考ldentif的'keycontaminants Iden the nature of posible remining contaminants as oo possible. The statemen hat the ous by Coomassier stained SDS-PAGE"Purity of 95%may be acceptable if the remaining 5%consists of harmless impurities.However,even minor impurities which may be biologically active could cause significant oved completely and re contamination problems. isbetter toverpurify than tounder-purify. Although h the number ed if sa ●m时 The need to mainta biological a ctivity must b first step. arget protei A downstream production process must achieve the required purity and mercial production the time ty or eto market Robustness and reliability are also of great concern since a batch failure can have major consequences. Special safety issues may be involved in purification of biopharma- ceuticals,such as derec It may be necessary to use analytical techniques targetted towards specific conta- minants in order to demonstrate that they have been removed to acceptable levels
12 Identify 'key' contaminants Identify the nature of possible remaining contaminants as soon as possible. The statement that a protein is >95% pure (i.e. target protein constitutes 95% of total protein) is far from a guarantee that the purity is sufficient for an intended application. The same is true for the common statement "the protein was homogenous by Coomassie™ stained SDS-PAGE". Purity of 95% may be acceptable if the remaining 5% consists of harmless impurities. However, even minor impurities which may be biologically active could cause significant problems in both research and therapeutic applications. It is therefore important to differentiate between contaminants which must be removed completely and those which can be reduced to acceptable levels. Since different types of starting material will contain different contaminant profiles they will present different contamination problems. It is better to over-purify than to under-purify. Although the number of purification steps should be minimised, the quality of the end product should not be compromised. Subsequent results might be questioned if sample purity is low and contaminants are unknown. Contaminants which degrade or inactivate the protein or interfere with analyses should be removed as early as possible. The need to maintain biological activity must be considered at every stage during purification development. It is especially beneficial if proteases are removed and target protein transferred into a friendly environment during the first step. A downstream production process must achieve the required purity and recovery with complete safety and reliability, and within a given economic framework. Economy is a very complex issue. In commercial production the time to market can override issues such as optimisation for recovery, capacity or speed. Robustness and reliability are also of great concern since a batch failure can have major consequences. Special safety issues may be involved in purification of biopharmaceuticals, such as detection or removal of infectious agents, pyrogens, immunogenic contaminants and tumorigenic hazards. It may be necessary to use analytical techniques targetted towards specific contaminants in order to demonstrate that they have been removed to acceptable levels
Define properties of target protein and critical impurities Goal:To determine 'stability window'for the targe otein for asier selection and optimisation of techniques and to avoid protein inactivation during purification. -Check target protein stability window for at least pH and ionic strength. All information oning the target protein and contaminant P erties will heln s and i ons fo purification.Database information for the target,or related proteins,may give size,isoelectric point(pl)and hydrophobicity or solubility data.Native one and two dimensional PAGE can indicate sample complexity and the properties of th major contaminants. articularly important is a e protein so tha ever and ion purification strategy ent target prote ein properti Table 1.Protein properties and their effect on development of purification strategies Sample and target protein properties Influence on purification strategy Temperature stability Need to work rapidl at lowered temperature oH stability reversed phase chromatography Organic solvents stability Selection of condtions for reversed phase atography Detergent requirement Salt (ionic strength) Co-factors for stability or activity Selection of additives.pH.salts.buffers Protease sensitivity oteases or addition of Sensitivity to metal ions Need to add EDTA or EGTA to buffers Redox sensitivity Need to add reducino agents Molecular weigh Selection of gel filtration media Charge pro Selection of ion exchange ecific affinity Selection of ligand for affinity medium Post translational modifications Selection of group-specific affinity medium Hydrophobicity Selection of m 13
13 Define properties of target protein and critical impurities Goal: To determine a 'stability window' for the target protein for easier selection and optimisation of techniques and to avoid protein inactivation during purification. Check target protein stability window for at least pH and ionic strength. All information concerning the target protein and contaminant properties will help to guide the choice of separation techniques and experimental conditions for purification. Database information for the target, or related proteins, may give size, isoelectric point (pI) and hydrophobicity or solubility data. Native one and two dimensional PAGE can indicate sample complexity and the properties of the target protein and major contaminants. Particularly important is a knowledge of the stability window of the protein so that irreversible inactivation is avoided. It is advisable to check the target protein stability window for at least