0.2
0.1
Hb
0.0
0.0
0.1
0.2
0.3
Concentration (mg/ml)
0.7
BSA
Absorbance (-)
0.6
BSA+Hb
0.5
0.4
y = 0.618x - 0.0066
R² = 0.9997
0.3
0.2
0.1
0.0
0.0
0.5
1.0
Concentration (mg/ml)
Figure S3. Analysis of BSA-Hb mixture using UV-Vis spectrophotometer. (A) Spectra for
BSA, Hb and BSA-Hb mixture, highlighting how Hb absorbs strongly at 416 nm, while BSA
does not. (B) Calibration curves for BSA (R2 = 0.9997) and Hb (R2 = 0.9995 and 0.9998)
obtained by reading absorbance values at 280 and 416 nm of different known amounts of
proteins.
7
1.5
Charged membrane filtration
Figure S4
Conductivity (mS/cm)
pH
25
Conductivity
pH
20
pH
15
pH 11.2
10
pH 11
pH 10.8
5
0
0
10
20
30
Time (min)
Conductivity
Equilibration
Filtration experiment
Figure S4. Operating conditions for Lys-RNase A filtration experiment carried out at pH 11
and under a conductivity gradient from 22.2 to 5.4 mS/cm.
8
Charged membrane filtration
Figure S5
A
B
SPMP
C=O stretching
0.10 M
0.08 M
0.06 M
Degree of grafting, DG (-)
C
0.04 M
0.20
0.15
0.02 M
0.10
0.01 M
0.05
Unmodified
0.00
0
0.02 0.04 0.06 0.08 0.1 0.12
SPMP concentration, CSPMP (M)
Figure S5. (A) ATR/FTIR analysis of SPMP degree of grafting, defined as the ratio of the
absorbance peak height at ~ 1727 cm-1 (carbonyl stretching) to ~ 1578 cm-1, a reference peak
of the benzene carbon-carbon double bond of PES membrane. (B) Magnification of the
peaks used for DG calculation and (C) DG values versus CSPMP for the different membranes.
9