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Affinity Purification

Affinity interaction chromatography is often the single most effective step in any protein purification procedure. Up to 95% purity can be achieved in one step, depending on the nature of the interaction and the starting composition of the protein solution. Well known examples of highly specific affinity interactions include antibodies and Protein A/G; multiple histidine tags and nickel; streptavidin and biotin; antibodies and antigens; and many others. Less specific interactions are also used for enrichment or depletion protocols, including albumin depletion on cibacron blue resin, glycoprotein enrichment on concavalin A resin, and capture of nucleic acid-binding proteins on heparin resin.

Antibody Purification

Purification Techniques

Antibody purification is performed to concentrate and enrich antigen-specific antibodies and lower background by removing any non-specific proteins. This step is performed when highly purified antibodies are required, such as in immunohistochemistry and immunocytochemistry. Affinity purification of monoclonal and polyclonal antibodies most commonly uses Protein A and Protein G chromatography. The Montage Antibody Purification Kit with PROSEP-A/G media is designed for simple and rapid antibody purification by centrifugation from serum, ascites, or cell culture supernatants.

Scale of Purification

Small Scale Purification

Montage Antibody Purification Kits

The Montage Antibody Purification Kit incorporates pre-packed Protein A or Protein G media plugs and spin columns, along with the buffers and filtration devices necessary for pre-clarification of the source material and concentration of the purified antibody. Spin-columns with PROSEP-A or PROSEP-G media are also available separately.

Antibody samples purified using Montage spin columns may be used in a wide range of laboratory procedures such as immunoprecipitation, immunofluorescence, Western blotting, ELISA, and others. The antibodies can be used for radiolabelling, conjugations (e.g., fluorescein), or preparation of immuno-affinity columns.

Advantages of Using the Montage Antibody Purification Kit with PROSEP®-A or-G Media

• Montage kits offer the researcher a total antibody purification process from the initial clarification stage to the final antibody concentration step.
• Montage kits use a spin column format designed to eliminate tedious chromatographic steps and expensive hardware normally associated with Protein chromatography.
• PROSEP media has a proven track record for biopharmaceutical therapeutic antibody production.
• Minimal hold-up volume of the Protein A or G resin plug ensures high antibody recovery.
• The reusable spin columns are sufficient for 10 purifications each and multiple samples can be processed simultaneously.
• The beaded supports offer excellent flow properties that, combined with the tapering of the spin column, provide uniform flow paths enabling optimal use of the media bed in swinging bucket rotors.

Contents of Montage Antibody Purification Kit with PROSEP-A or -G Media

The Montage Antibody Purification Kit with PROSEP Media contains:

• 2 media plugs containing either Protein A or Protein G
• Montage spin column devices (20 mL capacity in a swinging bucket rotor), qty. 2
• 50 mL centrifuge tubes with caps, qty. 4
• Amicon® Ultra-15 Centrifugal filter devices with 30,000 NMWL Ultracel® regenerated cellulose membrane, qty. 4 - Millipore Cat. No. UFC9 030 24
• Steriflip® sterile 50 mL disposable vacuum filtration devices with MBS/PP housing and 0.22 μm Millipore Express® membrane, qty. 4 - Millipore Cat. No. SCGP 005 25
• Binding Buffer A, qty. 2 bottles, 500 mL each
• Elution Buffer B2, qty. 1 bottle, 500 mL
• Neutralization Buffer C, qty. 1 bottle, 50 mL
• Plug insertion tool
• Laminated protocol card

Background Information

This section provides information that may be helpful when preparing to purify antibodies. Sections will include:

• Choosing between Protein A and Protein G Montage Spin Columns
• Protein A Affinity Media
• Protein G Affinity Media
• General Considerations for Selecting Optimal Binding Conditions for the Montage Antibody Purification Kit with PROSEP Media
• Binding Kinetics of Montage Spin Columns Choosing between Protein A and Protein G Montage Spin Columns

Choosing Between Protein A and Protein G Montage Spin Columns

Immunoglobulin G from most species consists of several subclasses with different biological properties. Four subclasses of IgG have been identified in human (IgG₁, IgG₂, IgG₃, and IgG₄) and in mouse (IgG₁, IgG_2a, IgG_2b and IgG₃). For immunological studies, it is often necessary to isolate one particular subclass of IgG from the other subclasses.

