rs and din 20 40 60 HiPrep 16/60 Buffer: ￥2 chome C.1 s300 8m 0.2 04 Fig14c.Resolution between IG,ovalbumin and cytochrome Cat different flow rates. If peaks are well separated at a low flow rate,increase the flow rate or sho the columr to save time.A se the sample volume and benefit from a higher capacity without significant loss of resolution. For group separations such as desalting,monitor the elution of protein at Aso and follow the elution of the salt peak using a conductivity monitor.Adjust flow rate and sample volume to balance speed of separation against an acceptable level of salt in the final sample. Recommended flow rates are given in the instructions supplied with each product. Flow rate is measured in simple volume terms,e.g.ml/min,but when comparing results between columns of different sizes it is useful to use the linear flow,cm/hour(see Appendix 5). Results obtained at the same linear flow on different size columns will be comparable as far as the effects of flow rate are concerned. Selecting a smaller particle size of the same medium (if available)can also help to achieve the the c ect balance be n flow rate and resolution.Smaller and may allow articles of the same medium can ease column efficie impro reso the use of higher ow o result in in may become restrictive if the intention is to scale up the separation. pressure and this factor 25
25 Fig. 14b. Resolution between two different concentrations of IGF-1 containing monomers and dimers at different flow rates. 0 0.5 1.5 1.0 2.0 Resolution, Rs 0 40 20 60 80 a b Flow, cm/h Column: HiLoad 16/60 Superdex 30 prep grade Buffer: 50 mM sodium acetate, 0.1 M NaCl, pH 5.0 Sample: IGF-1 containing monomers and dimers Sample volume: 1 ml (0.8% × Vt ) Sample concentration: a) 1.25 mg/ml b) 5 mg/ml Fig. 14c. Resolution between IgG, ovalbumin and cytochrome C at different flow rates. If peaks are well separated at a low flow rate, increase the flow rate or shorten the column to save time. Alternatively, increase the sample volume and benefit from a higher capacity without significant loss of resolution. For group separations such as desalting, monitor the elution of protein at A280 and follow the elution of the salt peak using a conductivity monitor. Adjust flow rate and sample volume to balance speed of separation against an acceptable level of salt in the final sample. Recommended flow rates are given in the instructions supplied with each product. Flow rate is measured in simple volume terms, e.g. ml/min, but when comparing results between columns of different sizes it is useful to use the linear flow, cm/hour (see Appendix 5). Results obtained at the same linear flow on different size columns will be comparable as far as the effects of flow rate are concerned. Selecting a smaller particle size of the same medium (if available) can also help to achieve the the correct balance between flow rate and resolution. Smaller particles of the same medium can increase column efficiency, improve resolution and may allow the use of higher flow rates. However, smaller particles can also result in increased back pressure and this factor may become restrictive if the intention is to scale up the separation. Resolution, Rs 0 1 0.4 2 0.0 0.2 3 0.6 0.8 1.0 1.2 Flow, ml/min S-300 S-200 S-100 HiPrep 16/60 Columns: HiPrep 16/10 Sephacryl S-100 HR HiPrep 16/10 Sephacryl S-200 HR HiPrep 16/10 Sephacryl S-300 HR Buffer: 50 mM NaPO4, 0.15 M NaCl, 0.02% NaN3, pH 7.0 Sample: IgG, ovalbumin, cytochrome C, 1:2:1 Sample volume: 2.4 ml (2% × Vt ) Total sample load: 8 mg
Indude a sat the end of ar to facilitate the removal of any molecules that may have been retained on the column,to prevent cross-contamination and to prepare the column for a new separation. Controlling flow Accurate,reproducible conrol of the flow rate is not only essential for good resolution, but also for reliability in routine preparative work and repeated experiments.A pump is used to control liquid flow for most gel filtration separations,although gravity feed has been used in the past(see Appendix 1). nhytehnaPei长p2ea四 utilize the high ity and r high r actionation.Always rather than c wing the buffer throuh the the pump belo w).This reduces he risk of bubble formation as a result of suction.If you have packe he column yourself always use a flow rate for separation that is less than the flow rate used for column packing. Use a syringe or pu ap with small prepacked columns such as HiTrap Desalting or gravity eed with PD-10 Des alting for group separations of small sar nple volur Gel filtration columns must not run dry.Ensure that there is sufficient buffer for long, unattended runs or that the pump is programmed to stop the flow after a suitable time Columns that run dry must be repacked since the packed bed has been destroyed. Reversing flow through a gel filtration column should only be considered under cases of severe contamination.There is a r the packed bed lea Professionally packed columns are less likely to be affected,but extreme care must be taken. Method development for high resolution fractionation Steps are given in order of priority 1.Select the medium that will give the best resolution of the target protein(s),see media selection guide,page 18. 