ProPac LC Columns for Protein and Charge Variant Characterization

Ultrahigh resolution of proteins, monoclonal antibodies, biosimilars, and charge variants

Achieve ultrahigh resolution and high efficiency separations of proteins and glycoproteins with a pI 3–10 and molecular weight over 10,000 units. Thermo Scientific ProPac Protein LC columns have a long-standing reputation for providing excellent reproducibility and durability under a broad range of pH, temperature, and mobile phase compositions.

ProPac columns along with Thermo Scientific MAbPac and Acclaim SEC 300 columns comprise our broader portfolio that address the challenges in biopharmaceutical separations.

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ProPac Ion Exchange columns

Unsure if you need anion or cation exchange column?

Separations are based on the accessible surface charge of the protein or glycoprotein with the column’s functional group. Unsure if you need an anion or cation exchange column? The protein’s pI can help to determine which column to use.

  • If your protein pI is < 6, start with anion exchange columns. You may need to try both SAX and WAX options to determine which offers the best resolution.
  • If your pI is > 6, start with cation exchange columns. The ProPac Elite is our newest innovation providing the ultrahigh resolution, speed and efficiently along with the ability to use pH gradients as well as salt gradients. If you are just starting to develop your method, consider starting with this column.
ProPac LC Columns for Protein and Charge Variant Characterization Decision Flowchart
 Click to enlarge

Recommended Ion Exchange Columns by Type

Column Type

Strong anion exchange (SAX) columns Weak anion exchange (WAX) columns Weak cation exchange (WCX) columns Strong cation exchange (SCX) columns

Column

ProPac SAX-10 ProPac WAX-10 ProPac Elite WCX 5μm ProPac WCX-10 MAbPac SCX

Functional group

Quaternary ammonium Tertiary amine Carboxylic acid Sulfonic acid

Sample pI

Typically for protein pI is < 6 Typically for protein pI is > 6

Notes

    Based on WCX-10 column

Ultrahigh resolution

Faster, more efficient, highly reproducible
Long standing gold-standard column Strong Cation Exchange columns offer an alternate selectivity to Weak Cation Exchange columns

Gradient

Use salt gradient WAX will buffer your phase more than SAX Use salt or pH gradients* Use salt gradient Use salt or pH gradients

* Using our CX-1 pH Gradient Buffers

Innovation inside ProPac Ion Exchange Columns

Gold standard for protein characterization in the biopharmaceutical industry
ProPac column nonporous particles covered by a hydrophilic polymer with grafted ion exchange functionality (strands).
Illustration: ProPac column nonporous particles covered by a hydrophilic polymer with grafted ion exchange functionality (strands).

 Click to enlarge
  • Resolves variants that differ by as little as one amino acid.
  • Eliminates hydrophobic interactions, resulting in very efficient peaks.
  • Nonporous 5 or 10µm particles are solvent-compatible ethylvinylbenzene cross-linked with 55% divinylbenzene polymer (DVB) substrate. 
  • Resin is covered with a highly hydrophilic, neutral polymer, to minimize nonspecific interactions between the surface and the biopolymer. 
  • A controlled polymer chain is grafted on the hydrophilic layer to introduce the ion exchange functionality containing the anion- or cation-exchange groups that impart selectivity and reproducible capacity. 
  • The selectivity differences between the various resin types provide flexibility in maximizing the resolution of closely related proteins. 

Use for

  • Characterization and quality control assessment of monoclonal antibodies and other proteins. 
  • Exceptionally high resolution and efficiency for separations of protein variants such as sequence variants, truncation variants, phosphorylation variants sialylation variants, deamidation variants, and other variants. 

ProPac & MAbPac hydrophobic interaction chromatography (HIC) columns

With many opportunities for post translational modifications (PTMs), it is often necessary to employ multiple column supports to detect, characterize, and quantify all structural variants and modifications and to monitor product stability. 

Separates proteins, peptides, and other biomolecules by their degree of hydrophobicity

HIC columns separate proteins and mAb molecules in order of increasing hydrophobicity under non-denaturing conditions. Analytes bind to the weakly hydrophobic stationary phase in the presence of high salt concentration and elute off the column as the salt concentration decreases. 

Nondenaturing

In contrast to reversed phase liquid chromatography, HIC typically preserves the biological activity of the protein, which is useful for downstream functional analysis such as binding and cell-based potency assays.

