Buffer Exchange, Desalting, and Concentration Using Microsep™ UF Spin Filter for Samples (1-3.5 mL) 4.4.2

Table 4.32

Typical Protein Recovery/Passage with Omega™ UF Membranes in a Nanosep® Centrifugal UF Device

		MWCO	3K	10K	30K	100K	300K
Solute	Solute MW (Kd)	Spin Time (min)	15	10	8	5	3
Vitamin B12	1,335	% Recovery	7	-	-	-	-
Aprotinin	6,200	% Recovery	99	51	11	-	-
Cytochrome C	12,400	% Recovery	100	89	77	1.8	-
Chymotrypsinogen A	25,000	% Recovery	-	97	94	2.1	-
Ovalbumin	45,000	% Recovery	-	97	92	3	-
BSA	67,000	% Recovery	-	-	100	26	1.5
Phosphorylase B	97,400	% Recovery	-	-	95	91	1
IgG	156,000	% Recovery	-	-	-	97	1.5
Thyroglobulin	677,000	% Recovery	-	-	-	100	91

Samples of 0.5 mL of a 1.0 mg/mL solution were centrifuged at 14,000 x g and were concentrated to a volume of 0.01-0.06 mL.

Application Guidelines for Microsep™ UF Spin Filter for Samples 1-3.5 mL

A simple guide to choosing the appropriate MWCO UF membrane in the Microsep device for a range of purification applications is summarized in Table 4.33. Full specifications of the Microsep UF device are summarized in Table 4.34 and a diagram in Figure 4.29. If low recovery of retentate samples is seen with these devices, an optional pre-treatment process to reduce potential non-specific binding to the membrane and device surfaces is recommended.

Table 4.33

Purification Application Guidelines on MWCO Selection

	MWCO UF Membrane
Application	10K	30K	100K
Buffer exchange or salt removal of chromatography eluates, gradient fractions	√
Concentrating dilute samples to enhance sensitivity for biological assay	√
Recovery of antibodies from cell culture			√ (IgM)
Recovery of low molecular weight compounds from fermentation broth	√	√
Natural products screening for medicinal chemistry	√	√	√
Virus concentration or removal			√

Table 4.34

Specifications of the Microsep™ Spin Filter

Specification	Parameter
UF Membrane	Omega™ membrane (low protein-binding, modified polyethersulfone on polyethylene substrate)
Materials of Construction      Device      Collection Tube	Styrene acrylonitrile (SANS) Polypropylene
Effective Membrane Area	0.46 cm2
Dimensions      Diameter      Overall Length (with Cap)	1.7 cm 9.9 cm
Capacities      Maximum Sample Volume      Final Retentate Volume      Final Receiver Volume      Hold-up Volume (Membrane/Support)	3.5 mL 0.030-0.05 mL 3.5 mL 0.02 mL
Operating Temperature Range	0-40 °C (32-104 °F)
pH Range	1-14
Maximum Centrifugal Force	7,500 x g
Centrifuge	Rotor accepting 1.7 x 10 cm tubes
Sanitization	70% ethanol

Figure 4.29

Components of the Microsep™ Centrifugal UF Device

 

Protocol for Microsep UF Spin Filter for Samples 1-3.5 mL

Omega™ membranes in Microsep devices contain trace amounts of glycerin and sodium azide (0.65 to 1.0 mg). If these chemicals interfere with an assay, they may be removed by filtering 3 mL high purity water or buffer through the membrane and repeat. If further flushing is required, start with 0.05N NaOH and repeat this procedure. Use device within 20 minutes to prevent irreversible membrane damage due to dehydration.

A. Materials Required

1. Microsep UF spin filters with Omega MWCO UF membrane and a collection tube. For specifications, see Table 4.34 and Figure 4.29.
2. Extra collection tubes for the Microsep UF spin filter
3. Degassed high purity water or buffer, such as phosphate buffered saline (PBS)

B. Basic Instructions for Use

1. Attach the filtrate receiver to bottom of sample reservoir. Pipettete 1.0 to 3.5 mL sample into upper reservoir. Place cap on reservoir to prevent evaporation.
   Tip: If pre-flushing to remove glycerin and sodium azide are required, add 3 mL of high purity water into the sample cup and process. Discard the filtrate and repeat with sample.
2. Place device into fixed angle centrifuge rotor that accepts 17 x 100 mm tubes.
   Tip: Always counterbalance the rotor with another Microsep spin filter containing equivalent sample volume.
3. Spin at 3,000-7,500 x g for the required length of time, typically 30-90 minutes to achieve desired concentrate volume. For optimal performance, it is recommended that spin time and g-force be determined for each application. See Table 4.35 and Table 4.36 to determine appropriate protocol.