2.To ensure reproducibility and high resolution,select a prepacked column that is best suited to volume of sample that needs to b e processed (see Chapter2for detailsof column. 4.Determine the maximum sample volume that can be loaded without reducing resolution Higher flow rates and viscous buffers yield higher operating pressures (remember that buffer viscosity increases when running at +4C).Check the maximum operating pressure of the packed column and set the upper pressure limit on the chromatography system accordingly
26 Include a wash step at the end of a run to facilitate the removal of any molecules that may have been retained on the column, to prevent cross-contamination and to prepare the column for a new separation. Controlling flow Accurate, reproducible control of the flow rate is not only essential for good resolution, but also for reliability in routine preparative work and repeated experiments. A pump is used to control liquid flow for most gel filtration separations, although gravity feed has been used in the past (see Appendix 1). Use a pump within a chromatography system (rather than a peristaltic pump or gravity feed) to fully utilize the high rigidity and excellent flow properties of Sephacryl, Superose or Superdex for high resolution fractionation. Always pump the buffer onto a column (rather than drawing the buffer through the column with the pump below). This reduces the risk of bubble formation as a result of suction. If you have packed the column yourself, always use a flow rate for separation that is less than the flow rate used for column packing. Use a syringe or pump with small prepacked columns such as HiTrap Desalting or gravity feed with PD-10 Desalting for group separations of small sample volumes. Gel filtration columns must not run dry. Ensure that there is sufficient buffer for long, unattended runs or that the pump is programmed to stop the flow after a suitable time. Columns that run dry must be repacked since the packed bed has been destroyed. Reversing flow through a gel filtration column should only be considered under cases of severe contamination. There is a risk that reversing the flow may cause channeling through the packed bed leading to poor resolution, loss of efficiency and the need to repack the column. Professionally packed columns are less likely to be affected, but extreme care must be taken. Method development for high resolution fractionation Steps are given in order of priority. 1. Select the medium that will give the best resolution of the target protein(s), see media selection guide, page 18. 2. To ensure reproducibility and high resolution, select a prepacked column that is best suited to the volume of sample that needs to be processed (see Chapter 2 for details of prepacked columns containing Superdex, Sephacryl or Superose). 3. Select the highest flow rate that maintains resolution and minimizes separation time. Check recommended flow rates supplied in the instructions for the specific medium and column. 4. Determine the maximum sample volume that can be loaded without reducing resolution. Higher flow rates and viscous buffers yield higher operating pressures (remember that buffer viscosity increases when running at +4 °C). Check the maximum operating pressure of the packed column and set the upper pressure limit on the chromatography system accordingly
If greater,nrease the bed height by containing the same medium in seric s.Altern tively,try a medium with the same or similar fractionation range,but with a smaller particle size. To process larger sample volumes,see scaling up on this page. Care of gel filtration media When a gel filtration medium has been in use for some time,it may be necessary to remove precipitated proteins or other contaminants that can build up.The need for cleaning may the appearance ofa band at top of space bet the upper ada nd the bed surfac at in ocedures for each gel filtra nedium are given 2.In all ases,prevention is better than cure and routine cleaning is recommended If an increase in back pressure is observed,either on the pressure monito or by seeing the ne medium move downwards,check that the problem is actually caused by the co before starting the ceanin procedure.Discomect one picce of quipment ata time (starting at the fraction collector),start the pump and check the pressure after each piece is disconnected.A dirty on-line filter is a common cause of increased back pressure Check back pressure at the same stage during each run,since the value can vary within a run during sample injection or when changing to a