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.
Pre-activated CIMmic™ Monolithic Columns are used as a basis for preparation of small volume affinity chromatographic columns as well as enzyme reactors. Small bed volume and flexible design makes them a powerful tool for screening purposes and immobilization protocol optimizations. Range of covalently bound ligands is wide and includes diverse set of proteins, peptides, nucleotides and other affinity ligands. The covalent nature of the bond between the ligand and matrix reduces leaching and improves stability and reusability. Reaction conditions must cater to their specific physiochemical nature.
Successful preparation of an affinity column includes a decision on the appropriate matrix chemistry and determination of an optimal immobilization protocol. Presented case study explores the basics of a coupling protocol optimization using covalent immobilization of Recombinant Prokaryotic Lectins (RPL-Gal1) on CIMmic CDI-0.1 and CIMmic ALD-0.1 columns, as an example. Carboxy imidazole (CDI) and aldehyde (ALD) activated CIMmic™ columns are used for covalent immobilization of amine or thiol containing molecules.
The application describes separation of Ni species by assembling four weak CIM DEAE anion-exchange disks into a monolithic column. The concentrations of the Ni species eluted from the column were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID)-ICP-MS. The Ni binding ligands eluted under the chromatographic peaks were identified off-line by tandem electro spray mass spectrometry (ESI-MS-MS), scanning for negative ions.
The mild chromatographic conditions of the CIM DEAE disks preserved chemical species and enabled separation of negatively charged Ni complexes.4 NH4NO3 was chosen as eluent since it enabled separation of Ni species and is compatible with ICP-MS and mass spectrometry detectors.
PEGylation involves the formation of a stable covalent bond between activated poly (ethylene glycol) polymers and polypeptidic drugs and molecules. This process causes a change in protein hydrophobicity and results in variance between the obtained conjugates. Despite this, hydrophobic interaction chromatography (HIC) is used less frequently for separation of PEGylation reaction products than other techniques. Separation of PEGylated conjugates of Ribonuclease A (RNase A) via HIC on monolithic supports was analysed in this work. The protein was PEGylated in the N-terminal amino group with 20 kDa methoxy poly (ethylene glycol) propionaldehyde.
Filamentous phage M13 is a rod shaped non-lytic bacterial virus. M13 genetic material is used for many recombinant DNA processes, and the virus has also been studied for its uses in nanostructures and nanotechnology. The phage has been intensively studied for purposes of phage display and as a delivery vehicle for gene therapy. Phage display was first demonstrated with M13 bacteriophages and the filamentous phage remains a workhorse for this technology. Because of its typical size and rod shape it is considered as a challenging for purification. With large and highly interconnected pores monolithic chromatographic supports are also bridging that problem.
The ability to improve the purification process of M13 and other phages can have a significant impact on the market. By using phages for gene therapy, there will be a decrease in manufacturing time and production costs while enhancing the gene insertion. For phage display, a quicker method for phage purification will allow this powerful tool, which shortens the new drug discovery path and illuminates the basic interactions between different proteins, to be used with higher frequency.
Bacteriophages are used in a broad range of applications, including phage therapy and phage display. With the growing problem of antibiotic resistance leading to untreatable bacterial infections, they are becoming very interesting as antimicrobial agents, not only in medicine, but also in veterinary medicine, food industry and agriculture. Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants.
Here we present efficient single step purification method for a Staphylococcus aureus phage VDX-10 from bacterial lysate on a CIM® QA Disk Monolithic Column (Figure 1). The described method can be used also on a larger scale using a CIM® QA-8 mL Tube Monolithic Column (Figure 2).
Bacteriophages, viruses that infect bacteria, are being used as antibacterial agents, in phage display screening, as gene therapy delivery systems, and for bacteria typing. To use phages in these applications, they must be free of all impurities. A purification and concentration process was recently developed using an ion exchange monolithic column . One of the key challenges faced in phage purification is the monitoring of genomic DNA (gDNA) released to the growth medium which can interfere with the various applications of phages. CIMac™ DEAE Analytical Columns can be used to monitor the fermentation process, evaluate the amount of degraded gDNA to determine the optimal fermentation endpoint and then to efficiently purify the phage particles.
A supernatant from Phanerochaete chrysosporium cultivation was loaded on CIM® QA Disk, and elution was effected by a linear gradient at a flow rate of 3 mL/min (9 CV/min). Baseline separation of isoenzymes H2, H6/H7, H8 and H10 was achieved in less than 3 minutes.