Rapid Analysis of Intact Antibody-Drug Conjugates (ADCs) By UPLC Q-TOF...

Rapid Analysis of Intact Antibody-Drug Conjugates (ADCs) By UPLC Q-TOF MS
Wei Jia1*, ChuanFei Yu2*, Kai Gao2
1 Waters corporation, Shang Hai, China; 2 China National institutes for Food and Drug Control, Bei Jing, China
INTRODUCTION
RESULTS
It is generally known that, antibody-drug conjugates (ADCs) are very important
tools for targeted therapy. The structure characterization of ADCs is very important
for its quality control, but there are many challenges due to its structure
complexity. In this work, using QTOF MS with a 12 min long LC method, lots of
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structure information of ADCs can be successfully acquired. Comparing to
traditional UV method for drug-antibody ratio (DAR) analysis, and fluorescence
The average mass error was 3.2Da (standard deviation 2.35Da) for all 36
peaks (each Dn group contains 4 different MW peaks due to glycosylation),
considering the sample’s huge complexity it is an astonishing result.
method for antibody glycan analysis, the LC QTOF MS method can give the DAR
value and glycan form information simultaneously in a short time, which is very
Because there was no deglycosylation procedure, the glycan information,
types of glycan and their ratio, can be found in MS data. It was found that, the
four main kinds of glycan were G0F&G0F, G0F&G1F, G1F&G1F (or G0F&G2F),
G1F&G2F. Their ratios were 19.14%, 36.58%, 33.31%, 10.97% respectively
(average value from group D0 to D8). The average mass difference of different
Glycosylation peaks in each group was 161.44Da (standard deviation 3.72Da),
which number is quite close to the theoretical value to value of Galactose. The
good precision of the data is very helpful for judging the glycosylation patterns.
important for effective acquiring reliable quality control data of ADCs.
METHODS
Instrument:
Deconvolution by BiopharmaLynx
ACQUITY UPLC®, Xevo® G2 QTof
UPLC Methods:
MassPREPTM Micro desalting column (Waters)
H2O with 0.1% FA (phase A),
In this work, using Xevo G2 QTof MS with a 12 min long LC method, the MS
raw data of Trastuzumab linked DM1 was successfully acquired. Then, the raw data
was treated by BiopharmaLynx to get the intact ADC molecular weight values.
Primarily, drug-antibody ratio (DAR) and glycan information were shown. There
were 9 groups of peak for Trastuzumab due to different number of conjugates. The
measured DAR value 3.6 by QTOF is quite similar to 3.5, which was measured by
traditional UV method.
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Comparing to traditional UV method for DAR analysis, and fluorescence
method for antibody glycan analysis, this robust LC QTOF MS method can give the
DAR value and glycan form information simultaneously in a short time, which is
very important for effective acquiring reliable quality control data of ADCs.
ACN with 0.1% FA (phase B)
0-0.5 min, Phase B 5%, 0.5 mL/min;
0.5-12min, Phase B 5-90%, 0.2 mL/min
MS Methods:
Full MS range 500m/z-3500m/z
Software:
BioPharmaLynxTM 1.3
CONCLUSION
Basing on the good performance of the benchtop MS system Xevo G2 QTof,
the reliable drug-antibody ratio and glycan information can be obtained in a very
shot time for antibody-drug conjugates analysis.