Solvent and Detergent Removal

Table 2.34
Properties of SDR HyperD® Resin

Specification	Parameter
Resin Structure	Spherical silica beads filled with a three-dimensional  hydrophobic polymer
Average Particle Size	40-100 μm
Nature of Polymer	Hydrophobic, long aliphatic chains bind solvents.
Typical Sample Load	2-3 times the column volume with residence  times of 5 minutes using lgG or ATIII treated solutions
Recommended Residence Time	5-15 minutes
Binding Capacity for Triton X-100	60-80 mg/mL*
Adsorption Buffer	Phosphate buffered saline (PBS)
Solvent/Detergent Elution Buffer	PBS/Ethanol (50/50) and EtOH or/and isopr
Operating pH Range	2-12
Resin Pressure Resistance	70 bar (7,000 kPa, 1,015 psi)

*Determined using 5 mg/mL Triton X-100 in PBS, pH 7.4, 10% breakthrough, 300 cm/h.

Figure 2.31

Schematic Interaction Mechanism of Triton X-100 and TnBP on SDR HyperD Resin

Protocol for SDR HyperD® Solvent and Detergent Removal Chromatography Resin

Triton X-100 interacts both with the silica surface (formation of hydrogen bonds between the silanols and the polyoxyethylene chain) and with the hydrophobic polymer moiety. TnBP interacts only with the hydrophobic polymer of the resin.

A. Materials Required

1. Container for SDR HyperD resin, one of the following:
   1. Empty, plastic, small-volume column with porous PE frits (disposable polypropylene column, e.g., Pierce PN 29922)
   2. Nanosep® MF centrifugal device with 0.45 μm GHP membrane (PN ODGHPC34)
   3. Choose either:
      • (1) AcroPrep™ 96 filter plate, 350 μL well, 0.45 μm GHP membrane (PN 5030); 0.45 μm Supor® membrane (PN 5029); 1.2 μm Supor membrane (PN 5039); or
      • (2) AcroPrep 96 filter plate, 1 mL well, 0.45 μm GHP membrane (PN 5054)
   4. Glass column 6.6 mm ID x 10 cm length, 1-2 mL volume (OmniFit PN 006CC- 06-10-AF)
2. Needed if filter plate is used:
   1. Collection plates [e.g., 96 well polypropylene V bottom, 0.5 mL (Axygen PN P96450V) or 1.64 mL round bottom (Axygen PN PDW20)]
   2. Adhesive plate sealing film
   3. AcroPrep 96-well plate sealing cap mat (PN 5230)
3. Separation apparatus (if filtration is used)
   1. Source of vacuum 25.4-50.8 cm Hg (10-20 in. Hg) (PN 5017); or
   2. Centrifuge fitted with a swinging bucket rotor
4. Degassed 50% (v/v) slurry of the SDR HyperD resin
5. Degassed suitable buffer, such as Phosphate buffered saline

Tips on Handling SDR HyperD Resin:
For packed columns, use only degassed liquids. Degassing is not necessary for batch mode methods. 

Some BioSepra® media are supplied as concentrated slurries and may be difficult to resuspend. Do not use magnetic stir bars with BioSepra media as they can damage the beads. Also, these resins are quite dense and settle quickly. When adding slurry to any device, mix well between additions. If it is necessary to prepare a 50% (v/v) slurry, use a clean spatula to remove some of the packed media and transfer to a graduated glass cylinder containing degassed water or buffer. DO NOT add any media back to the storage bottle to avoid contamination of the bulk media. Thoroughly mix and allow settling. Note the volume of settled resin. Decant the supernatant and add back an equal volume of water or buffer to make a 50% (v/v) slurry.

For packed columns, removal of fines may be necessary. Prepare the slurry in desired buffer, mix, and allow to settle for approximately 5 minutes or for enough time that the beads have settled but small particles are still in the solution.

