Methods for heat-assisted enzyme digestion

A method of digesting a sample comprising humanized IgG1K monoclonal antibody, the method comprising:

• adding the sample to an apparatus containing a buffer solution and a solid support, wherein a pH of the sample solution within the apparatus is between 5.6 and 7.0, wherein the solid support has a surface comprising a surface coating, wherein the surface coating comprises a first coating portion comprising immobilized trypsin and a second coating portion comprising a functionality to reduce interactions between the humanized IgG1K monoclonal antibody and the solid support surface, wherein the second coating portion renders the surface hydrophilic, the functionality comprising a diol obtained from hydrolysis of an epoxide-containing silane selected from the group consisting of a 3-glycidopropyl trifunctional silane and a 2-(3,4-epoxycyclohexyl)ethyl trifunctional silane, wherein the immobilized trypsin is conjugated to the solid support via reductive amination with an alkoxysilane aldehyde, wherein the surface coating is present at a total surface coverage of at least 5 μmoles/m2, and wherein the total surface coverage is the total of coverage of the first and the second coating portions;
• and
• heating the sample to perform a heat-assisted digestion of the humanized IgG1K monoclonal antibody with less than 15% missed cleavages and greater than 85% sequence coverage, wherein 90% or more of humanized IgG1K monoclonal antibody is digested within 10 minutes at a temperature of about 70° C.