Protein G binds to all major Ig classes except IgM and therefore has a wider reactivity profile than Protein A. However, the binding of IgGs to Protein G is often stronger, making elution and complete recovery of the immunoglobulin more difficult. Interestingly, due to the lower cost of Protein A compared to Protein G, researchers tend to experiment first with Protein A, then Protein G. Protein A withstands harsher conditions used in cleaning and regeneration.

The affinity of interaction of Protein A with mouse IgG subclasses varies. The most common subclass of mouse monoclonal antibodies is IgG₁. Customization of the purification strategy may be required for the affinity separation as mouse IgG₁ does not generally bind well to Protein A. However, as the affinity interaction is pH and salt dependent, under high salt regimes (2-3 M NaCl) and high pH (pH 8-9), the antibodies will bind to Protein A.

Immuoglobin Heavy Chain	Mean serum concentration (mg/mL)	Sedimen-tation constant	Molecular weight	Molecular weight of heavy chain	Number of heavy chain domains	% Carbohydrate
lgG1 g1	9	7S	146,000	51,000	4	2-3
lgG2 g2	3	7S	146,000	51,000	4	2-3
lgG3 g3	1	7S	170,000	60,000	4	2-3
lgG4 g4	0.5	7S	146,000	51,000	4	2-3
lgM μ	1.5	19S	970,000	65,000	5	12
lgA1 g1	3.0	7S	160,000	56,000	4	7-11
lgA2 a1	0.5	7S	160,000	52,000	4	7-11
slgA a1 or a2	0.05	11S	385,000	52-56,000	4	7-11
lgD d1	0.03	7S	184,000	69,700	4	9-14
lgE e1	0.00005	8S	188,000	72,500	5	12

Protein	A	G	Protein	A	G
Human IgG1	++	++	Rat lgM	+/-	-
Human IgG2	++	++	Rabbit lgG	++	++
Human IgG3	-	++	Hamster lgG	+	++
Human IgG4	++	++	Guinea Pig lgG	++	+
Human IgA	+	-	Bovine lgG	+	+
Human IgD	+	-	Sheep lgG	+/-	+
Human IgE	+	-	Goat lgG	+/-	+
Human IgM	+	-	Pig lgG	++	++
Mouse IgG1	+	+	Chicken lgG	-	+/-
Mouse IgG2a	++	++	Fragments
Mouse IgG2b	++	++	Human Fab	+	+
Mouse IgG3	+	++	Human F(ab’)2	+	+/-
Mouse IgM	+/-	-	Human ScFv	+	-
Rat IgG	++	++	Human Fc	+	-
Rat IgG1	+/-	+	Human K	-	-
Rat IgG2a	+/-	++	Human	-	+
Rat IgG2b	+/-	+
Rat lgG2C	+/-	+

Protein A Affinity Media

Protein A is a cell wall protein from Staphylococcus aureus with a molecular weight between 35-50 kDa. The quality of the Protein A media (or equivalent) is important to avoid leakage of Protein A during the elution procedure. PROSEP-A High Capacity media is based on a porous glass matrix that is fully incompressible yet highly porous, with a very high percentage of large, open-ended, interconnected pores. Its open pore structure allows very rapid mass transport, resulting in very high dynamic capacity for IgG. Immobilized Protein A binds specifically to the Fc region of immunoglobulin molecules of many mammalian species.

Protein A affinity chromatography is a rapid one-step purification, which removes most non-IgG contaminants and can achieve purities close to homogeneity. It is particularly useful for purifications of tissue culture supernatant, where 10- to 100-fold concentrations can be achieved.

