HEA and PPA HyperCel™ Sorbents product photo Primary L

HEA and PPA HyperCel™ Sorbents

Mixed-mode Chromatography for Protein Separation

Introduction

Pall 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).


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Sorbent - HEA HyperCel

List Price
$ 520.00
List Price
$ 143.00

Sorbent - PPA HyperCel

List Price
$ 520.00
List Price
$ 140.00
Description
Mixed-mode Chromatography for Protein Separation

Introduction

Pall 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).
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 Pall’s 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 (see Figure 1), which offer different selectivity and hydrophobicity options (refer to Figures 4 and 5).

 
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.

Figure 1
Structure of HEA and PPA Ligands

Chromatography Sorbents: Structure of HEA and PPA Ligands
Ligand density > 50 μmoles/mL
Critical working ligand pKa = 8

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) (See lysozyme binding to HEA HyperCel sorbent, Figure 7).

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 (see Figure 6).

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 (see Figure 3), 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 (see Figure 7).

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.

Figure 2
Pressure vs. Flow Rate

Chromatography Sorbents: HEA and PPA

Column: 16 mm I.D. x 20 cm height. HEA and PPA HyperCel sorbents provide linear pressure/flow behavior and have characteristics well suited to process-scale operations in low pressure columns.

Figure 3
Effect of Temperature on BSA Binding Capacity

HEA and PPA Chromatogragraphy Sorbents: Effect of Temperature on BSA Binding Capacity

Model protein: BSA (5 mg/mL) bound to HEA and PPA HyperCel sorbents (7 mL column, 100 cm/h) in PBS, pH 7.4.

This experiment, as shown in Figure 3, demonstrates the dominant hydrophobic interaction component of protein binding to HEA and PPA HyperCel sorbents. The binding of proteins by HIC is entropy-driven, and the interaction increases with rise of temperature, as shown by the binding capacity increase for BSA. In practice, for robustness and capacity optimization studies, special attention should be given to keep buffer and operation room temperatures consistent.

Figure 4
Comparison between HEA and PPA HyperCel, MEP HyperCel, Conventional HIC and Anion Exchange Sorbents

Comparison between HEA and PPA HyperCel, MEP HyperCel, Conventional HIC and Anion Exchange Sorbents 

A series of experiments with various standard proteins was carried out to show the selectivity differences of HEA and PPA HyperCel sorbents compared to MEP HyperCel sorbent (HCIC – Hydrophobic Charge Induction Chromatography), conventional HIC sorbents (Phenyl and Hexyl ligands), as well as anion exchange sorbents (DEAE). This figure illustrates the adsorption/ desorption of Bovine Serum Albumin (BSA), in PBS buffer, pH 7.4. Data shows that BSA is efficiently retained on both HEA and PPA HyperCel sorbents, but is poorly retained on MEP HyperCel sorbent. In PBS, without salt addition, low binding of BSA to both Phenyl and Hexyl HIC sorbents is observed. The anion exchange (DEAE) resin also did not bind BSA at these non-optimal conditions (pH 7.4 and too high salt concentration). 

Figure 5
Selectivity Difference between HEA and PPA HyperCel Sorbents: Adsorption/Desorption of α-chymotrypsinogen A

Selectivity Difference between HEA and PPA HyperCel Sorbents: Adsorption/Desorption of α-chymotrypsinogen A

HEA and PPA ligands are of different nature (respectively aliphatic and aromatic). This translates in differences in selectivity for various proteins. Figure 5 illustrates the binding and elution properties for α-chymotrypsinogen A (2 mg/mL, binding in PBS, pH 7.4, 0.14 M NaCl), elution in 0.02 M sodium acetate at pH 5.0, 4.0 or 3.0. Data suggests a stronger binding of the model protein to PPA HyperCel sorbent, as well as a good recovery (elution at pH 4.0 is the most efficient).

