Ion exchange columns and media

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1 GE Healthcare Ion exchange columns and media Product profile

2 Principle of Ion Exchange Chromatography Ion exchange (IEX) chromatography can separate molecules or groups of molecules that have only slight differences in charge. Separation is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. Typically, conditions are selected to ensure that the molecules of interest bind to the medium as they are loaded onto the column. Conditions are then altered so that the bound substances are eluted differentially. Elution is most often performed by a continuous gradient or a stepwise increase in ionic strength, most commonly using NaCl. Figure shows a typical high resolution gradient elution. A stepwise elution is illustrated in Figure 3. Proteins are built up of many different amino acids containing weak acidic and basic groups i.e. ionizable groups that can be titrated. Hence the net surface charge of a protein is highly ph dependent and will change gradually as the ph of the environment changes. Each protein has its own unique net charge versus ph relationship which can be visualized as a titration curve (Figure 2). This curve reflects how the overall net charge of the protein changes according to the ph of the surroundings. IEX can be repeated at different ph values to separate several proteins which have distinctly different charge properties. Figure 2 shows how selecting the correct ph is one of the most important parameters in achieving satisfactory separation. M [NaCl] equilibration unbound molecules elute before gradient begins C sample injection volume gradient elution C Column volumes [C] Figure. Typical IEX separation using gradient elution. Most acidic ph: all three proteins are below their isoelectric point, positively charged, and bind only to a cation exchanger. Proteins are eluted in the order of their net charge. Abs Abs Abs Abs wash high salt wash C Selectivity and buffer ph reequilibration tightly bound molecules elute in high salt wash C Most alkaline ph: all three proteins are above their isoelectric point, negatively charged, and bind only to the anion exchanger. Proteins are eluted in the order of their net charge. Choice of ion exchanger Begin with a strong exchanger (Q, S, SP) to enable development work to be performed over a broad ph range. Use a exchanger (Q) to bind the protein(s) of interest if their isoelectric point is below ph 7. or unknown. Use a strong exchanger in those cases where maximum resolution occurs at an extreme ph and the proteins of interest are stable at that ph. Consider using a weak exchanger (DEAE, ANX, CM) if the selectivity of the strong ion exchanger is unsatisfactory, but remember that the ion exchange capacity of a weak ion exchanger varies with ph. Multimodal ligands (MMC, Adhere) provide ionic interaction, hydrogen bonding and hydrophobic interaction. MMC behaves like a exchanger, but allows binding at high conductivity. Adhere has a exchanger behavior. Media selection Select the ion exchange medium according to the objective of the purification step and the condition of the starting material. Other factors such as sample stability, scale, speed, binding capacity and equipment available may also influence the final choice. Sample preparation Correct sample preparation is essential in order to achieve optimal separation and avoid deterioration in column performance. Samples must be clear and free from particulate matter. To remove particulate matter, filter (see buffer preparation for filter sizes) or centrifuge ( g for min). Desalt samples and transfer into the chosen start buffer. Use HiTrap Desalting ml (volumes up to. ml) or HiPrep 26/ Desalting (volumes up to ml). ery small sample volumes in high salt concentration and with no major contaminants, can be diluted with start buffer to lower the salt concentration to a level that does not interfere with binding to the medium. Column preparation Wash away storage solutions and preservatives before using any IEX medium. Use prepacked columns to ensure the best performance and reproducible results. The volume required for the packed bed is determined by the amount of sample to be purified and the binding capacity of the medium. Pack a column that will have approximately fold excess of the binding capacity required with a bed height up to cm. + Surface net charge Less acidic ph: blue protein is above its isoelectric point, negatively charged, other proteins are still positively charged. Blue protein binds to an anion exchanger and can be separated from the other proteins which wash through. Alternatively, red and green proteins can be separated on a cation exchanger and the blue protein washes through. Abs Abs Abs Abs Figure 2. Effect of ph on selectivity (elution patterns). Cation Anion ph Less alkali ph: red protein below its isoelectric point, positively charged. Red protein binds to cation exchanger and can be separated from the other proteins which wash through. Alternatively, blue and green proteins can be separated on an anion exchanger and the red protein washes through. Buffer preparation See Table for recommendations on volatile and nonvolatile buffer systems. Buffering ions should have the same charge as the functional groups on the IEX medium and a pka within.6 ph units of the working ph. Use a buffer concentration sufficient to maintain buffering capacity and constant ph, typically mm. Filter buffers after all salts and additives have been included. Use high quality water and chemicals. Use µm filters for particle sizes > µm,.4 µm filters for µm particles or.22 µm filters for particles sizes < µm or when sterile or extra clean samples are required. To avoid formation of air bubbles, ensure that column and buffers are at the same temperature. For samples with unknown charge properties, try the following: anion exchange (Q) start buffer: ph 8. elution buffer: start buffer including M NaCl, ph 8. cation exchange (S or SP) start buffer: ph 6. elution buffer: start buffer including M NaCl, ph 6.

