Stimmen - 4, Durchschnittliche Bewertung: 4
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Anleitung Zusammenfassung
In our experience, the CNBr coupling technique has a well-proven track record for the purification of therapeutic proteins. This, plus the performance of the matrix at large scale, makes the use of CNBr-activated Sepharose 4 Fast Flow particularly attractive for manufacturing applications in the biopharmaceutical industry. Furthermore, the medium is a member of the BioProcessTM media. BioProcess media are developed and supported for production scale chromatography. All BioProcess media are produced with validated methods and are tested to meet manufacturing requirements. Secure ordering and delivery routines give a reliable supply of media for production scale. Regulatory Support Files (RSF) are available to assist process validation and submissions to regulatory authorities. To ensure best performance and trouble-free operation, please read these instructions before using CNBr-activated Sepharose 4 Fast Flow. Table of contents 1. Product description 2 2. Coupling 3 3. Column packing guidelines 5 4. Evaluation of packing 7 5. Cleaning, Sanitization and Storage 10 6. Ordering information 11 1. Product description CNBr-activated Sepharose 4 Fast Flow is a bead-formed, highly crosslinked pre-activated matrix produced by reacting Sepharose 4 Fast Flow with cyanogen bromide (CNBr). This coupling makes the medium more rigid which in turn improves the pressure/flow characteristics. Proteins and other molecules containing primary amino groups can be coupled directly to the pre-activated medium. Multi-point attachment of proteins provides the immobilized product with good chemical stability. The resulting affinity medium can isolate a specific substance from a complex mixture, often achieving very high yield and purity in a single step. Many references demonstrate that binding affinity is frequently well maintained after CNBr coupling. A typical application of pre-activated affinity media like CNBr-activated Sepharose 4 Fast Flow is based on antigen-antibody reactions with immobilized monoclonal antibodies as ligands. In such cases, purification factors of 2 000–20 000 can be obtained. Table 1 summarizes the main characteristics of CNBr-activated Sepharose 4 Fast Flow. p. 2 Table 1. Medium characteristics. Table 1. Medium characteristics. Linear flowrate Base matrix 150–250 cm/h, 0.1 MPa (1 bar), XK 50/60 column, bed height 25 cm Swelling factor 4–5 ml drained medium/g Coupling capacity 13–26 mg .-chymotrypsinogen/ml pH stability* long term 3–11 short term (CIP) 3–11 * Refers to stability of coupling between ligand abd base matrix. Ligands can be less stable. Sepharose 4 Fast Flow matrix Sepharose 4 Fast Flow is a highly cross-linked agarose matrix. In its pre- activated CNBr form, it offers much improved performance when compared with the well established CNBr-activated Sepharose 4B. The Sepharose 4 Fast Flow matrix has higher rigidity and can thus be run at high flow rates (see Table 1). The higher mechanical strength of the cross-linked matrix makes it well- suited for use in large columns. Scaling up a purification developed on CNBractivated Sepharose 4 Fast Flow is therefore simple and more predictable. The coupled product is stable at low pH, which is often required for elution from some immunoadsorbents. For applications that require operation at high pH, note that the amide bond formed when using the companion product NHS-activated Sepharose 4 Fast Flow is stable up to pH 13 for normal use. 2. Coupling CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried in the presence of additives. These additives need to be washed away at low pH (pH-2–3) before coupling the desired ligand. The use of low pH (pH2–3) preserves the activity of the reactive groups, which otherwise hydrolyse at high pH. p. 3 In order to retain maximum binding capacity of CNBr-activated Sepharose 4 Fast Flow prior to coupling the ligand, use cold (0–4 °C) solutions. The time interval between washing and coupling must be minimised; therefore preparations of all required solutions prior to coupling is recommended. In order to retain maximum binding capacity of CNBr-activated Sepharose 4 Fast Flow prior to coupling the ligand, use cold (0–4 °C) solutions. The time interval between washing and coupling must be minimised; therefore preparations of all required solutions prior to coupling is recommended. . Prepare the coupling solution, i.e. dissolve the ligand to be coupled in a suitable coupling buffer, e.g. 0.1 M NaHCO3 pH 8.3 containing 0.5 M NaCl. For good coupling efficiency avoid unnecessarily dilute solutions (Recommended ratio of volumes, coupling solution/medium is 0.5:1). The coupling pH depends on the ligand. Normally pH in the range 7–9 is used. ppm sugar A-method: Add portions of 4 column volumes cold 1 mM HCl, stir and immediately remove the liquid. B-method: Add portions of 1 column volume cold 1 mM HCl, stir for approx. 5 min and then remove the liquid. 400 300 200 100 0 A-method B-method 0 102030405060 Gel vol. Fig 1. The c...