B. Packing SDR HyperD® Resin

1. Gravity flow column format
   1. Equilibrate column, degassed 50% gel slurry, and degassed buffer solution at room temperature.
   2. Secure a bottom cap on the column tip and clamp the column (1-5 mL bed volume column, e.g., Pierce PN 29922) upright in a laboratory stand.
   3. Add a sufficient volume of degassed buffer to the column to fill it up to the reservoir (wide-mouth) portion, and then gently tap the end and side of the column to dislodge any air bubbles.
   4. Float a porous disc on top of the liquid within the column.
   5. Using the reverse end of a Pasteur pipette or reverse end of a serum separator (e.g., Pierce PN 69710), push the disc evenly to the bottom of the column.
   6. Decant most of the liquid from the empty column, being sure to avoid getting air bubbles in the tip region of the column below the inserted disc. Place the column back in its stand with bottom cap still in place.
   7. Add sufficient volume of degassed gel slurry to obtain the desired settled gel volume of 1-2 mL.
   8. Allow gel to settle in the column for at least 5 minutes.
   9. Position a second porous disc on top of the settled gel bed by floating it on the liquid within the column and pushing it down to just above the settled gel. Leave 1-2 mm of space between the top of the gel bed and the top disc. Do not compress the gel bed.
   10. Wash the inside top part of the column with buffer to remove residual gel that may have remained along the sides during packing.
   11. Packed column is now ready for storage at 4 °C or immediate use.
   12.  l. Refer to Section C for use instructions.
       Tip: Store the packed column upright at 4 °C with the gel bed submerged under 1-2 mL of buffer and a top column cap securely in place. Sodium azide added to the storage buffer to a concentration of 0.02% (w/v) will help prevent microbial growth. Always remove the top cap before the bottom cap to avoid drawing air bubbles down into the gel bed. Prevent air bubbles from forming in the gel bed by using only degassed buffer and sample solutions. Degassing involves subjecting a solution to vacuum to remove excess dissolved air. Use of too high a vacuum can lead to evaporation of solvent from the solution. Check the final volume after degassing and, if necessary, add more solvent to return to original volume.
2. Nanosep® spin filter column format
   Tip: Adjust resin volume as needed for amount of detergent to be removed.
   1. Remove the spin filter from the Nanosep collection tube and place in a rubber stop per (with a suitable hole to form a tight fit to the spin filter) on the vacuum flask. Apply a low vacuum of 5-10 mm Hg.
   2. Mix the 50% (w/v) SDR HyperD slurry (washed in bulk with buffer to remove the 20% (v/v) ethanol preservative) and quickly pipette 0.4 mL of the slurry into the Nanosep device. Rapidly follow with a second and third volume of slurry.
      Tip: In between each addition of the slurry, allow the resin bed to settle.
   3. After final addition, allow the vacuum to remove the liquid from the resin bed which should now fill the Nanosep® device.
   4. Place the spin filter back into the collection tube to hold until sample addition.
      Tip: Because all the preservative has been removed from the resin in these devices, they should be used immediately or stored at 4 °C for no more than one week. If resin-filled devices will be stored, add just enough buffer so that the resin looks wet (~200 μL/well).
   5. Refer to Section C for use instructions.
3. AcroPrep™ multi-well filter plate format
   1. In bulk (e.g., a centrifuge tube) wash the SDR HyperD® media with 5 column volumes (CV) of buffer to remove the 20% (v/v) ethanol storage buffer.
   2. Place an AcroPrep filter plate (350 μL or 1.0 mL well volume) on a suitable vacuum manifold (PN 5017) with a collection plate underneath.
   3. Mix the 50% (v/v) SDR HyperD slurry and quickly pipette 0.4 mL of the slurry into the multi-well plate. Rapidly follow with a second and third volume of slurry if using the deep well plate. Resin volume should be adjusted according to need.
      Tip: In between each addition of the slurry, allow the resin bed to settle. Mix the slurry before each addition to the well to prevent settling.
   4. After final addition, allow the vacuum to remove the liquid from the resin bed which should partially fill the well of the AcroPrep 96 filter plate (~400 μL of resin).
      Tip: Visually inspect to ensure equivalent volume of resin in each well.
   5. The plate is ready for immediate use. To store the plate, add sufficient buffer/well to wet the resin, then cover. Excess buffer will need to be removed (by vacuum or centrifugation) immediately before use.
      Tip: Because all the preservative has been removed from the resin in these devices, they should be used immediately or stored at 4 °C for no more than one week.
   6. Refer to Section C  for use instructions.
4. Glass column format for packed bed chromatography applications
   Tip: Adjust bed height and resin volume to suit the specific application.
   1. Equilibrate column, degassed 50% gel slurry, and degassed buffer solution to room temperature.
   2. Attach the bottom end fitting on to the column and clamp upright in a laboratory stand.
   3. Add a 1 mL of degassed buffer to the column to cover the bottom frit, and then gently tap the end and side of the column to dislodge any air bubbles.
   4. Add sufficient volume of degassed gel slurry to obtain the desired settled gel volume of 1-2 mL.
   5. Allow gel to settle in the column for at least 5 minutes.
   6. Position the adjustable height top fitting on to the column. Gently screw the top fitting down on to the settled gel bed. This should displace air out of the top fitting in the column. Do not over compress the gel bed.
   7. Place the column on a suitable chromatography system and pump liquid up though the column at 1 mL/min for 2-3 minutes to displace any trapped air. Reverse the flow and equilibrate the column for at least 10 column volumes at up to 10 mL/min.
   8. Packed column is now ready for storage at 4 °C or immediate use.
   9. Refer to Section C below for use instructions.

