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
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 (Figure 1), 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.
Chemical Structure of CMM HyperCel Ligand
|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|
|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
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.
Protein Classification as a Function of Their Isoelectric Point and Hydrophobicity
Separation of a Mixture of Proteins on CMM HyperCel Sorbent
Contour Plots of Elution pH as a Function of pl and Hydrophobicity of the Model Proteins
A detailed study was performed to understand the selectivity of CMM HyperCel sorbent as a function of the binding conductivity. The six chosen model proteins were loaded as a mixture on a 1 mL column. The elution was performed with a mix of buffers to provide a near linear pH gradient from 4.5 to 10, at three conductivities: 5, 15 and 30 mS/cm.
Figure 4 shows the contour plots of pH elution as a function of the isoelectric point and hydrophobicity of the proteins. The data shows the sorbent is still able to separate proteins at high conductivity, expanding the operating range of the sorbent.
High binding capacity for protein capture
Mixed-mode sorbents are well-known to be able 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.
Figure 5 shows dynamic binding capacities (DBC) for two pure molecules (BSA, MAb) 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.
Dynamic Binding Capacity for Two Pure BSA and MAb at Two Conductivities
Column (0.5 cm ID x 7 cm)
High recovery and low elution volume
Recovery and Elution Volume Obtained on CMM HyperCel Sorbent
BSA and MAb elution was performed using 50 mM Tris at pH 7.5, 15 mS/cm and pH 8.5, 30 mS/cm respectively. The recovery was higher than 90% (even with elution volumes below 4 CV) for both BSA and MAb when loaded at 15 and 9 mS/cm.
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.
MAb Dynamic Capacity Binding Over Cycles
|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|
|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|