Mixed-Mode Chromatography

Novel biopharmaceutical targets require new selectivities to achieve high product purity. Simple process integration, as well as economic benefits, are additional drivers for the implementation of new modalities. 

Find the Right Solution for Your Chromatography Process

Mixed-mode chromatography commonly utilizes more than one interaction principle for purification. While a combination of hydrophobic interaction and ion exchange functionality is most common, the combination of cation exchange and metal affinity in HA Ultrogel is also well known. Mixed-mode resins exhibit unique separation characteristics and allow easy process integration reducing the requirement for additional process steps. 


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. 

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Introduction

HA Ultrogel® sorbent is a hydroxyapatite agarose composite sorbent for the separation of biomolecules from research and development scale to manufacturing.

Hydroxyapatite chromatography is considered to be a “pseudo-affinity” chromatography, or “mixed-mode” ion exchange. It has proven to be an effective purification mechanism in a variety of processes, providing biomolecule selectivity complementary to more traditional ion exchange or hydrophobic interaction techniques.

HA Ultrogel hydroxyapatite sorbent is composed of cross-linked agarose beads with microcrystals of hydroxyapatite entrapped in the agarose mesh. The particle size ranges between 60 and 180 µm. HA Ultrogel porosity is comparable to an agarose gel, with an exclusion limit for globular proteins of 5,000 kDa. This macroporosity avoids any moleculear sieving effect during the separation.
The sorbent is shipped in 1 M NaCl containing 20% ethanol and is available in a range of package sizes. Special packaging to meet specific manufacturing requirements is available on request.    


Features

  • Unique selectivity and separation mechanism
  • Protein purification in neutral, non-denaturating conditions
  • Laboratory scale to large-scale production columns

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

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

Chemical Stability

HA Ultrogel sorbent is stable in alkaline conditions, and can be treated with 0.1 M sodium hydroxide for regeneration and cleaning in place. HA Ultrogel must not be treated with pH <4 acidic solutions that dissolve the hydroxyapatite crystals.


Mechanical Stability

The recommended flow rates to be used with HA Ultrogel sorbent depend on the column geometry and on the separation phase (capture, elution or washing steps). At process scale, typical flow rates from 30 to 200 cm/h are currently applied with multi-liter column sizes.

HA Ultrogel sorbent is stable at high temperature (up to 121 °C). It can be sterilized by autoclaving without undergoing any changes to its chromatographic properties. However, the operation should be performed in buffered conditions at pH 7 to avoid the presence of phosphate which may precipitate. HA Ultrogel sorbent should never be frozen.

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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HA Ultrogel® Hydroxyapatite Chromatography Resin

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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. 

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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


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
BindingpH ~ 4 to 6; conductivity up to 50 mS/cm3
ElutionpH ~ 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

High selectivity to separate proteins with similar isoelectric point and /or hydrophobicity

The ability to separate acidic (e.g., ovalbumin) from basic proteins (e.g., MAbs), along with the power to separate hydrophobic proteins (e.g., MAbs) from more hydrophilic proteins (e.g., ß-lactoglobulin), illustrates the powerful selectivity of CMM HyperCel sorbent for a broad range of molecules.


High binding capacity for protein capture

Mixed-mode sorbents are well-known to resolve purification challenges which cannot be solved by ion exchangers, this is normally at the expense of reduced capacity. However, CMM HyperCel sorbent demonstrates capacity performance competitive with other chromatography technologies.

Dynamic binding capacities (DBC) for two pure molecules (BSA, MAb) were tested at two different conductivities. The DBC is higher than 60 mg/mL for both cases and remains high, even at 15 mS/cm for MAb, which facilitates process integration without the need for buffer exchange or dilution before loading. The pH for binding and elution is compatible for maintaining the integrity of MAb.


