Chapter 3 Three Phase Purification Strategy Principles Wth background informtion assays,and sample preparation and extraction procedures in place the Three Phase Purification Strategy can be applied(Figure 3).This strategy is used as an aid to the development of purification processes for therapeutic proteins in the pharmaceutical industry and is equally efficient as an aid when developing purification schemes in the research laboratory. Polishing Capture Preparation, Step Fig.3.Preparation and the Three Phase Purification Strategy Assign a specific objective to each step within the purification process. In the Three Phase Strategy a specific objective is assigned to each step.The purification problem associated with a particular step will depend greatly upon the properties of the starting material.Thus,the objective of a purification step will vary according to its position in the process,i.e.at the beginning for isolation of product from crude sample,in the middle for further purification of partially purified sample,or at the end for final clean up of an almost pure product. The Three Phase Strategy ensures faster method development,a shorter time to pure product and good economy. trate and stabilise the e pr which and transferred to an ll conserve ty.At best,significant removal of other critical contaminants can also be achieved 17
17 Chapter 3 Three Phase Purification Strategy Principles With background information, assays, and sample preparation and extraction procedures in place the Three Phase Purification Strategy can be applied (Figure 3). This strategy is used as an aid to the development of purification processes for therapeutic proteins in the pharmaceutical industry and is equally efficient as an aid when developing purification schemes in the research laboratory. Fig. 3. Preparation and the Three Phase Purification Strategy. Assign a specific objective to each step within the purification process. In the Three Phase Strategy a specific objective is assigned to each step. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process, i.e. at the beginning for isolation of product from crude sample, in the middle for further purification of partially purified sample, or at the end for final clean up of an almost pure product. The Three Phase Strategy ensures faster method development, a shorter time to pure product and good economy. In the capture phase the objectives are to isolate, concentrate and stabilise the target product. The product should be concentrated and transferred to an environment which will conserve potency/activity. At best, significant removal of other critical contaminants can also be achieved. Step Purity Capture Intermediate purification Polishing Preparation, extraction, clarification Achieve final high level purity Remove bulk impurities Isolate, concentrate and stabilise
During the intermediate purification phase the objectives are to remove most of the bulk ipurities,such as other proteins and nucleic acids,endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances.The objective is to achieve final purity. It should be noted that this Three Phase Strategy does not mean that all strategies must have three purification steps.For example.capture and intermediate as n nay intermediate purification gSimilarly demand so low that a rapid e step achie ve the ired result, of th be so high th starting material may proteins a purification step may be required to purity and safety demands. The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification process. Selection and Combination of Purification Techniques Re capacity,speed and recovery. Minimise sample handling Minimise number of steps Use different techniques at each step Goal:Fastest route to a product of required purity. For any chromatographic separation eachdifferent techniaue will offer different rfo capa city.A tech can be opt cus on one of these parameters,fo or example res olution,o two parameters,such as speed an d capacity. A separation optimised for one of these parameters will produce results quite different in appearance from those produced using the same technique,but focussed on an alternative parameter.See,for example,the results shown on page 49 where ion exchange is used for a capture and for a polishing step. 18
18 During the intermediate purification phase the objectives are to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity. It should be noted that this Three Phase Strategy does not mean that all strategies must have three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result, or the purity of the starting material may be so high that only a polishing step is needed. For purification of therapeutic proteins a fourth or fifth purification step may be required to fulfil the highest purity and safety demands. The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification process. Selection and Combination of Purification Techniques Every technique offers a balance between resolution, capacity, speed and recovery. Minimise sample handling Minimise number of steps Use different techniques at each step Goal: Fastest route to a product of required purity. For any chromatographic separation each different technique will offer different performance with respect to recovery, resolution, speed and capacity. A technique can be optimised to focus on one of these parameters, for example resolution, or to achieve the best balance between two parameters, such as speed and capacity. A separation optimised for one of these parameters will produce results quite different in appearance from those produced using the same technique, but focussed on an alternative parameter. See, for example, the results shown on page 49 where ion exchange is used for a capture and for a polishing step. Speed Recovery Capacity Resolution
Select a techniqueto meet the objectives for the purification step. Capacity,in the simple model shown,refers to the amount of target protein loaded during purification.In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration)or by large amounts of contaminants rather than the amount of the target protein. Speed is of the highest importance at the beginning of a purification where contaminants such as proteases must be removed as quickly as possible. Recovery becc mincreasingly importatas the purification proceed because of increased of. pur pr ed by dtructive proceses in the amplend ourons on the column Resolution is achieved by the selectivity of the technique and the efficiency of the romatograp Ic matr difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. Every technique offers a balance between resolution,speed,capacity and recovery and should be selected to meet the objectives for each purification step.In general,optimisation of any one of these four parameters can only be achieved at the expense of the others and a purification step will be a compromise.The pending on whethe a purification step ised for nediate purificati Thi vill steer the of the ical parameters,as well as the selection of the most suitable Proteins are purified usin eparatin to differences in shecifcation techniques Table 3.Protein properties used during purification. Protein property Technique Charge on exchange (IEX) Size Gel filtration (GF Biorecognition(ligand specificity) Affinity (AC) Charge,ligand specificity or hydrophobicity 19
