AEX Columns and Standards for Gene Therapeutics

AEX Columns and Standards for Gene Therapeutics

Develop charged base methods for a diverse range of gene therapies. Waters AEX columns provide versatile retention and selectivity to drug developers focused on synthetic oligonucleotides, CRISPR gene editing, mRNA, plasmid DNA, and viral vectors.

Develop charged base methods for a diverse range of gene therapies. Waters AEX columns provide versatile retention and selectivity to drug developers focused on synthetic oligonucleotides, CRISPR gene editing, mRNA, plasmid DNA, and viral vectors.
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Overview

AEX is a foundational technique for separating gene therapeutics, including antisense oligonucleotides, siRNA, CRISPR single guide RNA (sgRNA), ribonucleoprotein complexes, mRNA, DNA plasmids, and viral capsids. The mechanism of anion exchange is based on electrostatic interactions between negatively charged analytes and the positively charged stationary phase. The more strongly negative a biomolecule, the more tightly it adsorbs to the column’s resin. Analytes separate and elute based on charge density as the ionic strength or pH of the mobile phase changes.

Ensure optimum conditions for your analysis by choosing the best Waters AEX column and standard for your workflow.

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Download Characterizing AAVs eBook
Download Characterizing LNP mRNA eBook

Weak anion exchange columns for improved separations

Weak anion exchange columns for improved separations

Waters Gen-Pak FAX (4.6 x 100 mm) columns offer the highest resolution available in anion-exchange HPLC of nucleic acids. The Gen-Pak FAX column contains a weak anion exchanger based on DEAE functionalized non-porous resin. It contains 2.5 µm particles and is well-suited for analytical and micro-preparative applications.

  • Weak anion exchange non-porous polymer columns
  • High resolution separation of synthetic oligonucleotides and nucleic acids
  • Unique DEAE selectivity and a low retentivity needed for specific applications

Strong anion exchange columns for outstanding resolution in less time

Strong anion exchange columns for outstanding resolution in less time

Waters Protein-Pak Hi Res Q (4.6 x 100 mm) Ion-Exchange (IEX) column assists in the characterization of recombinant proteins, monoclonal antibodies, and other biological compounds. The non-porous, high compound binding capacity of these particles yields outstanding resolution of charged species in less time compared to use of many traditional porous IEX offerings.

  • Strong anion exchange non-porous polymer packed columns
  • High binding capacity with a quaternary amine ligand functionality
  • Compatible with a variety of biomolecules

Webinar: New Insights on Anion Exchange and Size Exclusion of Nucleic Acids

Developing new AEX and SEC methods for mRNA and AAV separations

The success of mRNA vaccines demonstrates the capabilities of nucleic acid-based drugs. More analytical characterization tools are needed to provide more information on stability, heterogeneity, drug design, and structure-function relationships. AEX and size-exclusion chromatography (SEC) techniques are well suited to providing high resolution separations of nucleic acids and their carriers. This webinar delves into a more robust platform method for multiple drug substances and sample types, including AAVs and mRNA.

Developing new AEX and SEC methods for mRNA and AAV separations

The success of mRNA vaccines demonstrates the capabilities of nucleic acid-based drugs. More analytical characterization tools are needed to provide more information on stability, heterogeneity, drug design, and structure-function relationships. AEX and size-exclusion chromatography (SEC) techniques are well suited to providing high resolution separations of nucleic acids and their carriers. This webinar delves into a more robust platform method for multiple drug substances and sample types, including AAVs and mRNA.

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Solutions

WAX on, WAX off

WAX on, WAX off
Gen-Pak FAX Columns

Under native conditions, oligonucleotides and longer nucleic acids carry a negatively charged polyanionic phosphate backbone. This functionality makes nucleic acids an ideal candidate for AEX. The Waters Gen-Pak FAX column is a non-porous 2.5 µm weak anion exchange resin designed for nucleic acids ranging from synthetic oligonucleotides to mRNA.

  • Synthetic Oligonucleotide Separations
  • Oligonucleotide Anion Exchange Separations
  • Empty/Full Viral Capsid Analysis

SAX to the MAX

SAX to the MAX
Protein-Pak Hi Res Q Columns

The Protein-Pak Hi Res Q column contains a non-porous polymeric resin with quaternary amine functionality. Its strong anion/cation exchange (SAX) applications are abundant and wide-reaching, including integrity analysis of mRNA, CRISPR single guide RNA (sgRNA) and ribonucleoprotein complex formation, isoform quantitation of plasmid DNA, and empty/full encapsidation analysis of viral vectored gene therapies. 

  • Oligonucleotide Anion Exchange Separations
  • Intact mRNA Analysis
  • Evaluating CRISPR Guide RNA and Ribonucleoprotein Complex Formation
  • Empty/Full Viral Capsid Analysis

Oligonucleotide & nucleic acid standards for AEX benchmarking

Oligonucleotide & nucleic acid standards for AEX benchmarking
Oligonucleotide & Nucleic Acid Standards

AEX methods are a powerful tool for characterization and confirming the quality of gene therapeutics. Having quality reference materials on hand that are chemically similar to your analytes can help speed up method development and provide a certified reference for future assays.

  • Lipid conjugated ASO
  • siRNA oligonucleotides
  • Single-Guide RNA (sgRNA)
  • DNA Ladder
  • Empty/Full Viral Capsid Analysis

Applications

There are many examples of the Gen Pak FAX weak anion exchange resin demonstrating superior resolving power, higher recovery, and unique selectivity for proteins, oligonucleotides, and nucleic acids. The lower retentivity of the column allows for mobile phase tuning for improved separation and achieving a unique deaminated isoform separation selectivity via a denaturing mobile phase condition. 