Efficient regeneration for a long service life

To test the efficiency of regeneration, MAb was purified from a clarified CHO cell culture supernatant. Five full purification cycles were performed. After each elution, the chromatography sorbent was regenerated with 1 N NaOH (1 hour contact time) and DBC at 10% breakthrough was tested. The DBC remained unchanged, confirming the efficient cleaning of the sorbent.

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 5x50 CMM HyperCel, 1 mL

PRCCMMHCEL1ML

PRC Prepacked Column 8x100 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

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*RoboColumn is a trademark of Repligen

CMM HyperCel™ Mixed-Mode Resin

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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. 

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Introduction

MEP HyperCel mixed-mode chromatography sorbent is a flexible chromatography sorbent designed for capture and purification of antibodies and various recombinant proteins from laboratory to manufacturing scale. It offers:

  • A unique separation mechanism and selectivity for protein separations 
  • A no-salt/low-salt alternative to Hydrophobic Interaction Chromatography (HIC)
  • Monoclonal and polyclonal IgG capture and intermediate purification (aggregate, DNA and HCP removal)
  • Enhanced process economics


MEP HyperCel sorbent brings a specific mixed-mode or multi-modal separation mechanism, different from conventional ion exchange or affinity mechanisms, and is particularly effective to replace conventional HIC. MEP HyperCel sorbent provides significant benefits when used at process scale; particularly, in contrast to conventional HIC, MEP HyperCel sorbent does not require massive addition of salt to promote protein binding, resulting in simplification of process operations, saving of unit operations (e.g., diafiltration or ultrafiltration), and better process economics. Due to its ligand structure, MEP HyperCel sorbent is immunoglobulin-selective. It can be used for direct capture or intermediate purification of IgG from various feedstocks in combination with other conventional methods such as cation exchange, HIC, or even following Protein A affinity capture for enhanced DNA clearance, HCP (Host Cell Proteins), and aggregate removal.


For “non-antibody” proteins (e.g., recombinant proteins, enzymes, etc.), MEP HyperCel sorbent also can be used for capture or intermediate steps in a purification sequence. When used at the capture step, feedstock is typically loaded directly without pH or ionic strength adjustments.


MEP HyperCel sorbent contributes to a simplification of process development, saving of unit operations such as diafiltration or ultrafiltration, and lower waste disposal; anticipated long service life is expected due to MEP HyperCel sorbent’s resistance to harsh cleaning-in-place methods (0.5 to 1 M NaOH, 30 to 60 minutes contact time) – all factors contributing to lower purification cost. 


Features and Benefits

Unique Separation Mechanism and Differentiated Selectivity

The mixed-mode mechanism allows purification of antibody or other proteins that cannot be easily achieved by conventional techniques such as ion exchange or conventional HIC; for example, playing on differences in hydrophobicity, and separation of proteins with very close isoelectric points.


Direct Immunoglobulin Capture from a Variety of Feedstocks

Due to its unique ligand structure, MEP HyperCel sorbent is immunoglobulin-selective. Antibody binding occurs at neutral pH and is largely independent of ionic strength. Concentration of dilute samples is not necessary (e.g., efficient capture is achieved even with feedstocks as dilute as 50 to 100 μg IgG/mL). Immunoglobulin purification from protein-free and protein-supplemented cell culture supernatants, transgenic milk, ascites fluids and serum has been reported. In contrast to Protein A affinity sorbents, IgG binding capacity on MEP HyperCel sorbent is essentially independent of subclass or species. “Weakly-binding” variants (e.g., murine IgG1 or Rat IgG) are well retained. MEP HyperCel sorbent contributes to HCP removal and virus clearance, and provides a very efficient one-step DNA clearance from cell culture supernatants. Note that the addition of Tween and Triton in feedstock or buffers is not recommended, because such surfactants may interfere with the binding of protein to MEP HyperCel sorbent.


