Rapid Antibody Digestion in Protease-Containing Membranes

Proteolytic digestion is often a vital step prior to mass spectrometry analysis of therapeutic antibodies and other proteins. However, complete in-solution proteolysis yields a complicated mixture of small peptides, some of which are not detectable. This reduces sequence coverage and decreases the probability of identifying unwanted posttranslational modifications such as oxidation or deamidation. Limited digestion to obtain fewer and larger peptides may increase sequence coverage, help to connect the sequences of smaller peptides in complete digestion, and determine correlations between posttranslational modifications. Thus we are investigating limited digestion in membranes containing immobilized enzymes. Digestion occurs in <1 minute and control over residence times at the msec level leads to large peptides (see Anal. Chem. 2015, 87, 10942–10949). This method readily reveals glycosylation patterns and antibody oxidation.

Workflow for spin digestion
Workflow for Spin Digestion

Most recently, we began working with spin membranes to develop convenient antibody digestion methods. The above figure shows the workflow for such digestions. Peptic digestion is especially convenient because reduction and digestion can occur under acidic conditions that do not require antibody alkylation. The entire process of reduction, digestion, and infusion analysis requires <30 min. Moreover, 96-well plates containing membranes will enable high-throughput digestion. Such products, which are based on this research, are now commercially available. Graduate student Weijing Liu is continuing the research. We are grateful to the US National Science Foundation (CHE-1506315) for their support of this work.

Dr. Yongle Pang

Yongle Pang, a recent graduate who now works with Covance, led much of the work on controlled antibody digestion.

Dr. Weijing Liu
Graduate student Weijing Liu