3 Method development and optimization (in priority order). Scout for optimum ph by testing a range of ph values within which the proteins of interest are known to be stable. If the isoelectric point of the target protein is known, then begin with a narrower ph range, for example,. ph unit away from the isoelectric point. 2. If required, scout for optimum selectivity (testing strong or weak exchangers) using automatic media scouting. 3. Scout for the steepest gradient that gives acceptable resolution at the selected ph. 4. Scout for the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates for the specific medium.. Scout for the maximum sample load that can be applied while maintaining satisfactory resolution. In general, loading 3% of the total binding capacity of the column gives optimal resolution with gradient elution. 6. For large scale purification, separation times and buffer consumption can be reduced by transfer to a stepwise elution as shown in Figure 3. M [NaCl] sample injection volume equilibration C Figure 3. Typical IEX separation using stepwise elution. Column cleaning unbound molecules elute elution of unwanted material C elution of target molecule C Column volumes [C] high salt wash Correct preparation of samples and buffers and application of a high salt wash ( M NaCl) at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants. It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be taken when changing a filter as this may affect the column packing and interfere with performance. C tightly bound molecules elute reequilibration C Alternatively,. Wash with 2 column volumes of 6 M guanidine hydrochloride (see Table 2 for recommended flow). 2. Wash immediately with at least column volumes of buffer at ph 78 (see Table 2 for recommended flow). 3. Rinse with at least 2 column volumes of distilled water (same flow as step 2) until the Ubaseline and eluent ph are stable. 4. Wash with at least 4 column volumes of start buffer or storage buffer (same flow as step 2) until ph and conductivity values have reached the required values. To remove lipids, hydrophobically bound proteins or lipoproteins Organic solvents or detergents may be required to completely remove contaminants of this type. Before using organic solvents, wash the medium with at least 4 column volumes of distilled water to avoid any salts precipitating on the column. When applying organic solvents or solutions it may be necessary to reduce the flow rate to avoid overpressuring the column. Use cleaning solutions such as up to % isopropanol, up to % methanol, up to % acetonitrile, up to 2 M NaOH, up to 7% acetic acid, up to % ethanol, ionic or nonionic detergents. Always check for solvent compatibility in the instructions supplied with the medium or column. Avoid anionic detergents with Q, DEAE and ANX charged groups. Avoid cationic detergents with S, SP and CM charged groups. Cleaning procedure: alternative. Wash with 4 column volumes of up to 7% ethanol or 3% isopropanol (see Table 2 for recommended flow). 2. Rinse with at least 2 column volumes of distilled water (see Table 2 for recommended flow) until the Ubaseline and eluent ph are stable. 3. Wash immediately with 3 column volumes of start buffer (same flow as step 2). Cleaning procedure: alternative 2. Wash with 2 column volumes of detergent in a basic or acidic solution, e.g...% nonionic detergent in. M acetic acid (see Table 2 for recommended flow). 2. Rinse with column volumes 7% ethanol to remove residual detergent (seetable 2 for recommended flow). 3. Rinse with at least 2 column volumes of distilled water (same flow as step ) until the Ubaseline and the eluent ph are stable. 4. Wash with 3 column volumes of start buffer (same flow as step ). Removal of common contaminants The following procedure should be satisfactory to remove common contaminants:. Wash with at least 2 column volumes of 2 M NaCl (see Table 2 for recommended flow). 2. Wash with at least 4 column volumes of M NaOH (same flow as step ). 3. Wash with at least 2 column volumes of 2 M NaCl (same flow as step ). 4. Rinse with at least 2 column volumes of distilled water (same flow as step ) until the Ubaseline and eluent ph are stable.. Wash with at least 4 column volumes of start buffer or storage buffer (same flow as step ) until ph and conductivity values have reached the required values. To remove precipitated proteins. Inject column volume of pepsin ( mg/ml in. M NaCl,. M acetic acid). Leave overnight at room temperature or for one hour at 37 C. 2. Rinse with at least 2 column volumes of distilled water (see Table 2 for recommended flow) until the Ubaseline and the eluent ph are stable. 3. Wash with at least 4 column volumes of start buffer or storage buffer, same flow as step 2, until eluent ph and conductivity have reached the required values.

