Phenyl Sepharose High Performance

2022-11-07 来源：步旅网

GE Healthcare

Data File 18-1172-87 AC

Hydrophobic interaction chromatography

Phenyl Sepharose High PerformanceButyl Sepharose High Performance

Phenyl Sepharose™ High Performance and Butyl Sepharose High Performance are hydrophobic interaction chromatography (HIC) media. Their performance is characterized by:

• High-resolution, high-capacity separations with high recovery• Reliable and reproducible

• High chemical stability for effective CIP and sanitization• Available in laboratory and BioProcess™ scale quantities• Easy to scale up

The media occupy a key position in the intermediate and final purification stages of a wide variety of proteins and peptides. Their availability in prepacked columns, such as, HiLoad™ and HiTrap™ plus laboratory and BioProcess pack sizes makes purification at all scales simple and convenient (Fig 1).

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography separates and purifies biomolecules based on differences in their surface hydrophobicity. The technique is versatile and offers

specific selectivity. Many proteins and peptides, as well as other hydrophobic biomolecules have sufficient numbers of exposed hydrophobic groups to allow interaction with hydrophobic ligands coupled to chromatographic matrices. Compared with Reversed Phase Chromatography (RPC) adsorbents, HIC media display milder elution conditions and consequently better retention of biological activity after separation. HIC is well suited for use in the middle or end of protein purification strategies that also employ other chromatographic techniques such as ion exchange and

imat work

Fig 1. Phenyl Sepharose High Performance and Butyl Sepharose High Performance, available in a variety of formats, allow high-resolution, high-capacity purification of biomolecules.

gel filtration. For example, HIC makes an ideal “next step” when purifying material that has been precipitated with ammonium sulfate or eluted in high salt concentrations during ion exchange.

HIC is usually performed in moderate to high concentrations of salts in the start buffer, which promotes binding and helps to stabilize the protein structure. The bound molecule is eluted by decreasing the salt concentration in a linear or stepwise manner. Continuous gradient elution is most frequently used when high resolution is needed and stepwise gradient elution is recommended for sample preparation and concentration. Several factors influence the behavior of proteins and peptides on HIC media. These include sample characteristics, flow rate, temperature, type and concentration of salt, and pH.

Table 1. Main characteristics of Phenyl Sepharose High Performance and Butyl Sepharose High Performance

Functional groupMatrix Average particle size Exclusion limit (Mr) Ligand concentration (µmol/ml medium) Binding capacity

Rec. linear flow rate Chemical stability

pH stability short term1

long term and working2 Storage 1

Phenyl Sepharose High Performance

Phenyl—O—

Highly cross-linked agarose 34 µm

4 × 106 (globular proteins) 25 µmol/ml medium 45 mg a-chymotrypsinogen /ml medium Up to 150 cm/h 1 M sodium hydroxide 1 M acetic acid 8 M urea 6 M guanidine hydrochloride 30% acetonitrile 70% ethanol 3 M (NH4)SO4 1 mM HCl 30% isopropanol 2% SDS 2 to 14 3 to 13 20% ethanol Butyl Sepharose High Performance

Butyl—O—(CH2)3—CH3Highly cross-linked agarose34 µm

4 × 106 (globular proteins)50 µmol/ml medium38 mg b-lactoglobulin /ml mediumUp to 150 cm/h1 M sodium hydroxide1 M acetic acid8 M urea6 M guanidine hydrochloride30% acetonitrile70% ethanol3 M (NH4)SO41 mM HCl30% isopropanol2% SDS2 to 143 to 1320% ethanol Short term pH stability: the pH interval to which the medium can be subjected for cleaning- or sanitization-in-place (accumulated 90–100 h at room temperature) without significant change in function.

Long term pH stability: the pH interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

Media characteristics

Phenyl Sepharose High Performance and Butyl

Sepharose High Performance are high-resolution, high-capacity media based on highly cross-linked, 34-µm agarose beads modified with phenyl or butyl groups via uncharged, chemically stable ether linkages. They are truly hydrophobic media displaying a minimum of ionic interactions. In addition, they tolerate detergents, polar organic solvents, and chaotropic agents, as well as agents for cleaning-in-place and sanitization. Table 1 lists the main characteristics of the two media.

