High-throughput ion exchange purification of positively charged recombinant
protein in the presence of negatively charged dextran sulfate
Lam Raga A. Markely1, Lutfiye Kurt1, Janet Lau1, Sarthak Mane1, Bing Guan1, Meisam
Bakhshayeshi2, John Pieracci2, Thomas Ryll1, Scott Estes1, and Shashi Prajapati1,3
Supplementary Information
Supplementary Figure 1. Supplementation of dextran sulfate significantly decreased SPXL
yield. We used SPXL to purify P1 from cell culture samples containing dextran sulfate, and
analyzed each fraction by SDS-PAGE. Lane 1 is the ladder, lane 2 is the SPXL load, lane 3 is the
SPXL flow-through, lanes 4, 5, 6 are the first, second, third SPXL washes, respectively, lane 7 is
the SPXL elution, lane 8 is the SPXL strip, and lane 9 is pure P1 control. Significant losses of P1
were observed in the flow-through (lane 3) and wash (lane 4). These effects occurred because
dextran sulfate bound to P1, thereby disrupting the ionic interaction between protein P1 and
SPXL resin.
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G
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Supplementary Figure 2. Optimization of SPXL purification by adjusting NaCl concentration
in washing buffer. Six concentrations of NaCl was used in the 50 mM Tris, pH = 8.5 washing
buffer: (A) 280 mM, (B) 250 mM, (C) 210 mM, (D) 180 mM, (E) 150 mM, and (F) 125 mM.
Reduced SDS-PAGE was used to analyze the performance of the SPXL purification. 13 µL of
each fraction of the SSP was taken for SDS-PAGE analysis. Lane 1 is the ladder, lane 2 is the
SPXL load, lane 3 is the SPXL flow-through, lanes 4, 5, 6 are the first, second, and third SPXL
washes, respectively, lane 7 is the SPXL elution, lane 8 is the SPXL strip, and lane 9 is pure
protein P1 control. Red arrows in (A) and (B) indicate protein P1 in the SPXL washes, and red
arrows in (E) and (F) indicate impurities in the SPXL elution. At 280 and 250 mM NaCl,
significant losses of protein P1 were observed in SPXL washes (lane 4, 5, and 6). At 180 and 150
mM NaCl, impurities were observed in SPXL elution (lane 7). 210 mM NaCl was chosen as the
optimum condition to ensure proper washing of impurities and minimize losses in the washing
step. (G) The % yield of SSP for each NaCl concentration was analyzed as described in
Materials and Methods section. Error bars: SD (n = 2).
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Supplementary Figure 3. The two-step SSP using QFF and SPXL resins successfully purify P1
from cell culture samples containing dextran sulfate. Here, 6 mL of conditioned cell culture
sample was loaded to 400 µL of QFF resin prepared in empty disposable PD-10 columns (GE,
17-0435-01). The flow-through was collected using Visiprep SPE vacuum manifold (Supelco,
57030-U), and diluted 4x with 50 mM phosphate buffer, pH = 7.0. The diluted QFF flowthrough was loaded to 800 µL of SPXL resin prepared also in empty disposable PD-10 columns.
The resin was washed with 5 x 1.2 mL 50 mM Tris Buffer, 210 mM NaCl, pH = 8.5. 1.2 mL of
50 mM Tris Buffer, 500 mM NaCl, pH = 8.5 was then added to elute the purified P1. The blue
and red curves correspond to duplicates of cell culture samples purified by the two-step SSP. The
yield of the SSP based on ELISA is 63 ± 0% (SD, n = 2). The chromatogram looks similar to that
shown in Fig. 4A. The chromatogram shown here has a shoulder because protein P1 in this cell
culture sample has lower level of glycan occupancy than the P1 shown in Fig. 4A.
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Supplementary Table 1. Optimization of QFF purification by adjusting NaCl concentration.
Solutions of P1 in 75 mM carbonate-bicarbonate buffer, pH = 9.0, (A) 200 mM, (B) 300 mM,
(C) 400 mM, and (D) 500 mM NaCl were mixed with dextran sulfate (DexS), purified by QFF,
and the flow-through was analyzed by SEC to calculate the purification yield. As controls, neat
pure P1 were also processed by QFF purification in each NaCl concentration.
[NaCl] in 75 mM sodium carbonate/bicarbonate, pH = 9.0
200 mM
300 mM
400 mM
500 mM
QFF Yield (%)
Pure P1 + DexS Pure P1 + water
27 ± 2
86 ± 0
44 ± 1
86 ± 0
63 ± 0
84 ± 0
83 ± 0
86 ± 0
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