pH and ionic strength. Table 1 shows how different target protein properties can affect a purification strategy. Table 1. Protein properties and their effect on development of purification strategies. Sample and target protein properties Influence on purification strategy Temperature stability Need to work rapidly at lowered temperature pH stability Selection of buffers for extraction and purification Selection of conditions for ion exchange, affinity or reversed phase chromatography Organic solvents stability Selection of conditions for reversed phase chromatography Detergent requirement Consider effects on chromatographic steps and the need for detergent removal. Consider choice of detergent. Salt (ionic strength) Selection of conditions for precipitation techniques, ion exchange and hydrophobic interaction chromatography Co-factors for stability or activity Selection of additives, pH, salts, buffers Protease sensitivity Need for fast removal of proteases or addition of inhibitors Sensitivity to metal ions Need to add EDTA or EGTA to buffers Redox sensitivity Need to add reducing agents Molecular weight Selection of gel filtration media Charge properties Selection of ion exchange conditions Biospecific affinity Selection of ligand for affinity medium Post translational modifications Selection of group-specific affinity medium Hydrophobicity Selection of medium for hydrophobic interaction chromatography
Develop analytical assays Goal:To follow the progress of a rification.to asse ss effectiveness(yield, biological activity,recovery)and to help during optimisation. Select assays which are fast and reliable. ng method develo ve access to wing assays .A rapid,reliable assay for the target protein .Purity determination The in hasise ofa reliable assay for thet arget protei for separation do not interfere with the assay.Purity of the target protein is most often estimated by sDS-PAGE.capillary electrophoresis.reversed phase chromatography or mass spectrometry.Lowry or Bradford assays are used most frequently to determine the total pro The Br les where there is a high lipid For large scale protein purification the need to assay for target proteins and critical impurities is often essential.In practice,when a protein is purified for for harmfuposes.oo e consning to denny and se up speenc ys A pra ro tO. and m s after a s dage per d to che Sample Extraction and Clarification Minimise sample handling Minimise use of additives Remove damaging contaminants early Definition:Prin isolati n of targe 2trnoicematerial val of particulate matter or other contaminants which are not compatible with chromatography
14 Develop analytical assays Goal: To follow the progress of a purification, to assess effectiveness (yield, biological activity, recovery) and to help during optimisation. Select assays which are fast and reliable. To progress efficiently during method development the effectiveness of each step should be assessed. The laboratory should have access to the following assays: • A rapid, reliable assay for the target protein • Purity determination • Total protein determination • Assays for impurities which must be removed The importance of a reliable assay for the target protein cannot be overemphasised. When testing chromatographic fractions ensure that the buffers used for separation do not interfere with the assay. Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed phase chromatography or mass spectrometry. Lowry or Bradford assays are used most frequently to determine the total protein. The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay. For large scale protein purification the need to assay for target proteins and critical impurities is often essential. In practice, when a protein is purified for research purposes, it is too time consuming to identify and set up specific assays for harmful contaminants. A practical approach is to purify the protein to a certain level, and then perform SDS-PAGE after a storage period to check for protease cleavage. Suitable control experiments, included within assays for bio-activity, will help to indicate if impurities are interfering with results. Sample Extraction and Clarification Minimise sample handling Minimise use of additives Remove damaging contaminants early Definition: Primary isolation of target protein from source material. Goal: Preparation of a clarified sample for further purification. Removal of particulate matter or other contaminants which are not compatible with chromatography
The need for sample preparation prior to the first chromatographic step is dependent upon sample type.In some situations samples may be taken directly to the first capture step.For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as SepharoseTM Fast Flow and may require only a minor adjustment of the pH or ionic strength.However,it is most often essential to perform some form of sample extraction and clarification procedure, If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used.It should be noted that a technique such as ar monly used in small scale,may be unsuitable for scale Choice of buffer and additives must e cases inexper ve b to the more co plex mpositions used i he lab ratory.It shou hat dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples. -For repeated purification,use an extraction and clarification technique that is robust and able to handle sample variability.This ensures a reproducible product for the next purification step despite variability in starting material. -Use additives only if essential for stabilisation of product or improved traction.Select thos e which ar easily remove Addi be removed in an extra purificati ion step ves may need to Use pre-packed columns of SephadexTM G-25 gel filratio edia,for rapid sample clean-up at laboratory scale,as shown in Table 2. Table 