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Affinity Chromatography

Affinity chromatography is used to capture molecules by specific interaction between the chromatography ligand and the target. This allows the removal of major contaminants to a low concentration in a single process step. Affinity chromatography is frequently used for downstream purification of proteins & vaccines. Sartorius offers a broad selection of resins and membranes for this purpose. 

Attributes  HA Ultrogel CMM Hypercel  MEP Hypercel HEA Hypercel  PPA Hypercel
Ligand  hydroxyapatite  aminobenzoic acid  mercaptoethylpyridine  hexylamine  phenylpropylamine 
Particle Size  60 – 180 µm  50 – 80 µm  80 – 100 µm  80 – 100 µm  80 – 100 µm 
Dynamic Binding Capacity  
(10% BT) 
> 7 mg/mL Cytochrome C  60 – 100 mg IgG  > 20 mg/mL hu IgG  40 – 60 mg/mL BSA  40 –60 mg/mL BSA 
Working pH  5 – 13  2 – 13  2 – 12  2 – 12  2 – 12 
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HA Ultrogel – hydroxyapatite

HA Ultrogel hydroxyapatite is a composite material of cross-linked agarose and microcrystalline hydroxyapatite enclosed in the agarose matrix. The material shows mixed mode functionality based on cation exchange and metal affinity in the hydroxyapatite structure. It is ideally suitable for general impurity removal. 

Description

Applications

Hydroxyapatite chromatography provides a gentle, neutral pH separation mechanism, different from other conventional methods such as ion exchange or affinity. The most well-known application of hydroxyapatite is the separation of basic proteins (cytochrome c, lysozyme, etc.) and phosphoproteins.

HA Ultrogel sorbent can be used for the separation of human serum proteins and plant proteins such as  lectins, glycoproteins, glycosidases, phospholipidases, sulfohydrolases, sphingomyelinases, transferases, trehalases and kinases.

As a phosphate-containing sorbent, HA Ultrogel can be used for the separation of phosphate-dependent proteins and enzymes as well as DNA-dependent enzymes.

Other key applications include :

  • Vaccine purification processes (e.g. Bordetella pertussis toxin)
  • Removal of aggregates in antibody purification
  • Separation of protein isoforms
  • Impurity removal during recombinant protein purification
  • Separation of phosphoproteins, enzymes, glycoproteins, EPO, receptors

Specifications

Main Properties of HA Ultrogel Sorbent

Particle Size 60 – 180 µm
Hydroxyapatite Content 40 %
Agarose (weight/volume) 4 %
Exclusion Limit > 5,000,000 dt
Working and Cleaning pH 5 – 13
Thermal Stability 4 – 121 °C
Capacity for Cytochrome c* > 7 mg/mL
Capacity for BSA** < 7 mg/mL

* Determined using 5 mg/mL cytochrome c diluted 50/50 in 10mM sodium phosphate buffer pH 6.9 at 30 cm/h
** Determined using 1mg/mL BSA diluted 50/50 in 10mM sodium phosphate buffer pH 6.9 at 12.5 cm/h

Performance

Ordering Information

HA Ultrogel Sorbent
Size Part Number
25 mL 24775-082
100 mL 24775-025
500 mL 24775-017
1 L 24775-041
10 L 24775-058
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CMM Hypercel – aminobenzoic acid

CMM Hypercel is a hydrophobic cation exchanger recommended for the capture of antibodies, antibody fragments, and recombinant proteins. It allows the separation of proteins with similar pIs by modulation of pH and conductivity. 

 

Description

Introduction

CMM HyperCel sorbent is composed of a rigid cellulose matrix that has flow properties compatible with the needs of manufacturing scale protein production.

The proprietary ligand, containing both a primary amine and a carboxyl group, confers cation exchange and hydrophobicity properties to the chromatography sorbent. At working pH (4 to 9), the amine group is never charged (pKa < 4). The carboxyl group is weakly charged at adsorption pH (4 to 6) to allow protein adsorption based on hydrophobicity. At elution pH (7 to 9), the carboxyl group is fully deprotonated and the elution will be based on negative charge repulsion. The flexibility of the ligand enables the separation of proteins with a large variety of isoelectric points and hydrophobicity levels, and multiple conditions can be employed to separate targeted molecules from contaminants.

