3 Product Guide Amersham Pharmacia Biotech manufactures a wide range of HICmedia suitable fo analytical,small scale preparative and process scale applications.The HIC product range is summarized in Table 4. Phenyl Sepharose 6 Fast Flow(low sub) Suitable for all initial and Phenyl Sepharose 6 Fast Flow(high sub) intermediate step purifications. oratory pack sizes and Phenyl Sepharose High Performance Suitable for all high resolution purifications. Available in laboratory pack sizes bulk quantities and as prepacke columns. Phenyl Sepharose CL-4B Traditional medium for all applications Octyl Sepharose CL-4B Available in laboratory pack sizes and bulk quantities. Alkyl Superose and For analytical and small scale Phenyl Superose preparative applications. Available as prepacked columns HIC Media Test Kit For screening different types of ligands products and for method development work at small scale. Five different HIC media as prepacked Pharmacia 1 ml columns Biotech. DM 21
21 BioProcess™Media Quality an performance from and regulatory support services from Pharmacia. production. Backed by full technical research through scale-up to BioProcess™Media Quality an performance from and regulatory support services from Pharmacia. production. Backed by full technical research through scale-up to 3 Product Guide Amersham Pharmacia Biotech manufactures a wide range of HIC media suitable for analytical, small scale preparative and process scale applications. The HIC product range is summarized in Table 4. * Octyl Sepharose 4 Fast Flow is currently (December 1992) only available as a CDM product (see p. 17), but will later be available as a standard catalogue product. Phenyl Sepharose 6 Fast Flow (low sub) Suitable for all initial and Phenyl Sepharose 6 Fast Flow (high sub) intermediate step purifications. Butyl Sepharose 4 Fast Flow Available in laboratory pack sizes and Octyl Sepharose 4 Fast Flow* bulk quantities. Phenyl Sepharose High Performance Suitable for all high resolution purifications. Available in laboratory pack sizes, bulk quantities and as prepacked columns. Phenyl Sepharose CL-4B Traditional medium for all applications. Octyl Sepharose CL-4B Available in laboratory pack sizes and bulk quantities. Alkyl Superose and For analytical and small scale Phenyl Superose preparative applications. Available as prepacked columns. HIC Media Test Kit For screening different types of ligands and for method development work at small scale. Five different HIC media as prepacked 1 ml columns. Table 4. HIC products available from Amersham Pharmacia Biotech
BioProcess Media BioProcessTM Media form a full range of separation mediaespecially designedtomeet the demandsoftoday's industrial production of biomolecules Productive: High flow rates,high capacity and high recovery lead to good process economy. Validated: Manufactured according to fully validated process with stric quality standards and complete documentation. Scaleable: Work equally well in laboratory and pilot production systems as well as in industrial operation. Cleanable: Very high chemical stability enables thorough cleaning and sanitization treatments that reduce the risk of contamination of the end product and increase the media lifetime. Documented: Regulatory Support Files give full details of approval support data suc stability age ata Support File is an invaluable starting point,especially for pharmaceutical process validations. Guaranteed supply:Large production capacity and guaranteed future supply Base matrices The BioProcess HICmedia range is based on the highly cross-linked beaded agarose matrices Sepharose Fast Flow and Sepharose High Performance.Their macrostructures containing polysaccharide chains arranged in bundles (Fig.6)are further strengthened by different degrees ofinter-chain cross-linking The resulting macroporou combine good capacities for molecules up to 4x105(6%agarose)and 2.7x107(4% agarose)in molecular mass with excellent flow properties and high physical and chemical stability. 22
22 Fig. 6. Structure of cross-linked agarose gels. BioProcess™ Media form a full range of separation media especially designed to meet the demands of today’s industrial production of biomolecules. Productive: High flow rates, high capacity and high recovery lead to good process economy. Validated: Manufactured according to fully validated process with strict quality standards and complete documentation. Scaleable: Work equally well in laboratory and pilot production systems as well as in industrial operation. Cleanable: Very high chemical stability enables thorough cleaning and sanitization treatments that reduce the risk of contamination of the end product and increase the media lifetime. Documented: Regulatory Support Files give full details of approval support data such as performance, stability (including leakage data), extractable compounds and analytical methods. A Regulatory Support File is an invaluable starting point, especially for pharmaceutical process validations. Guaranteed supply: Large production capacity and guaranteed future supply. BioProcess Media Base matrices The BioProcess HIC media range is based on the highly cross-linked beaded agarose matrices Sepharose Fast Flow and Sepharose High Performance. Their macrostructures containing polysaccharide chains arranged in bundles (Fig. 6) are further strengthened by different degrees of inter-chain cross-linking. The resulting macroporous structures combine good capacities for molecules up to 4x106 (6% agarose) and 2.7x107 (4% agarose) in molecular mass with excellent flow properties and high physical and chemical stability. BioProcess™Media Quality an performance from and regulatory support services from Pharmacia. production. Backed by full technical research through scale-up to
