Protein A Columns. MoBiTec GmbH, 2015 Page 1

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1 MoBiTec GmbH, 2015 Page 1 Protein A Columns

2 MoBiTec GmbH, 2015 Page 2 Content 1. Background... 3 Kit components Intended use Specifications Examples Elution Elution profile Protein recovery Procedure for immunoglobulin purification Sample preparation Column preparation Protocol Adsorption Wash Elution Column regeneration Cleaning Storage Sanitization Product life Precautions and safety procedures Quality assurance Buffers References Order information, shipping and storage Contact and support Our products are developed, designed and sold for research purposes only.

3 MoBiTec GmbH, 2015 Page 3 1. Background The compact affinity columns with immobilized Protein A facilitate highly efficient, cost effective purification for both monoclonal and polyclonal antibodies. These columns bind antibodies under binding buffer conditions, the antibodies are eluted from the column under elution buffer conditions, and the columns are then washed and stored under storage buffer. They can be stored and reused many times. The immobilization of Protein A has been developed to satisfy four critical factors: Orientation of the ligand Distribution of the ligand Stability of the immobilized ligand Elimination of non-specific surface interactions The protein is immobilized on controlled pore glass which is more rigid and durable than conventional polymeric (for example agarose) matrices. This glass also has a very uniform internal pore size distribution which falls within a very narrow range, resulting in uniform rates of diffusion and hence rapid transfer between solution and solid phase. The protein molecules are orientated and immobilized on the glass surface such that pairs of Protein A molecules can interact with the binding regions of each of the two Fc portions of the immunoglobulin at the same time. This results in very high binding capacities, especially for those immunoglobulins which typically do not bind well to conventional immobilized Protein A and where this bivalent binding is an essential prerequisite (for example mouse IgG 1 ). The increased capacity does not, however, result in a higher association constant (Ka) and therefore, elution conditions remain unaltered. The columns with immobilized Protein A have the following advantages: The Protein A columns are ideally suited for the efficient, cost effective and rapid purification of antibodies with minimal pre-treatment Protein A columns have good flow properties The capacity of these Protein A columns for murine IgG at physiological ph and salt concentration are greater than that for other Protein A adsorbents Protein A binds with high affinity to human IgG 1 and IgG 2 as well as mouse IgG 2a and IgG 2b, binds with moderate affinity to human IgM, IgA and IgE as well as to mouse IgG 3 and IgG 1, does not react with human IgG 3. Immobilized onto a solid glass support and packed into columns, Protein A purifies total IgG from crude protein mixtures such as serum or ascites fluid. Protein A (about 50 kda) is a cell wall surface protein of Staphylococcus aureus. It binds to immunoglobulins from many mammalian species (most notably IgGs), in particular to their Fc domain through interaction with the heavy chain. The biological function of this interaction is that, in serum, the bacteria will bind IgG molecules in the wrong orientation (with respect to normal antibody function) on their surface which disrupts opsonization and phagocytosis.

4 MoBiTec GmbH, 2015 Page 4 Kit components Order# Components Quantity Compact affinity columns with 500 µl Protein A porous glass matrix 2 Binding Buffer ph 7.4 (PBS tablet for 100 ml) 1 Elution Buffer (0.1 M Glycine/HCl ph 3.0) 5 ml PR-PAK002 Neutralization Buffer (1 M Tris/HCl ph 8.0) 1 ml Luer-lock caps ml reservoir 2 Ultrafiltration spin filters (10 kda) with tubes 2 2 ml tubes 10 Order# Components Quantity PR-PAK005 Compact affinity columns with 500 µl Protein A porous glass matrix 5 columns Order# Components Quantity PR-PAK010 Compact affinity columns 500 µl Protein A porous glass matrix 10 columns Order# Components Quantity Binding buffer for 10 applications (PBS tablets for 100 ml) 2 Elution buffer for 10 applications 25 ml Neutralization buffer for 10 applications 5 ml PR-PK001 Ultrafiltation spin filters (10 kda) and tubes 10 Luer-lock caps ml reservoir 10 2 ml tubes Intended use Columns with immobilized Protein A may be used for many applications including: Purification of monoclonal antibodies Selective purification of antibody fragments Purification or removal of polyclonal IgG from serum Separation of IgG sub-classes using a stepwise ph gradient The immobilized Protein A is manufactured by BioProcessing Ltd., Consett, England, under the registered trade mark PROSEP-A.

