Buffer Exchange, Desalting, and Concentration Using Macrosep® UF Spin Filter for Samples (3.5-15 mL) 4.4.3

Table 4.38

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 Macrosep™ UF Spin Filter for Samples 3.5-15 mL

A simple guide to choosing the appropriate MWCO UF membrane in the Macrosep device for a range of purification applications is summarized in Table 4.39. Full specifications of the Macrosep UF device are summarized in Table 4.40 and a diagram in Figure 4.31. 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 4.39

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 4.40

Specifications of the Macrosep™ Spin Filter

Specification	Parameter
UF Membrane	Omega™ membrane (low protein-binding, modified polyethersulfone on polyethylene substrate)
Materials of Construction      Device      Collection Tube	Polypropylene and polyethylene Polypropylene
Effective Membrane Area	10 cm2
Dimensions      Diameter      Overall Length (with Cap)	2.9 cm 10.9 cm
Capacities      Maximum Sample Volume      Final Retentate Volume      Final Receiver Volume      Hold-up Volume (Membrane/Support)	15 mL 0.5-1.5 mL 15 mL 0.03 mL
Operating Temperature Range	0-40 °C (32-104 °F)
pH Range	1-14
Maximum Centrifugal Force	5,000 x g
Centrifuge	Rotor accepting 50 mL volume tubes
Sanitization	70% ethanol

Figure 4.31

Components of the Macrosep™ Centrifugal UF Device

 

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

Each Macrosep spin filter consists of a sample reservoir, a sample reservoir cap, a membrane paddle, a concentrate cup and cap, a filtrate receiver, and a filtrate receiver cap. Centrifugation up to 5,000 x g provides the driving force for filtration, moving the sample towards the encapsulated Omega™ membrane. Biomolecules larger than the nominal MWCO of the membrane are retained in the sample reservoir. Solvent and low molecular weight molecules pass through the membrane into the filtrate receiver.

A. Materials Required

1. Macrosep UF spin filters with Omega MWCO UF membrane and a collection tube. For specifications, see Table 4.40 and Figure 4.31.
2. Extra collection tubes for the Macrosep UF device
3. Degassed high purity water or buffer, such as phosphate buffered saline (PBS)

B. Basic Instructions for Use

1. Insert the paddle firmly into the bottom of the sample reservoir. The “hooks” on the top part of the paddle must rest firmly in the notches on top of the sample reservoir (see Figure 4.32, Panel A). For best alignment, turn the reservoir upside down on the bench top and gently press the paddle into place. Once the paddle is firmly in place, there will still be a small gap in between the hooks of the paddle and the notches of the sample reservoir.

   Figure 4.32
   

   Details on Placement of Macrosep® Membrane “Paddle” in the Device and Centrifuge
   
   
   Tip: If pre-flushing to remove glycerin and sodium azide are required, add 15 mL of high purity water into the sample tube and process. Discard the filtrate and repeat with sample.
2. Attach the filtrate receiver to the bottom of the sample reservoir.
3. Pipette 5-15 mL of sample into the non-membrane side of the sample reservoir. Place cap on reservoir.
   Tip: If introducing a volume of 20 mL into the sample reservoir, make sure that the optional 20 mL filtrate receiver is attached.
4. Place the Macrosep spin filter into a bucket or rotor which accepts standard 50 mL tubes.
5. In a fixed-angle rotor, align the Macrosep spin filter so that one of the “hooks” faces the center of the centrifuge rotor (see Figure 4.32, Panel B). This prevents a buildup of macromolecules on the membrane paddle and allows the device’s dead-stop to function properly. A swinging-bucket rotor is self-aligning.
6. Spin at 1,000-5,000 x g, typically for 30 to 90 minutes, to achieve desired concentrate volume. It is recommended that spin time and g-force be determined for each application.
7. At the end of spin time, remove devices from the centrifuge. Concentrate recovery can be accomplished by one of the following methods:
   1. For concentrate volumes less than 1.5 mL
      • (1) Remove filtrate receiver and screw on the concentrate cup (see Figure 4.31). The center pin will cause the paddle to lift up and out of the bottom of the sample reservoir, allowing concentrate to flow into concentrate cup (see Figure 4.33).

