Capto Q Capto ViralQ Capto S
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1 GE Healthcare Data File AD Ion exchange chromatography Capto ViralQ Capto Q and are strong ion exchange media for packed bed chromatography that increase speed and throughput in capture and intermediate purification. They combine high capacity with high flow velocity and low backpressure to reduce process cycle times and increase productivity. Capto ViralQ is specifically intended for the purification of viruses., and Capto ViralQ are BioProcess media that meet the demands of largescale biopharmaceutical manufacturers by: Raising productivity with high dynamic binding capacity at high flow Increasing yield with rapid mass transfer Reducing process time with high volume throughput Cost-effective processing with smaller unit operations Important information Separating viral particles with products may require a license under United States patent 6,537,793 B2 and foreign equivalents owned by Centelion SAS. Such a license is not included with the purchase of but is included with the purchase of Capto ViralQ products. With the purchase of Capto ViralQ the customer is granted a free limited license under US patent 6,537,793 B2 and foreign equivalents owned by Centelion SAS to separate viral particles solely through use of the product purchased. Velocity (cm/h) Sepharose 6 Fast Flow Pressure (bar) Fig 1. The pressure-flow properties of Capto media compared to Sepharose 6 Fast Flow. Running conditions: BPG 3 (3 cm i.d.), open bed at settled bed height equal to 23 cm, with water at 2 C. Capto ViralQ is identical to in all aspects except for the addition of a license it provides for virus purification as described in the section Important information. All properties and applications described in this data file for are also valid for Capto ViralQ. Media characteristics High throughput in downstream purification requires separation media that combine mechanical strength of the matrix with a pore structure that allows fast mass transfer and higher capacity for the target molecules. Capto media are based on a highly rigid agarose base matrix that offers outstanding pressure/flow properties with optimized pore structure. Capto media are intended for general use in largescale bioprocess operations. The basic characteristics of and are summarized in Table 1. High flow and low backpressure in large-scale columns High flow velocities allow increased productivity of large-scale bioprocessing operations and processing of large volumes in one working shift. Shorter cycle times also reduce exposure
2 Table 1. Characteristics of and Matrix highly cross-linked agarose with dextran surface extender Ion exchange type strong anion, Q strong cation, S Charged group -N + (CH 3 ) 3 - -SO 3 Total ionic capacity mmol Cl - /ml medium mmol Na + /ml medium Particle size* 9 µm (d 5v ) 9 µm (d 5v ) Flow velocity at least 7 cm/h in a 1 m diameter column with 2 cm bed height at 2 C using process buffers with the same viscosity as water at < 3 bar (.3 MPa) Dynamic binding capacity > 1 mg BSA/ml medium > 12 mg lysozyme/ml medium ph stability short term working long term Working temperature 4 to 3 C 4 to 3 C Chemical stability all commonly used aqueous buffers 1 M acetic acid, 1 M NaOH, 8 M urea, 6 M guanidine hydrochloride, and 7% ethanol Storage 2% ethanol 2% ethanol +.2 M NaAc (sodium acetate) Avoid oxidizing agents, anionic detergents oxidizing agents, cationic detergents * d 5v is the median particle size of the cumulative volume distribution. Dynamic binding capacity at 1% breakthrough as measured at a residence time of 1 minute, 6 cm/h in a Tricorn 5/1 column with 1 cm bed height in a 5 mm Tris-HCl buffer, ph 8.. Short term ph: ph interval that the medium can be subjected to, for cleaning- or sanitization-in-place (accumulated 9 3 h at room temperature) without significant change in function. Working ph: ph interval where the medium binds protein as intended or is needed for elution, without adverse long-term effect. Long term ph: ph interval where the medium can be operated without significant change in function. Low temperatures can decrease capacity for. No significant change in ionic capacity and carbon content after 1 week storage in 1 M NaOH at 4 C. of the target protein to proteases. Typical flow velocities for Capto media in a 1 m diameter column with 2 cm bed height are over 7 cm/h, with a backpressure below 3 bar. Figure 1 compares the pressure/flow