ProPac HIC-10
  • Ideal for separation of protein/peptides including complex mixtures 
  • Non-denaturing conditions to preserve biological activity 
  • Excellent resolution and high efficiencies 
  • High capacity column 
  • Compatible with organic solvents and can be used in separations of very hydrophobic proteins 
  • Broad range of applications

Also see our Thermo Scientific MAbPac HIC columns

MAbPac HIC-10 LC Column MAbPac HIC-20 LC Columns MAbPac HIC-Butyl LC Columns
  • High purity, spherical, wide-pore (1,000Å), 5μm silica particles
  • Functionalized with proprietary alkyl amide groups
  • Advanced surface bonding technology
  • Excellent chemical stability, high efficiency, and unique selectivity optimized for mAbs
  • Different from other HIC phases
  • High resolution silica based
  • Based on ProPac HIC-10 column
  • Unique chemistry for excellent separation of mAbs and mAb variants such as oxidized mAb variants
  • Compatible with both organic solvent and aqueous mobile phase
  • Rugged column stability
  • Hydrophilic, nonporous, 5μm polymer particles
  • Functionalized with butyl groups
  • Density of butyl group optimized for high resolution separation and biocompatibility

About ProPac & MAbPac biopharma HPLC columns

The biotechnology and biopharmaceutical industry has an extensive development pipeline for protein and monoclonal antibody (mAb) therapeutics. We understand that mAbs have very complex structures, with many possible site-specific variations that can change safety, efficacy, and stability profiles. With such potential for post translational modifications (PTMs), it is essential to detect, characterize, and quantify structural variants and modifications and to monitor product stability.

We see it as our job to develop and deliver innovative and efficient analytic tools during mAb development and production to prove safety, efficacy, and stability to regulatory agencies. Look to Thermo Scientific HPLC and LC/MS instruments and HPLC columns as your essential tools for the characterization of mAb and other protein biotherapeutics. They have been designed to address your challenges.

Thermo Scientific BioLC columns for mAbs, mAb variants, and other proteins

  • ProPac ion exchange columns provide exceptionally high resolution and efficiency for separations of protein variants. These columns are capable of resolving isoforms that differ by as little as one charged residue.
  • MAbPac columns, specifically designed for the separation of monoclonal antibodies, complement ProPac columns. 
Analysis Description Columns & Buffers

Charge variant analysis

Routine charge variant profiling/screening, including lysine truncation, deamidation & acylation

Methionine & tryptophan oxidation

Targeted analysis of methionine and tryptophan oxidation

Antibody drug conjugate (ADC) analysis using MS

ADC DAR analysis/ Drug to Antibody ratio and intact mass

Intact mAb & fragment analysis

Light chain (LC) and heavy chain (HC) analysis
Fragment (Fab and Fc) analysis
scFc and F(ab’)2 analysis

ProPac Ion Exchange columns

Unsure if you need anion or cation exchange column?

Separations are based on the accessible surface charge of the protein or glycoprotein with the column’s functional group. Unsure if you need an anion or cation exchange column? The protein’s pI can help to determine which column to use.

  • If your protein pI is < 6, start with anion exchange columns. You may need to try both SAX and WAX options to determine which offers the best resolution.
  • If your pI is > 6, start with cation exchange columns. The ProPac Elite is our newest innovation providing the ultrahigh resolution, speed and efficiently along with the ability to use pH gradients as well as salt gradients. If you are just starting to develop your method, consider starting with this column.
ProPac LC Columns for Protein and Charge Variant Characterization Decision Flowchart
 Click to enlarge

Recommended Ion Exchange Columns by Type

Column Type

Strong anion exchange (SAX) columns Weak anion exchange (WAX) columns Weak cation exchange (WCX) columns Strong cation exchange (SCX) columns

Column

ProPac SAX-10 ProPac WAX-10 ProPac Elite WCX 5μm ProPac WCX-10 MAbPac SCX

Functional group

Quaternary ammonium Tertiary amine Carboxylic acid Sulfonic acid

Sample pI

Typically for protein pI is < 6 Typically for protein pI is > 6

Notes

    Based on WCX-10 column

Ultrahigh resolution

Faster, more efficient, highly reproducible
Long standing gold-standard column Strong Cation Exchange columns offer an alternate selectivity to Weak Cation Exchange columns

Gradient

Use salt gradient WAX will buffer your phase more than SAX Use salt or pH gradients* Use salt gradient Use salt or pH gradients

* Using our CX-1 pH Gradient Buffers

Innovation inside ProPac Ion Exchange Columns

Gold standard for protein characterization in the biopharmaceutical industry
ProPac column nonporous particles covered by a hydrophilic polymer with grafted ion exchange functionality (strands).
Illustration: ProPac column nonporous particles covered by a hydrophilic polymer with grafted ion exchange functionality (strands).

 Click to enlarge
  • Resolves variants that differ by as little as one amino acid.
  • Eliminates hydrophobic interactions, resulting in very efficient peaks.
  • Nonporous 5 or 10µm particles are solvent-compatible ethylvinylbenzene cross-linked with 55% divinylbenzene polymer (DVB) substrate. 
  • Resin is covered with a highly hydrophilic, neutral polymer, to minimize nonspecific interactions between the surface and the biopolymer. 
  • A controlled polymer chain is grafted on the hydrophilic layer to introduce the ion exchange functionality containing the anion- or cation-exchange groups that impart selectivity and reproducible capacity. 
  • The selectivity differences between the various resin types provide flexibility in maximizing the resolution of closely related proteins. 

Use for

  • Characterization and quality control assessment of monoclonal antibodies and other proteins. 
  • Exceptionally high resolution and efficiency for separations of protein variants such as sequence variants, truncation variants, phosphorylation variants sialylation variants, deamidation variants, and other variants. 

