Digestion of IgM

Site-specific Digestion of Human IgM

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.

IgM is the first class of antibodies produced by the human immune system in the response to an infection or foreign substance. IgM commonly constitutes a complex of five or six monomers capable of binding ten or twelve antigens, respectively. Each IgM monomer consists of two heavy chains comprising one variable and four constant regions (VH-CH1-CH2-CH3-CH4) and two light chains comprising one variable and one constant region (VL-CL). The monomers are linked together via disulfide bonds and the pentamer also contains a joining (J) chain.

The high avidity binding, engagement of the complement system, and agglutination properties of the IgM qualifies it as an interesting candidate for future immunotherapy. Nevertheless, the size of the IgM multimers, approx. 1000 kDa, and large number of N-glycans present make them particularly difficult to analyze. While existing techniques have been important for advancing the understanding of IgM, there is a need for improved tools for further characterization and deeper insight into the function of this antibody isotype.

IgMBRAZOR Digestion

Site-specific Digestion of IgM

IgMBRAZOR digests human IgM at one specific site below the CH2 domain, generating 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.., Fig. 1). 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. 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.

Figure 1. 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.

IgMBRAZOR Digestion

Isotype-specific Digestion using IgMBRAZOR™

IgMBRAZOR is isotype-specific towards human IgM antibodies. Here, all four subclasses of human IgG along with IgA and IgM were incubated with IgMBRAZOR Lyophilized. No digestion was observed for any of the IgG subclasses or IgA, while IgM was successfully digested into F(ab’)2 and Fc fragments in 30 minutes (Fig. 2). This shows the high specificity of IgMBRAZOR and demonstrates the possibility of using the enzyme in complex samples such as serum, plasma and blood samples without the risk of off-target digestion.

Figure 2. Isotype specificity of IgMBRAZOR. Human IgG1 (trastuzumab), IgG2 (panitumumab), IgG3 (from human serum; polyclonal), IgG4 (nivolumab) and IgA (from human serum; polyclonal) were incubated with IgMBRAZOR Lyophilized for 2 h at 37°C. IgM (from human serum; polyclonal) was incubated with IgMBRAZOR Lyophilized for 30 minutes or 2h at 37°C. All samples were analyzed reduced with SDS-PAGE.

IgMBRAZOR Digestion

Digestion of IgM in Serum

IgM constitutes around 5% of the total amount of immunoglobulins in normal human serum, and the ability to selectively digest IgM can be of great value in research and diagnostics as well as in bioprocessing and therapeutic applications. The specificity of IgMBRAZOR makes it an excellent tool for such isotype-specific digestion of IgM in human serum samples. Here, human serum was incubated with IgMBRAZOR Lyophilized and/or FabRICATOR Lyophilized for 30 minutes at 37°C before analysis with SDS-PAGE and western blot (Fig. 3). IgM was successfully digested using IgMBRAZOR and IgG using FabRICATOR. Both enzymes were isotype-specific, and no interference with the FabRICATOR activity was observed when combining the two enzymes.

Figure 3. Activity of IgMBRAZOR in human serum. a) IgMBRAZOR Lyophilized was added to human serum and incubated for 30 minutes at 37°C . The digested sample and control samples of human serum, human IgM, and human IgG were separated on two reducing SDS-PAGE gels. One of the gels was stained, and the other transferred to a membrane. After blocking of the membrane in a casein solution, alkaline phosphatase conjugated α-human IgM HC antibody was added and incubated for 1 h at room temperature. The membrane was thoroughly washed before addition of the chromogenic substrate BCIP/NBT. b) IgMBRAZOR Lyophilized and FabRICATOR Lyophilized were incubated in human serum for 30 minutes at 37°C, both individually and in combination. The digested samples and controls were prepared as in (a), except for the primary antibody solution used; alkaline phosphatase conjugated α-human IgG HC. Black arrows indicate the expected molecular weight of IgM HC that due to the low concentration is not visible in the serum samples.