Characterizing and Quantitating Therapeutic Tethered Multimeric Antibody Degradation Using Affinity Capture Mass Spectrometry

There
are many pharmacokinetic challenges associated with administering
protein therapeutics, including biotransformation via clipping, deamidation,
isomerization, oxidation, etc. In the case of engineered multivalent
tethered antibody formats, proteolysis or deconjugation at the fusion
or conjugation site present further issues. Unlike degradations associated
with antibody drug conjugates, such biotransformations of tethered
antibody formats usually result in degraded products with large mass
differences. These large differences can result in processing or mass
spectrometry response bias among the resulting product species that
can lead to inaccurate stability quantitation. Herein, we describe
an assay strategy for characterizing and quantitating degradations
accurately for multivalent antibodies by incorporating response bias
corrections. For the multivalent tethered antibody molecules selected,
an ∼30–80% difference in response, compared to the cleaved
product, was observed. To correct for the response bias, selected
tethered multivalent antibodies and an IgG antibody (representing
the stable intact and the degraded product species, respectively)
were spiked in serum at known ratios for analysis. Following affinity
capture, we generated calibration curves (five-parameter logistic
fit p < 0.05) by plotting the measured ratios
of the MS ion responses against the known spiked-in ratios (CVs <
8% for calibration standards). The qualified calibration curve (accuracy
within 8% and 2% for measuring degradations of 5% and 15% product,
respectively) was then used, through interpolation, to determine stability
profiles for the same multivalent tethered antibody formats from both in vitro serum and pharmacokinetic study samples.