5 MoBiTec GmbH, 2015 Page 5 Table 1. Relative affinity of Protein A and Protein G. Protein A G Human IgG Human IgG Human IgG Human IgG Human IgA + - Human IgD + - Human IgE + - Human IgM + - Mouse IgG Mouse IgG 2a Mouse IgG 2b Mouse IgG Mouse IgM +/- - Rat IgG Rat IgG 1 +/- + Rat IgG 2a +/- ++ Rat IgG 2b +/- + Rat IgG 2c +/- + Rat IgM +/- - Rabbit IgG Hamster IgG + ++ Guinea pig IgG ++ + Bovine IgG + + Sheep IgG +/- + Goat IgG +/- + Pig IgG Chicken IgG - +/- Fragments Human Fab + + Human F(ab ) Human scfv + - Human Fc + + Human κ - - Human λ = strong affinity + = moderate/slight affinity +/- = requires evaluation - = no affinity

6 MoBiTec GmbH, 2015 Page 6 3. Specifications Matrix Controlled Porous Glass with particle size between 75 and 125 µm Pore size 1000 Å ph range Stable from ph 1.0 to ph 9.0 Leakage Flow rate Life Binding capacity Less than 15 ng Protein A/mg of mouse IgG 1 purified (determined using an EIA system sensitive to 1 ng/ml). 2 ml per minute. Control flow rate by using a syringe or pump At least 20 cycles without significant loss of capacity using the recommended procedures Table 2 shows typical capacities for a range of polyclonal and monoclonal antibodies Table 2. Maximal binding capacity of a column with 500 µl immobilized Protein A matrix. Monoclonal Antibodies Polyclonal Antibodies Subclass Capacity (mg/ml) IgG Species Capacity (mg) Mouse IgG 1 κ 6 Human >20 Mouse IgG 2a 15 Bovine 13 Mouse IgG 2b 11 Goat 8 Mouse IgG 3 18 Guinea Pig 19 Mouse IgG 3 λ 14 Mouse 16 Mouse IgG 1 7 Porcine 19 Rabbit 18 Monoclonal antibodies were adsorbed from 0.1 M borate buffer at ph 8.5 containing 0.15 M NaCl and eluted with 0.1 M citrate at ph 6.0 to 3.0. Rat 6 Sheep 6 Polyclonal antibodies were adsorbed from PBS at ph 7.4 (page 14) and eluted with 0.1 M glycine/hcl at ph 3.5 to 3.0.

7 MoBiTec GmbH, 2015 Page 7 4. Examples 4.1 Elution The elution yield is determined by SDS-PAGE gel analysis of the washing and elution steps. Fig. 1.: SDS-PAGE analysis of washing and elution steps. M: protein markers, C: loading, Flow: flow-through 1 and 2, Wash: washing steps 1, 2 and 3, Elution: elution steps 1, 2, 3, 4 and 5. 1:4 diluted rabbit serum was loaded onto the column (C) and the flow-through analyzed (Flow). For washing (Wash), about 5 ml PBS buffer (10 column volumes) was loaded onto the column and the flow-through was collected in 1.5 ml aliquots; 3 aliquots were loaded on the gel. For elution (Elution), 5 ml elution buffer 0.1 M Glycine/HCl ph 3.0 was loaded onto the column and the flow-through was collected in 0.5 ml aliquots; the first 5 aliquots were loaded on the gel. The large amount of the dominant 65 kda protein runs through the column and does not bind. The antibody heavy chain (50 kda) does bind to the column (band present in lane C but not in Flow and Wash lanes). The antibody heavy (50 kda) and light (22 kda) chains elute in elution aliquots 2 to 4 and mainly in elution step Elution profile The elution profile was determined by measuring the OD 280 of flow-through, washing and elution samples. 0.5 ml rabbit serum was loaded onto the Protein A column and washed with 10 ml PBS (page 14). Protein was eluted from the Protein A columns by applying 5 times 400 µl elution buffer (page 14). After the second elution step, over 85% of bound protein was eluted from the Protein A column (Fig. 2).

