Capto * Blue and Capto Blue (high sub)

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1 GE Healthcare Life Sciences Data file AC Affinity chromatography Capto * Blue and (high sub) and (high sub) are affinity chromatography media (resins) for the capture of human serum albumin, as well as purification of HSA fusion proteins, blood coagulation factors, enzymes, and recombinant proteins at laboratory and process scales. Developed from Blue Sepharose* 6 Fast Flow, products are more chemically stable and have a more rigid agarose base matrix than their predecessor. These improvements allow the use of faster flow rates and larger sample volumes, leading to higher throughput and improved process economy. and (high sub) offer the following benefits: Excellent chemical stability ensures tolerance of harsh solvents used in repeated cleaning-in-place and sanitization procedures Highly rigid agarose base matrix allows high flow rates and processing of large sample volumes Ligand functionality may be modified through the use of appropriate buffer salts and buffer conductivity to increase selectivity for desired targets Excellent choice for the removal or purification of proteins at both laboratory and process scales Principles and (high sub) are media for affinity chromatography that can be used in the same way as Blue Sepharose 6 Fast Flow. Binding of albumin occurs near ph 5.5 and elution is performed by increasing the conductivity using sodium chloride and increasing ph. The Cibacron Blue ligand contains sulfonic groups that can take part in ion exchange interactions and also groups that can bind to the target molecule by hydrophobic interactions. (high sub) is part of the Custom Designed Media line and available by request. Fig 1. and (high sub) are affinity media for the capture of albumin, as well as purification of HSA fusion proteins, enzymes, and recombinant proteins at laboratory and process scales. Depending on the target molecule, the effect of these groups can be enhanced or weakened by the choice of buffer salt and conductivity. To increase yield or to regenerate the chromatography medium, elution with salt can be complemented by adding an organic solvent such as ethylene glycol. A typical protocol for purification is described as follows: 1. Pack a column with (or use the prepacked HiScreen* ) or (high sub). 2. Wash the medium bed with starting buffer. 3. Adjust the sample ph to that of the starting buffer (e.g., binding of human serum albumin, HSA, occurs at ph 5.5). 4. Filter the sample through a.22 to.45 μm filter. 5. Load the sample. 6. Wash the medium bed with starting buffer to remove weakly bound proteins. 7. Optimize elution conditions to attain maximum purity and throughput of the captured proteins. Bound proteins may be eluted, for example, by the addition of acl.

2 Characteristics of the chromatography medium media are based on a highly rigid agarose base matrix that offers outstanding pressure and flow properties, allowing rapid processing of large sample volumes. The Cibacron Blue ligand is attached to the base matrix via a hydrophilic spacer and is immobilized with a stable amine bond (Fig 2). The main characteristics of and (high sub) are shown in Table 1. A) B) O O OH O OH OH Fig 2. (A) medium and (B) (high sub) showing the base matrix, spacer, and ligand. R1 R2 R1 R2 Binding capacity and recovery The binding capacity for HSA was evaluated in terms of 1% breakthrough (Q B1, the point that 1% of unbound HSA is detected in collected fractions). The conditions used in the evaluation of binding capacity are shown in Table 2. Table 2. Conditions used in the evaluation of binding capacity of and (high sub) for HSA at 1% breakthrough compared with Blue Sepharose 6 Fast Flow Column Tricorn* 5/1 (1 cm bed height) Sample HSA, 1 mg/ml Binding buffer 2 mm citric acid, ph 5.5 Elution buffer 5 mm sodium phosphate, 2 M acl, ph 7 Flow velocity 15 cm/h Retention time 4 min Figure 3 shows the breakthrough curves for the three products. The results show that the dynamic binding capacity for (high sub) is 25% higher than that of. and Blue Sepharose 6 Fast Flow are comparable. Considering that the Capto base matrix tolerates a much higher flow rate and bed height, there is much to gain in terms of productivity, especially in equilibration, wash, elution and CIP. Table 1. Physical and performance characteristics of and (high sub) Matrix Highly cross-linked agarose Average particle size 75 µm Ligand Cibacron Blue Ligand density ~18 μmol/ml, (high sub) ~13 μmol/ml, Dynamic binding capacity of 3 mg/ml at 4 min residence time for HSA at 1% breakthrough (high sub) 24 mg/ml at 4 min residence time for Maximum flow velocity At least 6 cm/h in a 1 m column with 2 cm bed height at 2ºC using process buffers with the same viscosity as water; corresponds to a residence time of 2 min ph stability operational during CIP 3 to 13 2 to 13.5 Cibacron Blue binds targets through a combination of aromatic and electrostatic interactions, which makes the medium suitable for many different applications. The ligand has structural similarities to naturally occurring molecules such as the cofactor AD +, which enables it to bind a wide range of proteins. mau Blue Sepharose 6 FF (high sub) ml Fig 3. Breakthrough curves for Blue Sepharose 6 Fast Flow (FF),, and (high sub). Figure 4 shows how the dynamic capacity increases with an increased residence time until a plateau is reached. A more concentrated feed also resulted in a higher binding capacity (data not shown). In a recovery study, 7% of Q B1 was loaded. The study shows similar results for and Blue Sepharose 6 Fast Flow. (high sub) binds harder. To improve recovery for (high sub), 2% ethylene glycol was added to the elution buffer (Table 3). With 5% ethylene glycol, recovery in the elution peak was 95% AC

