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.
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.
Process Analytical Technology (PAT) is of crucial importance in the process of IgM manufacturing, especially in its optimization where fast and reliable analytical methods capable of quantitation of the corresponding recombinant IgM concentration levels in the upstream processes are required.
Convective Interaction Media CIM® strong anion exchange monolithic columns have a great advantage in comparison to particle related methods due to their separation capability based on the convective flow mechanism that proved to be particularly efficient in the separation of large IgM molecules.