8 MoBiTec GmbH, 2015 Page 8 Fig. 2. Extinction at 280 nm of flow-through, washing and elution steps. The column is washed 10 times with 0.5 ml PBS, then 5 times with 1 ml PBS (blue curve). Flow-through of every step was collected and measured at 280 nm. ODs were measured in diluted solution, the obtained values were used to calculate the OD in the undiluted solution. The OD 280 of flowthrough and washing samples varied between and Elution is done with 400 µl elution buffer 0.1 M Glycine/HCl ph 3.0 in 5 steps (12 to 14 ml, red curve). In the third step, the OD 280 was Protein recovery Different amounts (0.25, 0.5 and 1 ml) of rabbit serum were loaded onto Protein A columns to determine protein recovery. After washing and elution, the eluates were pooled and concentrated by ultrafiltration cartridges. Protein recovery was determined by measuring the extinction at 280 nm using a spectrophotometer. In Figure 3 protein recovery of these amounts of serum are shown. It illustrates that an input of 1 ml rabbit serum yielded in an antibody recovery of 7.9 mg. Fig. 3. Protein recovery from different amounts of serum. 0.25, 0.5, and 1 ml of rabbit serum were loaded onto a Protein A column and protein recovery was detected at 280 nm by a spectrophotometer. Protein recovery ranges from 3.5 mg out of 0.25 ml serum to 7.9 mg out of 1 ml serum.

9 MoBiTec GmbH, 2015 Page 9 5. Procedure for Immunoglobulin Purification 5.1. Sample Preparation Monoclonal Antibodies Cell culture supernatant should be centrifuged, or filtered at pore size 0.2 μm, to remove cell debris before loading onto the column at physiological ph and salt concentration. Adjustment to ph 8.5 or diluting 50:50 (v/v) with binding buffer 0.1 M borate, 0.15 M NaCl, ph 8.5 (page 15) is preferable for increased binding when the antibody concentration is greater than 1 mg/ml. Concentration of antibody by, for example, ultrafiltration or buffer exchange is not required. Ascites should be diluted 50:50 (v/v) with 1 M glycine 0.3 M NaCl, ph 8.6 (page 15) and left at 4 C for 24 hours. Any precipitate should be removed by centrifugation before loading onto the column Polyclonal Antibodies Plasma or serum should be de-lipidized by overnight dialysis at 4 C against 25 mm sodium acetate at ph 5.2, 10 mm NaCl. The centrifuged supernatant should then be dialyzed against, or diluted 50:50 (v/v) with binding buffer PBS (page 14) before loading onto the column. Partial purification of IgG by ammonium sulfate or polyethylene glycol is not required Column preparation The column is supplied in storage buffer containing 100 mm sodium acetate at ph 5.0 1% benzyl alcohol Since the storage buffer contains 1% benzyl alcohol, mouth pipetting should be avoided. Put the Protein A column in vertical flow position and let the matrix material settle down slowly. Then wash the column with 5 ml binding buffer PBS (page 14). Running dry During use, the Protein A column may run dry resulting in air bubbles within the matrix material. In this case, refill the column with buffer, close the column, turn the column upside down once and let the matrix material settle down again slowly. Then, the Protein A column can be used again Protocol 1. Wash column with 5 ml binding buffer. 2. Load Column with feedstock (serum, substrate, plasma) containing antibody (1:1 diluted). 3. Wash column with 5 ml binding buffer. 4. Elute in 5 steps with 400 µl elution buffer each into tubes containing 100 µl neutralization buffer. 5. Regenerate the Column, wash with 5 ml binding buffer and store in storage buffer.