3 Q B 1 % (mg HSA / ml) Relation between residence time and dynamic capacity Residence time (min) Fig 4. Dynamic binding capacity relative to residence time for (high sub). The dynamic binding capacity increases with increased residence time until a plateau is reached. Table 3. Comparative breakthrough capacity and recovery of Blue Separose 6 FF,, and (high sub). Blue Sepharose 6 FF 1 Capto Blue 1 (high sub) 1 (high sub) 2 Q B1 (mg/ml) Load (mg) Eluate (mg) Recovery (%) CIP (mg) , Recovery (%) Elution buffer: 5 mm ah M acl, ph 7 2 Elution buffer: 5 mm ah M acl, ph 7 + 2% (w/w) ethylene glycol Binding buffer: 2 mm citric acid, ph 5.5 CIP: 1 mm aoh (Blue Sepharose 6 FF),.5 M aoh ( products) Load: HSA in binding buffer ~1 mg/ml Figure 5 shows the chromatograms from the recovery study. Flowthrough and wash were equal with baseline and were not analyzed for all runs. However they were analyzed sporadically to verify that they contained very little protein. Small-scale format provides fast screening and method development Using small-scale format to screen for the most suitable chromatography process conditions in the early stages of process development saves both time and sample. is available in the small, prepacked HiScreen column format (4.7 ml). Together with a chromatography system, such as ÄKTA* avant, prepacked HiScreen columns are convenient to use when developing an efficient and robust separation method. Further development and optimization using HiScale* columns then permits straightforward scale-up. Basic characteristics of HiScreen prepacked columns are summarized in Table 4. Table 4. Main characteristics of prepacked HiScreen columns Column volume Column dimensions Maximum flow velocity Recommended flow velocities Maximum back-pressure over the packed bed Maximum pressure over the hardware Purification of HSA from plasma Column: Equilibration: Load: Wash: Elution: 4.7 ml.77 1 cm 6 cm/h 15 3 cm/h.3 MPa (3 bar).8 MPa (8 bar) The selectivity of and (high sub) was verified in a one-step purification of HSA from serum (Fig 6). The loaded, washed and eluted samples were analyzed by SDS-PAGE (Fig 7). The selectivity for HSA was high and the eluted fraction had purity similar to the reference material. Tricorn 1/1 (1 cm bed height) 1 column volumes (CV) of 5 mm Tris, 5 mm acl, ph 8, 535 cm/h 2.5 ml of filtered human serum, 3 cm/h 1 CV of 5 mm Tris, 5 mm acl, ph 8, 535 cm/h 7.5 CV of 5 mm Tris, 2 mm sodium thiocyanate (asc), ph 8, 3 cm/h mau 25 2 Elution peaks CIP peaks Blue Sepharose 6 FF (high sub) mau ml Fig 5. Comparative chromatograms from the recovery study involving Blue Sepharose 6 FF,, and (high sub). Elution conditions as in footnote 1 in Table 3. F2 Waste F3 Waste ml Fig 6. Purification of HSA from serum using (high sub). The total load of HSA corresponds to 5% of the maximum capacity. Similar results were obtained when using AC 3