        Figure 4.33
        

        Concentrate Recovery in Macrosep® UF Device
        
         
        
      • (2) Place the “hooks” into the notches on the sample reservoir. Replace filtrate receiver.
      • (3) Place the Macrosep device back into the centrifuge and spin at 1,000- 5,000 x g for 1 to 3 minutes. Remove the device and unscrew the concentrate cup.
   2. For concentrate volumes greater than 1.5 mL
      • (1) Pour off concentrate to reduce volume to 1.5 mL or less, then perform Step B7a above.
      • (2) Attach a clean filtrate receiver to the sample reservoir.
      • (3) Lift the membrane paddle out and turn so that the “hooks” rest on the lip of the sample reservoir, not in the notches. This allows concentrate to flow into the filtrate receiver.
      • (4) Spin at 1,000-5,000 x g for maximum concentrate recovery. 

C. Buffer Exchange of Purification Samples (3.5-15 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 4.39 and 4.40) to retain the molecule of interest, buffer exchange can be achieved within 2-3 cycles of processing in the Macrosep spin filter.

1. Select the Macrosep spin filter 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 C4.
3. Add 15 mL of high purity water into the non-membrane side of the sample reservoir. Place cap on reservoir. Centrifuge as described in Steps B5-B6 above. Discard the filtrate.
4. Add up to 15 mL of the sample and centrifuge as described in Steps B5-B6. 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 mL of new buffer to the non-membrane side of the sample reservoir. Mix using a pipette (cycle up and down) to thoroughly mix the retentate with the new buffer solution. Place cap on reservoir. Re-centrifuge as described in Steps B5-B6.
6. Usually three cycles will achieve optimal 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 as described in Steps B7a-B7b.

D. Desalting of Purification Samples (3.5-15 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 which can interfere with later purification steps or may inhibit biological activity in an assay. Detergents at concentrations above their critical micelle points (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 UFbased 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 Macrosep® 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 15 mL of high purity water into the non-membrane side of the sample reservoir. Place cap on reservoir. Centrifuge as described in Steps B5-B6 above. Discard the filtrate.
4. Add up to 15 mL of the sample and centrifuge as described in Steps B5-B6. At this stage it is important to achieve concentration of the sample to < 1 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 mL of high purity water to the non-membrane side of 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 B5-B6.
6. Usually three cycles will achieve optimal desalting.
   Tip: Multiple desalting steps can decrease overall yields.
7. Recover the retained sample as described in Steps B7a-B7b.

E. Concentration of Samples (3.5-15 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 Macrosep® 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 E4.
3. Add 15 mL of high purity water into the non-membrane side of the sample reservoir. Place cap on reservoir. Centrifuge as described in Steps B5-B6 above. Discard the filtrate.
4. Add up to 15 mL of the sample and centrifuge as described in Steps B5-B6. 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 Steps B7a-B7b.

Troubleshooting for Macrosep UF Spin Filter for Samples 3.5-15 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 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 Macrosep® UF Spin Filter for Samples 3.5-15 mL

Macrosep Centrifugal Devices, Omega™ Membrane

Part Number	Description	Pkg
OD001C36	1K, yellow	6/pkg
OD001C37	1K, yellow	24/pkg
OD001C38	1K, yellow	100/pkg
OD003C36	3K, gray	6/pkg
OD003C37	3K, gray	24/pkg
OD003C38	3K, gray	100/pkg
OD010C36	10K, blue	6/pkg
OD010C37	10K, blue	24/pkg
OD010C38	10K, blue	100/pkg
OD030C36	30K, red	6/pkg
OD030C37	30K, red	24/pkg
OD030C38	30K, red	100/pkg
OD050C36	50K, green	6/pkg
OD050C37	50K, green	24/pkg
OD050C38	50K, green	100/pkg
OD100C36	100K, clear	6/pkg
OD100C37	100K, clear	24/pkg
OD100C38	100K, clear	100/pkg
OD300C36	300K, orange	6/pkg
OD300C37	300K, orange	24/pkg
OD300C38	300K, orange	100/pkg
OD990C36	1000K, purple	6/pkg
OD990C37	1000K, purple	24/pkg
OD990C38	1000K, purple	100/pkg

References for Macrosep® UF Spin Filter for Samples 3.5-15 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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