performance of Capto with Sepharose 6 Fast Flow in a representative large-scale situation with a BPG 3 column that gives negligible wall support. Although the bead and pore sizes are similar between the two matrices, the pressure/flow properties of Capto is significantly better. This is a result of the exceptional mechanical stability of the Capto base matrix. A Dynamic binding capacity (mg/ml) Q Sepharose Fast Flow Residence time (min) Strong anion and cation exchangers with fast mass transfer and high dynamic binding capacities uses a quaternary amine group and uses a sulfonate group for ion exchange. The groups are linked to a high flow agarose base matrix modified with a dextran surface extender which further increases capacities and mass transfer properties. High dynamic binding capacity is important for obtaining high yield of the target protein. It also contributes to shortening the overall processing time as the total number of cycles may be reduced. The dynamic binding capacities of and at different residence times are shown in Figure 2. B Dynamic binding capacity (mg/ml) SP Sepharose Fast Flow Residence time (min) Fig 2. Dynamic binding capacity as a function of residence time for: (A) and bovine serum albumin (BSA), (B) and α-chymotrypsin. For Sepharose Fast Flow media, residence times below 2 minutes are not possible in large-scale columns. 2 Data File AD
3 Agarose assures protein friendliness and high chemical stability Agarose is a natural polymer with hydrophilic properties that minimizes structural changes of the target molecule and unspecific adsorption to the matrix. It is compatible to the broad range of buffer systems commonly used in bioprocess chromatography for protein purification. Therefore, optimized conditions for binding and elution can be applied, assuring mild treatment of the target molecules. The chemical stability of agarose prolongs media lifetime even if harsh cleaning procedures are applied. Characteristics such as capacity, elution behavior and pressure/flow properties are not affected by common cleaning solutions in process chromatography. These properties make agarose media well accepted by regulatory authorities and have resulted in proven track records in biotherapeutic manufacturing. Rigid media for cost-effective purification The rigidity of Capto products allows improved process economics. and characteristics allow a wider working range of flow velocities, bed heights and sample viscosities, all of which affect processing costs in a positive way. High flow velocities increase volume throughput and reduce process time, longer bed heights means smaller equipment and reduced footprint, and high flow processing of viscous samples means less dilution and shorter cycle times. The available degree of freedom in process design for a medium can be illustrated as its window of operation. Figure 3 shows schematically the ranges for key operating variables for Capto and Sepharose 6 Fast Flow. Given a maximum allowed pressure, it predicts the allowable combinations of column bed heights and operating velocities. The pressure limits, shown as blue and red curves, are based on a 1 m diameter column and calculated from 2 cm bed height and maximum operating velocities of 7 and 25 cm/h, respectively. At this point, the pressure is 3 bar for Capto and 1 bar for Sepharose 6 Fast Flow. For Sepharose 6 Fast Flow, 1 bar represents the highest recommended pressure. For Capto, 3 bar corresponds to the maximum pressure for many low-pressure systems; the medium as such can normally be run to the maximum pressure rating of low and medium pressure columns. Velocity (cm/h) Capto Sepharose 6 Fast Flow Bed height (cm) Fig 3. The highly rigid Capto base matrix allows a much larger window of operation (area below the curves) at large-scale than Sepharose 6 Fast Flow. This is particularly true at bed heights of 25-3 cm and above. Data correspond to a 1 m diameter column, at 2 o C and viscosity of water. Red and blue curves correspond to pressure limits of 1 and 3 bar, respectively. Green contours give the residence time in the column in minutes. The size of the area below the pressure limit curves represents the window of operation, or the available operating range for the respective medium. As shown in Figure 3, this is significantly larger for Capto than for Sepharose 6 Fast Flow based media, especially when bed heights increase to 2-3 cm or more. At these bed heights, Capto can still