Chapter 1 Purification Strategies -a simple approach Apply a systematic approach to development of a purification strategy The first step is to describe the basic scenario for the purification.General considerations answer questions such as:What is the intended use of the product? What kind of starting material is available and how should it be handled?What are the purity issues in relation to the source material and intended use of the final product?What has to be removed?What must be removed completely? What will be the final scale of purification?If there is a need for scale-up,what ces will this have on he choser n purification techr are the straints and what reso urces nd equipment the product from one technique into conditions suitable to perform the next technique.Each step in the process will cause some loss of product.For example, if a yield of 80%in each step is assumed,this will be reduced to only 20% overall yield after 8 processing steps as shown in Figure 1.Consequently,to reach the targets for yield and purity with the minimm number of steps and the simplest possible design,it is not efficient to add one step to another until purity requirements have be en fulfilled.Occasionally when a sample is readily available can be achieved by simply adding or repeating ste s.Howev rience s that n for the challens ved efficier ntly in fe ewe r than fo s app and vield and steps.Techniques shou organised in logical the nee d for hniques selected appropriately to Limit the number of steps a purification procedure 7
7 Chapter 1 Purification Strategies - a simple approach Apply a systematic approach to development of a purification strategy. The first step is to describe the basic scenario for the purification. General considerations answer questions such as: What is the intended use of the product? What kind of starting material is available and how should it be handled? What are the purity issues in relation to the source material and intended use of the final product? What has to be removed? What must be removed completely? What will be the final scale of purification? If there is a need for scale-up, what consequences will this have on the chosen purification techniques? What are the economical constraints and what resources and equipment are available? Most purification protocols require more than one step to achieve the desired level of product purity. This includes any conditioning steps necessary to transfer the product from one technique into conditions suitable to perform the next technique. Each step in the process will cause some loss of product. For example, if a yield of 80% in each step is assumed, this will be reduced to only 20% overall yield after 8 processing steps as shown in Figure 1. Consequently, to reach the targets for yield and purity with the minimum number of steps and the simplest possible design, it is not efficient to add one step to another until purity requirements have been fulfilled. Occasionally when a sample is readily available purity can be achieved by simply adding or repeating steps. However, experience shows that, even for the most challenging applications, high purity and yield can be achieved efficiently in fewer than four well-chosen and optimised purification steps. Techniques should be organised in a logical sequence to avoid the need for conditioning steps and the chromatographic techniques selected appropriately to use as few purification steps as possible. Limit the number of steps in a purification procedure
Yield (% 95%/step 90%/step 85%/step 20 75%/6部 0 234567 8Number of steps Fig.1.Yields from multi-step purifications. Preparation The need to obtain a protein,efficiently,economically and in sufficient purity and quantity,applies to every purification.It is important to set objectives for purity, quantity and maintenance of biological activity and to define the economical and time framework for the work.All information concerning properties of the targe protein and contaminants will help during purification development.Some simple experiments to characterise the sa ole and target molecule are excellent inve f fast eliable of the purific preparation an iabedevdlopedprioroheirsi With background information,assays and sample preparation procedures in place the Three Phase Purification Strategy can be considered. Three Phase Purification Strategy In the Three Phase Strategy specific objectives are assigned to each step within the process: In the capture phase the objectives are to isolate,concentrate and stabilise the target product. During the intermediate burification phase the obiective is to remove most of the bulk impurities such as other proteins and nucleic acids,endotoxins and viruses In the polishing phase the objective is to achieve high purity by removing any remaining trace impurities or closely related substance timum development.a shorter time to pure product and good economy. g
8 Fig.1. Yields from multi-step purifications. Preparation The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to every purification. It is important to set objectives for purity, quantity and maintenance of biological activity and to define the economical and time framework for the work. All information concerning properties of the target protein and contaminants will help during purification development. Some simple experiments to characterise the sample and target molecule are an excellent investment. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess its effectiveness. Sample preparation and extraction procedures should be developed prior to the first chromatographic purification step. With background information, assays and sample preparation procedures in place the Three Phase Purification Strategy can be considered. Three Phase Purification Strategy Imagine the purification has three phases Capture, Intermediate Purification and Polishing. In the Three Phase Strategy specific objectives are assigned to each step within the process: In the capture phase the objectives are to isolate, concentrate and stabilise the target product. During the intermediate purification phase the objective is to remove most of the bulk impurities such as other proteins and nucleic acids, endotoxins and viruses. In the polishing phase the objective is to achieve high purity by removing any remaining trace impurities or closely related substances. The selection and optimum combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial to ensure fast method development, a shorter time to pure product and good economy. 10 80 60 40 20 0 1 2 3 4 5 6 7 8 Number of steps 95% / step 90% / step 85% / step 80% / step 75% / step Yield (%)