C. Detergent Removal

1. Gravity flow column format
   1. Prepare a column as described above.
   2. Wash the SDR HyperD® resin with 5 CV of buffer to remove the 20% (v/v) ethanol storage buffer.
   3. Allow the liquid to drain from the column and load the sample, up to 2 mL, onto the column. (If more volume is preferred, column size can be increased. This will depend on amount of detergent to be removed.)
   4. Collect the column effluent in 1 mL fractions. Measure absorbance at 280 nm to locate the protein peak.
      Tip: Protein rapidly elutes from the column and should be found in the first three fractions. Some slight dilution of the sample will occur during elution. If necessary, the sample can be concentrated with a centrifugal UF spin filter, such as a Nanosep® centrifugal device, with a 10K MWCO UF membrane.
   5. After unretained protein has been eluted, discard the resin.
2. Nanosep spin filter column format
   1. Prepare the spin column as described above.
   2. Centrifuge the spin column in a swinging bucket rotor at 1000 x g for 2 minutes to remove excess fluid from the packed bed.
   3. Remove the filtrate from the collection tube.
   4. Very carefully pipette the sample (0.05-0.2 mL, maximum volume depends on resin capacity vs detergent concentration, and well size) onto the top of the dry packed bed in the Nanosep device. Replace in the collection tube.
   5. Centrifuge the spin column in a swinging bucket rotor at 1000 x g for 4 minutes to pass the sample through the SDR resin bed.
   6. Filtrate in the collection tube will be detergent depleted.
3. AcroPrep™ multi-well filter plate format
   Tip: For prolonged incubations or incubations using solutions that reduce surface tension (e.g., detergents, alcohols, and acetonitrile) in 96-well plates, it may be necessary to seal the bottom of the plate to prevent leakage. This can be tested in advance using just the solutions to be evaluated loaded into the chosen plates.
   
   An adhesive plate sealer can be used on the top of the wells to prevent cross-contamination during vigorous shaking or evaporation during prolonged or warm incubations. For sealing, you can use a plastic, self-adhesive plate sealer (e.g., Sigma EASYseal^♦) or a cap-mat (PN 5230).
   1. Prepare the packed multi-well filter plate as described above.
   2. Centrifuge the multi-well plate and collection plate in a suitable swinging bucket rotor at 1000 x g for 2 minutes to remove excess fluid from the packed bed.
      Tip: As an alternative to centrifuging, a vacuum manifold can be used. See Section 6.3 of the Appendix for more detail.
   3. Remove the filtrate from the collection tube.
   4. Very carefully pipette the sample (0.05-0.2 mL, maximum volume depends on resin capacity vs detergent concentration, and well size) onto the top of the dry packed bed in the Nanosep® device.
      Tip: At this stage, sample-loading conditions should be optimized. The amount of sample loaded should be < 50% of the static binding capacity of the resin.
   5. Centrifuge the multi-well plate and collection plate in a swinging bucket rotor at 1000 x g for 4 minutes to pass the sample through the SDR resin bed.
   6. Detergent free samples are recovered from the collection plate.
4. Packed bed chromatography column format, pumped system
   1. Prepare the column as described above.
   2. Load the sample up to a 2 mL volume (maximum volume depends on resin capacity vs detergent concentration) onto the column at 1 mL/min flow rate. Monitor effluent at 280 nm.
   3. Collect the column effluent in 1 mL fractions. Measure A₂₈₀ to locate the protein peak.
      Tip: Protein rapidly elutes from the column and should be found in the first three fractions. Some dilution of the sample will occur during elution. If necessary, the sample can be concentrated with a centrifugal UF spin filter, such as a Nanosep centrifugal device, with a 10K MWCO membrane.
   4. After unretained protein has been eluted, the resin can be regenerated to remove retained detergent.
      Tip: If re-use is desired, the retained detergents can be eluted with 10 CV of PBS/ethanol (50/50 mix) followed by 10 CV of 95% (v/v) ethanol, followed by re-equilibration with buffer. If sanitization or depyrogenation is required, two methods are recommended:
      • Alcohol/acid treatment. Wash with at least 3 CV of degassed 20% (v/v) ethanol containing 1 M Acetic acid (Note: monitor volume during degassing). Allow the resin to soak in the presence of the solution (recycle effluent manually or using a pump) for an exposure time of 1 hour. Then re-equilibrate with buffer.
      • Diethyl pyrocarbonate/ethanol treatment. Wash with 2 CV of PBS in 50% (v/v) ethanol and 5% (v/v) diethyl pyrocarbonate. Allow the resin to soak in the presence of this solution (recycle effluent manually or using a pump) for an exposure time of 1 hour. Wash the column with 3 CV of 3M pyrogen free sterilized NaCl/1 M acetic acid to remove all pyrogens. Then re-equilibrate with sterile pyrogen-free buffer.