There are many examples of the Gen Pak FAX weak anion exchange resin demonstrating superior resolving power, higher recovery, and unique selectivity for proteins, oligonucleotides, and nucleic acids. The lower retentivity of the column allows for mobile phase tuning for improved separation and achieving a unique deaminated isoform separation selectivity via a denaturing mobile phase condition. 

Application Notes

Gen Pak

Cas protein forms a ribonucleoprotein (RNP) complex with sgRNA. It is critical to evaluate the efficiency and strength of the RNP complexation during drug development. It is also important to test the purity of the related drug substances. A new approach highlights how these three components can be readily separated based on native charge by coupling anion and cation exchange. 

Cas protein forms a ribonucleoprotein (RNP) complex with sgRNA. It is critical to evaluate the efficiency and strength of the RNP complexation during drug development. It is also important to test the purity of the related drug substances. A new approach highlights how these three components can be readily separated based on native charge by coupling anion and cation exchange. 

Application Notes

Protein

AEX provides a direct measurement for separating and quantifying plasmid isoforms for purity testing. AEX separates supercoiled (SC), open circular (OC), and linear (L) plasmids based on differences in charge densities present in the plasmid’s conformation. For example, supercoiled DNA has a higher negative charge density than a more relaxed open circular or linear form, resulting in stronger interaction with the Protein-Pak Hi Res Q positively charged stationary phase.

AEX provides a direct measurement for separating and quantifying plasmid isoforms for purity testing. AEX separates supercoiled (SC), open circular (OC), and linear (L) plasmids based on differences in charge densities present in the plasmid’s conformation. For example, supercoiled DNA has a higher negative charge density than a more relaxed open circular or linear form, resulting in stronger interaction with the Protein-Pak Hi Res Q positively charged stationary phase.

Application Notes

AEX

Monitoring the encapsulation efficiency of the DNA payload in a viral capsid, such as an AAV gene therapy, is imperative during biomanufacturing. A high percentage of capsids may not contain the desired transgene and no corresponding therapeutic benefits. Anion exchange can functionally separate empty and full capsids based on the increased negative charge imparted by the transgenes present within the vector. 

Monitoring the encapsulation efficiency of the DNA payload in a viral capsid, such as an AAV gene therapy, is imperative during biomanufacturing. A high percentage of capsids may not contain the desired transgene and no corresponding therapeutic benefits. Anion exchange can functionally separate empty and full capsids based on the increased negative charge imparted by the transgenes present within the vector. 

Application Notes

AAV

The mRNA renaissance has resulted in a renewed need for sensitive and robust analytical methods to characterize the biophysical properties of RNA drug and vaccine candidates. For intact mRNA analysis, an alkyl ammonium salt gradient may be employed at low temperature to preserve the self-structure of mRNA allowing heterogeneity to be evaluated along with a quick determination of the sample’s concentration. These techniques can also be optimized to monitor the progress of an in vitro transcription reaction.

The mRNA renaissance has resulted in a renewed need for sensitive and robust analytical methods to characterize the biophysical properties of RNA drug and vaccine candidates. For intact mRNA analysis, an alkyl ammonium salt gradient may be employed at low temperature to preserve the self-structure of mRNA allowing heterogeneity to be evaluated along with a quick determination of the sample’s concentration. These techniques can also be optimized to monitor the progress of an in vitro transcription reaction.

Application Notes

stock-molecule-MRNA-strand

The data speaks for itself

The data speaks for itself

sgRNA was observed to be present in slight excess following complex formation with Cas9 protein. Complexation was monitored at both 260 (black) and 280 nm (red).

UV chromatograms (260 nm) obtained for an oligo dT ladder using various AEX columns. Salt gradient separations were performed at 30 ˚C with a 0.72 mL/min flow rate, 20 mM Tris pH 8 buffer mobile phase, and a 10 minute gradient running from 300 to 600 mM NaCl.

ΦX174 plasmid isoform (L, O, SC) separation on a Waters Protein-Pak Hi Res Q column. 20 mM Tris pH 7.4, 1.69–1.75 M tetramethylammonium chloride in 10 min. Flow rate: 0.4 mL/min.

Quantification of % empty capsid in various empty and full capsid mix using the optimized AEX method.

Ion exchange separations of Cas9 mRNA (A) and EPO mRNA (B) using an AEX column, a Tris buffered mobile phase, and sodium chloride gradient combined with a series of different column temperatures ranging from 30 to 60 °C.

Separation of an 18-mer oligonucleotide on the Gen-Pak FAX (WAT015490) highlighting efficient separation of N-1, N+1, and suspected deaminated impurities (X) with an optimized high organic method. Nercessian, L., et al. (2024). Robustness evaluation of weak anion exchange chromatography method for the purity analysis of therapeutic oligonucleotides. Journal of Chromatography A, 1736, 465412.

Webinars and Resources

  • How-to Video

New Insights on Anion Exchange and Size Exclusion of Nucleic Acids

New Insights on Anion Exchange and Size Exclusion of Nucleic Acids
Watch Webinar
  • eBook

Characterizing Antibody Conjugates Guidebook

Characterizing Antibody Conjugates Guidebook
Download
  • eBook

eBook: Characterizing LNP mRNA Consumables

eBook: Characterizing LNP mRNA Consumables
Download
  • Infographic

Separation Solutions to Accelerate Next Generation Gene Therapy Development

Separation Solutions to Accelerate Next Generation Gene Therapy Development
Download

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Learn more about AEX Columns and Standards for Gene Therapeutics.

Learn more about AEX Columns and Standards for Gene Therapeutics.
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