Buffer Exchange, Desalting, and Concentration Using Jumbosep™ UF Spin Filter for Samples (15-60 mL) 4.4.4

Table 4.41

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 Jumbosep™ UF Spin Filter for Samples 15-60 mL

A simple guide to choosing the appropriate MWCO UF membrane in the Jumbosep device for a range of purification applications is summarized in Table 4.42. Full specifications of the Jumbosep UF device are summarized in Table 4.43 and a diagram in Figure 4.34. 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 4.42

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 4.43

Specifications of the Jumbosep™ Spin Filter

Specification	Parameter
UF Membrane	Omega™ membrane (low protein-binding, modified polyethersulfone on polyethylene substrate)
Materials of Construction      Device      Collection Tube	Polysulfone and polyethylene Polypropylene
Effective Membrane Area	15.2 cm2
Dimensions      Diameter      Overall Length (with Cap)	6.0 cm 11.3 cm
Capacities      Maximum Sample Volume      Final Retentate Volume      Final Receiver Volume      Hold-up Volume (Membrane/Support)	60.0 mL 3.5-4.0 mL 60.0 mL 0.02 mL
Operating Temperature Range	0-40 °C (32-104 °F)
pH Range	1-14
Maximum Centrifugal Force	3,000 x g
Centrifuge	Rotor accepting 250 mL bottles
Sanitization	70% ethanol

Protocol for Jumbosep UF Spin Filter for Samples 15-60 mL

Centrifugation up to 3,000 x g provides the driving force for filtration, moving samples toward the highly selective, low protein-binding Omega UF membrane. Macromolecules larger than the membrane’s nominal MWCO are retained in the sample reservoir. Solutes and macromolecules smaller than the MWCO of the membrane pass through the membrane surface into the membrane insert and through the filtrate port into the filtrate receiver. See Figure 4.34 for a diagram showing the components of the Jumbosep centrifugal UF device.

A. Materials Required

1. Jumbosep UF devices with Omega MWCO UF membrane and a collection tube. For specifications, see Table 4.43 and Figure 4.34.
2. Extra filtrate receiver bottles for the Jumbosep UF device
3. Degassed high purity water or buffer, such as phosphate buffered saline (PBS)

Figure 4.34

Components of the Jumbosep™ Centrifugal UF Device

 

B. Basic Instructions for Use

1. Separate the filtrate receiver from the sample reservoir.
2. Remove the protective plastic from the membrane insert.
   Tip: The color of the button on the top of the membrane insert indicates the MWCO of the membrane.
3. Hold the membrane insert by the edge with the filtrate port facing down and drop the insert into the sample reservoir.
4. Place the sample reservoir on a hard surface and, with both thumbs placed on the colored button in the middle of the membrane, press down firmly on the membrane insert. The membrane insert rests on the knobs at the bottom of the sample reservoir. 
5. Attach the empty filtrate receiver to the bottom of the sample reservoir.
6. Add 15-60 mL of sample to the sample reservoir. Place the cap on top of the reservoir to prevent evaporation during centrifugation.
7. Place the device in a swinging bucket rotor that accepts standard 250 mL bottles. Remove any bottle adapters to ensure that the bottom of the bucket is flat. Presence of the adapters might cause deformation of the bottom of the Jumbosep device’s filtrate receiver.
   Tip: Always counterbalance the rotor with another Jumbosep device containing an equivalent sample volume.
8. Spin at 1,000-3,000 x g, typically for 15 to 40 minutes, to achieve the desired retentate volume. It is recommended that spin times and g-force be determined experimentally for each application.
   Tip: Maximum g-force is 3,000 x g. Higher g-forces may cause retentate leakage into the filtrate.
9. At the end of spin time, stop the centrifuge and remove the Jumbosep devices.
10. Separate the sample reservoir from the filtrate receiver in the following manner. Hold the device so that both palms are placed on the filtrate receiver, with both thumbs placed side by side on the sample reservoir, press upward.
11. To recover retentate, pour off the retentate into a storage vessel. Some retentate will remain under the membrane insert. To remove the remaining retentate, twist the insert release onto the sample reservoir. Turn the sample reservoir sideways (taking care that the retentate remains in the sample reservoir). Slide a pipette tip under the dislodged membrane insert and remove the remaining retentate. 
12.  For application specific protocol, see Section 2.4.2.1.

C. Buffer Exchange of Purification Samples (15-60 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 Table 4.41) to retain the molecule of interest, buffer exchange can be achieved within 2-3 cycles of processing in the Jumbosep™ device.

1. Select the Jumbosep device 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 C4.
3. Add 15-60 mL of high purity water into the sample reservoir. Centrifuge as described in Steps B7-B8 above. Discard the filtrate.
4. Add up to 15-60 mL of the sample and centrifuge as described in Steps B7-B8. Transfer the filtrate into a clean tube and retain in case the protein of interest was not retained by the UF membrane. At this stage, it is important that the retentate volume be < 4.0 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 15-60 mL of new buffer to the sample reservoir. Mix using a pipette (cycle up and down) to thoroughly mix the retentate with the new buffer. Place cap on reservoir. Re-centrifuge as described in Steps B7-B8.
6. Usually three cycles of buffer exchange will remove over 99% of the original components and achieve buffer exchange.
   Tip: Multiple buffer exchange steps can decrease overall yields.
7. Recover the retained sample as described in Step B11.

D. Desalting of Purification Samples (15-60 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™) or 5 mg/mL heparin (see Heparin HyperD® F). 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 them 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 Jumbosep™ device 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 15-60 mL of high purity water into the sample reservoir. Centrifuge as described in Steps B7-B8 above. Discard the filtrate.
4. Add up to 15-60 mL of the sample and centrifuge as described in Steps B7-B8. Transfer the filtrate into a clean tube and retain in case the protein of interest was not retained by the UF membrane. At this stage, it is important that the retentate volume be < 4.0 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 desalting by adding 15-60 mL of high purity water to the sample reservoir. Mix using a pipette (cycle up and down) to thoroughly mix the retentate with the water. Place cap on reservoir. Re-centrifuge as described in Steps B7-B8.
6. Usually three cycles of desalting will remove over 99% of the original low molecular weight salts and achieve the necessary desalting.
   Tip: Multiple desalting steps can decrease overall yields.
7. Recover the retained sample as described in Step B11.

E. Concentration of Samples (15-60 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 Jumbosep device with a 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 E4.
3. Add 15-60 mL of high purity water into the sample reservoir. Place cap on reservoir. Centrifuge as described in Steps B7-B8 above. Discard the filtrate.
4. Add up to 15-60 mL of the sample and centrifuge as described in Steps B7-B8. 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 as described in Step B11.

Troubleshooting for Jumbosep™ UF Spin Filter for Samples 15-60 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-subunit proteins or proteinprotein 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 Jumbosep™ UF Spin Filter for Samples 15-60 mL

Jumbosep Centrifugal Devices

Part Number	Description	Pkg
FD003K65	3K, starter kit, gray	4/pkg
OD003C65	3K, membrane insert, gray	12/pkg
FD010K65	10K, starter kit, blue	4/pkg
OD010C65	10K, membrane insert, blue	12/pkg
FD030K65	30K, starter kit, red	4/pkg
OD030C65	30K, membrane insert, red	12/pkg
FD100K65	100K, starter kit, clear	4/pkg
OD100C65	100K, membrane insert, clear	12/pkg
FD300K65	300K, starter kit, orange	4/pkg
OD300C65	300K, membrane insert, orange	12/pkg

References for for Jumbosep UF Spin Filter for Samples 15-60 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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