Miniaturised immobilised enzymatic reactors can be used for small scale digestion of proteins. There is need for such devices; small scale devices are used either for processing of analytical sample quantities, or as proof of concept before protein digestion at larger scale. This application note compares the performance of a flow through miniaturised immobilised enzymatic reactor (μIMER) with in-solution batch digestion of simple proteins and complex matrices. Automation of peptide analysis by coupled LC-MS is explored as an option to increase throughput. In the cases evaluated, the miniaturised immobilised enzymatic reactor offered comparative results to overnight in-solution digestion, within less than 10 minutes.
Pre-activated CIMmic™ monolithic columns with 100 μL bed volume were immobilised with trypsin from bovine pancreas. This small format allows coupling to HPLC for on-line protein digestion, as well as syringe (manual) operation of the IMER. Pre-treated samples (denatured, alkylated and ultra-filtered) are injected into the column, and the eluate (tryptic digests) are subjected to LC-ESI-MS-MS analysis for protein identification and post-translational modification (PTM) determination.
A purification of synthetic oligonucleotides by using CIM™ monolith was evaluated. In this case study, the CIM™ anion exchange column had the capability to resolve oligonucleotides with small difference in comparative chain length.
A crude reaction mixture of synthetic oligonucleotide was loaded onto the CIM™ anion exchange column. Sample elution was achieved by salt concentration gradient. In comparison with conventional media, CIM™ monolith indicated higher resolution for major impurities.
Advantages of the characteristic properties of the CIM™ monolith were evaluated based on the high throughput purification of oligonucleotides under the identified gradient separation conditions. Over 99 % HPLC purity for the target oligoDNA was achieved by one-step purification from the crude reaction mixture.
Pre-activated CIMmic™ monolithic columns are cost efficient tools for screening of immobilisation conditions and small scale proof-of-concept testing of custom affinity columns and enzymatic reactors. Each column is assembled from a dedicated housing and discs containing a chromatography medium. With a bed volume of 100 μL, sample requirements are minimal, while inserting multiple discs in the housing adapts the column volume to application requirements. Different surface modifications of the discs enable immobilisation of a wide variety of ligands.
The increasing demand for messenger RNA (mRNA) as a therapeutic product requires larger production scales, and in turn more efficient extraction techniques. One of the most convenient techniques for its extraction is the use of oligo deoxythymidine (dT) coupled to a solid support . Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT, while other contaminant impurities (proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes) lack the poly-A moiety and do not adhere to the solid support.
The increasing demand for messenger RNA (mRNA) as therapeutic product requires larger production scales, and in turn more efficient extraction techniques. Messenger RNA can be produced by in vitro transcription reactions (IVT) or isolated from eukaryotic cells. One of the most convenient techniques for its extraction is the use of oligo deoxythymine (dT) coupled to a solid support. Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT. Contaminant impurities, such as proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes lack the poly-A moiety and are not retained on the solid support.
Chromatography using a solid phase consisting of large channels, such as monoliths, allows high flow rates and low shear forces. This can have a positive impact on recovery and productivity in purification of biologics. In addition, chromatography offers a closed system to minimise the risk of cross-contamination or exposure to RNase degradation, and an easily scalable platform.
CIMmultus™ Oligo dT is a chromatography column with Oligo dT ligands covalently bound on its surface. The sample containing poly-adenylated mRNA is loaded onto the column in a high salt concentration buffer. Salt ions screen the electrostatic repulsion between the negatively charged backbones and allow interaction between the Oligo dT and poly-adenylated tail of mRNA. Before product elution, a wash step at reduced salt concentration removes unspecifically bound contaminants. Elution of messenger RNA occurs under mild conditions in low conductivity buffer at neutral pH. In the absence of salt, electrostatic repulsion between the negatively charged backbones of Oligo dT and poly-adenine destabilises the T–A pairs and releases mRNA from the column.
Coupling trypsin enzyme onto chromatographic supports provides a platform to reuse the enzyme and automate the hydrolysis process. A monolithic chromatographic support, such as Convective Interaction Media (CIM®), enables mass transfer of molecules within its channels exclusively by convective flow. This results in enzymatic conversion which is not limited by diffusion, making CIM® monoliths ideal for the preparation of immobilised monolith enzymatic reactors (IMERs). BIA Separations offers CIMac™ trypsin IMER with a bed volume of 0.1 mL as analytical platform for mass spectrometry (MS)-based proteomics. Larger volume IMERs (up to 80 mL) are available for industrial scale production of protein hydrolysates. The following example describes the enzymatic production of β-Lactoglobulin (β-Lg) hydrolysates using monoliths with 2 μm (N1) or 6 μm (N2) average channel diameter.
A mixture of 8mer, 10mer, 12mer, 14mer, 15mer and 16mer Oligodeoxynucleotides was loaded on CIM® DEAE Disk and eluted in linear gradient mode at a flow rate of 6 mL/min (17 CV/min). Separation of all nucleotides could be accomplished within 60 seconds.