Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor

Abstract

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion’s unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.

Sequencing a Bispecific Antibody by Controlling Chain Concentration Effects When Using an Immobilized Nonspecific Protease

“…Conjugation of aspergillopepsin I to 1 μm aldehyde/sulfate solid sphere beads was performed as previously described . Briefly, ∼10 μg of aldehyde functionalized beads were conjugated with aspergillopepsin I.…”

“…The beads were washed with water between each step and stored dry at 4 °C. Beads were benchmarked against previous ∼1 s digestions of 0.2 μg/μL apomyoglobin, as previously described …”

“…Individual protein samples were prepared by evaporating an aliquot to dryness. Samples were reduced by reconstituting in 15 mM TCEP in 8 M urea and 0.5% acetic acid in water and alkylated with 10 mM NAEM in 8 M urea and 0.5 M ammonium acetate in water at pH ∼7 . The samples were then brought to the desired concentration and pH ∼4 using digestion buffer.…”

“…Nonspecific proteases are a novel alternative to specific proteases for antibody analysis. , Aspergillopepsin I is a nonspecific aspartic protease that functions in 8 M urea and acidic conditions . Our lab demonstrated the implementation of an immobilized aspergillopepsin I enzyme reactor to digest monospecific antibodies and analyze them in a single LC-MS/MS analysis. , Although the enzyme is nonspecific, the resulting chromatographic profiles and peptide coverage maps are highly reproducible under consistent working conditions (i.e., digestion time, sample concentration, buffer conditions, etc.). Aspergillopepsin I is bound to beads and packed into a column to control the amount of time the protein is in contact with the enzyme.…”

“…Digesting a protein with aspergillopepsin I for hundreds of milliseconds results in full characterization of the protein from a single LC-MS/MS analysis, reducing preparation and analysis time. Multiple overlapping peptides are observed within this single analysis and provide unambiguous sequence coverage of a molecule . Varying the flow rate through the column dictates the peptide size distribution; as flow rate increases, the interaction time between enzyme and protein decreases, resulting in fewer cleavages and longer peptides that overlap in sequence …”