Site-specific Digestion of IgM

Site-specific IgM digestion under physiological conditions, producing homogeneous fragments for precise middle-level LC-MS characterization.

IgMBRAZOR is a unique IgM-specific enzyme that digests human IgM at one specific site below the CH2 to generate F(ab’)2 and Fc fragments, which allows for middle-level analysis of IgM.

To demonstrate the site-specificity of IgMBRAZOR, human myeloma IgM was digested with IgMBRAZOR Lyophilized for 30 minutes at 37°C and compared to undigested IgM. To facilitate data interpretation, the N-glycans were removed using PNGase F under denaturing and reducing conditions. The homogenous fragments observed in the mass spectra after IgMBRAZOR and PNGase F digestion and the masses of the detected fragments were consistent with digestion at one single site (..VPDQDT / AIRVFA..).

Highly specific digestion of IgM at one single site

The digestion using IgMBRAZOR is fast, easy, and carried out under physiological conditions, and the specificity of the enzyme prevents overdigestion of IgM and hydrolysis of other proteins, challenges otherwise commonly associated with other proteolytic enzymes.

Fast and efficient digestion with no risk of overdigestion

Middle-level analysis allows for the generation of high resolution mass data with minimal sample preparation and data analysis, which can be efficiently used for characterization, quality control, stability testing and production monitoring of therapeutic antibodies.

Simplified characterization IgM using middle-level analysis

Site-specific digestion of IgM. IgM was digested with IgMBRAZOR Lyophilized for 30 minutes at 37°C, where after the Fc N-glycans were cleaved off using PNGase F Lyophilized under reducing and denaturing conditions to facilitate data interpretation. a) Schematic illustration of the sample preparation workflow. b) Deconvoluted mass spectra of the intact deglycosylated heavy chain and the VH-CH1-CH2 and CH3-CH4 fragments generated after IgMBRAZOR and PNGase F digestion. The inserts (top and bottom spectra) display the two peaks with and without the loss of the C-terminal tyrosine. The analysis was performed by reversed-phase LC-MS using a Waters™ BioAccord™ LC-MS system.

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