Application Data for SDR HyperD® Solvent and Detergent Removal Chromatography Resin

Dynamic Binding Capacity Data for SDR HyperD Resin in a Gravity Flow Column Format Efficient binding of various detergents in various formats

Removal of detergents from protein samples can be achieved in a gravity flow format for samples in the 1-2 mL volume range. To estimate the detergent removal capacity of the resin, a panel of packed columns (1-2 mL) were loaded with a 1 mL sample of 5 mg/mL human serum albumin (HSA) in 1% (w/v) detergent and allowed to drain into the bed, followed by 1% detergent solution. 1 mL fractions were collected. The protein was measured at 280 nm and then dye binding reagent was added to the effluent to visualize detergent in the fractions. When the column was saturated with detergent, a "break through" curve was recorded. The dynamic binding capacity was calculated as follows; detergent capacity (mg/mL) = [Volume to breakthrough peak (# of fractions x Vol) – volume to protein elution] x 10/Column volume A breakthrough curve and estimate of the dynamic binding capacity for ASB-14 (see Figure 2.32 for chemical structure) is illustrated in Figure 2.33 below. A range of detergents have been evaluated with SDR HyperD resin and their dynamic binding capacity data is summarized in Table 2.35 below. Under these conditions, SDR HyperD resin exhibits a high binding capacity for zwitterionic detergents such as ASB-14 and CHAPS. SDS, an anonic detergent, is influenced by the ionic strength of the sample. Addition of 0.1 M NaCl to 1% SDS (w/v) enhanced the SDR HyperD resin binding capacity by 80-100%. Protein recovery was > 95% in a volume of 1.5-2.0 mL (50-100% volume increase). This diluted sample can be rapidly concentrated with a Nanosep® centrifugal UF spin filter with a 10K MWCO UF membrane if necessary (see Section 2.4).

Figure 2.32

Chemical Structure and Properties of Detergent ASB-14

A zwitterionic amidosulfobetaine detergent useful for solubilizing proteins to be analyzed by 2Delectrophoresis. The increased solubilization produced by this detergent enables the identification pf previously undetected membrane proteins.

Figure 2.33

Dynamic Binding Capacity (mg/mL Media) for ASB-14 Removal by SDR HyperD® Resin in a Gravity Flow Column Format

SDR HyperD resin (1 mL packed volume) packed in a gravity flow column as described in the Protocol section was loaded with 1 mL of a 5 mg/mL albumin solution in 1% ASB-14 detergent in water. The sample was allowed to enter the resin followed by 1% ASB-14 in water (no protein) to elute the protein detergent. Effluent fractions of 1 mL were collected. Protein was monitored at 280 nm and detergent detection was measured interference in the BioRad dye binding assay read at 595 nm.

