The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information
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.
Electron
transfer dissociation (ETD) is an analytically useful
tool for primary structure interrogation of intact proteins, but its
utility is limited by higher-order reactions with the products. To
inhibit these higher-order reactions, first-generation fragment ions
are kinetically excited by applying an experimentally tailored parallel
ion parking waveform during ETD (ETD-PIP). In combination with subsequent
ion/ion proton transfer reactions, precursor-to-product conversion
was maximized as evidenced by the consumption of more than 90% of
the 21 kDa Protein G precursor to form ETD product ions. The employment
of ETD-PIP increased sequence coverage to 90% from 80% with standard
ETD. Additionally, the inhibition of sequential electron transfers
was reflected in the high number of complementary ion pairs from ETD-PIP
(90%) compared to standard ETD (39%).
Previous
work employing five SARS-CoV-2 spike protein receptor-binding
domain (RBD) constructs, comprising versions originally developed
by Mt. Sinai or the Ragon Institute and later optimized in-house,
revealed potential heterogeneity which led to questions regarding
variable seropositivity assay performance. Each construct was subjected
to N-deglycosylation and subsequent intact mass analysis, revealing
significant deviations from predicted theoretical mass for all five
proteins. Complementary tandem MS/MS analysis revealed the presence
of an additional pyroGlu residue on the N-termini of the two Mt. Sinai
RBD constructs, as well as on the N-terminus of the full-length spike
protein from which they were derived, thus explaining the observed
mass shift and definitively establishing the spike protein N-terminal
sequence. Moreover, the observed mass additions for the three Ragon
Institute RBD constructs were identified as variable N-terminal cleavage
points within the signal peptide sequence employed for recombinant
expression. To resolve this issue and minimize heterogeneity for further
seropositivity assay development, the best-performing RBD construct
was further optimized to exhibit complete homogeneity, as determined
by both intact mass and tandem MS/MS analysis. This new RBD construct
has been validated for seropositivity assay performance, is available
to the greater scientific community, and is recommended for use in
future assay development.
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