4 Technical information Table. Nonvolatile and volatile buffer systems. Buffers for anion exchange chromatography ph interval Substance conc. (mm) Counterion pka (2 C) NMethylpiperazine Cl Piperazine Cl or HCOO LHistidine Cl BisTris Cl BisTris propane Cl BisTris propane Cl Triethanolamine Cl or COO Tris Cl NMethyldiethanolamine 2 SO NMethyldiethanolamine Cl or COO Diethanolamine at ph 8.4 Cl 8.88 at ph Propane,3diamino Cl Ethanolamine Cl Piperazine Cl Propane,3diamino Cl..6.6 Piperidine Cl.2 Handbook of chemistry and physics, 83 rd edition, CRC, 23. Buffers for cation exchange chromatography ph interval Substance conc. (mm) Counterion pk a (2 C).42.4 Maleic acid Na Methyl malonic acid Na + or Li Citric acid Na Lactic acid Na Formic acid Na + or Li Succinic acid Na Succinic acid Na Acetic acid Na + or Li Methyl malonic acid Na + or Li MES Na + or Li Phosphate Na HEPES Na + or Li BICINE Na Handbook of chemistry and physics, 83 rd edition, CRC, 23. olatile buffer systems ph range Buffer system Counterion pkavalues for buffering ions 3..3 Formic acid H ; Pyridine/formic acid HCOO 3.7; ; Trimethylamine/formic acid HCOO 4.7; Pyridine/acetic acid COO 4.7; ; Trimethylamine/acetic acid COO 4.7; ; Ammonia/formic acid HCOO 3.7; ; Ammonia/acetic acid COO 4.7; ; Trimethylamine/carbonate 2 CO 3 6.3; ; Ammonium bicarbonate HCO 3 6.3; ; Ammonium carbonate/ammonia CO 3 6.3; ; Ammonium carbonate 2 CO 3 6.3; : Nethylmorpholine/acetate HCOO 4.7; 7.72 Ref: Handbook of chemistry and physics, 83 rd edition, CRC, 23. ÄKTAdesign chromatography systems provide advice on buffer recipes for each ph and medium. The BufferPrep function, available in selected systems, compensates automatically for changes in ph caused by changes in salt concentration and temperature, ensuring constant ph throughout the separation.

5 Selection Guide Ion Exchange Media Selecting an anion or cation exchanger Ion exchange separates proteins on the basis of differences in their net surface charge in relation to ph of the surroundings. The figure here illustrates how the net charge of a protein can vary with ph. Every protein has its own charge/ph relationship. Will Bind Cation Exchanger Will Bind Anion Exchanger Net Charge of Protein Isoelectric point (pi) Select Anion Exchange at System ph Above pi System ph Select Cation Exchange at System ph Below pi ph If isoelectric point (pi) of the target protein is known: select an anion exchanger (Q, DEAE, ANX) with a buffer ph above the pi. select a cation exchanger (S, SP, CM) with a buffer ph below the pi. Polishing Remove trace impurities or closelyrelated substances Sample condition: almost pure Intermediate purification Remove bulk impurities Sample condition: partially purified High resolution Easy scaleup Highest resolution µg/run Highest resolution mg/run High resolution High throughput Easy scaleup High purity and yield at high sample loads for large molecules. MiniBeads MonoBead s SOURCE SOURCE 3 Sepharose High Performance (Q or SP) MacroCap (SP) Use for intermediate purification if column capacity is sufficient and no scaleup is required. Use for intermediate purification if column capacity is sufficient and no scaleup is required. Can be used for capture steps if sample is free from particulate matter. Use SOURCE when resolution is top priority. Use SOURCE 3 when speed is top priority. Use HiTrap columns prepacked with Sepharose High Performance, Sepharose XL and Sepharose Fast Flow