10 MoBiTec GmbH, 2015 Page 10 In general, the flow rate should not exceed 2 ml/minute. For buffers, see chapter 12, page Adsorption As each antibody has different adsorption and elution kinetics, it is preferable to optimize the conditions (buffers, loading concentration, flow rate) for your particular applications. Start using the buffer you normally use for antibody binding to Protein A. However, please note that high salt concentration will result in a high binding capacity but also increases the nonspecific binding; lower salt concentrations increase the specificity of the binding. We therefore suggest to lower the salt concentration to a value where binding capacity and selectivity are optimal. 1. Monoclonal antibodies: We found good binding properties using 1 M glycine, 0.15 M NaCl at ph 8.6 or 0.1 M borate, 0.15 M NaCl at ph 8.5 (page 15). 2. Polyclonal antibodies: We obtained good results with 1 M glycine, 0.15 M NaCl at ph 8.6 or 0.1 M borate, 0.15 M NaCl at ph 8.5 for mouse and rat (page 15) and PBS (page 14) for human and other species. Binding buffers with ph greater than ph 8.6 or containing TRIS should not be used. TRIS appears to interfere with antibody binding on the Protein A column and increases nonspecific binding. Load the Protein A column with feedstock (serum, plasma, solution) containing antibody using a syringe or pump. The columns have excellent flow characteristics. Therefore, limit the flow rate to 2 ml/min (i.e. 33 μl/second, or 1 drop per second). Wash the column with 5 ml binding buffer at 2 ml/minute. The matrix does not exhibit cracking of the column bed as with soft matrices and is unaffected if accidently allowed to run dry during operation; neither does the matrix expand or contract under differing conditions of ionic strength Wash The wash step removes unbound or weakly bound material from the column. Wash buffers usually have the same buffer components as the loading buffer to ensure the captured product remains bound to the column while non-binding contaminants are washed off. As the ligand/ligate binding is a reversible equilibrium process, large volumes (>20 column volumes) of wash buffer should be avoided as this may decrease the dynamic capacity of the separation, especially for weakly binding antibodies such as murine monoclonal antibodies. No more than column volumes of wash buffer should normally be required to wash the Protein A columns. Protein A columns may exhibit non-specific binding due to certain process conditions. Such non-specific binding is characterized as the binding of any non-immunoglobulin species present in the feed which then co-elutes with the IgG. In general, the levels of non-specific binding are very low and the purity of the eluate can routinely exceed 98%. In cases where non-specific binding is observed, it is generally due to either ionic or hydrophobic interaction with the base matrix or the immobilized ligand coupling.

11 MoBiTec GmbH, 2015 Page 11 In the case of non-specific binding due to ionic interactions, adding salt (1 M NaCl) in the post-load wash has been found to be effective. If the non-specific binding is not addressed by high salt wash, it may be due to hydrophobic interaction, and lowering the ionic strength of the wash buffer or alternatively adding detergent in the post-load wash, for instance % Tween or Triton X-100, may be effective. A combination of salt and detergent can be effective if the non-specific binding is due to a combination of ionic and hydrophobic interactions. Reducing the ph of the wash buffer may also help to remove other contaminants Elution To preserve biological activity and reduce IgG denaturation it is preferable to avoid extremes of ph during elution. The following elution buffers were found optimal for different antibodies Monoclonal Antibodies All subclasses of mouse IgG can be purified from these columns by using a stepwise ph gradient from ph 6.0 to 3.0 to separate each subclass. We recommend: 0.1 M citrate ph 6.0 to 3.0 (page 14) Polyclonal Antibodies We recommend (for buffers see chapter 12, page 14): Elution 0. 1 M glycine ph 3.5 to 3.0 Species elution ph Porcine 3.5 Human and other 3.0 Following elution the immunoglobulin solution should be neutralized to ph 6.8 to 7.4 to reduce the possibility of denaturation due to low ph. The recommended method is to elute directly into tubes provided with neutralization buffer (1 M Tris/HCl ph 8.0). This reduces the risk of precipitation and denaturation.