Tricorn™ and XK ranges are available in a variety of sizes for users who want to pack their own columns. For a complete overview of the HIC High Performance product line, refer to the Ordering information.

Prepacked columns

By providing added speed, convenience, and reproducibility, prepacked columns extend the usefulness of Phenyl Sepharose High Performance and Butyl Sepharose High Performance. The columns can be used with simple pump configurations or a wide variety of systems. ÄKTAdesign™ include preset method templates based on these prepacked columns, which further improves separation results,

particularly reproducibility, and the speed at which they are achieved. Phenyl Sepharose High Performance is supplied in four types of prepacked columns, HiLoad 16/10 Phenyl Sepharose High Performance (20 ml), HiLoad 26/10 Phenyl Sepharose High Performance (53 ml) and HiTrap Phenyl HP, 1 ml and 5 ml. Butyl Sepharose High Performance is supplied in prepacked HiTrap Butyl HP, 1 ml and 5 ml.

Laboratory and BioProcess pack sizes

Phenyl Sepharose High Performance and Butyl Sepharose High Performance are supplied in laboratory pack sizes as well as BioProcess pack sizes for flexible scale-up and process development. The laboratory packs are for those who prefer packing columns of their choice and include straightforward instructions and tips for packing, operation, and maintenance. Empty high-resolution columns from the

2 Data File 18-1172-87 AC

Table 2. HiLoad Phenyl Sepharose High Performance characteristics

Bed diameter 16 or 26 mm Bed height 10 cm Bed volume

20 ml (16/10) 53 ml (26/10) Theoretical plates >12 000/m

Rec. flow rate

Up to 150 cm/h (5 ml/min for 16/10, 13 ml/min for 26/10) Max. back pressure over the packed bed during operation 3 bar (43 psi, 0.3 MPa) HiLoad column hardware pressure limit 5 bar (73 psi, 0.5 MPa) Storage

20% ethanol

Table 3. Characteristics of HiTrap Phenyl HP and HiTrap Butyl HP

Column dimensions 0.7 × 2.5 cm (1 ml), 1.6 × 2.5 cm (5 ml)Column volumes 1 ml and 5 ml

Rec. flow rate 1.0 ml/min (1 ml), 5 ml/min (5 ml) Max. flow rate1

4.0 ml/min (1 ml), 20 ml/min (5 ml) Max. back pressure 3 bar (43 psi, 0.3 MPa) Storage

20% ethanol

Room temperature, aqueous buffers.

HiLoad 16/10 and 26/10 Phenyl Sepharose High Performance

HiLoad prepacked columns are XK laboratory columns prepacked with Phenyl Sepharose High Performance. The columns are constructed from precision bore borosilicate glass. Dead volumes are less than 0.1% of the total column volume. A thermostatic jacket is fitted as standard. HiLoad Phenyl Sepharose High Performance is available in two sizes, 16- or 26-mm diameter, both with a bed height of 10 cm and giving column volumes of 20 ml and 53 ml, respectively. In addition to the built-in benefits of high-resolution, high capacity preparative separations, HiLoad columns offer great reproducibility and convenience. Every HiLoad column is packed under stringent control and tested individually for bed volume, efficiency (theoretical plates/m), asymmetry factor, and flow characteristics. They can be used in a wide variety of systems, such as ÄKTAdesign. Flanged tubing and M6 connectors for connection to FPLC™ are supplied as standard, as are connectors for ÄKTAdesign systems. Table 2 lists the key characteristics of HiLoad 16/10 and 26/10 Phenyl Sepharose High Performance columns.

HiTrap Phenyl HP and HiTrap Butyl HP columns

HiTrap Phenyl HP and HiTrap Butyl HP are small, prepacked columns made of biocompatible polypropylene. The columns are delivered with stoppers on the inlets and a snap-off ends on the outlets. All necessary connectors

are included for connection to different systems as well as to a laboratory pump and a simple syringe. Note that HiTrap columns cannot be opened or repacked. Two sizes are available: 1 ml and 5 ml. The 1-ml column is often used for method screening to quickly establish optimal binding and elution conditions for specific applications. Its fast and simple operation is well-suited to this role, as well as to small-scale purifications. The larger 5-ml column is an excellent choice when the purification method has been established and larger amounts of protein need to be purified. Two or three columns can be connected in series for easy scale-up. Table 3 lists key characteristics of HiTrap Phenyl HP and HiTrap Butyl HP columns.