be run at flow velocities of 3-4 cm/h or more. Thus, the high mechanical stability of Capto allows practical and cost-effective use of smaller diameter columns. A large window of operation also allows flexibility even if the viscosity of the feed is high. Doubling viscosity halves the operational velocity. For a feedstock with a viscosity of 2 cp at a bed height of 3 cm, the flow velocity of Capto is 235 cm/h compared to 8 cm/h for Sepharose 6 Fast Flow. Figure 3 also shows contours of the residence time in the column. A long residence time allows for better utilization of the full equilibrium capacity. It is possible to increase the residence time by either decreasing the flow velocity, or increasing the column bed height. For Capto, increasing bed heights assures longer residence times even under high flow conditions. 3 Data File AD
4 HiTrap columns for fast screening Time and sample can be saved in the early stages of development by using HiTrap columns to screen for the most suitable media and to develop the basic separation method (Fig 4 and Fig 5). In combination with an automated system, such as ÄKTAexplorer, it is easy and fast to scout for optimum selectivity, ph, ionic strength etc. Capto media are available as HiTrap prepacked 1 ml and 5 ml columns. Basic characteristics of HiTrap columns are summarized in Table 2. Table 2. Characteristics of HiTrap columns Column volumes Column dimensions Maximum flow rates Recommended flow rates Maximum backpressure 1 ml and 5 ml.7 x 2.5 cm (1 ml); 1.6 x 2.5 cm (5 ml) HiTrap 1 ml: 4 ml/min, HiTrap 5 ml: 2 ml/min HiTrap 1 ml: 1ml/min, HiTrap 5 ml: 5 ml/min.3 MPa, (3 bar) Column: HiTrap, 1 ml Sample: GFP in E. coli homogenate Buffer A: 5 mm Tris/HCl, ph 8.2 Buffer B: 5 mm Tris/HCl, ph M NaCl Flow: 1 ml/min (156 cm/h) Gradient: 1% B, 15 column volumes System: ÄKTAexplorer 1 A 28nm (mau) A 49nm (mau) 4 Column: HiTrap, 1 ml Sample: α-chymotrypsin in E. coli homogenate Buffer A: 5 mm Sodium acetate, ph 4.8 Buffer B: 5 mm Sodium acetate, ph M NaCl Flow: 1 ml/min (156 cm/h) Gradient: 1% B, 1 column volumes System: ÄKTAexplorer 1 A 28nm (mau) Q Sepharose XL 15 SP Sepharose XL Q Sepharose Fast Flow 25 SP Sepharose Fast Flow ml Volume (ml) Fig 4. The selectivity and separation ability of for GFP compared to Q Sepharose Fast Flow and Q Sepharose XL media in 1 ml HiTrap columns. Fig 5. The selectivity and separation ability of for α-chymotrypsin compared to SP Sepharose Fast Flow and SP Sepharose XL media in 1 ml HiTrap columns. 4 Data File AD
5 Applications Recent developments in upstream processing have resulted in larger feed volumes and increased protein expression levels. The combination of high volume throughput and high capacity makes and the optimal choices for processing large amounts of protein in a fast and efficient way. As strong ion exchangers, their behavior can be easily controlled and application areas predicted by buffer choice and pi of the target proteins. The ligand is a quaternary amine identical to the Q ligand used in other strong anion exchange media. The ligand is a sulfonic group identical to SP and S ligands used in other strong cation exchange media. Despite this, minor differences in selectivity can occur between the media as illustrated in Figures 4 and 5. Improved productivity based on high flow features Scale-up modelling and productivity calculations based on experimental data at small and pilot scale indicate that it is possible to capture and recover >1 kg of green fluorescent protein (GFP) from an Escherichia coli homogenate in 24 h using in a 1.6 m i.d. column at 2 cm bed height (equivalent to 4 L of medium). Assuming the same process conditions, using Q Sepharose Fast Flow would require a 3 m i.d. column or 14 L medium (in practice, this means three separate columns would be needed). This example supports the argument that is being particularly suitable for high throughput and high productivity capture purification. For further details see Application Note Similar calculations indicate that it is possible to capture and recover >1 kg of α-chymotrypsin from E. coli homogenate in 24 hours with in a.8 m i.d. column at 2 cm bed height (equivalent to 1 L of medium). Assuming the same process cycle conditions, using SP Sepharose Fast Flow would require a 1.2 m i.d. column at 2 cm bed height (equivalent to 25 L medium). This example also indicates that is suitable for high throughput and high productivity capture purification. For further