   Table 4.35
   

   Typical Protein Recovery/Passage with Omega™ UF Membranes in a Microsep™ Centrifugal UF Device
   
   

   Microsep Device MWCO	Recommended g-force	MWt. Range
   1K, Yellow	5,000-7,000 x g	3K-10K
   3K, Gray	5,000-7,000 x g	10K-20K
   10K, Blue	5,000-7,000 x g	30K-90K
   30K, Red	5,000-7,000 x g	90K-180K
   50K, Green	1,000 x g	150K-300K
   100K, Clear	1,000 x g	300K-900K
   300K, Orange	1,000 x g	900K-1800K
   1000K, Purple	1,000 x g	>3000

   Table 4.36
   

   Processing Times for Microsep UF Devices
   
   

   		Time to “Dead Stop” (min)
   MWCO	Solute	3,000 x g	5,000 x g	7,500 x g
   1K	Cytochrome C (1 mg/mL)	200	130	100
   3K	Cytochrome C (1 mg/mL)	180	120	90
   10K	Albumin (1 mg/mL)	55	40	25
   30K	Albumin (1 mg/mL) IgG (0.1 mg/mL)	40 60	30 30	30 20
   50K	Albumin (1 mg/mL) IgG (0.1 mg/mL)	25 45	15 30	10 30
   100K	Apoferritin (1 mg/mL) IgG (0.1 mg/mL)	40 45	35 30	30 30
   300K	Yeast (0.5%)	20	14	--
   1000K	Yeast (0.5%)	8	5	--

4. At the end of spin time, stop centrifugation and remove devices. Using a pipette, transfer concentrated sample to concentrate cup. To pipette concentrated sample, slowly siphon the concentrate by moving the pipette tip around the perimeter of the plastic ring at the bottom of the sample reservoir (See Figure 4.30). 

   Figure 4.30
   

   Recovery of Retentate from Microsep™ UF Device
   
    

5. Cap storage cup containing concentrated sample, and store. Filtrate collected in filtrate receiver can also be stored for further analysis.
6. For application-specific protocol, see Section 2.4.2.1.

C. Buffer Exchange of Purification Samples (1-3.5 mL)

In purification it is a common occurrence that serial process steps are not always compatible and require buffer exchange to adjust pH or ionic strength without loss of sample. Using a suitable MWCO UF membrane (see Tables 4.35 and 4.36) to retain the molecule of interest, buffer exchange can be achieved within 2-3 cycles of processing in the Microsep spin filter.

1. Select the Microsep spin filter with an MWCO three times smaller than the MWt. of the protein to be retained.
2. If the devices have been pre-treated, proceed directly to the Step C4.
3. Add 3.5 mL of high purity water to the retentate cup and centrifuge at 3,000-7,500 x g for 30-90 minutes depending on the MWCO membrane used (see Table 4.36). Discard the filtrate.
4. Add up to 3.5 mL of the sample and centrifuge as described in Step B3. At this stage, it is important to achieve concentration of the sample to < 0.05 mL to achieve efficient buffer exchange. Transfer the filtrate into a clean tube and retain in case the protein of interest was not retained by the UF membrane.
   Tip: A pilot experiment is usually necessary to confirm that > 99% of the protein target is retained before using this MWCO membrane for buffer exchange.
5. Commence buffer exchange by adding 3.5 mL of the second buffer to the retentate cup. Mix using a pipette (cycle up and down) to thoroughly mix the retentate with the new buffer solution. Re-centrifuge as described in Steps B3-B4.
6. Usually 2-3 cycles of buffer exchange will remove over 99% of the original components of the sample and achieve buffer exchange. Monitor pH and conductivity after each step to follow the progress of buffer exchange.
   Tip: Multiple buffer exchange steps can decrease overall yields.
7. Recover the retained sample with a pipette tip (see Figure 4.30). To maximize recovery, rinse the retentate cup twice with 0.01-0.02 mL new buffer.