IgG Elution in Mild Conditions and Separation of Contaminants

IgG is typically eluted in the pH 5.5 to 4.0 range, depending on isoelectric points and contaminants profiles. This milder elution compared to Protein A affinity may contribute to reduced aggregate formation and better preservation of the antibody biological activity. Moreover, MEP HyperCel sorbent’s pH-dependent elution mechanism allows a separation of HCPs, DNA, antibody aggregates and misfolds from the monomeric, IgG based on differences of hydrophobicity. In some cases, the addition of arginine in MEP HyperCel elution buffers (0.1 to 1.0 M ) reduces the risk of antibody aggregation and prevents the loss of solubility encountered at acidic pH with many antibodies and allows even milder pH elution (around pH 7.0).


Protein Binding in No-salt or Low-salt Conditions

Several families of “non-antibody” recombinant proteins have been purified using MEP HyperCel sorbent. In contrast to conventional HIC (e.g., Phenyl or Butyl ligands), protein binding to the sorbent does not require the massive addition of salt such as ammonium sulphate or other lyotrope. This results in lower process costs and waste disposal benefits. Product can be recovered in dilute buffer, and unit operation steps such as ultrafiltration or diafiltration are minimized, contributing to better process flow and enhanced process economics.

Methods Screening and Scale Up

The physical and chemical properties of MEP HyperCel sorbent are well suited to both laboratory, pilot and process scale use. MEP HyperCel sorbent is compatible with systems routinely used for low or medium-pressure process chromatography. For challenging separations of proteins and impurities, it is recommended to screen MEP HyperCel sorbent along with HEA HyperCel and PPA HyperCel mixed-mode sorbents carrying aliphatic and aromatic synthetic ligands that provide additional chromatographic selectivities. 


At Laboratory Scale or for Methods Development

Efficient separations can be achieved using 96-well filter plates or PRC prepacked columns. The 1 mL and 5 mL PRC prepacked columns demonstrate a high packing efficiency (>2500 plates/meter), can be directly connected to commonly used laboratory chromatography systems, and deliver optimal and consistent performance. Further scale up (up to 900 mL sorbent volume) can be achieved by packing the sorbent in laboratory empty glass tube columns. 


Pilot and Process Scale Applications

MEP HyperCel sorbent has been designed to meet pilot to manufacturing-scale requirements in protein purification, and is currently used in columns of multiliter up to several hundred liter volumes. Specific packing protocols and technical support for large-scale column packing are available. A comprehensive validation package and Regulatory Support File (RSF) are also available to assist users in the development of validation procedures.


For manufacturing scale, Sartorius offers a complete range of Resolute® columns from 28 cm to 2 m diameter.

MEP HyperCel sorbent is composed of a proprietary rigid cellulose matrix to which 4-Mercapto-Ethyl-Pyridine (4-MEP) is linked. The cellulose bead confers high porosity, chemical stability and low non-specific interaction. An 80 to 100 μm average bead diameter allows excellent flow properties at low column backpressures, compatible with large-scale production. MEP HyperCel sorbent can be used from laboratory to hundred-liter production-scale columns. The sorbent is available in a variety of packaging configurations as well as convenient PRC prepacked columns of 1 mL and 5 mL designed for sorbent screening, fast methods optimization and scale up. MEP HyperCel sorbent is supplied in 1 M NaCl containing 20% ethanol, and custom packaging is available on request.


Table 1
Main Properties of MEP Hypercel Sorbent

Particle Size (average)

80 - 100 µm

Dynamic binding capacity for hu IgG* (10% breakthrough)

> 20 mg/mL

Ligand 

4-Mercapto-Ethyl-Pyridine

Ligand density

80-125 µmol/mL

Working pH (long term)

2 - 12 

Cleaning pH (less than 6 hours)

2 - 14 
Pressure Resistance 

< 3 barg (44 psig)

Typical Working Pressure

< 1 barg (14 psig)


Determined using 5 mg/mL human IgG in PBS, 6 minute residence time (flow rate 70 cm/h).