details see Application Note Process cycle times and productivity data for both examples are summarized in Table 3. A A 28nm (mau) A 49nm (mau) B Column: Tricorn 5/1, 1 cm bed height (column volume 2 ml) Medium: Sample: GFP in E. coli homogenate, 12 ml Buffer A: 5 mm Tris/HCl, ph 8.2 Buffer B: 5 mm Tris/HCl, ph M NaCl Flow rate: 3 cm/h Gradient: 1% B (6 CV), 33% B (6 CV), 1% B (4 CV) System: ÄKTAexplorer Volume (ml) A 28nm (mau) A 49nm (mau) Column: FineLINE 7, 2 cm bed height (column volume 88 ml) Medium: Sample: GFP in E. coli homogenate, 4835 ml Buffer A: 5 mm Tris/HCl, ph 8.2 Buffer B: 5 mm Tris/HCl, ph M NaCl Flow rate: 6 cm/h Gradient: 1% B (6 CV), 33% B (6 CV), 1% B (4 CV) System: ÄKTApilot TM Volume (ml) Fig 6. A 4-fold scale up using (A) Tricorn 5/1 and (B) FineLINE 7. Table 3. Results from scale-up modelling and productivity calculations for and, as described in the text Target protein Cycle time Productivity Media volume for & medium (min) (kg/h, m 3 ) 1 kg/24h (L) GFP Q Sepharose Fast Flow α-chymotrypsin SP Sepharose Fast Flow Data File AD
6 A B Column: Tricorn 5/1 (bed height 9.7 cm, CV=1.9 ml) Medium: Sample: α-chymotrypsin in E. coli homogenate, 4 mg/ml 5 ml Loading buffer: 5 mm NaAc, ph 4.8 Elution buffer: 5 mm NaAc, ph M NaCl Fluid velocity: 285 cm/h Gradient: 1% CV, 1% 5 CV System: ÄKTAexplorer 1 Residence time: 2 minutes mau ms/cm I nle t Wash Column: XK 16/4 (bed height 2.7 cm, CV= 41.5 ml) Medium: Sample: α-chymotrypsin in E. coli homogenate, 4 mg/ml 14 ml Loading buffer: 5 mm NaAc, ph 4.8 Elution buffer: 5 mm NaAc, ph M NaCl Fluid velocity: 624 cm/h Gradient: 1% CV, 1% 5 CV System: ÄKTAexplorer 1 Residence time: 2 minutes mau ms/cm I nle t Wash C Column: AxiChrom 5 (bed height 22 cm, CV= 431 ml) Medium: Sample: α-chymotrypsin in E. coli homogenate, 4 mg/ml 1.8 L Loading buffer: 5 mm NaAc, ph 4.8 Elution buffer: 5 mm NaAc, ph M NaCl Fluid velocity: 645 cm/h Gradient: 1% CV, 1% 5 CV System: ÄKTApilot Residence time: 2 minutes mau ms/cm min min Wash E lutio n E lutio n E lutio n C I P C I P C I P R e-equilibratio n R e-equilibratio n R e-equilibratio n Operation Fast method development Fast method development can easily be achieved on ÄKTAexplorer with quick initial screening of selectivity and process conditions using prepacked HiTrap columns. Further optimization and method development using Tricorn or XK columns allow straightforward scale-up. UNICORN software on ÄKTAdesign systems makes it simple to transfer the optimized method to a production scale process system. For more information about method development and optimization, consult the handbook, Ion Exchange Chromatography & Chromatofocusing: Principles and Methods, ( ). Fully scalable and belong to the BioProcess range of media that are developed and supported for production-scale chromatography. This includes validated manufacturing methods, secure supply and Regulatory Support Files (RSF) to assist process validation and submission to regulatory authorities. Scale-up is typically done by keeping bed height and flow velocity constant, while increasing column bed diameter and flow rate. However, since optimization is preferentially done with small column volumes, to save sample and buffer, some parameters such as the dynamic binding capacity may be optimized using shorter bed heights than those being used in the final scale. As long as the residence time is constant, the binding capacity for the target molecule remains the same. Other factors, like clearance of critical impurities, may change when column bed height is changed and should be validated using the final bed height. To utilize the full potential of and, we recommend bed heights of 2 cm and higher at large scale. A process was developed and optimized for purification of recombinant GFP using (Fig 6). The process was performed in a Tricorn 5/1 column with 1 cm bed height and then scaled-up 4-fold in a FineLINE 7 column with 2 cm bed height. To maintain constant residence time (2 min) at both scales the flow velocity was doubled in the FineLINE column since it had twice the bed height of the Tricorn column. GFP was eluted in ~2.5 CV in pilot scale and recovery exceeded 9%. A similar scale-up experiment was conducted for (Fig 7) using an optimized process for α-chymotrypsin with a constant residence time of 2 min. From Tricorn 5/1 the bed height was doubled to 2 cm in a XK 16/4 column (CV=4 ml). From the XK 16/4 column further scale up was conducted on an AxiChrom 5 (CV=4 ml) by increasing bed diameter to give a 2-fold scale up min Fig 7. A 2-fold scale up using (A) Tricorn 5/1, (B) XK 16/4 and, (C) AxiChrom columns. 6 Data File AD