The final purification process should ideally consist of sample preparation, including extraction and clarification when required,followed by three major purification steps,as shown in Figure 2.The number of steps used will always depend upon the purity requirements and intended use for the protein. Polishing Capture Preparation 6s69gentrat Step Fig.2.Preparation and the Three Phase Purification Strategy
9 The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by three major purification steps, as shown in Figure 2. The number of steps used will always depend upon the purity requirements and intended use for the protein. Fig. 2. Preparation and the Three Phase Purification Strategy Step Purity Capture Intermediate purification Polishing Preparation, extraction, clarification Achieve final high level purity Remove bulk impurities Isolate, concentrate and stabilise
Guidelines for Protein Purification The guidelines for protein purification shown here can be applied to any purification process and are a suggestion as to how a systematic approach can be applied to the development of an effective purification strategy.As a reminder these guidelines will be highlighted where appropriate throughout the following chapters. Define objectives for purity,activity and quantity required of final product to avoid over or under developing a method Define properties of target protein and critical impurities to simplify technique selection and optimisation fastetecionof proten activity/recovery and critical contaminants says Minim e sample handling at every stage to avoid lengthy procedures which risk losing activity/reducing recovery Minimise use of additives additives may need to be removed in an extra purification step or may interfere with activity assays Remove damaging contaminants early for example,proteases Use a different technique to tak each step (iecharge.hydrophobiity.ph can be used for separation e cha Minimise nun mber of steps extra steps reduce yield and increase time,combine steps logically KEEP IT SIMPLE! 10
10 Guidelines for Protein Purification The guidelines for protein purification shown here can be applied to any purification process and are a suggestion as to how a systematic approach can be applied to the development of an effective purification strategy. As a reminder these guidelines will be highlighted where appropriate throughout the following chapters. Define objectives for purity, activity and quantity required of final product to avoid over or under developing a method Define properties of target protein and critical impurities to simplify technique selection and optimisation Develop analytical assays for fast detection of protein activity/recovery and critical contaminants Minimise sample handling at every stage to avoid lengthy procedures which risk losing activity/reducing recovery Minimise use of additives additives may need to be removed in an extra purification step or may interfere with activity assays Remove damaging contaminants early for example, proteases Use a different technique at each step to take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity) Minimise number of steps extra steps reduce yield and increase time, combine steps logically KEEP IT SIMPLE!
Chapter 2 Preparation Before You Start tion, n of an extrac recombinant pro s important to set objectives for purity and quantity, tivity and economy in terms of money and time. Purity requirements must take into consideration the nature of the source material,the intended use of the final product and any special safety issues.For example,it is important to differentiate between contaminants which must be removed and those which can be tolerated.Other factors can also influence the prioritisation of objectives.High yields are usually a key objective,but may be less crucial in cases where a sample is readily available or product is required only in small quantities.Extensive method development may be impossible without resources such as an AKTATMdesign chromatography system.Similarly,time essur mbined with a slow ay turnaround will steer towards less extensive uti and op ation.All info ng p erties of the ants will help dur ent,allo rge avoiding conditions Development of fast and reliable analytical assays is essential to foll low the progress of the purification and assess effectiveness(yield,biological activity, recovery). Define objectives Goal:To set minimum objectives for purity and quantity,maintenance of biological activity and economy in terms of money and time. Define purity requirements according to the final use of the product. Purity requirement examples are shown below Extremely high >99% Therapeutic use.in vivo studies High 95-99% Heyeiacaanngmostpmysieo-chenmal Moderate<95% 11
11 Chapter 2 Preparation Before You Start The need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to any purification, from preparation of an enriched protein extract for biochemical characterisation to large scale production of a therapeutic recombinant protein. It is important to set objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time. Purity requirements must take into consideration the nature of the source material, the intended use of the final product and any special safety issues. For example, it is important to differentiate between contaminants which must be removed and those which can be tolerated. Other factors can also influence the prioritisation of objectives. High yields are usually a key objective, but may be less crucial in cases where a sample is readily available or product is required only in small quantities. Extensive method development may be impossible without resources such as an ÄKTA™design chromatography system. Similarly, time pressure combined with a slow assay turnaround will steer towards less extensive scouting and optimisation. All information concerning properties of the target protein and contaminants will help during purification development, allowing faster and easier technique selection and optimisation, and avoiding conditions which may inactivate the target protein. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess effectiveness (yield, biological activity, recovery). Define objectives Goal: To set minimum objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time. Define purity requirements according to the final use of the product. Purity requirement examples are shown below. Extremely high > 99% Therapeutic use, in vivo studies High 95- 99 % X-ray crystallography and most physico-chemical characterisation methods Moderate < 95 % Antigen for antibody production N-terminal sequencing