Application Examples

Application 1 - Purification of Rat IgG from a “Protein-rich” Feedstock: Principle of Elution Optimization Using Decreasing pH Steps

A protein-rich feedstock (rat IgG in 15% Fetal Bovine Serum content) was selected to illustrate the impact of elution pH on antibody purity. In a first series of experiments, the IgG fraction was eluted at pH 4.0; however, a broad range of impurities was also eluted at pH 4.0, including a truncated form of free light chain (TFLC), leading to medium (around 75%) purity of the target IgG. Then, a pH-step-elution was conducted at pH 5.5, 5.2, 4.6, 4.0 and 3.0. Using pH 5.5 elution, the IgG eluted purity was increased to 95% (the fraction contained 4% TFLC and was remarkably free of other impurities [Lane 4]). When pH was reduced to 5.2, desorption of an increased concentration of TFLC was prompted (Lane 5).

When the pH was reduced to 4.6 and then to pH 4.0 (Lanes 6 and 7), impurity components were eluted. Finally, TFLC was eluted at pH 3.0 (Lane 8). Based on these findings, optimal recovery of the target antibody would be conducted at pH 5.5.


Data Courtesy of J. Ford and D. Conrad, Virginia Commonwealth University


Application 2 - Laboratory Scale Purification of Monoclonal IgG from Ascites Fluid

MEP HyperCel sorbent was used to purify IgG from ascites fluid. In order to reduce viscosity, the sample was diluted with an equal volume of equilibration buffer prior to loading. The chromatogram, IgG was 83% pure with 79% yield. Purity of the IgG fraction could be increased by anion exchange chromatography on DEAE Ceramic HyperD™ F sorbent.


Application 3 - One-step Capture of Monoclonal Mouse IgG1 from “Protein-rich” (Albumin Containing) CHO (Chinese Hamster Ovary) Cell Culture Supernatant (CCS)

MEP HyperCel sorbent can achieve one-step IgG purification with similar levels of purity and yield than Protein A sorbents, even when the CCS contains high amounts of albumin as major contaminant. 


Application 4 - Contaminant (HCP and DNA) Removal During a MAb Capture Step from CHO Cell Culture on MEP HyperCel Sorbent

MEP HyperCel sorbent was used to capture a MAb from a protein-free CHO cell culture supernatant. Results (Table 3 below) demonstrate a very efficient DNA removal (> 4.7 Log) and a 100-fold reduction in HCP. Further chromatographic step using ion exchange chromatography on CM Ceramic HyperD F cation exchange sorbent did reduce the HCP content further (data not shown).


Application 5 - Evaluation of MEP HyperCel Sorbent as a HIC Alternative for the Purification of an E. coli Recombinant Protein: Summary of Process Benefits

MEP HyperCel sorbent was used as a replacement of a Butyl resin in an E. coli recombinant protein purification sequence. Results summarized (Table 4 below) demonstrate that used at either step 2 or step 3 in the process, MEP HyperCel sorbent could reduce the amount of salt required for protein binding, resulted in better capacity and purity, and eliminated the need for the final time-consuming size exclusion step needed in the conventional first generation process.


Table 3
Contaminant Removal from CHO Cell Culture

Fraction

IgG Recovery (%)

IgG (ng/mL)

HCP (ppm)

HCP (Log 10 reduction)

HCP (ng/mL)

DNA (ppm)

DNA (Log10 reduction)

Start  Feedstock

100

92000

102000

-

705

781

-

MEP HyperCel Sorbent

93

8600

1200

1.9

<0.1

<0.014

>4.7

DNA assay using Quant-IT PicoGreen dsDNA assay kit (Invitrogen); HCP assay using ELISA kit (Cygnus Technologies).


