Chromatography is a versatile tool used in bioprocessing to purify and analyse biomolecules. In our previous blog we have explored the basics of chromatography and its different types.
This blog will delve into the specific applications of chromatography for:
mRNA Purification Techniques
mRNA requires gentle purification to avoid degradation. We recommend using techniques that effectively separate mRNA from impurities, exploiting its unique properties such as the poly A tail and its charge. At Sartorius BIA Separations, we use various techniques to successfully separate mRNA from various impurities in the mixture.
- Affinity Chromatography: A part of the mRNA molecule called the poly A tail binds to the Oligo dT18 ligand.
- Multimodal Chromatography: This technique combines hydrogen bonding and anion exchange properties to retain mRNA. Molecules with predominantly negative charges are retained on the column. Remaining impurities are separated from mRNA in the elution gradient.
- Ion Pair Reverse Phase Chromatography: Used for the removal of double-stranded RNA by exploiting differences in the hydrophobicity of different RNA species.
- Hydrophobic Interactions: Used for the removal of protein impurities.
Plasmid DNA (pDNA) Separation
When it comes to pDNA, we apply similar principles.
- Ion Exchange (Anion Exchange): DNA is a negatively charged molecule and binds to our positive ion exchangers, allowing for the elution of impurities.
- Hydrophobic Interactions: These allow us to separate pDNA isoforms and to further remove genomic DNA.
Adeno-Associated Virus (AAV) Purification
For the purification of AAVs, we employ a variety of techniques:
- Hydrophobic Interactions: Used as an optional pre-capture step to reduce host cell impurities.
- Ion Exchange (Cation and Anion Exchange): Cation exchange is used as a capture step to remove most of the host cell impurities, while anion exchange separates empty and full capsids.
- Multimodal chromatography: By employing combination of different mechanisms multimodal ligands are an alternative to anion exchange chromatography for Empty-Full separation.
Extracellular Vesicles (EV) Isolation
For extracellular vesicles (EVs), we use a single chromatography technique:
- Anion Exchange: Negatively charged molecules on the surface of exosomes, called phospholipids, bind to the stationary phase and EVs are later eluted due to the increase in conductivity.
Chromatography offers a range of techniques for the purification and analysis of various biomolecules. In the final part of this blog series, we will discuss the key parameters that influence chromatography.
