Technical Notes

CIM® SDVB is a polystyrene divinylbenzene monolith used for reversed phase (RP) purification of large biologics. It is used in purification of nucleic acids for size fractionation. In addition to length, the degree of base pairing influences selectivity and can enable separation between double-stranded and single-stranded species of similar length. As a preparative tool, CIMmultus® SDVB gives its best results as a polishing method. It can be used for fast, low pressure and room temperature purification of RNA, including RNA exceeding 10 kb (such as self-amplifying RNA).

CIMmultus® SDVB is commonly but not exclusively used with an ion-pair reagent. The ion-pair reagent neutralises the negative charge on the RNA backbone to improve binding of RNA or other nucleic acid to the monolith. Elution is achieved by increasing concentration of organic solvent such as acetonitrile.

The ligand in CIM® PrimaS contains a series of hydrogen donors and hydrogen acceptors, combined with a weak anion exchange character. Its hydrogen bonding ability makes its selectivity entirely distinct from traditional anion exchangers. PrimaS purifies large single-stranded mRNA (ssRNA) under aqueous conditions at ambient temperature. Contaminants are removed by means of a hight salt wash, while ssRNA is recovered from the column with a pH gradient. It can be used for purification or analytics of ssRNA, including RNA exceeding 10 kb (such as self-amplifying RNA). In a purification process, it is most often employed for capture of RNA in crude samples such as IVT mixtures.

CIMmultus® PrimaS combines anion exchange and hydrogen bonding properties. It binds molecules with predominantly negative charge and repels molecules with a predominantly positive charge. Samples are applied at neutral pH. A high salt wash is used to remove most contaminants, while ssRNA is eluted by increasing pH, which reduces the binding ability of the PrimaS column.

The ligand in CIM® C4 HLD monoliths is a hydrophobic butyl group. RNA binds on the column under hydrophobic conditions. The majority of DNA and short transcripts elute earlier than intact ssRNA, while most of the proteins and aggregates bind very strongly and are eliminated by a cleaning step with NaOH. C4 HLD can be used to produce research grade ssRNA from in vitro transcription (IVT) mixtures but gives its best results as a polishing method.

The sample is applied at elevated concentrations of salts that precipitate RNA. The high salt concentration enables binding on the column. A decreasing salt gradient, or a step elution can be used to elute RNA from the column. Fractionation of the elution peak is recommended to analyse for purity (fragments).

The ligand in CIM® Oligo dT18 is an 18-mer deoxythymidine chain covalently immobilised on the monolith with a carbon linker. RNA with a poly(A) tail binds on the ligand by hybridisation between adenine (A) bases on the RNA tail and deoxythymine (T) bases of the ligand. Species which do not contain a poly(A) tail do not bind on the column and are found in the unbound (flow through) fraction. CIM® Oligo dT18 can be used for purification or analytics or ssRNA, including RNA exceeding 10 kb (self-amplifying RNA). In a purification process, it is most often employed for capture of RNA in crude samples such as IVT reaction mixtures.

The sample is applied at elevated ionic strength to suppress charge repulsion between the phosphatidic acid residues on the ligand and the target RNA. RNA is recovered by reducing ionic strength, which restores charge repulsion sufficiently to dissociate the hydrogen bonds.

CIMac PrimaS is a new member of BIA’s family of high performance chromatography products for analysis and purification of mRNA. Its positive charge gives it some anion exchange behavior but hydrogen bonding makes its selectivity is entirely distinct from traditional anion exchangers. QA and DEAE anion exchangers need to be heated into the range of 50–70°C for large mRNA to elute. Large mRNA elutes from CIMac PrimaS at ambient temperature in an ascending pH gradient.

CIMac PrimaS™ is a new member of BIA’s family of high performance monoliths for analysis and purification of mRNA. Its positive charge gives it some anion exchange behavior but hydrogen bonding makes its selectivity is entirely distinct from traditional anion exchangers. QA and DEAE anion exchangers need to be heated into the range of 50–70°C for large mRNA to elute. PrimaS elutes mRNA in a pH gradient, well separated from DNA and dsRNA. New experimental data show that NaCl shifts the elution profile to lower pH values.