Consortium for Top-Down Proteomics Study on mAbs

Direct fragmentation of the intact NISTmAb Light Chain confirms 89 % of the sequence in a single spectrum.

• Intact mass or subunit mass determinations after, e.g., IdeS digestion provide a very fast and direct access to information suitable to confirm a reference sequence and includes quantitative information about PTMs such as N-terminal pyroglutamylation, C-term Lys-loss and the glycosylation profile.

• Subunit mass analysis (after IdeS digestion and reduction of the HC; Middle-Up, MU) proved particularly useful as monoisotopic peak picking at the 25 kDa range can be quite reliable if the isotopic distribution of subunit peaks is well represented in the spectra. In addition high sequence coverages of the subunits (Middle-Down, MD) up to 80 % were achieved. Enough to confirm terminal modification status and the sequence.  
  
• Combination of MU and MD analysis is particularly fast and information rich as the accurate subunit mass can narrow down questions left open by MD. (see also these 2 papers about Cetuximab and Natalizumab sequence confirmation and curation using the MU-MD approach).
  

• Consistently, subunit mass accuracy and reliability was highest on the QTOF platform (we used a Bruker maXis II ultra-high resolution QTOF for the lab #1 contribution), as trap based instruments typically suffered limitations in the size of the ion population, which increased uncertainties in picking the correct monoisotopic peak in less well-defined isotopic patterns.

• As a surprise in the MD mAb analysis, the use of the MALDI ionization technique (TOF and FT-ICR alike) provided consistently high sequence coverages for the subunits of all mAbs in the 70-80 % range. MALDI-TOF analyses required offline-LC separations for best results, exemplified by an LC-MALDI-ISD setup consisting of a spotOn MALDI fraction collector and a rapifleX MALDI-TOF/TOF instrument (Bruker). Misassignments of monoisotopic masses as well as charge deconvolution and peak overlaps - common in ESI measurements - weren´t an issue with MALDI. Just single charged fragments were observed, which renders data validation and automation of the analysis straight forward.

• MD analysis in general required careful reduction, deglycosylation and the chromatographic separation of the subunits to reduce peak misassignment in these relatively complex datasets. It was recognized in the study that experience in this field was helpful for providing best results, which we certainly agree with.

• The MU and MD data that we  analyzed and contributed to the study as lab #1 were largely analyzed automatically in the BioPharma Compass^® software (BPC, Bruker).  This included all steps from dataset import and generating results to reports and sequence-annotated spectra and chromatograms. The MAM Protein Screening and the Top-Down MALDI workflows in BPC were used for all our data provided in this study.

Essential for the acceptance of intact mass, MU and MD approaches is a streamlined software environment that provides a fair degree of automation to reduce hands-on analysis time during biopharmaceutical characterization in the Biotech industry and CROs. In our view, this condition is met with the technologies and software that we employed to this study´s tasks. Crucial for automation is the high ruggedness of monoisotopic peak assignment and the high sequence

coverage provided by the technologies we used, together with the software designed for routine biopharmaceutical characterization. BioPharma Compass^® reduces the analysis time drastically and renders MU/MD analysis a new and complementary approach to tryptic digests for the biotech industry, which reduces analysis time at increased confidence and reduced costs.