12 MoBiTec GmbH, 2015 Page Column regeneration After use, regenerate the column with 5 ml of HCI at ph 1.5 followed by 5 ml of binding buffer PBS (page 14). 6. Cleaning Cleaning is the removal of residual protein and contaminants from the column. The use of a low ph (ph 1.5) regeneration with phosphoric or hydrochloric acid solution after every cycle is very effective at removing strongly bound material at Protein A media. Both protein A and the controlled pore glass base matrix are stable to prolonged exposure at low ph. In contrast, both protein A and the glass base matrix exhibit limited stability at high ph. Where acid alone is insufficient and a stronger cleaning regime is required, a periodic treatment with 2-6 M guanidine hydrochloride (page 14) is recommended (5 column volumes). Frequency of treatment depends on the degree of fouling but typically every 5-10 cycles is found to be effective. If discoloration of the media due to very dirty feeds is experienced, the use of ethanolic acetic acid has been found to be useful. A wash beginning with 20% ethanol/0.5 M acetic acid is recommended. It is possible to use higher concentrations of ethanol if necessary. Other useful cleaning solutions include: 1-3% Tween or Triton X % ethanol 20% ethanol/2 M acetic acid 0.1 M imidazole/20 % ethanol, ph 7.5 Other detergents may be effective including: 1% sodium dodecyl sulfate 3% sodium deoxycholic acid 2% octyl-b-d glucopyranosid Note: Do not clean with NaOH!!! 7. Storage During use it may be stored in PBS (page 14) containing an antibacterial agent such as 1% benzyl alcohol (see under Sanitization). Store at 2 C to 8 C. If the storage buffer contains Thimerosal, mouth pipetting should be avoided.

13 MoBiTec GmbH, 2015 Page Sanitization On a regular basis sanitize the Protein A column with 5 ml of one of the following solutions before the HCl regeneration: 6 M Guanidine HCl in distilled water (freshly prepared, solutions of Guanidine HCl must not be stored prior to use), or Chloroform/Methanol/H 2 O (50:40:10 (v/v)) 6 M Urea 10% Acetic acid 10% Formic acid Chloroform Ethanol Note: Do not sanitize with NaOH!!! 9. Product life Under storage conditions the column will retain binding capacity for at least six months. Subject to the recommended handling procedure, the column will not show any marked deterioration in binding capacity after usage in 20 cycles. 10. Precautions and safety procedures 1. The column is supplied in buffer containing benzyl alcohol. Therefore, mouth pipetting should be avoided. 2. The presence of proteases in the feedstock, particularly serum or plasma, could have a serious adverse affect on product performance due to Protein A digestion by the proteases. Where protease contamination is suspected or highly probable, the feedstock should be treated with a protease inhibitor prior to loading on the column. 3. Do not expose the column to ph outside of the range of 1.0 to Quality assurance Each batch of immobilized Protein A is tested to determine the binding capacity with standard solutions of human polyclonal IgG and mouse monoclonal IgG1. Leakage of Protein A is also determined for each batch.