All Sepharose based matrices have virtually no non-specific adsorption properties and are also resistant to microbial degradation due to the presence of the unusual sugar 3,6-anhydro-L-galactose. Coupling The HIC ligands are coupled to the monosaccharide units by stable ether linkages. The structures of the coupled ligands are shown in Fig.3. Chemical stability BioProcess HICMedia are stablein allcommonly used aqueous buffers andsolvents in the pH range 2-14.When these media were challenged by storage for7daysat 40C in the solutions listed in Table 5,no significant change in chromatographic function was seen. Of special interest is their stability in alkaline solutions,as cleaning and sanitization with NaOH solt tions are preferred in process applications.The functional stabiliry and recommended pH ranges are summarized in Table 6. The ligand leakage of BioProcess HIC Media at different pH values has been tested and generally found to be extremely low (43).The pH range 2-14 can be used for cleaning-in-place(CIP) on-in-place(SIP) ee"Cleaning,sanitization and sterilization procedures",page 63.BioProcess HIC Media are stable at high tempe- ratures and can be sterilized by autoclaving at 120C for 20 min. Table 5.Chemical stability test of BioProcess HIC Media Tested media Test solutions 1 M NaOH mM HCL (NH).SO.ol GuHCIM Urea (n.t.) (n.t.) + X + X X X (n.t.) (n.t) X X X X (n.t) (n L) (n.t) High (nt) X.Functio Long term stability and recommended working pH range: 3-13 Table 6 Short term stability and recommended CIP and SIP pH range: 2-14 ability and recommended CmeorBioProcess Recommended long term storage: 0.01M 20H or ethanol. 23
23 All Sepharose based matrices have virtually no non-specific adsorption properties and are also resistant to microbial degradation due to the presence of the unusual sugar 3,6-anhydro-L-galactose. Coupling The HIC ligands are coupled to the monosaccharide units by stable ether linkages. The structures of the coupled ligands are shown in Fig. 3. Chemical stability BioProcess HIC Media are stable in all commonly used aqueous buffers and solvents in the pH range 2-14. When these media were challenged by storage for 7 days at 40o C in the solutions listed in Table 5, no significant change in chromatographic function was seen. Of special interest is their stability in alkaline solutions, as cleaning and sanitization with NaOH solutions are preferred in process applications. The functional stability and recommended pH ranges are summarized in Table 6. The ligand leakage of BioProcess HIC Media at different pH values has been tested and generally found to be extremely low (43). The pH range 2–14 can be used for cleaning-in-place (CIP) and sanitization-in-place (SIP), see ‘‘Cleaning, sanitization and sterilization procedures’’, page 63. BioProcess HIC Media are stable at high temperatures and can be sterilized by autoclaving at 120o C for 20 min. Long term stability and recommended working pH range: 3–13 Short term stability and recommended CIP and SIP pH range: 2–14 Recommended long term storage: 0.01 M NaOH or 20% ethanol. Table 6. Stability and recommended pH ranges for BioProcess HIC Media. Tested media Test solutions 1 M acetic 3 M 70% 30% 1 M NaOH acid 1 mM HCL (NH4 ) 2 SO4 ethanol isopropanol 6 M GuHCl 8 M Urea Phenyl Sepharose 6 Fast Flow (low sub) X (n. t.) (n. t.) X X X X X Phenyl Sepharose 6 Fast Flow (high sub) X (n. t.) (n. t.) X X X X X Butyl Sepharose 4 Fast Flow X (n. t.) X (n. t.) X X X (n. t.) Phenyl Sepharose High Performance X X (n. t.) (n. t.) X X X X X = Functionally stable when tested for 7 days at +40°C (n. t.) = Not tested Table 5. Chemical stability test of BioProcess HIC Media
Physical stability The highly cross-linked structures of Sepharose Fast Flow and Sepharose High Performance matrices are physically stable resulting in very good flow properties.This is illustrated by the pressure-flow rate curves for Phenyl Sepharose 6 Fast Flow shown in Fig.7.In columns with 5 cm inner diameter and a bed height of 15 cm,flow rates /can be used without exceeding a back pressure of 1 bar. The optima working flow rate during elution is normally 50-150 cm/h but during equilibration, regeneration,and also often during sample application,higher flow rates of 200- 300 cm/h can be used.These higher flow rates reduce cycle times. Flow rate(cm/h) Fig.7. ha 700 (low sub)and Phenyl Sepharose 6 600 Fast Flow(high sul in an XK 500 Amersham Pharmacia Biotech, 400 Uppsala,Sweden). 300 200 100. 0.2 04 0.6 0.8 1.012 Pressure (bar) Binding capacity One of the major features of BioProcess HIC Media is the high binding capacity which results in high throughput and productivity even at relatively low salt concentrations.Fig.8 shows the total dynamic binding capacities of human serum albumin and human IgG at different concentrations of ammonium sulphate as determined by frontal analy is.Phen rose 6 Fast Flow(high sub)showed the highest capacities for both hIgG and HSA.Phenyl Sepharose High Performance had higher capacity for hIgG compared with HSA while Butyl Sepharose 4 Fast Flow showed the reverse,indicating the difference in selectivity.The protein recoveries when eluting with low salt buffer were all 80% or more. The dynamic binding capacity will decrease with increasing linear flow rates.This is especially important to consider when optimizing initial separation steps where large volumes need to be processed.Productivity may be higher at high flow rates even though the binding capacity is decreased. 24