The sorbent is available in a variety of configurations: 200 and 600 μL ScreenExpert RoboColumnsu for initial resin screening, and convenient 1 mL and 5 mL PRC prepacked columns for rapid method optimization, selectivity screening or small preparative work. CMM HyperCel sorbent is also supplied as a slurry/suspension in 1 M NaCl containing 20% (v/v) ethanol, or as a moist cake for process-scale applications. The moist cake sorbent facilitates the sorbent transfer, avoiding the agitation and suspension of large material volumes.

CMM HyperCel sorbent has a chemical stability that ensures simple clean-in-place (CIP) and storage. For standard CIP, 0.5 to 1 M NaOH treatment is recommended, while long-term storage in 10 to 100 mM NaOH is possible.

Features

  • Industry-scalable cation exchange mixed-mode sorbent for high performance capture and impurity removal at moderate salt conductivity
  • Superior separation performance, typical of a mixed-mode resin, without the traditional limitations commonly associated with this sorbent class
  • Ability to separate proteins with similar isoelectric point and/or hydrophobicity at low or high conductivity
  • High dynamic binding capacity over repeated purification cycles
  • High yield of recovery, low elution volume
  • Easy regeneration
  • Designed for capturing monoclonal antibodies (MAbs), Fab antibody fragments and recombinant proteins from challenging samples

Specifications

Main Properties

Particle size range 50-80 μm
Ligand description Aminobenzoic acid
Ligand density Av. 70 μeq/mL
Dynamic binding capacity
BSA1 > 50 mg/mL at pH 4.5, 15 mS/cm
IgG2 > 60 to 100 mg/mL at pH 4.0 to 5.0, 4 to 12 mS/cm
Working conditions
Binding pH ~ 4 to 6; conductivity up to 50 mS/cm3
Elution pH ~ 4 to 9; conductivity up to 50 mS/cm3
Working pressure at 1,000 cm/hr4 ~ 1.0 bar g
Working pH 2 to 13
Cleaning pH 1 to 14
Cleaning in place 1 M NaOH – 1 hour contact time – 5 CV
1 4 g/L BSA in 50 mM Na acetate complemented with NaCl, 7 minute residence time
2 5 g/L IgG in 50 mM Na acetate complemented with NaCl, 2 minute residence time
3 Conductivity adjustment with NaCl (~ 0 to 0.5 M)
4 Determined using 50 mM Na acetate, pH 5.0 on laboratory scale column of 15 mm I.D. x 200 mm length

Performance

Ordering Information

Bottled Sorbent
Description Part Number
CMM HyperCel, 25 mL 20270-025
CMM HyperCel, 100 mL 20270-031
CMM HyperCel, 1 L 20270-041
CMM HyperCel, 5 L 20270-055
CMM HyperCel, 10 L 20270-066
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Columns
Description Part Number
PRC Prepacked Column 5×50 CMM HyperCel, 1 mL PRCCMMHCEL1ML
PRC Prepacked Column 8×100 CMM HyperCel, 5 mL PRCCMMHCEL5ML
ScreenExpert RoboColumn* CMM HyperCel 200 μL, row of 8 SR2CMM
ScreenExpert RoboColumn* CMM HyperCel 600 μL, row of 8 SR6CMM
Request a Quote
*RoboColumn is a trademark of Repligen

MEP Hypercel- mercaptoethylpyridine

MEP Hypercel provides unique selectivity for monoclonal antibody capture or polishing to remove aggregates, HCP, and DNA. The ligand facilitates the binding of target proteins at neutral pH under moderate salt concentrations. 

 

Description

Introduction

CMM HyperCel sorbent is composed of a rigid cellulose matrix that has flow properties compatible with the needs of manufacturing scale protein production.

The proprietary ligand, containing both a primary amine and a carboxyl group, confers cation exchange and hydrophobicity properties to the chromatography sorbent. At working pH (4 to 9), the amine group is never charged (pKa < 4). The carboxyl group is weakly charged at adsorption pH (4 to 6) to allow protein adsorption based on hydrophobicity. At elution pH (7 to 9), the carboxyl group is fully deprotonated and the elution will be based on negative charge repulsion. The flexibility of the ligand enables the separation of proteins with a large variety of isoelectric points and hydrophobicity levels, and multiple conditions can be employed to separate targeted molecules from contaminants.