2.Pre-packed columns for sample clean-up. Pre-packed column Sample volume Samnle volume Code No. loading per run recovery per run HiTrap TM Desaltin ng26/10 25-15ml 75.20ml 17.5087-01 025.15m 1.0-2.0m 17.1408.01 esalting PC 3.2/10 0.05-0.2ml 0.2-0.3m 17-0774-0 1.5-2.5m 2.5-3.5m 17-0851-0 Sephadex G-25 gel filtration media are used at laboratory and production scale for sample preparation and clarification of proteins >5000.Sample volumes of up to 30%or in ome cases,40%of the total column volume are loaded.In a single step,the sample is desalted,exchanged into a new buffer,and low molecular weight mate als are removed.The high volume ble very lare relative insensitivity to sample ad of thi ple volum be pro ap nly.Using a highs ts in separati al sample dilution (approximately1:1. apter 8 contains further details on sample storage,extraction and clarification procedures. 15
15 The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose™ Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure. If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used. It should be noted that a technique such as ammonium sulphate precipitation, commonly used in small scale, may be unsuitable for very large scale preparation. Choice of buffers and additives must be carefully considered if a purification is to be scaled up. In these cases inexpensive buffers, such as acetate or citrate, are preferable to the more complex compositions used in the laboratory. It should also be noted that dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples. For repeated purification, use an extraction and clarification technique that is robust and able to handle sample variability. This ensures a reproducible product for the next purification step despite variability in starting material. Use additives only if essential for stabilisation of product or improved extraction. Select those which are easily removed. Additives may need to be removed in an extra purification step. Use pre-packed columns of Sephadex™ G-25 gel filtration media, for rapid sample clean-up at laboratory scale, as shown in Table 2. Table 2. Pre-packed columns for sample clean-up. Pre-packed column Sample volume Sample volume Code No. loading per run recovery per run HiPrep™ Desalting 26/10 2.5 -15 ml 7.5 - 20 ml 17-5087-01 HiTrap™ Desalting 0.25 - 1.5 ml 1.0 - 2.0 ml 17-1408-01 Fast Desalting PC 3.2/10 0.05 - 0.2 ml 0.2 - 0.3 ml 17-0774-01 PD-10 Desalting 1.5 - 2.5 ml 2.5 - 3.5 ml 17-0851-01 Sephadex G-25 gel filtration media are used at laboratory and production scale for sample preparation and clarification of proteins >5000. Sample volumes of up to 30%, or in some cases, 40% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity, relative insensitivity to sample concentration, and speed of this step enable very large sample volumes to be processed rapidly and efficiently. Using a high sample volume load results in a separation with minimal sample dilution (approximately 1:1.4). Chapter 8 contains further details on sample storage, extraction and clarification procedures
Sephadex G-25 is also used for sample conditioning,e.g.rapid adjustment of pH, buffer exchange and desalting between purification steps. Media for consideration Sephadex G 25 gel filtration For fast group separations between high and low molecular weight substances Typical flow velocity 60 cm/h(Sephadex G-25 Superfine,Sephadex G-25 Fine), 150 cm/h(Sephadex G-25 Medium). Combine Sample Clean-up and Capture in a single step If large sample volumes will be handled or the method scaled-up in the future consider using STREAMLINETM expanded bed adsorption.This technique is suited for large scale re ombinant protein and mo noclonal antibody containing lied to the expa n. are p ow all y de gn in S geth needed for high producti cation beds.The technique requires no sample clean up and so combines sample e l particulate matter,whole cells,and contaminants pass through.Flow is reversed and the target proteins are desorbed in the elution buffer. Media for consideration: STREAMLINE UIEX AC.Hic For sample clean-up and capture direct from crude sample. STREAMLINE adsorbents are designed to handle feed directly from both ock from cell cultu re/fermentation at Note: cmlb:flow velocity (linear flow rate)=volumetric flow ratelcross sectional area of column. 16
16 Sephadex G-25 is also used for sample conditioning, e.g. rapid adjustment of pH, buffer exchange and desalting between purification steps. Media for consideration: Sephadex G 25 gel filtration For fast group separations between high and low molecular weight substances Typical flow velocity 60 cm/h (Sephadex G-25 Superfine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium). Combine Sample Clean-up and Capture in a single step If large sample volumes will be handled or the method scaled-up in the future, consider using STREAMLINE™ expanded bed adsorption. This technique is particularly suited for large scale recombinant protein and monoclonal antibody purification. The crude sample containing particles can be applied to the expanded bed without filtration or centrifugation. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. Together they enable the high flow rates needed for high productivity in industrial applications of fluidised beds. The technique requires no sample clean up and so combines sample preparation and capture in a single step. Crude sample is applied to an expanded bed of STREAMLINE media. Target proteins are captured whilst cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target proteins are desorbed in the elution buffer. Media for consideration: STREAMLINE (IEX, AC, HIC) For sample clean-up and capture direct from crude sample. STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application. Note: cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column