Table 4
Purification of E. coli Recombinant Protein Using MEP HyperCel Sorbent as HIC (Hydrophobic Interaction Chromatography) Alternative (Replacement of a Butyl Ligand)

Conventional Process Including a HIC (Butyl) Step

Process Including MEP HyperCel Sorbent as HIC Replacement

Number of Chromatographic Steps in the Process

4 (including final size exclusion)

3 (saves the final size exclusion)

Salt Concentration Required for Protein Binding

3.5 M NaCl

2 M NaCl

Binding Capacity

Low

Good (10X higher than the HIC
conventional resin)

Robustness

Not applicable

Excellent (11 fermentation runs)

Purity (C4 HPLC Assay)

Requires final SEC after the HIC step

High


HIC = Hydrophobic Interaction Chromatography
SEC = Size Exclusion Chromatography

Description

Size

Part Number

MEP HyperCel

25 mL

12035-010

MEP HyperCel

100 mL

12035-028

MEP HyperCel

1 L

12035-036

MEP HyperCel

5 L

12035-040

MEP HyperCel

10 L

12035-044

PRC Column 5x50 MEP HyperCel

Prepacked 1 mL of
sorbent

PRC05X050MEPHCEL

PRC Column 8x100 Prepacked
MEP HyperCel
Prepacked 5 mL of
sorbent

PRC08X100MEPHCEL

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Description

Part Number

ScreenExpert RoboColumn MEP HyperCel 200 μL, row of 8

SR2MEP

ScreenExpert RoboColumn MEP HyperCel 600 μL, row of 8

SR6MEP

96-well RoboColumn array plate

SR96WAP

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MEP HyperCel™ Mixed-Mode Chromatography Resin - Hydrophobic Charge Induction Chromatography | HCIC

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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. 

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HEA and PPA HyperCel sorbents are industryscalable chromatography sorbents designed for protein capture and impurity removal in a biopharmaceutical environment. Operating on a “mixed-mode” mechanism, their behavior is based on a combination of electrostatic and hydrophobic interactions of the proteins with the ligands.

HEA and PPA HyperCel sorbents provide unique selectivities, different from those given by ion exchange or conventional HIC (hydrophobic interaction chromatography), that can be screened to facilitate process development.

For example, the mixed-mode interaction mechanism can be exploited to achieve discrimination of proteins isoforms, or proteins having similar or very close isoelectric points, separations which usually cannot be achieved by conventional methods. The sorbents’ mechanical stability allows their use at high flow rates in laboratory to production-scale columns.

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

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.


Principles of Operating Mechanism and General Guidelines

(Refer to product insert for details on column packing, buffers and recommendations.)


Protein Binding

Protein binding is usually achieved at neutral pH (i.e., PBS, pH 7.4), principally by hydrophobic interaction. Binding of very basic proteins may require increased pH (pH 9.0).

At salt concentrations recommended for binding, there is limited ion exchange binding. Unlike traditional HIC, binding occurs at low ionic strength, in “physiologicallike” conditions. In general, no addition of lyotropic or other salt is required; however in some cases, the addition of moderate quantities of salt (e.g., 0.5 M ammonium sulphate) promotes protein adsorption.

PPA HyperCel sorbent carries an aromatic ligand and has a stronger hydrophobicity than HEA HyperCel sorbent. The binding capacity is a function of the protein. For protein models like BSA, typical capacities of 40 to 60 mg/mL for HEA and PPA HyperCel sorbents are obtained (PBS, pH 7.4, 0.14 M NaCl buffer, flow rate 100 cm/h). The factors that affect capacity include temperature, residence time, isoelectric point, hydrophobicity of the target protein, and the quality of column packing. (Using PRC prepacked columns is recommended for screening).


Protein Elution

Protein elution is driven by electrostatic charge repulsion, as pH is reduced to values below the pI of the protein and below the pKa of the ligand. Elution is triggered by reducing the pH (from 7 to 2) because some proteins can be eluted without any change in pH just by decreasing the salt concentration. At laboratory scale, optimization can be achieved by descending salt gradient elution experiments; while stepwise elution will be selected at process-scale. This approach can also serve to resolve the target protein from impurities whose hydrophobic characteristics differ. Basic proteins will desorb earlier in the pH gradient or step-elution sequence, followed by more acidic proteins.