D. Desalting of Purification Samples (1-3.5 mL)

During purification steps, samples are frequently eluted from chromatography surfaces with high salt (up to 3 M NaCl) or biospecific eluates, such as 200 mM imidazole (see IMAC HyperCel™ resin), or 5 mg/mL heparin resin (see Heparin HyperD® F resin). These samples need to be desalted to remove reagents that can interfere with later purification steps or may inhibit biological activity in an assay. Detergents at concentrations above their critical micelle concentrations (CMC), such as Triton^♦-X100, Tween-20, CHAPS, or SDS, are more difficult to remove by size exclusion since they are present in solution as large micelles. The micellar state of these detergents prevents then from being easily resolved from the molecule of interest. For this application, SDR HyperD F resin (see Section 2.3.1) is highly recommended. If the detergent to be desalted is present lower than its CMC, then it may be possible to remove these low molecular weight materials by UF-based desalting. On removal of a detergent, sample solubility can change and may lead to aggregation or precipitation. It may be necessary to carry out exchange (see Section 2.4) to place the sample into a new buffer system, to maintain sample solubility. It is highly recommended to carry out some pilot experiments to confirm that detergent in its non-micellar state can be removed from the sample without compromising its solubility.

1. Select the Microsep™ spin filter with an MWCO three times smaller than the MWt. of the protein to be retained.
2. If the devices have been pre-treated, proceed directly to Step D4.
3. Add 3.5 mL of high purity water to the retentate cup and centrifuge at 3,000-7,500 x g for 30-90 minutes depending on the MWCO membrane used (see Table 4.36). Discard the filtrate.
4. Add up to 3.5 mL of the sample and centrifuge as described in Step B3. At this stage, it is important to achieve concentration of the sample to < 0.05 mL to achieve efficient desalting. Transfer the filtrate into a clean tube and retain in case the protein of interest was not retained by the UF membrane.
   Tip: A pilot experiment is usually necessary to confirm that > 99% of the protein target is retained before using this MWCO membrane for desalting.
5. Commence the desalting process by adding 3.5 mL of high purity water to the retentate cup. Mix using a pipette (cycle up and down) to thoroughly mix the retentate with the new buffer solution. Re-centrifuge as described in Steps B3-B4.
6. Usually 2-3 cycles of desalting will remove over 99% of the salt from the sample.
   Tip: Multiple desalting steps can decrease overall yields.
7. Recover the retained sample with a pipette tip (see Figure 4.30). To maximize recovery, rinse the retentate cup twice with 0.01-0.02 mL high purity water.

E. Concentration of Samples (1-3.5 mL)

Samples eluting from chromatographic processes are frequently more dilute than the original starting sample. In many cases, the samples recovered are too dilute for the next step of processing or for detection in a biological assay. Re-concentrating dilute samples is a key application for UF membrane devices in purification processes. This process can efficiently remove solvent and retain samples of interest up to very high protein concentrations (> 20 mg/mL have been achieved with BSA). At these high levels, some protein-protein aggregation and, in extreme cases, precipitation can occur. It is highly recommended that some pilot studies be carried out to ascertain whether there are any protein-protein interactions and set some limits on the concentration target for the filtrate in this UF membrane-based process.

1. Select the Microsep™ spin filter with an MWCO three times smaller than the MWt. of the protein to be retained (see Tables 4.35 and 4.36).
2. If the devices have been pre-treated, proceed directly to Step E3.
3. Add 3.5 mL of high purity water to the retentate cup and centrifuge at 3,000-7,500 x g for 30-90 minutes depending in the MWCO membrane used (see Table 4.36). Discard the filtrate.
4. Add up to 3.5 mL of the sample and centrifuge as described in Step B3. Transfer the filtrate into a clean tube and retain in case the protein of interest was not retained by the UF membrane.
5. Recover the retained sample with a pipette tip (see Figure 4.30). To maximize recovery, rinse the retentate cup twice with 0.01-0.02 mL new buffer or water.

Application Data for Microsep UF Spin Filter for Samples 1-3.5 mL

Pall centrifugal UF spin filters are ideal for the removal or exchange of buffers and salts. Desalting by dialysis is time-consuming and does not concentrate dilute samples and results in further dilution of the original sample. A single round of protein concentration using UF results in a sample with essentially the same buffer or salt composition as the starting material. To remove salts or exchange buffers, the concentrated sample is diluted with the new buffer or water and centrifuged a second time (this process is called discontinuous diafiltration). The dilution/concentration steps can be repeated until the required amount of salt is removed or exchanged. The results for the removal of 0.5 M NaCl from a 5 mg/mL human serum albumin (HSA) solution are summarized in Table 4.37 for a Microsep spin filter. The ionic strength of the sample retentate was monitored by conductivity measurement with a Horiba Model B173 miniature device. This device gave a linear conductivity response from 1.0 M down to 10-100 μm NaCl in high purity water. Protein was monitored by absorbance at 280 nm. The result showed a high recovery of protein and > 99.9% removal of NaCl after three cycles. Total elapsed time was 90 minutes for the Microsep spin filter. After one 10-30 minute spin, > 98% of the NaCl was removed in the filtrate. Two more spins achieved complete desalting.