Protein G Affinity Media

Protein G is a cell wall protein from Streptococci with a molecular weight of 22 kDa. The covalent immobilization of the Protein G is important to avoid leakage of Protein G during the elution procedure. PROSEP-G consists of recombinant Protein G covalently immobilized on porous glass that is more rigid and durable than conventional polymeric matrices. Porous glass also has a very high uniform internal pore size distribution that falls within a very narrow range, resulting in uniform rates of diffusion and hence rapid mass transfer between solution and solid phase. The Protein G that is immobilized is a recombinant protein from which the albumin binding region has been deleted. Immobilized Protein G binds specifically to the Fc region of immunoglobulin molecules of many mammalian species. In many instances antibodies bind more strongly to Protein G than Protein A. PROSEP-G can be used to purify antibodies that may be difficult or impossible to purify on PROSEP-A High Capacity Media (for example, Rat IgG2b and Human IgG3). Excellent yields have been established using PROSEP-G resin with polyclonal rat, rabbit, and human antibodies.

Protein G affinity chromatography is a rapid one-step purification, which removes most non-IgG contaminants and can achieve purities close to homogeneity. It is particularly useful for purifications of tissue culture supernatant, where 10- to 100-fold concentrations can be achieved.

General Considerations for Selecting Optimal Binding Conditions for the Montage Antibody Purification Kit with PROSEP Media

Any antibody-containing sample, such as a crude biological extract, a cell culture supernatant, serum, or ascites can be used in the Montage spin column. It is important that the sample is first filtered through a 0.22 :m Steriflip-GP filter unit to remove particulates that could clog the media flow channels. This is critical for re-use of the device. All samples should be filtered just prior to loading even if they have been filtered several days before the chromatographic run. Aggregation precipitation of proteins is common during storage and repeated freeze/thaw cycles in sera, ascites and tissue culture supernatants. Lipids, which can be found at high levels in serum or ascites, should also be removed (see Delipidation Procedure). Millipore recommends that samples from only one source be run on any given column. In PROSEP separations, the sample should simply be diluted 1:1 (v/v) in 1 x binding buffer.

Eluting the Antibody from a PROSEP-G Spin Column

The most common elution conditions for Protein G affinity separations involve a reduction in pH to pH 2.5. It is important to appreciate that a few proteins (e.g., some monoclonal antibodies) are acid-labile and they can lose their activity at very low pH values. Above all, the elution conditions must preserve the integrity and activity of the target protein. Most observed denaturation is caused by harsh elution conditions. Acidic pH is known to reduce the antibody titre, decrease immunoreactivity and distort the antibody structure. It is therefore critical that the pH is restored to neutrality immediately after elution.

Binding Kinetics of Montage Spin Columns

Controlling the flow rate through an affinity chromatography support is important in achieving binding. Flow rate through the column support is inextricably related to the efficiency of the separation; too fast a flow will cause the mobile phase to move past the beads faster than the diffusion time necessary to reach the internal bead volume. Our studies demonstrate that the large internal surface area of the Montage media bed compensates completely for the velocity of the mobile phase through the column support when the centrifugal speed does not exceed 1,000 Hg* for PROSEP-G or 1,500 xg* for PROSEP-A. The PROSEP media chemistries used in the Montage spin column have sufficiently rapid association kinetics between the protein molecule and the immobilized ligand to allow for optimal diffusional flow through the internal bead structure. Traditionally, gravity flow chromatography is very slow and resolution of the protein separation can be adversely affected by secondary diffusion effects.

Purifying Antibodies

This section outlines the information necessary to purify antibodies using the Montage Antibody Purification Spin Columns with PROSEP Media, including information for those not using the kit about preparing the buffers. Other information will include:

• Loading the Media Plugs into the Spin Columns
• Pre-equilibration
• Sample Clarification
• Sample Loading
• Washing
• Elution
• Regeneration
• Desalting and Concetrating

Protein A or G Kit Buffer Formulation

Use the following recipes to prepare the buffers supplied with the Montage Antibody Purification Kit with PROSEP-G Media. All buffers contain 0.1 % sodium azide as a preservative and can be stored at room temperature.