ProPac & MAbPac hydrophobic interaction chromatography (HIC) columns

With many opportunities for post translational modifications (PTMs), it is often necessary to employ multiple column supports to detect, characterize, and quantify all structural variants and modifications and to monitor product stability. 

Separates proteins, peptides, and other biomolecules by their degree of hydrophobicity

HIC columns separate proteins and mAb molecules in order of increasing hydrophobicity under non-denaturing conditions. Analytes bind to the weakly hydrophobic stationary phase in the presence of high salt concentration and elute off the column as the salt concentration decreases. 

Nondenaturing

In contrast to reversed phase liquid chromatography, HIC typically preserves the biological activity of the protein, which is useful for downstream functional analysis such as binding and cell-based potency assays.

ProPac HIC-10
  • Ideal for separation of protein/peptides including complex mixtures 
  • Non-denaturing conditions to preserve biological activity 
  • Excellent resolution and high efficiencies 
  • High capacity column 
  • Compatible with organic solvents and can be used in separations of very hydrophobic proteins 
  • Broad range of applications

Also see our Thermo Scientific MAbPac HIC columns

MAbPac HIC-10 LC Column MAbPac HIC-20 LC Columns MAbPac HIC-Butyl LC Columns
  • High purity, spherical, wide-pore (1,000Å), 5μm silica particles
  • Functionalized with proprietary alkyl amide groups
  • Advanced surface bonding technology
  • Excellent chemical stability, high efficiency, and unique selectivity optimized for mAbs
  • Different from other HIC phases
  • High resolution silica based
  • Based on ProPac HIC-10 column
  • Unique chemistry for excellent separation of mAbs and mAb variants such as oxidized mAb variants
  • Compatible with both organic solvent and aqueous mobile phase
  • Rugged column stability
  • Hydrophilic, nonporous, 5μm polymer particles
  • Functionalized with butyl groups
  • Density of butyl group optimized for high resolution separation and biocompatibility

About ProPac & MAbPac biopharma HPLC columns

The biotechnology and biopharmaceutical industry has an extensive development pipeline for protein and monoclonal antibody (mAb) therapeutics. We understand that mAbs have very complex structures, with many possible site-specific variations that can change safety, efficacy, and stability profiles. With such potential for post translational modifications (PTMs), it is essential to detect, characterize, and quantify structural variants and modifications and to monitor product stability.

We see it as our job to develop and deliver innovative and efficient analytic tools during mAb development and production to prove safety, efficacy, and stability to regulatory agencies. Look to Thermo Scientific HPLC and LC/MS instruments and HPLC columns as your essential tools for the characterization of mAb and other protein biotherapeutics. They have been designed to address your challenges.

Thermo Scientific BioLC columns for mAbs, mAb variants, and other proteins

  • ProPac ion exchange columns provide exceptionally high resolution and efficiency for separations of protein variants. These columns are capable of resolving isoforms that differ by as little as one charged residue.
  • MAbPac columns, specifically designed for the separation of monoclonal antibodies, complement ProPac columns. 
Analysis Description Columns & Buffers

Charge variant analysis

Routine charge variant profiling/screening, including lysine truncation, deamidation & acylation

Methionine & tryptophan oxidation

Targeted analysis of methionine and tryptophan oxidation

Antibody drug conjugate (ADC) analysis using MS

ADC DAR analysis/ Drug to Antibody ratio and intact mass

Intact mAb & fragment analysis

Light chain (LC) and heavy chain (HC) analysis
Fragment (Fab and Fc) analysis
scFc and F(ab’)2 analysis

Related products

CX-1 pH Gradient Buffers
  • Robust, reproducible pH gradients for a wide range of mAb charge variants
  • Ready to use with existing LC columns and systems
  • No need to formulate mobile phases
  • Save time in method development 
  • Save time in method transfer to QA/QC 

See all pH gradient buffers


Featured ProPac videos

Learn how to easily produce linear pH gradients for fast and robust therapeutic protein separations pH gradient buffer kits with ion exchange columns.

Learn about this simple pH gradient/ion-exchange chromatography workflow for the characterization of charge variants in monoclonal antibodies.


Educational webinars

Taking charged variant analysis of therapeutic proteins to the next level

Learn how to optimize the challenging characterization of proteins, mAbs, biosimilars, and their charge variants using new ProPac Elite weak cation exchange column chemistry.

Join scientific experts from NIBRT in Dublin, Ireland and Thermo Fisher Scientific for the latest analytical advances for intact protein characterization.


Biopharma community & collaboration

National Institute for Bioprocessing Research and Training (NIBRT)

National Institute for Bioprocessing Research and Training (NIBRT)

Collaboration between NIBRT and Thermo Fisher Scientific is designed to enhance structural knowledge, accelerate innovations, and ensure drug safety and quality.

AppsLab Library of Analytical Applications

AppsLab Library of Analytical Applications

Unique applications compendium from NIBRT and Thermo Fisher Scientific focused on simplified, faster methods that deliver highly informative characterization data.

Visit Biopharma Characterization Resource Center

 Visit Biopharma Characterization Resource Center

Learn more about the innovative technologies for charge variant analysis.

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