The sorbent is available in a variety of configurations: 200 and 600 μL ScreenExpert RoboColumnsu for initial resin screening, and convenient 1 mL and 5 mL PRC prepacked columns for rapid method optimization, selectivity screening or small preparative work. CMM HyperCel sorbent is also supplied as a slurry/suspension in 1 M NaCl containing 20% (v/v) ethanol, or as a moist cake for process-scale applications. The moist cake sorbent facilitates the sorbent transfer, avoiding the agitation and suspension of large material volumes.

CMM HyperCel sorbent has a chemical stability that ensures simple clean-in-place (CIP) and storage. For standard CIP, 0.5 to 1 M NaOH treatment is recommended, while long-term storage in 10 to 100 mM NaOH is possible.

Features

  • Industry-scalable cation exchange mixed-mode sorbent for high performance capture and impurity removal at moderate salt conductivity
  • Superior separation performance, typical of a mixed-mode resin, without the traditional limitations commonly associated with this sorbent class
  • Ability to separate proteins with similar isoelectric point and/or hydrophobicity at low or high conductivity
  • High dynamic binding capacity over repeated purification cycles
  • High yield of recovery, low elution volume
  • Easy regeneration
  • Designed for capturing monoclonal antibodies (MAbs), Fab antibody fragments and recombinant proteins from challenging samples

Specifications

Main Properties

Particle size range 50-80 μm
Ligand description Aminobenzoic acid
Ligand density Av. 70 μeq/mL
Dynamic binding capacity
BSA1 > 50 mg/mL at pH 4.5, 15 mS/cm
IgG2 > 60 to 100 mg/mL at pH 4.0 to 5.0, 4 to 12 mS/cm
Working conditions
Binding pH ~ 4 to 6; conductivity up to 50 mS/cm3
Elution pH ~ 4 to 9; conductivity up to 50 mS/cm3
Working pressure at 1,000 cm/hr4 ~ 1.0 bar g
Working pH 2 to 13
Cleaning pH 1 to 14
Cleaning in place 1 M NaOH – 1 hour contact time – 5 CV
1 4 g/L BSA in 50 mM Na acetate complemented with NaCl, 7 minute residence time
2 5 g/L IgG in 50 mM Na acetate complemented with NaCl, 2 minute residence time
3 Conductivity adjustment with NaCl (~ 0 to 0.5 M)
4 Determined using 50 mM Na acetate, pH 5.0 on laboratory scale column of 15 mm I.D. x 200 mm length

Performance

Ordering Information

Bottled Sorbent
Description Part Number
CMM HyperCel, 25 mL 20270-025
CMM HyperCel, 100 mL 20270-031
CMM HyperCel, 1 L 20270-041
CMM HyperCel, 5 L 20270-055
CMM HyperCel, 10 L 20270-066
Request a Quote
Columns
Description Part Number
PRC Prepacked Column 5×50 CMM HyperCel, 1 mL PRCCMMHCEL1ML
PRC Prepacked Column 8×100 CMM HyperCel, 5 mL PRCCMMHCEL5ML
ScreenExpert RoboColumn* CMM HyperCel 200 μL, row of 8 SR2CMM
ScreenExpert RoboColumn* CMM HyperCel 600 μL, row of 8 SR6CMM
Request a Quote
*RoboColumn is a trademark of Repligen

HEA Hypercel – hexylamine

HEA Hypercel is a mixed-mode ligand with a less hydrophobic hexylamine chain allowing protein binding at a moderate salt concentration under neutral pH. 

 

Description

Applications

Take advantage of the benefits mixed mode sorbents present to:

  • Purify proteins at low ionic strength by direct hydrophobic capture
  • Separate challenging mixtures with new ligand selectivities
  • Be orthogonal to ion exchange or other chromatography steps

Specifications

HEA and PPA HyperCel sorbents are members of Sartorius’ family of chromatography mixed-mode sorbents, complementing MEP HyperCel sorbent (Hydrophobic Charge Induction). HEA and PPA HyperCel sorbents carry synthetic ligands, currently used in large columns up to 500 L for the production of immobilized on HyperCel sorbent, a mechanically-stable base matrix currently used in > 100 L columns for the production of proteins. The ligands include aliphatic (HEA – hexylamine) and aromatic (PPA – phenylpropylamine) amines, which offer different selectivity and hydrophobicity options.