• Binding Buffer A (1.5 M Glycine/NaOH buffer, 3 M NaCl, pH 9.0) Add 112.6 g glycine (free base; MW 75.07), 175.3 g NaCl (MW 58.44), 1.0 g sodium azide to 800 mL distilled water. Titrate with 5 M NaOH to pH 9.0. Make up final volume to 1 L with distilled water.
• Elution Buffer B1 (0.1 M Sodium citrate buffer pH 5.5) Add 23.44 g citric acid (trisodium salt, dihydrate; MW 294.1), 3.872 g citric acid (anhydrous; MW 192.1), 1.0 g sodium azide to 900 mL distilled water.
• Make up final volume to 1 L with distilled water.
• Elution Buffer B2 (0.2 M Glycine/HCl buffer pH 2.5) Add 15.0 g glycine (free base; MW 75.07), 1.0 g sodium azide to 900 mL distilled water. Titrate with 5 M Hcl to pH 2.5. Make up final volume to 1 L with distilled water.
• Neutralization Buffer C (1 M Tris/HCl buffer pH 9.0) Add 103.72 g Tris base (MW 121.1), 22. 72 g Tris hydrochloride (MW 157.6), 1.0 g sodium azide to 800 mL distilled water. Make up final volume to 1 L with distilled water.

Loading the Media Plug into the Spin Column

1. Unwrap the sealing film from both ends of the plug.
2. Remove top and bottom caps. Note: The dot on the top cap is red for PROSEP-A media plugs and yellow for PROSEP-G plugs. Once the caps are removed, the top end can be identified because it is recessed (approximately 2 mm deep).
3. Insert the plug into the spin column with the top (recessed) end uppermost.
4. Push the plug fully into the tapered end of the spin column using the insertion tool. To remove the plug from the spin column, insert the tool into the bottom of the spin column and push upward.

Antibody Purification Protocol

After loading the plug into the spin column and placing the spin column into a centrifuge tube, follow the procedure below.

Pre-Equilibration

1. Equilibrate the PROSEP media with 10 mL Binding Buffer A by centrifuging the spin column at 500 xg for 5 minutes.
   
   Note:If using one spin column, ensure that the spin column is counter balanced with a unit of equal weight (adjusted with distilled water; without a plug).

Clarification of Sample

2. Pre-filter the sample (e.g., tissue culture supernatant, serum or ascites) through a 0.22 :m Steriflip-GP device to remove any debris immediately before loading the sample. Protein precipitation is common during storage and when repeated freeze/thaw cycles in ascites, sera and tissue culture supernatants occur. Newly formed aggregates and precipitating protein complexes can foul the PROSEP media plug and result in significantly slower flow rates.
   Ensure that the samples are filtered immediately before loading, using filters with pore sizes no greater than 0.22 :m. It is critical to the optimal performance of these devices that these instructions are rigorously followed.
   Note: See P35824, the Steriflip Filter Unit User Guide, for complete information about using the Steriflip filter unit.

Sample Loading

3. Dilute the filtered sample 1:1 v/v in Binding Buffer A. (For example, add 10 mL filtered sample to 10 mL buffer.) Pipette the 20 mL sample into the spin column. Centrifuge the spin column at 100-150 xg for 20 minutes. Ideal sample loading conditions are obtained using a flow rate of less than 1 mL/min. It may be necessary to increase the spin time or spin speed if any sample remains on top of the plug. Alternatively, a flow rate slower than expected is indicative of a partially clogged plug resulting from incomplete filtration of the sample.
   Note: See P35824, the Steriflip Filter Unit User Guide, for complete information about using the Steriflip filter unit.

Washing

4. Wash the spin column to remove unbound contaminants by adding 10 mL Binding Buffer A and centrifuging the spin column for 2 minutes at 500 Hg. Add another 10 mL Binding Buffer A and centrifuge for 2 more minutes at 500 Hg. The unbound wash will contain non-immunoglobulin components. A flow rate slower than expected is indicative of a partially clogged plug resulting from incomplete filtration of the sample. In the unlikely event that flow rates are significantly slower than those expected, increase the centrifugal speed to 1000 xg (1000-1500 xg for PROSEP-A) with 5 minute spin time bursts.

Elution

5. PROSEP-A only (for PROSEP-G go to step 6) Elute the bound IgG with 10 mL Elution Buffer B1 directly into a fresh centrifuge tube containing 1.3 mL Neutralization Buffer C to bring the sample to neutral pH. Centrifuge the spin column for 5 minutes at 500 xg.
6. Elute the bound IgG with 10 mL Elution Buffer B2 directly into a fresh centrifuge tube containing 1. 3 mL Neutralization Buffer C to bring the sample to neutral pH. Centrifuge the spin column for 5 minutes at 500 x g. A flow rate slower than expected is indicative of a partially clogged plug resulting from incomplete filtration of the sample. In the unlikely event that flow rates are significantly slower than those expected, increase the centrifugal speed to 1000 x g with 5 minute spin time bursts.

