Technical Notes

CIMmultus® QA HR column is anion exchange chromatography monolithic column. One of its main applications is enrichment of AAV full particles. Empty and full AAV capsids have small difference in pI, which can be used for separation using its charge difference for elution. After samples are bound to the column at high pH and low conductivity, they are eluted with increasing linear salt gradient. Empty and full capsids elute at different conductivities due to their charge differences.

CIMmultus® QA HR can be used for polishing step of AAV samples. Polishing step is used as a second AAV chromatography step after capture step. In capture step we remove majority of impurities, which could co-elute with full AAV capsids in polishing step and reduce its purity.

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.

Chromatographic applications in diagnostics call for automated systems and high-throughput analyses in order to cope with the large numbers of samples. Sartorius BIA Separations offers differently modified monoliths (ion exchanging, affinity, hydrophobic…) in 96-well plate format to follow the increasing needs of DIAGNOSTIC laboratories. The example described here is an affinity-based CIM® Protein G 96-monolithic plate (Pores 2 um), which enables efficient and robust capture of different antibodies from complex samples. The application describes the capture of immunoglobulin G (IgG) from human plasma with subsequent IgG glycosylation studies. The stability of the column for at least 70 isolation steps and proof of no cross-contamination are shown.

CIMac™ pDNA-0.3 Analytical Columns (Pores 1.4 μm) were introduced by BIA Separations d.o.o. in 2010. In the following years they were recognised by the biotechnological and analytical community as excellent choice for fast and exact plasmid DNA (pDNA) monitoring and quantification. Due to high sensitivity of the column to small changes in chromatographic conditions we publish this technical note, how to exploit the column’s excellent chromatographic characteristics to the highest possible extent.
A standard testing protocol for the columns is analytical separations of open circular (oc) and supercoiled (sc) pDNA isoforms of a 4.7 kbp large plasmid DNA molecule. The concentration of the pDNA sample used was 23μg/mL. The sample contained between 21 and 22% of the oc pDNA isoform and an exact amount of uracil as unbound tracer. The separation of the isoforms was performed in a linear gradient of NaCl with the temperature of the whole system controlled to 15.0±0.5 °C. The typical experimental conditions together with an example of chromatogram are shown in Figure 1.

The stability of QA, DEAE and SO3 CIMac™ Analytical Columns was tested according to the CIP (Cleaning In Place) procedures described in the respective Product Specification Sheets (see Table 1). We compared the separation of a mixture of test proteins, the dynamic binding capacity for BSA and the pressure drop after 50 and 100 CIP procedures with the initial characteristics of the columns.

CIMac™ SO3 Analytical Column is a strong cation exchanger based on a monolithic support. This material provides many advantages over conventional stationary phases for the separation of biomacromolecules, such as: low back-pressure, flow-unaffected chromatographic properties and a high resolution power due to a convective flow transport within open large-diameter (1.5 μm) channels.

Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target.

H-Bond ADC is the first of a new class of chromatography ligands from BIA Separations that exploits hydrogen bonding as the dominant mode of biomolecule retention. The ligand consists of a terminal series of hydrogen donors grafted to a root series of hydrogen acceptors.

H-Bond ADC brings a unique new selectivity to all fractionation tasks but is especially distinctive in its ability to retain large biomolecules more strongly than small ones. This can be of great value for removing fragments, aggregates, and viruses from protein products, or for removing proteins and other small contaminants from large biologics like viruses and extra-cellular vesicles.

Coupling trypsin directly onto a Convective interaction media (CIM®) monolithic chromatographic support makes it possible to reuse the enzyme for multiple process cycles and to automate hydrolysis processes. BIA Separations offers CIM trypsin immobilised enzyme reactors (trypsin IMERs) based on monoliths with volumes from 0.1 mL up to 80 mL) and with channel sizes of 2 µm or 6 µm to meet the needs of various applications. Common applications include sample preparation for mass spectrometry (MS)-based proteomics or industrial scale production of protein hydrolysates.