different buffer. .Always use filtered buffers and samples to reduce the need for additional column maintenance.See appendix 3 for further details on sample preparation. Always use well degassed buffers to avoid the formation of air bubbles in the packed column during a run. coumns and samples should be kept at the same temperature to bubbles for ming in the column Filter cleaning solutions before use and always re-equilibrate the column with 23colum of bufer before the next separation Equipment selection Appendix 4 provides a guide to the selection of systems recommended for gel filtration separation. Scaling up oup separation ona small column,it may be 宝 larger sample volumes in a single Maintain Increase Bed height Column diameter Linear flow rate Volumetric flow rate Sample composition Sample volume 27
27 If greater resolution is required, increase the bed height by connecting two columns containing the same medium in series. Alternatively, try a medium with the same or similar fractionation range, but with a smaller particle size. To process larger sample volumes, see scaling up on this page. Care of gel filtration media When a gel filtration medium has been in use for some time, it may be necessary to remove precipitated proteins or other contaminants that can build up. The need for cleaning may show itself as the appearance of a colored band at top of the column, as a space between the upper adaptor and the bed surface, as a loss in resolution or as a significant increase in back pressure. Detailed cleaning procedures for each gel filtration medium are given in Chapter 2. In all cases, prevention is better than cure and routine cleaning is recommended. If an increase in back pressure is observed, either on the pressure monitor or by seeing the surface of the medium move downwards, check that the problem is actually caused by the column before starting the cleaning procedure. Disconnect one piece of equipment at a time (starting at the fraction collector), start the pump and check the pressure after each piece is disconnected. A dirty on-line filter is a common cause of increased back pressure. Check back pressure at the same stage during each run, since the value can vary within a run during sample injection or when changing to a different buffer. • Always use filtered buffers and samples to reduce the need for additional column maintenance. See Appendix 3 for further details on sample preparation. • Always use well degassed buffers to avoid the formation of air bubbles in the packed column during a run. • Buffers, prepacked columns and samples should be kept at the same temperature to prevent air bubbles forming in the column. • Filter cleaning solutions before use and always re-equilibrate the column with 2–3 column volumes of buffer before the next separation. Equipment selection Appendix 4 provides a guide to the selection of systems recommended for gel filtration separation. Scaling up After establishing a high resolution or group separation on a small column, it may be preferred to pack a larger column in order to process larger sample volumes in a single step. General guidelines for scaling up are shown below. Maintain Increase Bed height Column diameter Linear flow rate Volumetric flow rate Sample composition Sample volume
When scaling up a gel filtration column,follow the points below: 1.Optimize the separation at small scale(see method development page 26) 2.Maintain the sample volume:column volume ratio and the sample concentration. 3.Increase the column volume by increasing the cross sectional area of the column 4.Maintain the bed height. Appen Refer to Appendix 1 for column selection and column packing. Different equipment factors may affect performance after scale-up.If the large scale column has a less efficient flow distribution system,or the large scale system introduces large dead volumes,peak broadening may occur.This will cause extra dilution of the product fraction or even loss of resolution if the application is sensitive to variations in efficiency. For certain media,e.g.Superdex,Superose or Sephadex,it is usually recommended to select a larger pa icle igh r n fractionation,pack a small col conta ing eany optimization that may be needed 新62 ard and well- in processes for removing endotoxins from albumin,and preparative steps during the production of vaccines.Figure 15 shows an example of a large scale buffer exchange step used during the production of albumin and IgG from human plasma. BPS40a/0075