Regeneration

7. Wash the media with 10 mL Elution Buffer B2 by centrifuging the spin column at 500 Hg for 5 minutes. Re-equilibrate the media with 5 mL of Binding Buffer A by centrifuging the spin column at 500 Hg for 2 minutes. For immediate reuse, wash with 5 mL of Binding Buffer A, or store the plugs without their end caps in 5 mL of Binding Buffer A in a 15 mL screw-capped tube for further use.

Desalting and Concentrating

8. If necessary, desalt and concentrate the antibody preparation using the Amicon Ultra-15 centrifugal filter device with 30,000 NMWL, supplied in the Montage kit. Add 0.05-0.5% w/v sodium azide if the antibodies are to be stored at 2—8°C. We recommend freezing the antibodies in small aliquots in 50%25 glycerol at —20°C for long term storage.
   Note: See P36375, the Amicon Ultra-15 Centrifugal Filter Device User Guide, for complete information about using Amicon Ultra Centrifugal Filter Devices.

Delipidation Procedure

All Protein A and G affinity columns are affected by the presence of lipids and lipoproteins, especially in antibody samples derived from ascites fluid. For end users who have antibody solutions that they need to delipidate, the following protocol is a gentle and easy method for removing lipids and lipoproteins.

1. Add 0.04 mL 10% dextran sulphate solution and 1 mL 1 M calcium chloride per mL sample.
2. Mix for 15 minutes.
3. Centrifuge at 10,000 x g for 10 minutes.
4. Discard the precipitate.
5. Exchange the sample into TBS (Tris Buffered Saline) using an Amicon Ultra-15 device (Millipore Cat. No. UFC9 030 24), dialysis, or a desalting column. Do not buffer exchange into a phosphate-containing buffer such as PBS.

Optional Quantitation Procedure

By using the Beer-Lambert law, A = e.c.l, the concentration of IgG (mg/mL) in the sample can be measured by multiplying the absorbance at 280 nm by 0.72. If IgM or IgA are purified, multiply the absorbance at 280 nm by 0.84 or 0.94, respectively. These antibody concentrations are only estimates as other contaminating proteins can also contribute to the absorbance reading. However, they can provide a reliable and quantitative method for determining the concentrations of pure antibody solutions. Most researchers use different sandwich ELISA assays to accurately measure antibody concentrations within ranges of 0.01 mg/mL to 20 mg/mL.

Product Performance

The antibodies are monitored for purity by SDS-PAGE under reducing or non-reducing conditions. Note that IgG appears in a reducing SDS-PAGE as 25 kDa and 50-55 kDa bands. Recovery of immunoglobulins can be quantified by a standard protein assay, scanning densitometry of reducing or non-reducing SDS-polyacrylamide gels or ELISA. Antigen binding parameters can be measured for both affinity and avidity.

PROSEP Media Plug Specifications

Source: Recombinant Protein A or G expressed in E. coli Supporting matrix: Porous glass Media particle size: 74 :m-125 :m Media bed volume: 1. 6 mL Recommended working pH: pH 2.0-9.0 Binding capacity: for PROSEP-A typically >15 mg rabbit IgG and for PROSEP-G typically >8 mg rabbit IgG Solubility in water: Insoluble Maximum volume in swinging bucket rotor: 20 mL

Chemical Compatibility of the Montage Spin Columns

All resins are susceptible to oxidative agents. Avoid high temperatures. Protein G is resistant to limited exposure to 8 M urea pH 10.5 and extremes of pH (e.g. pH 1.0 and pH 11). It lacks the robustness of Protein A with respect to 1 M NaOH and it is denatured under these aggressive conditions. However, Protein G is stable to treatment with 0.1 M NaOH.

Affinity Purification with Ultrafree-MC Centrifugal Filters

We show the applicability of the device for purification of rabbit IgG on PROSEP-A resin and His-tagged C-RP protein on three different commercial metal-chelate resins.