Operating HiTrap Phenyl HP and HiTrap Butyl HP

Using HiTrap Phenyl HP and HiTrap Butyl HP columns is easy. Complete, easy-to-follow instructions are included for fast start-up and method optimization. Whether you use a syringe and the provided Luer adapter, a peristaltic pump, or a chromatography system such as ÄKTAdesign, operation is straightforward.

HIC Selection Kit

HiTrap Phenyl HP and HiTrap Butyl HP are two components of the HiTrap HIC Selection Kit. This kit comprises seven HIC media with different hydrophobic characteristics, each prepacked in a 1-ml HiTrap column. The remaining five media are: Phenyl Sepharose 6 Fast Flow (low sub), Phenyl Sepharose 6 Fast Flow (high sub), Butyl Sepharose 4 Fast Flow, Butyl-S Sepharose 6 Fast Flow, and Octyl Sepharose 4 Fast Flow. The kit helps users screen factors that have an influence on the behavior of proteins and peptides, and select the most appropriate HIC medium to use for a specific separation. The HiTrap HIC Selection Kit is well suited to this task (Fig 2). More information about the kit is available in Data File 18-1143-21.

Chemical stability

Good chemical stability (Table 1) allows the use of effective cleaning-in-place (CIP) schemes that result in high

recoveries over many purification cycles. Regular CIP and sanitization hinders microbial growth and maintains a high level of hygiene to promote good economy. For CIP, regular washing with 0.5 to 1.0 M sodium hydroxide should be sufficient to remove most contaminating material,

although very hydrophobic molecules may bind so tightly that they must be eluted with organic solvents agents like 70% ethanol or 30% isopropanol, or strong detergents. CIP and sanitization protocols for Phenyl Sepharose High Performance and Butyl Sepharose High Performance are included in their respective packages. Note that specific protocols should be developed according to the nature and condition of the starting material.

Data File 18-1172-87 AC 3

Phenyl Sepharose High PerformancemAU 800 2.33 600 400 200 14.84 00.05.010.015.020.025.0ml 0 18.27 20.42100150mS/cm200Butyl Sepharose High PerformancemAU 800 18.02 20.39 2.37mS/cm200150 600 400 200 00.05.010.015.020.025.0ml100 50 15.23 50 0Butyl Sepharose 4 Fast FlowmAU 800150 600 400 200 14.77 00.05.010.015.020.025.0ml 0 2.05 17.40 19.29100mS/cm200Octyl Sepharose 4 Fast FlowmAU 800150 600 400 200 00.05.010.015.020.025.0ml 2.11 17.48100mS/cm200Sample:

Column volume: Sample volume: Sample load: Flow rate:

Start buffer (A):

Elution buffer (B): Gradient: System: Cytochrome C, Ribonuclease A, Lysozyme, α-chymotrypsinogen

6 mg protein/ml, (1:3:1:1) in start buffer1 ml, HiTrap HIC Selection Kit1 ml

6 mg protein/ml medium1.0 ml/min, (150 cm/h)

0.1 M Na2HPO4, 1.7 M (NH4)2SO4, pH 7.0

0.1 M Na2HPO4, pH 7.0

0%–100% Elution buffer in 10 mlÄKTAFPLC™

50 50 0Phenyl Sepharose 6 Fast Flow (low sub)mAU 800150 600 2.13 400 200 00.05.010.015.020.025.0ml 18.52100mS/cm200Butyl-S Sepharose 6 Fast FlowmAU 800150 600 400 200 00.05.010.015.020.025.0ml 1.97 16.61100mS/cm200Phenyl Sepharose 6 Fast Flow (high sub)mAU 800150 600 400 2.54 200 16.43 00.05.010.015.020.025.0ml 21.21100mS/cm200 50 0 50 0 50 0Fig 2. Comparison of the selectivity characters of the different media in HiTrap HIC Selection Kit. Elution volumes are shown at each peak.

Applications

Screening

The separation result shown in Figure 2 demonstrates how important method screening is when working with HIC.

Model proteins were separated using the same method and buffers. After sample injection, the bound proteins were eluted with a decreasing gradient over 10 ml.