Particle Size 80-100 µm (avg)
Bead Composition High porosity crossed-linked cellulose
Dynamic Binding Capacity for BSA
(10% breakthrough)¹
40-60 mg/mL
Ligand: Aliphatic (HEA) Hexylamine
               Aromatic (PPA)
Hexylamine
Phenylpropylamine
BSA Recovery >90%
Working pH 2 – 12
Cleaning pH 1 – 14
Pressure Resistance < 3 bar (44 psi)
Typical Working Pressure < 1 bar (14 psi)

¹Determined using 5 mg/mL BSA in PBS, flow rate: 100 cm/h.

Performance

Ordering Information

HEA HyperCel
Size Part Number
25 mL 20250-026
100 mL 20250-033
1 L 20250-041
5 L 20250-042
10 L 20250-056
1 mL PRC Prepacked Column, 5 mm ID x 50 mm PRC05X050HEAHCEL
5 mL PRC Prepacked Column, 8 mm ID x 100 mm PRC08X100HEAHCEL
Request a Quote
Description Part Number
ScreenExpert RoboColumn HEA HyperCel 200 μL, row of 8 SR2HEA
ScreenExpert RoboColumn PPA HyperCel 200 μL, row of 8 SR2PPA
ScreenExpert RoboColumn HEA HyperCel 600 μL, row of 8 SR6HEA
ScreenExpert RoboColumn PPA HyperCel 600 μL, row of 8 SR6PPA
96-well RoboColumn array plate SR96WAP
Request a Quote

PPA Hypercel – phenylpropylamine

PPA Hypercel exhibits stronger hydrophobicity in comparison to HEA Hypercel and thus provides a different selectivity in protein purification. 

Description

Applications

Take advantage of the benefits mixed mode sorbents present to:

  • Purify proteins at low ionic strength by direct hydrophobic capture
  • Separate challenging mixtures with new ligand selectivities
  • Be orthogonal to ion exchange or other chromatography steps

Specifications

HEA and PPA HyperCel sorbents are members of Sartorius’ family of chromatography mixed-mode sorbents, complementing MEP HyperCel sorbent (Hydrophobic Charge Induction). HEA and PPA HyperCel sorbents carry synthetic ligands, currently used in large columns up to 500 L for the production of immobilized on HyperCel sorbent, a mechanically-stable base matrix currently used in > 100 L columns for the production of proteins. The ligands include aliphatic (HEA – hexylamine) and aromatic (PPA – phenylpropylamine) amines, which offer different selectivity and hydrophobicity options.

Particle Size 80-100 µm (avg)
Bead Composition High porosity crossed-linked cellulose
Dynamic Binding Capacity for BSA
(10% breakthrough)¹
40-60 mg/mL
Ligand: Aliphatic (HEA) Hexylamine
               Aromatic (PPA)
Hexylamine
Phenylpropylamine
BSA Recovery >90%
Working pH 2 – 12
Cleaning pH 1 – 14
Pressure Resistance < 3 bar (44 psi)
Typical Working Pressure < 1 bar (14 psi)

¹Determined using 5 mg/mL BSA in PBS, flow rate: 100 cm/h.

Performance

Ordering Information

HEA HyperCel
Size Part Number
25 mL 20250-026
100 mL 20250-033
1 L 20250-041
5 L 20250-042
10 L 20250-056
1 mL PRC Prepacked Column, 5 mm ID x 50 mm PRC05X050HEAHCEL
5 mL PRC Prepacked Column, 8 mm ID x 100 mm PRC08X100HEAHCEL
Request a Quote
Description Part Number
ScreenExpert RoboColumn HEA HyperCel 200 μL, row of 8 SR2HEA
ScreenExpert RoboColumn PPA HyperCel 200 μL, row of 8 SR2PPA
ScreenExpert RoboColumn HEA HyperCel 600 μL, row of 8 SR6HEA
ScreenExpert RoboColumn PPA HyperCel 600 μL, row of 8 SR6PPA
96-well RoboColumn array plate SR96WAP
Request a Quote

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