Table 2.35

Dynamic Binding Capacity of SDR HyperD Resin in a Gravity Flow Format (1 mL Volume)

Detergent	Detergent Dynamic Binding Capacity (mg/mL)
ASB-14	60.0
ASB-14 + 6M Urea/2M Thiourea	70.0
CHAPS	75.0
SDS	15.0
SDS + 0.1 M NaCl	28.0

Dynamic Binding Capacity Data for SDR HyperD® Resin in a Spin Column Format

A spin column format was developed to process smaller volume samples (<– 0.2 mL) in a shorter time frame using a modified centrifugal desalting procedure. In this study, SDRHyperD resin was first washed with water to bring the conductivity down to < 0.045 mS/cm and then packed into empty Nanosep® GHP devices under a low vacuum facilitated flow conditions (see Protocol Section B). The packed bed was then "dewatered" by spinning at 1,000 x g in a swinging bucket rotor for 2 minutes. Samples (0.1 mL) were carefully added to the top of the dewatered resin bed and then spun at 1,000 x g in the above rotor for 4 minutes. The resulting filtrate was placed in a multi-well plate and 0.2 mL of the diluted dye binding reagent was added (BioRad). The presence of detergent correlated with development of a blue color after the addition of the dye. The results are summarized in Table 2.36 below.

Table 2.36

Detergent Binding Capacity of 1 mL SDR HyperD Resin in a Spin Column Format

Sample	# of Cycles*	Volume 1% (w/v) Detergent	SDR/Device
ASB-14	22	2.2 mL	22 mg
ASB-14 + 6 M  Urea/2 M Thiourea	7	0.7 mL	7 mg
CHAPS	24	2.4 mL	24 mg
CHAPS + 6 M  Urea/2M Thiourea	12	1.2 mL	12 mg
SDS	20	2.0 mL	20 mg
SDS + 0.1 M NaCl	24	2.4 mL	24 mg

*Detergent binding capacity was measured by adding 0.1 mL volumes of the detergent to challenge the device, followed by centrifugation as described in the Protocol section. This was repeated until detergent “break through” was seen with the protein assay. The volume of detergent was then calculated from volume 1% detergent = # cycles x 0.1.

Dynamic Binding Capacity Data for SDR HyperD Resin in a Pumped Packed Bed Column Format

The high specific surface area (200 m2/g) of the porous silica of this resin allows a high capacity for Triton X-100 and TnBP. The dynamic binding capacities obtained are 60-80 mg/mL for Triton X-100 and 40-50 mg/mL for TnBP at 100 cm/hr (initial concentrations of Triton X-100 and TnBP in bovine plasma are respectively 10 and 5 mg/mL). Examples of removal efficiencies from various samples are summarized in Table 2.37.  

Table 2.37

Solvent-Detergent Depletion Example

Solution	Surfactant	Before Depletion	After Depletion	Removal Efficiency
lgG	TnBP  Triton X-100	5,000 ppm  10,000 ppm	<0.4 ppm <10 ppm	99.9% 99.9%
ATIII	TnBP  Triton X-100	5,000 ppm  10,000 ppm	<0.4 ppm  <10 ppm	99.9% 99.9%
Bovine Serum	TnBP  Triton X-100	5,000 ppm  10,000 ppm	<0.4 ppm  <10 ppm	99.9% 99.9%

Sample volume: 3.6 CV, flow rate: 150 cm/h; column length: 10 cm; residence time: 4 minutes.

Tip: Removal efficiency is also dependent on flow rate and column loading: i.e. when using a 10 cm column at 150 cm/h (2 CV load of bovine serum containing Triton X-100 or TnBP), a removal of 95.5% was observed for Triton X-100. This removal efficiency was decreased to 80% when 8 CV loads were used.

In summary, the SDR HyperD® resin is an excellent tool for removal of detergent from protein samples and can be used in a range of device formats to achieve rapid processing with high recovery of sample.

Ordering Information for SDR HyperD Solvent and Detergent Removal Chromatography Resin

Part Number	Description	Pkg
20033-065	SDR HyperD	5 mL
20033-031	SDR HyperD	25 mL
20033-023	SDR HyperD	100 mL
20033-015	SDR HyperD	1000 mL

References for SDR HyperD Solvent and Detergent Removal Chromatography Resin

1. Guerrier, L., et al. (1995). Specific sorbent to remove solvent-detergent mixtures from virus-inactivated biological fluids. J. Chromatogr. B., (664), 119.

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