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Ph.D.
(Science)
CHARACTERIZATION OF CHARGED HETEROGENEITY OF ONCOLOGICAL
MONOCLONAL ANTIBODIES USING LIQUID CHROMATOGRAPHY AND
ONLINE HIGH-RESOLUTION MASS SPECTROMETRY
Ph.D. Scholar : Bhatt Mithun
Research Supervisor : Dr. Bhushan Kulkarni & Dr. Sandesh Chibber

Regi. No.: 19276481001
Abstract :
Post-translational modifications (“PTMs”) in monoclonal antibodies (mAbs) contribute to
charge variant distribution, which will affect biological efficacy and safety. For the
characterization of mAbs, charge variants are used as a critical quality attribute for
product quality, stability consistency and effectiveness. Charge variants in mAbs are
characterized by a time-consuming and a multistep process starting from cation/anion
exchange chromatography, acidic/basic fractions collection and subsequent reverse
phase (RP) liquid chromatography, coupled with mass spectrometry (MS) analysis.
Hence, an alternative characterization approach that would be highly selective for ion
exchange chromatography-based charge variant analysis, which is compatible with on-
line MS detection, is needed in the biopharma industry. Against this backdrop, multiple
studies are being conducted to develop a simple straight on-line charge variant analysis
method. In this regard, we apply the current study, which aims to develop a charge variant
analytical method, based on volatile buffers with low ionic strength that can be used for
on-line MS detection of charge variants of mAbs. This would enable the detection on
“PTMs” using low ionic strength mobile phase compatible with MS. Hence, fruitful data
can be obtained with a single chromatography run without any test sample preparation,
eliminating the need for multiple steps of analysis, time-consuming process and multiple
sample preparation steps. Thus, Charge Variant Analysis-MS technique will allow the
characterization of charge-related PTMs on the intact protein stage. In this regard, this
study is about development of a method having combination of chromatography and
volatile mobile phase for mass spectrometry detection of mAbs being analyzed in native
form. The method is qualified considering pharmacopeia guidelines because the ultimate
aim is to transfer this method for Quality Control (QC) release testing of a monoclonal
antibody, which is critical for batch release and the regulatory point of view. Acidic and
basic variants have been separated with high resolution peak profile.

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