Sepharose Fast Flow media are based on a matrix of 90 μm particles made from 6% agarose and highly cross-linked for chemical and physical stability. ANX Sepharose 4 Fast Flow (high sub) is based on 4% agarose to form a medium that maintains a high binding capacity when separating large molecules such as thyroglobulin (Mr = 650 000), particularly suitable for large scale production when total binding capacity becomes economically significant.
Sepharose Fast Flow matrices are substituted with a range of ion exchange groups (Q, DEAE, ANX, SP and CM) giving the opportunity to test and use different selectivities (see Chapter 1 for an explanation of strong and weak ion exchangers). Ion exchangers containing strong ion exchange groups (Q and SP) maintain their charge over a broad pH range, allowing selection of the most suitable pH for each application.
Ion exchangers containing weak ion exchange groups (DEAE, CM and ANX) offer alternative selectivities, but over a narrower pH working range. Figure 55 illustrates how the selectivity of Sepharose Fast Flow media changes according to the anion exchange group.
Particle size and bed volumes remain stable, despite changes in ionic strength or pH, to ensure fast separations at high flow rates with good resolution. Methods can be easily scaled up from columns such as HiTrap Q FF (1 mL, prepacked with Q Sepharose Fast Flow) through to large scale columns such as FineLINE. The performance of Sepharose Fast Flow media is well documented and there are many examples of the smooth transfer from the laboratory to pilot scale and on to production.
Figure 55.Separation of conalbumin (I), a-lactalbumin (II) and soya bean trypsin inhibitor (III) on a range of anion exchange HiTrap columns demonstrates the difference in selectivity according to the anion exchange group.
Figure 56.Sepharose Fast Flow media, with a range of selectivities, are available prepacked in HiTrap and HiPrep columns and in media packs.
*See Appendix 5 to convert linear flow (cm/hour) to volumetric flow rates (mL/min) and vice versa.
**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.
***Maximum operating back pressure refers to the pressure above which the medium begins to compress.
Select a production column such as BPG or Chromaflow for larger volumes.
Using 1 mL HiTrap columns the most suitable matrix and charged group for a separation can be quickly and easily selected before optimization and scale-up. In Figure 57 a comparison of elution profiles for the same sample separated under identical conditions on three different media illustrates the differences in selectivity and resolution that can result from changing the charge group and the particle size. The most suitable medium can be selected and conditions optimized according to the requirements for the separation, for example to isolate a single, well-resolved peak or to maximize resolution between several peaks of interest.
Begin by scouting on the strong ion exchangers (Q, S or SP) in order to find the greatest differences in charge between the molecules of interest.
Figure 57.Media scouting: separation of ribonuclease A (I), cytochrome C (II) and lysozyme (III) on HiTrap CM Sepharose Fast Flow 1 mL, HiTrap SP Sepharose Fast Flow 1 mL and HiTrap SP XL 1 mL.
Figure 58.A HiPrep 16/10 DEAE Sepharose Fast Flow column is used as the capture step to concentrate rPhosphatase and remove most of the contaminants.
Figure 59 shows the ease with which separations can be scaled up on columns prepacked with Sepharose Fast Flow media. Beginning with a 1 mL HiTrap column the reproducibility of the separation has been maintained through a 20-fold scale-up.
Figure 59.5-fold and 20-fold scale-up using prepacked Q Sepharose Fast Flow columns.
Figure 60.Optimization and scale up on DEAE Sepharose Fast Flow.
When the most suitable medium has been selected for a separation, conditions can be optimized further by adjusting parameters such as pH. Figure 61 shows how increasing the pH on a column prepacked with CM Sepharose Fast Flow (HiPrep 16/10 CM FF) significantly improved resolution of a mixture of model proteins.
Figure 61.Selecting optimal pH for separation of standard proteins on HiPrep 16/10 CM FF.
It can be an advantage to concentrate a sample prior to gel filtration in order to minimize sample volume and facilitate a rapid, high resolution size separation. HiTrap columns offer a convenient, ready to use solution for sample concentration. Table 7 on page 89 gives examples of the high concentration factors achieved when concentrating proteins from very dilute starting material using HiTrap columns prepacked with Sepharose HP medium.Similar results can be achieved with HiTrap columns prepacked with Sepharose Fast Flow or Sepharose XL media.
Guidelines for selection of media, buffer, pH and ionic strength conditions and method optimization are given in Chapter 2. Use the instructions given here as a basis from which to optimize a separation.
For samples with unknown charge properties, try the following:
If selectivity is not satisfactory when using a strong ion exchanger (Q or SP), try a weak ion exchanger (DEAE, ANX or CM) instead.
Users of ÄKTAdesign systems with BufferPrep functionality can select one of the buffer recipes recommended for anion exchange chromatography at pH 8 or cation exchange chromatography at pH 6.
Flow: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL) or at 150 cm/h for Sepharose Fast Flow packed in larger columns. Collect fractions throughout the separation.
Flow: 1 mL/min (HiTrap 1 mL), 5 mL/min (HiTrap 5 mL), 5 mL/min (HiPrep 20 mL) or at 150 cm/h for Sepharose Fast Flow packed in larger columns. Collect fractions throughout the separation.
Correct preparation of samples and buffers and application of a high salt wash (1 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.
The following procedure should be satisfactory to remove common contaminants:
To remove precipitated proteins, lipids, hydrophobically bound proteins or lipoproteins, refer to Appendix 1.
Composition:
*Long term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography performance.
Short term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.
All ranges are estimates based on the experience and knowledge within Cytiva.
For daily use, Sepharose Fast Flow media are stable in all common, aqueous buffers, 1 M NaOH, denaturing agents (8 M urea, 6 M guanidine hydrochloride), with additives such as non-ionic detergents, 70% ethanol, 1 M acetic acid and 30% isopropanol. Sepharose Fast Flow can be used with organic solvents such as dimethylsulfoxide, dimethylformamide, tetrahydrofuran, acetone, chloroform, dichloromethane, dichloroethane and dichloroethane/pyridine (50:50) as well as polar solvents and aqueous/organic isolutions. The water in the medium can be exchanged by the alternative solvent with very little effect on the pore size of the matrix.
Avoid cationic detergents with SP or CM Sepharose Fast Flow. Avoid anionic detergents with Q, DEAE or ANX Sepharose Fast Flow. Avoid oxidizing agents.
For column storage, wash with 2 column volumes of distilled water followed by 2 column volumes of 20% ethanol. Include 0.2 M sodium acetate in the 20% ethanol solution for SP Sepharose Fast Flow. Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid over-pressuring the column. Store at room temperature or, for long periods, store at +4 °C to +8 °C. Ensure that the column is sealed well to avoid drying out. Whenever possible, use the storage and shipping device if supplied by the manufacturer. Store unused media at +4 °C to +30 °C in 20% ethanol. Do not freeze.
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