7 Cleaning and sanitization Cleaning-in-place (CIP) is a cleaning procedure that removes contaminants such as lipids, precipitates, or denatured proteins that may remain in the packed column after regeneration. Regular CIP also prevents the build-up of these contaminants in the media bed and helps to maintain the capacity, flow properties and general performance of the media. A specific CIP protocol should be designed for each process according to the type of contaminants present. The frequency of CIP depends of the nature and the condition of the starting material, but one CIP cycle is generally recommended every 1 to 5 separation cycles. and withstand all standard CIP procedures, e. g. 1 M NaOH, 2 M NaCl or 7% ethanol. Equipment and can be used together with most equipment available for chromatography from lab scale to production scale. Due to the high rigidity of the medium, packing procedures are slightly different compared to Sepharose 6 Fast Flow based media. For details on packing lab-scale columns, see Instruction manuals, and for packing process-scale columns see Application notes. Appropriate columns from GE Healthcare are shown in Table 4. Table 4. Appropriate columns Column family Range (bed diameters) Tricorn 5 mm, 1 mm XK 16 mm, 26 mm FineLINE 35 mm 35 mm BPG 1 mm 3 mm 1 BioProcess LPLC 1 mm 12 mm Chromaflow 4 mm 2 mm 1 The pressure rating of BPG 45 is too low to use it with and. Storage Store unused media and prepacked columns at 4 to 3 C in 2% in ethanol. Store unused media and prepacked columns at 4 to 3 C in 2% in ethanol +.2 M sodium acetate. Ordering information, Capto ViralQ and are available as bulk media and in prepacked HiTrap columns. Product Pack size Code No 25 ml ml l l l l Capto ViralQ 25 ml Capto ViralQ 1 ml Capto ViralQ 1 l HiTrap 5 x 1 ml HiTrap 5 x 5 ml HiTrap Capto ViralQ 5 x 5 ml Product Pack size Code No. 25 ml ml l l l l HiTrap 5 1 ml HiTrap 5 5 ml Literature Code No. Ion Exchange Chromatography & Chromatofocusing: Principles and Methods Capture of Green Fluorescent Protein on application note Methods for packing in production scale columns application note High productivity capture of α-chymotrypsin on cation exchanger Screening and optimization of loading conditions on cation exchanger for post-protein A purification of monoclonal antibodies Data File AD 7
8 GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden ÄKTAdesign, ÄKTAexplorer, ÄKTApilot, AxiChrom, BioProcess, BPG, Capto, Chromaflow, Dropdesign, FineLINE, HiTrap, Sepharose, Tricorn, and UNICORN are trademarks of GE Healthcare companies. GE, imagination at work, and GE monogram are trademarks of General Electric Company. Separating viral particles with products may require a license under United States patent 6,537,793 B2 and foreign equivalents owned by Centelion SAS. Such a license is not included with the purchase of but is included with the purchase of Capto ViralQ products. With the purchase of Capto ViralQ the customer is granted a free limited license under US patent 6,537,793 B2 and foreign equivalents owned by Centelion SAS to separate viral particles solely through use of the product purchased. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifi cations and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your local GE Healthcare representative for the most current information. 26 General Electric Company All rights reserved. GE Healthcare Bio-Sciences AB, a General Electric Company. GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare UK Ltd Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp 8 Centennial Avenue, P.O. Box 1327 Piscataway, NJ , USA GE Healthcare Bio-Sciences KK Sanken Bldg , Hyakunincho Shinjuku-ku, Tokyo , Japan Asia Pacific Tel Fax Australasia Tel Fax Austria Tel 1/ Fax 1/ Belgium Tel Fax Canada Tel Fax Central, East, & South East Europe Tel Fax Denmark Tel Fax Finland & Baltics Tel +358 () Fax +358 () France Tel Fax Germany Tel: Fax Greater China Tel Fax Italy Tel Fax Japan Tel Fax Latin America Tel Fax Middle East & Africa Tel Fax Netherlands Tel Fax Norway Tel Fax Portugal Tel Fax Russia & other C.I.S. & N.I.S Tel +7 (495) Fax +7 (495) Spain Tel Fax Sweden Tel Fax Switzerland Tel Fax UK Tel Fax USA Tel Fax imagination at work AD 1/26
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