Unlike traditional HIC, the target protein is recovered in dilute buffer, reducing the need for intermediate diafiltration, saving unit operations and contributing to better process economics.

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

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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

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HEA and PPA HyperCel™ Resins Mixed-mode Chromatography for Protein Separation

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PPA Hypercel - phenylpropylamine

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

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HEA and PPA HyperCel sorbents are industryscalable chromatography sorbents designed for protein capture and impurity removal in a biopharmaceutical environment. Operating on a “mixed-mode” mechanism, their behavior is based on a combination of electrostatic and hydrophobic interactions of the proteins with the ligands.

HEA and PPA HyperCel sorbents provide unique selectivities, different from those given by ion exchange or conventional HIC (hydrophobic interaction chromatography), that can be screened to facilitate process development.

For example, the mixed-mode interaction mechanism can be exploited to achieve discrimination of proteins isoforms, or proteins having similar or very close isoelectric points, separations which usually cannot be achieved by conventional methods. The sorbents’ mechanical stability allows their use at high flow rates in laboratory to production-scale columns (see Figure 2 for pressure vs. flow rate data).

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

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.



Principles of Operating Mechanism and General Guidelines


Protein Binding

Protein binding is usually achieved at neutral pH (i.e., PBS, pH 7.4), principally by hydrophobic interaction. Binding of very basic proteins may require increased pH (pH 9.0) (See lysozyme binding to HEA HyperCel sorbent).

At salt concentrations recommended for binding, there is limited ion exchange binding. Unlike traditional HIC, binding occurs at low ionic strength, in “physiologicallike” conditions. In general, no addition of lyotropic or other salt is required; however in some cases, the addition of moderate quantities of salt (e.g., 0.5 M ammonium sulphate) promotes protein adsorption.

PPA HyperCel sorbent carries an aromatic ligand and has a stronger hydrophobicity than HEA HyperCel sorbent. The binding capacity is a function of the protein. For protein models like BSA, typical capacities of 40 to 60 mg/mL for HEA and PPA HyperCel sorbents are obtained (PBS, pH 7.4, 0.14 M NaCl buffer, flow rate 100 cm/h). The factors that affect capacity include temperature, residence time, isoelectric point, hydrophobicity of the target protein, and the quality of column packing. (Using PRC prepacked columns is recommended for screening).


Protein Elution

Protein elution is driven by electrostatic charge repulsion, as pH is reduced to values below the pI of the protein and below the pKa of the ligand. Elution is triggered by reducing the pH (from 7 to 2) because some proteins can be eluted without any change in pH just by decreasing the salt concentration. At laboratory scale, optimization can be achieved by descending salt gradient elution experiments; while stepwise elution will be selected at process-scale. This approach can also serve to resolve the target protein from impurities whose hydrophobic characteristics differ. Basic proteins will desorb earlier in the pH gradient or step-elution sequence, followed by more acidic proteins.

Unlike traditional HIC, the target protein is recovered in dilute buffer, reducing the need for intermediate diafiltration, saving unit operations and contributing to better process economics.

 

Size

25 mL

20260-025

100 mL

20260-030

1 L

20260-040

5 L

20260-045

10 L

20260-052

1 mL PRC Prepacked Column, 5 mm ID x 50 mm

PRC05X050PPAHCEL

5 mL PRC Prepacked Column, 8 mm ID x 100 mm

PRC8X100PPAHCEL

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Part Number

Description

SR2HEA

ScreenExpert RoboColumn HEA HyperCel 200 μL, row of 8

SR2PPA

ScreenExpert RoboColumn PPA HyperCel 200 μL, row of 8

SR6HEA

ScreenExpert RoboColumn HEA HyperCel 600 μL, row of 8

SR6PPA

ScreenExpert RoboColumn PPA HyperCel 600 μL, row of 8

SR96WAP

96-well RoboColumn array plate

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HEA and PPA HyperCel™ Resins Mixed-mode Chromatography for Protein Separation

PDF | 1.6 MB

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