for media selection and ph scouting. Use MacroCap to purify PEGylated proteins and other large biomolecules. RESOURCE Q, ml SOURCE 3Q (XK6 mm) Mini Q (column 4.6 mm) ml... Mono Q (column mm) MacroCap Sample: Pegylated Cytochrome C Resolution If pi is unknown: test for selectivity using a strong ion exchanger, Q, S or SP. Strong ion exchangers maintain their charge over a wider ph range than weak ion exchangers and are suitable for most applications. Easy scaleup Broad choice of selectivity, including alternatives to Q or S ion exchange media Sepharose Fast Flow (Q, SP, DEAE, CM, ANX) Try weak ion exchangers such as DEAE, CM or ANX if the selectivity of Q or S is unsatisfactory. min 3 4 Q Sepharose HP (XK 6 mm) A typical purification strategy has three phases: Capture, Intermediate Purification and Polishing (Cipp). Each phase has a specific objective, dependent largely on the properties of the starting material. Select the appropriate ion exchange medium according to the objective of your purification step and the condition of your starting material. Start here Capture Isolate, concentrate and stabilize target protein(s) Sample condition: clarified or nonclarified High volume throughput and high capacity Easy scaleup High binding capacity for selected proteins Easy scaleup Large scale, viscous samples Capto (Q, S, DEAE, adhere, MMC) Sepharose XL (Q or SP) Sepharose Big Beads (Q or SP) Use high bed heights for increased productivity. Use MMC for high salt feed. Use Sepharose Q XL virus licensed as an alternative to cesium chloride gradients for purification of viruses, including adenovirus, or viral vectors. Use with step elution. Q Sepharose FF (XK 6 mm) Q Sepharose XL.... olume (l) Sample: Recombinant amylase Pilot scale: begins after l Product Prepacked columns/bulk media Ordering Information Code No. Column dimensions diam. (mm)/h(mm)/pack size Bed volume (ml) approx. Recommended working flow range Maximum flow Maximum operating back pressure ) (MPa/psi) ( MPa = bar) ph working range 2) ph stability 3) (long term) ph stability 4) (short term, cleaning) Type of ion exchanger Nominal bead size (µm) Functional group Examples of dynamic binding capacities mg/ml medium Mini Q PC 3.2/3 Mini S PC 3.2/3 Mini Q 4.6/ PE Mini S 4.6/ PE Mono Q / GL Mono Q / GL Mono Q 4.6/ PE Mono Q HR 6/ Mono S / GL Mono S / GL Mono S 4.6/ PE Mono S HR 6/ RESOURCE Q, ml RESOURCE Q, 6 ml SOURCE Q 4.6/ PE RESOURCE S, ml RESOURCE S, 6 ml SOURCE S 4.6/ PE SOURCE Q SOURCE S SOURCE 3Q SOURCE 3S HiTrap Q HP, ml HiTrap Q HP, ml HiTrap SP HP, ml HiTrap SP HP, ml HiLoad 6/ Q Sepharose High Performance HiLoad 26/ Q Sepharose High Performance HiLoad 6/ SP Sepharose High Performance HiLoad 26/ SP Sepharose High Performance Q Sepharose High Performance SP Sepharose High Performance MacroCap SP HiTrap IEX Selection Kit ) HiTrap Q FF, ml HiTrap Q FF, ml HiTrap SP FF, ml HiTrap SP FF, ml HiTrap DEAE FF, ml HiTrap DEAE FF, ml HiTrap ANX FF (high sub), ml HiTrap ANX FF (high sub), ml HiTrap CM FF, ml HiTrap CM FF, ml HiPrep 6/ Q FF HiPrep 6/ SP FF HiPrep 6/ ANX FF (high sub) HiPrep 6/ CM FF HiPrep 6/ DEAE FF Q Sepharose Fast Flow SP Sepharose Fast Flow DEAE Sepharose Fast Flow ANX Sepharose 4 Fast Flow (high sub) CM Sepharose Fast Flow HiTrap Capto Q, ml HiTrap Capto Q, ml HiTrap Capto iralq, ml HiTrap Capto S, ml HiTrap Capto S, ml HiTrap Capto DEAE, ml HiTrap Capto DEAE, ml HiTrap Capto adhere, ml HiTrap Capto adhere, ml HiTrap