28 When scaling up a gel filtration column, follow the points below: 1. Optimize the separation at small scale (see method development page 26). 2. Maintain the sample volume: column volume ratio and the sample concentration. 3. Increase the column volume by increasing the cross sectional area of the column. 4. Maintain the bed height. 5. Run the separation at the same linear flow rate as used on the smaller column (see Appendix 5). Refer to Appendix 1 for column selection and column packing. Different equipment factors may affect performance after scale-up. If the large scale column has a less efficient flow distribution system, or the large scale system introduces large dead volumes, peak broadening may occur. This will cause extra dilution of the product fraction or even loss of resolution if the application is sensitive to variations in efficiency. For certain media, e.g. Superdex, Superose or Sephadex, it is usually recommended to select a larger particle size. For high resolution fractionation, pack a small column containing the larger particles and repeat the separation to facilitate any optimization that may be needed to achieve the same resolution on the larger column. Scaling up on Sephadex G-25, even to production scale, is a straightforward and wellestablished process. Well known examples of commercial applications include buffer exchange in processes for removing endotoxins from albumin, and preparative steps during the production of vaccines. Figure 15 shows an example of a large scale buffer exchange step used during the production of albumin and IgG from human plasma. Elution volume A280 Conductivity 1 2 1 2 1 2 1 2 1 2 Column: BPSS 400/600, 75 l Sample: 10 l human plasma 1: sample application 2: eluent application Eluent: 0.025 M sodium acetate Flow: 240 l/h Fig. 15. Chromatogram of the buffer exchange step on Sephadex G-25 Coarse during production of albumin and IgG from human plasma
BioProcess Media for large-scale production Specific BioProcess Media have been designed for each chromatography stage in a process from Capture to Polishing.Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity,at the right place and at the right time.Amersham Biosciences can assure future supplies of BioProcess Media,making them a safe investment for long-term production.The media are produced following validated methods and tested under strict c trol to fulfill high performance specifications.A certificate of analysis is nalytical oint for poing p rm sions to regulatory authorities.Using BioProcess M a for every stage results in an easily validated process.High flow rate,high capacity and high recovery contribute to the overal economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitization procedures.Packing methods are established for a wide range of scales and compatible large-scale columns and equipment are available. Troubleshooting colu The diagrams in a chromatogram may deviate from ide chavior during their remedies. Highly acidic or basic substances at low ionic strength or aromatic materials may behave differently during gel filtration,interacting with the matrix.For some applications this can tides and other substances that differ only slightly aadtdonephades.However ths is not a true gel filration weight can b separation 29
29 BioProcess Media for large-scale production Specific BioProcess™ Media have been designed for each chromatography stage in a process from Capture to Polishing. Large capacity production integrated with clear ordering and delivery routines ensure that BioProcess media are available in the right quantity, at the right place and at the right time. Amersham Biosciences can assure future supplies of BioProcess Media, making them a safe investment for long-term production. The media are produced following validated methods and tested under strict control to fulfill high performance specifications. A certificate of analysis is available with each order. Regulatory Support Files (RSF) contain details of performance, stability, extractable compounds and analytical methods. The essential information in these files gives an invaluable starting point for process validation, as well as providing support for submissions to regulatory authorities. Using BioProcess Media for every stage results in an easily validated process. High flow rate, high capacity and high recovery contribute to the overall economy of an industrial process. All BioProcess Media have chemical stability to allow efficient cleaning and sanitization procedures. Packing methods are established for a wide range of scales and compatible large-scale columns and equipment are available. Troubleshooting This section focuses on practical problems that may occur when running a chromatography column. The diagrams indicate how a chromatogram may deviate from ideal behavior during a gel filtration separation. The following pages contain suggestions of possible causes and their remedies. Highly acidic or basic substances at low ionic strength or aromatic materials may behave differently during gel filtration, interacting with the matrix. For some applications this can be an advantage. For example aromatic peptides and other substances that differ only slightly in molecular weight can be separated on Sephadex. However this is not a true gel filtration separation