Figure 6
BSA Binding Capacity of HEA and PPA HyperCel Sorbents in the Presence of 1.7 M ammonium sulphate

BSA Binding Capacity of HEA and PPA HyperCel Sorbents in the Presence of 1.7 M ammonium sulphate

As shown in Figure 6, HEA and PPA HyperCel sorbents can be used in “conventional” hydrophobic interaction conditions, in the presence of lyotropic salt-like ammonium sulphate or other salt. Data shows that BSA binding capacity in these conditions is close to the capacity of a conventional HIC sorbent (phenyl resin). Lower concentrations of salt (0.5 M ammonium sulphate) have been used to promote stronger adsorption of recombinant proteins (data not shown).
Performance

Application Examples

Application 1 - Separation of a Protein Mix on HEA and PPA HyperCel Sorbents

HEA and PPA HyperCel sorbents have different retentivities and selectivities for proteins and should be screened at early stages during process development. The example in Figure 7 illustrates the chromatographic profiles obtained with a reference protein mixture applied to columns of 1.1 cm I.D. x 7 cm length. A step-elution sequence was performed followed by gradient elution from pH 5.4 to 2.6; all conducted using sodium phosphate/citrate buffers. Data shows that a very basic protein – lysozyme – does not bind to PPA HyperCel sorbent at pH 7.4, and is found in the flowthrough; in contrast, when raising the binding pH to 10.0 by reducing the ionic repulsion, lysozyme can be retained on HEA HyperCel sorbent.

In practice, for a protein of unknown pI and hydrophobicity, screening of the two ligands at different pH and salt concentrations is needed, and can be conveniently done by using ready-to-use 1 mL or 5 mL PRC prepacked columns (refer to Pall Product Note USD 2492a and User Guide USD 2596).

Figure 7
Sample volume: 1 mL; Column volume: 7 mL; Proteins: BSA, ovalbumin, lysozyme, α-chymotrypsinogen A, at a concentration of 2 mg/mL each.

HEA HyperCel Sorbent
Binding buffer: Carbonate-bicarbonate, pH 10 + 150 mM NaCl


HEA HyperCel Sorbent: Binding buffer: Carbonate-bicarbonate, pH 10 + 150 mM NaCl

PPA HyperCel Sorbent
Binding buffer: PBS, pH 7.4


PPA HyperCel Sorbent: Binding buffer: PBS, pH 7.4

Application 2 - Separation of Recombinant GST (glutathione S transferase) from filtered E.coli Lysate on HEA HyperCel PRC Prepacked Columns of 1 mL, 2 mL and 2 x 5 mL Connected in Series

The example in Figure 8 illustrates the reproducibility of a real feedstock separation using PRC prepacked columns of different volumes (1 mL and 5 mL) and connected in series (2 x 5 mL). The lysate was diluted 10-fold with 50 mM Tris-HCl pH 8.0 buffer, and elution carried by pH steps at pH 5.0, 4.0 and 3.0. Data shows that the chromatograms are perfectly overlayed, illustrating the consistency of the separations on the three PRC columns or assemblies tested. Purity analysis by SDS-PAGE showed an equivalent purity level for the eluted fractions in all cases (see Figure 9).

Figure 8
Scale up of rGST on PRC Prepacked Columns

Scale up of rGST on PRC Prepacked Columns

Figure 9
SDS-PAGE of the Elution Fractions (NuPAGE Novex 4 – 12% Bis-Tris gel, Coomassie staining)

SDS-PAGE of the elution fractions  

Application 3 - Separation of Partially-purified Polyclonal IgG from Major Plasma Impurities on HEA HyperCel Sorbent

The objective of this experiment was to separate a target IgG from contaminants present in the feedstream. Three different mixed-mode sorbents – HEA, PPA, and MEP HyperCel sorbents – were tested. Figure 10 shows only the chromatogram obtained with HEA HyperCel sorbent, which gave the best results in this case; the IgG (main elution, E2) was well separated from HSA (elution E3). Additionally, molecular-mass based analysis using mass spectrometry (data not shown) demonstrated resolution from lower molecular weight contaminants (elution E1), as well as a partial separation of IgA, co-eluting with the HSA peak.