Capto MMC, ml HiTrap Capto MMC, ml Capto Q Capto iralq Capto S Capto DEAE Capto adhere Capto MMC HiTrap Q XL, ml 6) HiTrap Q XL, ml 6) HiTrap SP XL, ml HiTrap SP XL, ml HiPrep 6/ Q XL 6) HiPrep 6/ SP XL Q Sepharose XL virus licensed 6) Q Sepharose XL 6) SP Sepharose XL Q Sepharose Big Beads SP Sepharose Big Beads /3 3.2/3 4.6/ 4.6/ / / 4.6/ 6/ / / 4.6/ 6/ 6.4/3 6/3 4.6/ 6.4/3 6/3 4.6/ ml ml ml ml ml ml ml ml 6/2 6/2 6/ 26/ 6/ 26/ 7 ml 7 ml 2 ml ml 6/2 6/2 6/2 6/2 6/2 6/ 6/ 6/ 6/ 6/ 2 ml 2 ml 2 ml ml 2 ml ml 2 ml ml 6/2 6/2 6/2 6/2 6/2 6/2 2 ml ml 2 ml ml 2 ml ml 2 ml ml 2 ml ml 2 ml ml 6/2 6/2 6/ 6/ 2 ml l l ml/min.. ml/min.2. ml/min.2. ml/min.3 ml/min 26 ml/min.3 ml/min up to ml/min.3 ml/min 26 ml/min.3 ml/min up to ml/min. ml/min.6 ml/min.2. ml/min. ml/min.6 ml/min.2. ml/min cm/h cm/h 3 cm/h 3 cm/h up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to 3 ml/min up to ml/min up to 3 ml/min up to cm/h up to cm/h < cm/h up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min up to ml/min 4 cm/h 4 cm/h 4 cm/h 3 cm/h 4 cm/h ml/min ml/min ml/min ml/min ml/min ml/min ml/min.ml/min 2.ml/min ml/min ml/min <6cm/h <6cm/h up to ml/min up to ml/min up to ml/min up to ml/min cm/h cm/h cm/h up to cm/h up to cm/h ml/min ml/min 2 ml/min 2 ml/min 3 m/min ml/min 3 ml/ml ml/min 3 ml/min ml/min 3 ml/ml ml/min ml/min 6 ml/min ml/min ml/min 6 ml/min ml/min 8 cm/h 8 cm/h cm/h cm/h ml/min 3 ml/min ml/min 3 ml/min cm/h cm/h < cm/h ml/min ml/min ml/min ml/min ml/min 7 cm/h 7 cm/h 7 cm/h 4 cm/h 7 cm/h 4ml/min ml/min >7 cm/h ) >7 cm/h ) >7 cm/h ) >7 cm/h ) >6 cm/h ) >6 cm/h ) ml/min ml/min 7 cm/h 7 cm/h 7 cm/h 8 cm/h 8 cm/h /4 /4 8/26 8/26 4/8 4/8 4/8 3/43 4/8 4/8 4/8 3/43./2.6/87 4/8./2.6/87 4/8./72./72./72./72./22./22./22./22./22./22./ ) ) ) ) ) ) Strong anion Strong cation Weak anion Multimodal exchanger Multimodal exchanger Strong anion Strong anion Strong cation Weak anion Multimodal exchanger Multimodal exchanger (monosized) (monosized) (monosized) (monosized) (monosized) (monosized) 3 (monosized) 3 (monosized) ) O CHOH N + H( O CHOH O CHOH N + H( O COO O CHOH O CHOH N + H( O COO O CHOH N + H( O CHOH N + H( O CHOH O CHOH N + H( O COO O CHOH O O N + H( (O CH(OH) )( C 6 H )( OH) (O CH(OH) S( CH(COO )NHCOC 6 H O CHOH O CHOH O O N + H( (O CH(OH) )( C 6 H )( OH) (O CH(OH) S( CH(COO )NHCOC 6 H αamylase (M r 49 ) Trypsin inhibitor (M r ) Ribonuclease (M r 3 7) Lysozyme (M r 4 3) αamylase (M r 49 ) Trypsin inhibitor (M r ) Ribonuclease (M r 3 7) Lysozyme (M r 4 3) αlactalbumin (M r 4 3) IgG (human) (M r 6 ) Ribonuclease (M r 3 7) Lysozyme (M r 4 ) Lysozyme (M r 4 ) Lysozyme (M r 4 ) Ribonuclease (M r 3 7) Ribonuclease (M r 3 7) Ribonuclease (M r 3 7). to.3 mmol H+/ml medium Ribonuclease A (M r 3 7) Ribonuclease A (M r 3 7) Ribonuclease (M r 3 7) Ribonuclease (M r 3 7) αlactalbumin (M r 4 3) IgG (human) (M r 6 ) Bovine COHb (M r 69 ) Ribonuclease (M r 3 7) αlactalbumin (M r 4 3) Ribonuclease (M r 3 7) mg BSA/ml medium mg lysozyme/ml medium mg ovalbumin/ml medium N/A 8) 4mg BSA/ml medium at 3 ms/cm mg BSA/ml medium mg BSA/ml medium mg lysozyme/ml medium mg ovalbumin/ml medium N/A 8) 4mg BSA/ml medium at 3 ms/cm Lysozyme (M r 4 3) Lysozyme (M r 4 ) Lysozyme (M r 4 ) tested for specific application only tested for specific application only ) mg/ml mg/ml mg/ml mg/ml 2 mg/ml 7 mg/ml 7 mg/ml 4 mg/ml 4 mg/ml mg/ml mg/ml mg/ml 6 mg/ml mg/ml mg/ml mg/ml mg/ml 43 mg/ml mg/ml mg/ml mg/ml 43 mg/ml mg/ml mg/ml mg/ml 3 mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml 43 mg/ml mg/ml >3 mg/ml >6 mg /ml >3 mg/ml >6 mg/ml >3 mg/ml >6 mg/ml All ranges quoted are estimates based on our knowledge and experience. RESOURCE is a series of prepacked columns containing SOURCE media for fast separation and high resolution. ) Maximum operating back pressure refers to the pressure above which the medium begins to compress. 