Column: Sample:

Start buffer:

Elution buffer: Flow rate: HiLoad 16/10 Phenyl Sepharose High Performance (VT: 20 ml)400 ml (10 mg protein) from previous steps diluted to 600 ml with 3 M ammonium sulfate. The starting material for the whole procedure was lysed E. coli (90 g cells) containing rHIV RT10 mM Tris-HCl, 1 M ammonium sulfate, 10% glycerol, 1 mM DTT, pH 8.0

10 mM Tris-HCl, 10% glycerol, 1 mM DTT, pH 8.03 ml/min (90 cm/h)

A280 nm0.5

Partial purification of HIV reverse transcriptase

High resolution at high sample loads and flow rates, plus first class chemical and physical stability, make Phenyl Sepharose High Performance and Butyl Sepharose High Performance natural choices for preparative separations during the intermediate and final steps of a protein

purification scheme. As pointed out previously, HIC is an ideal technique to further purify material that has been precipitated with ammonium sulfate or eluted in high salt concentrations during ion exchange.

Figure 3 shows HiLoad 16/10 Phenyl Sepharose High

Performance used to concentrate and purify recombinant HIV reverse transcriptase (rHIV RT). This step, which follows ammonium sulfate precipitation and ion exchange, is used prior to final purification on a Mono Q™ column.

0.25

650700750800 (ml)

Fig 3. Concentration and purification of recombinant HIV reverse

transcriptase on HiLoad 16/10 Phenyl Sepharose HP. Work by T. Unge, B. Strandberg, K. Brobäck and S. Lövgren (Inst. for Molecular Biology, Uppsala University, Sweden), and R. Bhikhabhai (GE Healthcare, Uppsala, Sweden).

4 Data File 18-1172-87 AC

Column:Sample: Tricorn 5/100

IgG

1 purified by affinity (protein A) and anion exchange Sample volume: chromatography 1 ml

Sample load: 1.37 mg/ml

Start buffer:Elution buffer: 50 mM sodium phosphate, 3 M sodium chloride, pH 6.850 mM sodium phosphate, pH 6.8Flow rate:Gradient: 0.6 ml/min (180 cm/h)

0–100% elution buffer, linear gradient in 20 CVSystem: ÄKTAexplorer™ 100

mAU100NaCl8060Butyl Sepharose HPPhenyl Sepharose HP40200

020406080100Fig 4. Separation of monomeric MAb from truncated MAb and other Time (min)

impurities on Butyl Sepharose High Performance and Phenyl Sepharose High Performance.

MAb Purification

Butyl Sepharose High Performance is an effective second or third step in a MAb purification process due to its ability to separate monomeric MAb from aggregates and other impurities. In this case, a partly purified IgG1 antibody eluted from MabSelect SuRe™ was purified on Butyl

Sepharose High Performance and Phenyl Sepharose High Performance. Both media were packed in Tricorn 5/100 and tested under similar conditions. Figure 4 shows that Butyl Sepharose High Performance and Phenyl Sepharose High Performance remove impurities equally well but with slightly different elution volumes.

Process development and scale-up

The excellent performance of Phenyl Sepharose High Performance and Butyl Sepharose High Performance for laboratory-scale preparative applications can be extended to process development and scale-up of HIC separations. The media are well supported for these tasks, with special services and documentation to facilitate the development, scale-up, and routine operation of production applications. Validated manufacture, secure supply, and regulatory support comprise just part of this package. For more information, please contact your GE Healthcare representative.

Ordering information

Prepacked columns

Product

Quantity

Code no.

HiTrap Butyl HP

5 × 1 ml 5 × 5 ml 28-4110-01 28-4110-05HiTrap Phenyl HP 5 × 1 ml 17-1351-01

5 × 5 ml 17-5195-01HiLoad16/10 Phenyl Sepharose HP 1 (20 ml) 17-1085-01HiLoad 26/10 Phenyl Sepharose HP

1 (53 ml)

17-1086-01

Bulk Media

Product

Quantity

Code no.

Butyl Sepharose High Performance 25 ml 200 ml 17-5432-01 17-5432-02 1 l 17-5432-03

5 l 17-5432-04Phenyl Sepharose 75 ml 17-1082-01 High Performance 1 l 17-1082-03

5 l

17-1082-04

全部栏目

Phenyl Sepharose High Performance