19 Select a technique to meet the objectives for the purification step. Capacity, in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein. Speed is of the highest importance at the beginning of a purification where contaminants such as proteases must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and unfavourable conditions on the column. Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. Every technique offers a balance between resolution, speed, capacity and recovery and should be selected to meet the objectives for each purification step. In general, optimisation of any one of these four parameters can only be achieved at the expense of the others and a purification step will be a compromise. The importance of each parameter will vary depending on whether a purification step is used for capture, intermediate purification or polishing. This will steer the optimisation of the critical parameters, as well as the selection of the most suitable media for the step. Proteins are purified using chromatographic purification techniques which separate according to differences in specific properties, as shown in Table 3. Table 3. Protein properties used during purification. Protein property Technique Charge Ion exchange (IEX) Size Gel filtration (GF) Hydrophobicity Hydrophobic interaction (HIC), Reversed phase (RPC) Biorecognition (ligand specificity) Affinity (AC) Charge, ligand specificity or hydrophobicity Expanded bed adsorption (EBA) follows the principles of AC, IEX or HIC
Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. Minimise sample handling between purification steps by combining techniques to avoid the need for sample conditioning. A guide to the suitability of each purification technique for the stages in the Three Phase Purification Strategy is shown in Table 4. Fechnigwe M台in fealures Captre n修 驰 EX ★★★ ★★★ ★★★ ★★ ★★★ ★ ★★★ ★★★ ★★ o GF ghsae ★★★★ ★★★★ Table 4.Suitability of purification techniques for the Three Phase Purification Strategy Avoid additional sampe conditionin stes The product should be eluted from the first column in conditions suitable for the start conditions of the next column. The start conditions and end conditions for the techniques are shown in Table 4. For le,if the nle has a low ionic IEX the app lied to an IEX h an e applied to a HIC th alt can e a dded).In c essary t can be st,if e is eluted ikely to be n h igh salt and will ution or a bu exchange step in order to further decrease the ionic strength to a level suitable for IEX.Thus it is more straightforward to go from IEX to HIC than vice-versa. Ammonium sulphate precipitation is a common sample clarification and concentration step at laboratory scale and in this situation HIC(which requires high salt to enhance binding to the media)is ideal as the capture ncentration and the total sar ple volume will be significantly the hic column.Dilution of the fra ated sa step
20 Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step. Minimise sample handling between purification steps by combining techniques to avoid the need for sample conditioning. A guide to the suitability of each purification technique for the stages in the Three Phase Purification Strategy is shown in Table 4. Table 4. Suitability of purification techniques for the Three Phase Purification Strategy Avoid additional sample conditioning steps. The product should be eluted from the first column in conditions suitable for the start conditions of the next column. The start conditions and end conditions for the techniques are shown in Table 4. For example, if the sample has a low ionic strength it can be applied to an IEX column. After elution from IEX the sample will usually be in a high ionic strength buffer and can be applied to a HIC column (if necessary the pH can be adjusted and further salt can be added). In contrast, if sample is eluted from a HIC column, it is likely to be in high salt and will require dilution or a buffer exchange step in order to further decrease the ionic strength to a level suitable for IEX. Thus it is more straightforward to go from IEX to HIC than vice-versa. Ammonium sulphate precipitation is a common sample clarification and concentration step at laboratory scale and in this situation HIC (which requires high salt to enhance binding to the media) is ideal as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a Sephadex G-25 desalting column will prepare it for the next IEX or AC step. Technique Main features Capture Intermediate Polish Sample Start Sample End condition condition IEX high resolution low ionic strength high ionic high capacity sample volume strength or high speed not limiting pH change concentrated HIC good resolution high ionic strength low ionic good capacity sample volume strength high speed not limiting concentrated AC high resolution specific binding specific high capacity conditions elution high speed sample volume conditions not limiting concentrated GF high resolution limited sample buffer using Superdex™ volume (<5% total exchanged (if column volume) required) and flow rate diluted range RPC high resolution requires organic in organic solvents solvent, risk loss of biological activity concentrated ★★★ ★★★ ★★★ ★ ★★ ★★★ ★★★ ★★★ ★ ★★ ★★★ ★ ★★★
GF is well suited for use after any of the concentrating techniques (IEX,HIC,AC)since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions). Selection of the final strategy will always depend upon specific sample properties and the required level of purification.Logical combinations of techniques are shown in Figure 4. Crude samplersample in highsatcorio mode Capture Polish GF GF GF he他d Clear orvery dilute samples Capture AC IEX E Resolubilise Polishing GF Fig.4.Logical combinations of chromatographic steps For any capture step,select the technique showing the strongest binding to the target protein while binding as few of the contaminants as possible, ie.the technique with the highest selectivity and/or capacity for the protein of interest
21 GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions). Selection of the final strategy will always depend upon specific sample properties and the required level of purification. Logical combinations of techniques are shown in Figure 4. Fig. 4. Logical combinations of chromatographic steps. For any capture step, select the technique showing the strongest binding to the target protein while binding as few of the contaminants as possible, i.e. the technique with the highest selectivity and/or capacity for the protein of interest. Clear or very dilute samples AC IEX IEX Precipitation (e.g. in high ionic strength) HIC Resolubilise GF GF GF Treat as for or or sample in high salt RPC RPC concentration Capture Intermediate Purification Polishing Crude sample or sample in high salt concentration GF GF GF desalt mode desalt mode desalt mode AC IEX HIC IEX dilution may be needed IEX HIC GF GF GF GF Sample clarification* Capture Intermediate Purification Polish * Alternatively samples can be filtered and, if required, their ionic strength can be reduced by dilution