Table 4.37

Diafiltration to Remove NaCl from a BSA Solution in Microsep™ Spin Filters Using a 10K MWCO Omega™ Membrane

	Starting Sample (0.5 M NaCl)	Cycle #1 (% Desalting)	Cycle #2 (% Desalting)	Cycle #3 (% Desalting)
Conductivity of Retentate (mS/cm)	9.25	0.69	0.048	0.008
% Desalted	100%	98.4%	99.8%	99.9%
Protein Recovery				94.3%

A BSA sample (5 mg/mL) in 0.5 M NaCl was processed for desalting. After each cycle of concentration, 0.45 mL or 3.5 mL of high purity water (conductivity < 0.002 mS/cm) was added to the retentate and mixed with a pipette (5x up-down cycles). A sample (0.1 mL) was removed and measured for conductivity on the Horiba model B173 meter. After measurement, the sample was recovered and returned to the retentate cup of the Microsep UF spin filter. The retentate was then re-mixed with a pipette as described above. After the final cycle of desalting, 0.2 mL of water was added to the retentate, mixed with a pipette and transferred to a microcentrifuge tube. An additional 0.2 mL of water was then used to rinse the device with a pipette, then removed and added to the original retentate. The pooled volume was then measured by aspiration up into a 1 mL pipette and the protein content measured by absorbance at 280 nm.

Troubleshooting for Microsep UF Spin Filter for Samples 1-3.5 mL

1. Common variables that increase molecule passage:
   • Molecular shape, at the same MWt. A molecule can exhibit a different hydrodynamic shape or Stokes radii in the linear or globular states.
   • High trans-membrane pressure created by too high a g-force in centrifugal concentrators. (Especially important in the case of linear molecules, for example DNA fragments. Decreasing the g-force can increase retention of molecules by a membrane.)
   • Buffer composition that leads to dissociation of multi-sub-unit proteins or protein-protein complexes to yield individual sub-units.
   • pH and ionic conditions that induce conformational changes in a molecule leading to a small apparent hydrodynamic shape.
2. Common variables that decrease molecule passage:
   • Buffer conditions that induce molecular aggregation.
   • Presence of other molecules that increase sample concentration.
   • Lower trans-membrane pressure (in the case of centrifugal concentrators, too low a g-force).
   • Non-specific adsorption to the membrane or device.
   • Low temperature (4 °C versus 24 °C) which can increase solution viscosity or lead to aggregation due to changes in solubility.

Ordering Information for Microsep™ UF Spin Filter for Samples 1-3.5 mL

Microsep Centrifugal Devices, Omega™ Membranes

Part Number	Description	Pkg
OD001C41	1K, yellow	24/pkg
OD001C46	1K, yellow	100/pk
OD003C41	3K, grey	24/pkg
OD003C46	3K, grey	100/pk
OD010C41	10K, blue	24/pkg
OD010C46	10K, blue	100/pk
OD030C41	30K, red	24/pkg
OD030C46	30K, red	100/pk
OD050C41	50K, green	24/pkg
OD050C46	50K, green	100/pk
M OD100C41	100K, clear	24/pkg
OD100C46	100K, clear	100/pk
OD300C41	300K, orange	24/pkg
OD300C46	300K, orange	100/pk
OD990C41	1000K, purple	24/pkg
OD990C46	1000K, purple	100/pk

References for Microsep UF Spin Filter for Samples 1-3.5 mL

1. Vollmers, H.P., Wozniak, E., Stepien-Botsch, E., Zimmermann, U., & Muller-Hermelink, H.K. (1996). A rapid method for purification of monoclonal human IgM from mass culture. Hum Antibodies Hybridomas, 7(1), 37–41.
2. Van Oss, C.J., & Bronson, P.M. (1970, August). Removal of IgM from serum by ultrafiltration. Anal Biochem., 36(2), 464–469.

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