24 Physical stability The highly cross-linked structures of Sepharose Fast Flow and Sepharose High Performance matrices are physically stable resulting in very good flow properties. This is illustrated by the pressure-flow rate curves for Phenyl Sepharose 6 Fast Flow shown in Fig. 7. In columns with 5 cm inner diameter and a bed height of 15 cm, flow rates up to 500 cm/h can be used without exceeding a back pressure of 1 bar. The optimal working flow rate during elution is normally 50–150 cm/h but during equilibration, regeneration, and also often during sample application, higher flow rates of 200– 300 cm/h can be used. These higher flow rates reduce cycle times. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 100 200 300 400 500 600 700 high sub low sub Pressure (bar) Flow rate (cm/h) Binding capacity One of the major features of BioProcess HIC Media is the high binding capacity, which results in high throughput and productivity even at relatively low salt concentrations. Fig. 8 shows the total dynamic binding capacities of human serum albumin and human IgG at different concentrations of ammonium sulphate as determined by frontal analysis. Phenyl Sepharose 6 Fast Flow (high sub) showed the highest capacities for both hIgG and HSA. Phenyl Sepharose High Performance had higher capacity for hIgG compared with HSA while Butyl Sepharose 4 Fast Flow showed the reverse, indicating the difference in selectivity. The protein recoveries when eluting with low salt buffer were all 80% or more. The dynamic binding capacity will decrease with increasing linear flow rates. This is especially important to consider when optimizing initial separation steps where large volumes need to be processed. Productivity may be higher at high flow rates even though the binding capacity is decreased. Fig. 7. Typical pressure/flow rate curves for Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) in an XK 50/30 Column, bed height 15 cm, mobile phase 0.1 M NaCl. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden)
otion capacities of Phenyl 20 hlgG and Butyl S narose media for as a fu n of sulphate in the equilibration buffer 6 Fast Flov 20 Fast Flow(low sub),4=Butyl 10 Work from 0.39 0.45 0.57 068 Concn.of ammonium sulphate (M) HSA 96 4 23 10 A 0.6 0.9 12 15 Concn.of ammonium sulphate(M) Phenyl Sepharose 6 Fast Flow (low sub) Phenyl Sepharose 6 Fast Flow(high sub) Phenyl Sepharose 6 Fast Flow(lowsub)and Phenyl Sepharose6 Fast Flow(high sub) are based on highly cross-linked 6%agarose with phenyl ligands coupled via stable ether linkages.The media characteristics are summarized in Table 7. Phenyl Sepharose 6 Fast Flow(low sub)and Phenyl Sepharose 6 Fast Flow(high sub) were initially developed and tested in cooperati on with leading pharmaceutical manufacturers.They are ideal for initial or intermediate step purification of proteins Table 7. Characteristics of Phenyl Sepharose6 Read structure cross-linked agarose.6%.spherical rticle size eny w(high sub) artic 45-165 Degree of substitution opney1g0pange40mighsub) in Data File 2040 (Code No.18-1020-53) 25
25 Fig. 8. Total adsorption capacities of Phenyl and Butyl Sepharose media for human IgG and HSA as a function of the concentration of ammonium sulphate in the equilibration buffer. 1=Phenyl Sepharose 6 Fast Flow (high sub), 2=Phenyl Sepharose High Performance, 3=Phenyl Sepharose 6 Fast Flow (low sub), 4=Butyl Sepharose 4 Fast Flow. (Work from Amersham Pharmacia Biotech, Uppsala, Sweden). Phenyl Sepharose 6 Fast Flow (low sub) Phenyl Sepharose 6 Fast Flow (high sub) Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) are based on highly cross-linked 6% agarose with phenyl ligands coupled via stable ether linkages. The media characteristics are summarized in Table 7. Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub) were initially developed and tested in cooperation with leading pharmaceutical manufacturers. They are ideal for initial or intermediate step purification of proteins ▲ ▲ ▲ ▲ ▲ ▲ ▲ 30 20 10 0.39 0.45 0.57 0.68 0.6 0.9 1.2 1.5 Concn. of ammonium sulphate (M) Adsorption capacity (mg h IgG/ml gel) Concn. of ammonium sulphate (M) 30 20 10 Adsorption capacity (mg HSA/ml gel) hIgG HSA 1 2 3 4 1 4 3 2 Bead structure cross-linked agarose, 6%, spherical Mean particle size 90 µm Particle size range 45–165 µm Degree of substitution approx. 20 (low sub) and 40 (high sub) µmol phenyl groups/ml gel Further information is available in Data File 2040 (Code No. 18-1020-53). Table 7. Characteristics of Phenyl Sepharose 6 Fast Flow (low sub) and Phenyl Sepharose 6 Fast Flow (high sub). BioProcess™Media Quality an performance from and regulatory support services from Pharmacia. production. Backed by full technical research through scale-up to