4 (Mr) Lane 1. Low Molecular Weight Marker 2. Serum 3. Flowthrough 4. Eluate 5. HSA reference sample 6. Low Molecular Weight Marker Fig 7. SDS-PAGE using PhastGel* Gradient 1 15 and PhastSystem* analysis of whole human serum before application and eluate from (high sub) under reducing conditions (similar results were obtained when using ). ote that most HSA binds to the (high sub) (lane 3) and that the purity (lane 4) is similar to the reference sample (lane 5). Cleaning-in-place and sanitization A cleaning or sanitization protocol should be designed for each application in order to prevent the accumulation of contaminants over time. The tolerance of (high sub) to cleaning with aoh was evaluated by studying binding capacity for HSA after repeated exposure to aoh. Each purification cycle consisted of equilibration, loading of plasma, elution, and cleaning-in-place (CIP) with.5 M aoh for 3 min. After every tenth cycle, the total binding capacity of the medium for HSA was determined. Figure 8 shows that the binding capacity for HSA over 5 cycles of CIP remained similar to the initial measurement and that.5 M aoh can therefore be used for routine CIP. is expected to show similar results. Chemical stability and recommended storage To evaluate chemical stability, and (high sub) were stored at room temperature for three weeks in the storage solutions listed in Table 5. o change in ligand density was observed following storage in any of the storage solutions. Although exposure to.1 M HCl did not decrease ligand density, this solution may hydrolyze the base matrix over extended periods of contact and is thus not recommended for prolonged storage. and (high sub) are supplied, pre-swollen, in a solution of.1 M KH 2 and 2% ethanol, ph 8. The medium should be stored in this solution at 2 C to 8 C. Table 5. Storage solutions used in the study of chemical stability of and (high sub).1 M KH 2 + 2% ethanol, ph 8 8 M urea.1 M aoh + 2% ethanol 6 M guanidine-hcl.1 M HCl.1 M HCl.1 M aoh.5 M aoh 1 Binding capacity (% of reference) umber of CIP cycles Fig 8. Cleaning-in-place with.5 M aoh on (high sub). 1 (high sub) can also be sanitized by autoclaving, which is particularly appropriate if microbial contamination is suspected. When (high sub) was autoclaved 1 times (121ºC, 2.5 to 2.7 bar), no decrease in ligand density was recorded. has not been exposed to autoclaving but is expected to have the same tolerance as (high sub) AC

5 Ordering information Product Quantity Code no. 25 ml ml L HiScreen ml (high sub) 25 ml (high sub) 5 ml (high sub) 5 L ote: (high sub) is made available through Custom Design Media and is only produced on customer demand. Thus delivery time is typically longer than for standard media, such as. Both media have validated manufacturing methods and are supported with a Regulatory Support File AC 5

6 For local office contact information, visit GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden imagination at work GE, imagination at work, and GE monogram are trademarks of General Electric Company. * ÄKTA, Capto, HiScale, HiScreen, PhastGel, PhastSystem, Sepharose, and Tricorn are trademarks of GE Healthcare companies. The Tricorn column and components are protected by US design patents USD5856, USD56261, USD5555, USD4956 and their equivalents in other countries. All third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published April 28. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9A, UK GE Healthcare Europe GmbHMunzinger Strasse 5 D Freiburg, Germany GE Healthcare Bio-Sciences Corp. 8 Centennial Avenue, P.O. Box 1327, Piscataway J , USA GE Healthcare Japan Corporation Sanken Bldg., , Hyakunincho, Shinjuku-ku, Tokyo Japan AC 6/211