2) Working ph range refers to the ph interval where the medium binds protein as intended or as needed for elution, without adverse long term effects. 3) ph stability (long term) refers to the ph interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. 4) ph stability (cleaning) refers to the ph interval for regeneration, cleaninginplace and sanitization procedures. ) HiTrap IEX Selection Kit includes: HiTrap Q FF ml, HiTrap SP FF ml, HiTrap DEAE FF ml, HiTrap CM FF ml, HiTrap ANX FF (high sub) ml, HiTrap Q XL ml and HiTrap SP XL ml. 6) Important information: separating viral particles with Q Sepharose XL or Capto Q products may require a license under United States patent 6,37,793 B2 and foreign equivalents owned by Gencell SAS. Such a license is not included with the purchase of Q Sepharose XL or Capto Q, but is included with the purchase of Q Sepharose XL virus licensed and Capto iralq products. Purchasers of these products are granted a free limited license under US patent 6,37,793 B2 and foreign equivalents owned by Gencell SAS to separate viral particles solely through use of the product purchased. 7) Capto MMC is a multimodal exchanger and the ligand is charged in the ph interval 6. Outside this ph interval other interactions are still active. 8) Capto adhere is in antibody processes used in flowhtrough mode, and the loading can be in the region 3 mg/ml. The loading conditions should be optimized in each specific case. 9) The maximum pressure of the media has not been tested for more than.3 MPa (3 bar). ) The maximum flow rate of the media has not been tested for more than 7 or 6 cm/h. Maximum flow velocity (linear flow) is calculated from measurement in packed columns as follows: Sepharose Big Beads, ANX Sepharose 4 Fast Flow (high sub) XK, bed height 2 cm Sepharose XL and Sepharose Fast Flow: XK, bed height cm Sepharose High Performance: BioPilot 6, bed height 3 cm SOURCE 3: FineLINE, bed height cm SOURCE : RESOURCE, bed height 3 cm Tricorn prepacked columns ensure highest resolution with excellent reproducibility and durability. Tricorn columns are manufactured in glass (GL) and in PEEK (PE), for example Mono Q / GL and Mini 4.6/ PE. HiTrap columns are convenient and simple to use with a syringe, peristaltic pump or chromatography system. HiLoad columns are prepacked for preparative, high resolution purification at laboratory scale. HiPrep columns are prepacked for preparative purification at laboratory scale. Labpacks are available for column packing.

6 handbooks from Ge healthcare GST Gene fusion System Handbook 878 Affinity Chromatography Antibody Purification Handbook Percoll Methodology and Applications 869 ion exchange chromatography & chromatofocusing 42 Purifying challenging Proteins 2893 Gel filtration 8228 recombinant Protein Purification Handbook 8427 Protein Purification Handbook hydrophobic interaction and reversed Phase chromatography 269 2D electrophoresis using immobilized ph gradients microcarrier cell culture 8462

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