Technical Notes

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.

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. New experimental data show that large mRNA can also be eluted from CIMac PrimaS at ambient temperature in a pyrophosphate gradient. Resolution between in vitro transcription (IVT) contaminants and mRNA can be controlled with pH.

Chromatographic applications in diagnostics call for automated systems and high-throughput analyses in order to cope with the large numbers of samples. 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.