Figure 10
HEA HyperCel Sorbent Chromatogram

HEA HyperCel Chromatography Sorbent Chromatogram

Column: 0.66 cm I.D. x 7 cm height; Sorbent volume: 2.4 mL. Run at 100 cm/h; Equilibration in PBS, pH 7.4; Loading 5 mL of a partially-purified human polyclonal IgG (60% purity) at 3.8 mg/mL, pH 8.4, and 8.3 mS/cm; Wash with 5 CV in PBS; Elution in 0.2 M sodium phosphate/100mM citric acid, pH 7.0 (Elution 1), pH 5.4 (Elution 2), pH 4.4 (Elution 3), pH 3.4 (Elution 4), pH 2.6 (Elution 5); Regeneration in 1M NaOH.

Application 4 - Separation and Resolution of Low Molecular Weight Recombinant Protein Isoforms on PPA HyperCel Sorbent

Sample Courtesy of Dr. Giovanni Magistrelli, NovImmune, Plan les Ouates, Switzerland

This example illustrates the unique resolution power of mixed-mode chromatography sorbents (PPA HyperCel sorbent in this case) to capture and discriminate isoforms of recombinant proteins expressed at low concentrations in cell culture supernatants.

The supernatant was loaded on the PPA HyperCel sorbent column at pH 7.4 (PBS), and fractions eluted at pH 5.0, 4.0 and 2.6.

Figure 11 shows a SELDI-MS analysis (confirmed by ELISA assays, not shown) indicating that a first population of isoforms with molecular weight (MW) between 8.0 and 8.3 kDa are eluted first and another population of isoforms with MW > 8.5 kDa are eluted next. Proteins with minor differences can therefore be at least partially separated using mixed-mode chromatography.

Figure 11
SELDI Molecular Mass Analysis (Da) of the Fractions Eluted at 115 mL (blue) and 125 mL (red) from HEA HyperCel Sorbent

SELDI Molecular Mass Analysis  

Application 5 - Purification of Recombinant F(ab’)2 Fragment on HEA HyperCel Sorbent

Courtesy of X.Santarelli and J.Pezzini, ESTBB, Bordeaux, France

HEA HyperCel sorbent was used as a capture chromatography step to purify a recombinant F(ab’)2 fragment obtained through baculovirus expression in SF9 insect cells. Sample was loaded on HEA HyperCel sorbent at pH 6.0, 4.0 and 2.0, and analysis done by SDS-PAGE, ELISA and BCA assays. Results shown in Figure 12 indicate that no protein was found in the flowthrough or elution at pH 6.0. The F(ab’)2fragment eluted at pH 4.0 (recovery 82%, 39-fold purification factor), while HCP (Host Cell Proteins) eluted at pH 2.0.

Figure 12
Analysis of Load and Elution

HEA and PPA HyperCel Chromatography Sorbents: Analysis of Load and Elution
The fractions identified on the chromatogram during load and elution were analyzed using ELISA (not shown) and SDS-PAGE to identify and quantify the F(ab’)2.
Ordering Information
 
Size HEA HyperCel PPA HyperCel
25 mL 20250-026 20260-025
100 mL 20250-033 20260-030
1 L 20250-041 20260-040
5 L 20250-042 20260-045
10 L 20250-056 20260-052
1 mL PRC Prepacked Column, 5 mm ID x 50 mm PRC05X050-HEAHCEL01 PRC05X050-PPAHCEL01
5 mL PRC Prepacked Column, 8 mm ID x 100 mm PRC08X100-HEAHCEL01 PRC8X100-PPAHCEL01
 

 
Product No. Description
96WP3MIXM50 AcroPrep ScreenExpert Plates filled with MEP, HEA and PPA HyperCel Sorbents
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
 
Application
Sorbents