14 MoBiTec GmbH, 2015 Page Buffers 12.1 Buffer formulation All formulations given are for the preparation of one liter buffer Binding buffers for polyclonal antibodies 10 mm Phosphate Buffered Saline, ph 7.4 (PBS) Disodium hydrogen orthophosphate (Na 2 HPO 4 x 12 H 2 O): Potassium di-hydrogen orthophosphate (KH 2 PO 4 ): Potassium chloride: Sodium chloride: Distilled water: 3.0 g 0.2 g 0.2 g 8.0 g 10 mm Phosphate Buffered Saline, ph 7.4 (PBS) containing 0.5 M glycine Disodium hydrogen orthophosphate (Na 2 HPO 4 x 12 H 2 O): Potassium di-hydrogen orthophosphate (KH 2 PO 4 ): Potassium chloride: Sodium chloride: Glycine: Distilled water: 3.0 g 0.2 g 0.2 g 8.0 g 37.5 g Elution buffers 0.1 M Glycine/HCl ph Glycine: Distilled water: adjust ph to with concentrated HCl 7.5 g 0.1 M Citrate ph Citric acid monohydrate: Distilled water: Adjust ph to with 5 M NaOH 21 g Neutralization buffer 1 M Tris/HCl ph 8.0 Tris Distilled water: adjust ph to 8.0 with concentrated HCl g Cleaning buffer HCl ph 1.5 Concentrated hydrochloric acid Distilled water: check ph before use 3.0 ml

15 MoBiTec GmbH, 2015 Page 15 6 M Guanidine HCl Guanidine hydrochloride: Distilled water: g Alternative binding buffers for monoclonal antibodies 1 M Glycine, 0.3 M NaCl, ph 8.6 Glycine Sodium chloride Distilled water 5 M NaOH sufficient to adjust ph to g 17.6 g 1 M Glycine, 0.15 M NaCl, ph 8.6 Glycine Sodium chloride Distilled water 5 M NaOH sufficient to adjust ph to g 8.8 g 0.1 M Borate, 0.15 M NaCl, ph 8.5 Boric acid Sodium chloride Distilled water 5 M NaOH sufficient to adjust ph to g 8.8 g 13. References Bennel, M.A. & Watson, D.L., Microbiol. Immunol. 24 (1989) Beyzavi, K. & Wood, H.C., In Separation For Biotechnology Vol 2 eds Pyle D.L. Elsevier Applied Science, 1990 pp Chalon, M.P., Milne, R.W. & Vaerman, J.P., Scad J Immunol. 9 (1979) Groudswaard, J., Van der Doonk, J.A. & Noordzij, A., Scad J Immunol. 8 (1978) Hector Juarez-Salinas L. & Gray, S. OH L., United States Patent no. 4,704,366 Nov Kronvall, G., Grey, H.M. & Williams, R.C., J. Immunol 1052 (1970) Lindmark, R., Thoren-Tolling, K. & Sjoquist, J., J Immunol. Methods 62 (1983) Mackenzie, M.R., Warner, N.L. & Mitchel, G.F., J Immunol 120 (1978) Scott, R.W., Duffy, S.A., Moellering, B.J. & Prior, C., Biotech. Progress 3 (1987) Skvaril, F., Roth-Wicky, B. and Barandum, S., Vox Sang. 38 (1980) Steppes, J.R., Lee, J.M. and Wigeon-Smith., Anal Biochem. 142 (1984)

16 MoBiTec GmbH, 2015 Page Order information, shipping and storage Order# Product Quantity PR-PAK002 Protein A Starter Kit, 2 columns, buffers, 2 ultrafiltration spin filters and tubes Kit PR-PAK005 Protein A Kit with 5 columns Kit PR-PAK010 Protein A Kit with 10 columns Kit PR-PK001 Buffers for Protein A/G Kits for 10 applications with ultrafiltration spin filters and tubes Kit shipped at RT; store at 4 C Empty columns (order# M105035F) are available from MoBiTec. For information on other immobilized biomolecules or custom immobilization service please contact MoBiTec directly. 15. Contact and support MoBiTec GmbH Lotzestrasse 22a D Goettingen Germany Customer Service General inquiries & orders Technical Service Product information phone: +49 (0) phone: +49 (0) fax: +49 (0) fax: +49 (0) order@mobitec.com info@mobitec.com MoBiTec in your area: Find your local distributor at