1. IntroductionIn preclinical and clinical trials of therapeutic monoclonal antibodies (mAbs), the

construction of a measurement method for the drug concentration in blood or disease

tissue is essential for its pharmacokinetic (PK) evaluation because overall PK pictures

showing the correlation between antibody drug efficacy and clinical indicators is still

insufficient. While ligand-binding assays have been used historically, alternative LC/MS/MS

platforms have emerged. One such platform is LC/MS/MS quantitation based on nano-

surface and molecular-orientation limited (nSMOL) proteolysis (Iwamoto N et al., Analyst,

2014). It is designed to keep antibody specificity with minimizing sample complexity and

simplify a sample preparation protocol, thus delivering robust quantitation with higher

sensitivity (Figure 1). Immunoglobulin (IgG) fractions containing therapeutic antibodies

were collected from human plasma with IgG collection resin (diameter of pore: 100 nm).

The IgGs immobilized on the inside of the pores are proteolyzed with trypsin-immobilized

nanoparticles on the surface (diameter: 200 nm). Because Fc is immobilized to the pore,

Fab oriented towards the solution is predominantly reacted with trypsin. Thus, nSMOL

proteolysis minimize the sample complexity by excluding both tryptic fragments other than

those around the Fc region and trypsin enzyme, allowing a selective quantification of target

mAb peptides with higher efficiency and selectivity in combination with MRM measurement.

The practical validation dataset of several mAbs acquired by nSMOL method meets the

validation guideline criteria issued by the U.S. FDA for Bioanalytical Method Validation of

LCMS bioanalysis for low MW drug compounds (Figure 3, Reference II – VII). This fact

demonstrates that the standard workflow for nSMOL improves robustness of analysis. It is

dramatically lowered the matrix-derived background noise and increased the reproducibility

of quantitative data with the standard sample preparation protocol which is significantly

simplified over the state-of-art technologies in the field of quantitative proteomics.

In this presentation, we will introduce the analytical workflow using the new nSMOL

reagent kit, and discuss for the application into the regulated LCMS bioanalysis of mAbs in

human plasma.

2. MethodsnSMOL™ Antibody BA Kit is a ready-to-use providing almost of all reagents and the protocol required for nSMOL

workflow. Five μL of plasma is treated according to standard protocol of nSMOL (Figure 2). The workflow requires just

three steps; 1) Capturing the IgGs from the plasma sample, 2) nSMOL proteolysis, and 3) quantitation using LC-MS/MS

in MRM mode. After the capturing steps, the nSMOL reaction solution or the enhanced nSMOL reaction solution were

added together with internal standards and FG beads Trypsin DART™. Following nSMOL proteolysis, the reaction

solution was centrifuged and the supernatant injected into a triple quadrupole mass spectrometer (LCMS-8060/8050,

Shimadzu Corporation, Japan), and monitored the signature peptides with the complementarity-determining region (CDR)

corresponding to each mAb.

3. Result The data shown below are the results of practical bioanalysis of therapeutic antibodies in human plasma using

nSMOL™ Antibody BA Kit (Figure 4, 5).

4. ConclusionsnSMOL proteolysis is our SHIMADZU original technology with a proven track

record for the analysis of a variety of antibodies. nSMOL™ Antibody BA Kit provides

a total strategy combining with a high-sensitivity triple quadrupole mass spectrometer

LCMS-8060 / LCMS-8050 which can surpass the conventional methods of

monoclonal antibody analysis, including ligand binding assay and standard

proteomics.

This original approach can maximize the use of LC/MS/MS in the development of

antibody drugs or clinical studies. LC/MS/MS can also accelerate the speed of

development for antibody pharmaceuticals just as it did in small molecule drug

development. In addition, PK of therapeutic antibodies by nSMOL has the potential of

becoming a standard approach for therapeutic drug monitoring (TDM).

Feature of nSMOL™ Antibody BA Kit

Excellent reproducibility achieved by "selectivity of nSMOL proteolysis" and

“effortless workflow".

Quick method development and lower initial cost.

Wide applicability regardless of varieties of therapeutic antibodies.

Multiplex bioanalysis using nSMOL™ Antibody BA Kit

5. ReferenceI. Iwamoto N et al, Analyst. 2014 139(3):576-80

II. Iwamoto N et al., Analytical Methods. 2015; 21:9177-9183.

III. Iwamoto N et al., Drug Metab. Pharmacokinet. 2016 31(1): 46-50

IV. Iwamoto N et al., J Chromatogr. B 2016 1023-24:9-16

V. Iwamoto N et al., Biol. Pharm. Bull. 2016 39(7): 1187-94

VI. Iwamoto N et al., Bioanalysis, 2016 8(10):1009-20

VII. Iwamoto N et al., Clin. Pharmacol. Biopharm. 2016, 5-4

VIII. Jenkins et al., AAPS Journal. 2015, Vol 17, No.1, DOI: 10.1208/s12248-014-9685-5

Disclaimer : nSMOL™ Antibody BA Kit and LCMS-8060/8050 is intended for

Research Use Only (RUO). Not for use in diagnostic procedures.

AcknowledgementThe applications of nSMOL™ Antibody BA Kit in this presentation are the results

of cooperative research between Shimadzu’s Technology Research Laboratory

and research agencies, primarily the National Cancer Center Japan. We are

deeply grateful to Drs. Akinobu Hamada and Hitoshi Nakagama for providing us

the useful discussions and kind supports.

Peptide MRM transition Objectives

P14R (IS)

512.1>292.3 (b3+)

512.1>389.3 (b4+)

512.1>660.4 (b6+)

Quantifier

Qualifier

Qualifier

Peptide MRM transition Objectives

IYPTNGYTR

542.8>404.7 (y7++)

542.8>808.4 (y7+)

542.8>610.3 (y5+)

Quantifier

Qualifier

Qualifier

Set conc.

[μg/ml]

Average

(N=15)

Accuracy

(%)

CV

(%)

2.93 2.58 88.1 8.2

200 211 106 5.6

Quantitation range in human plasma: 0.0610 to 250 ug/mLAveraged accuracy: 100.7 %

0 50 100 150 200 Conc.

0.0

1.0

2.0

3.0

Area ratio

Figure 5. Multiplex nSMOL bioanalysis using 10 antibody drugs in human plasma.

TR: Trastuzumab, BV: Bevacizumab, CTX: Cetuximab, RTX: Rituximab, NV: Nivolumab, IPL:

Ipilimumab, RAM: Ramucirumab, BRX: Brentuximab vedotin, IFX: Infliximab, ADL: Adalimumab

Calibration CurveRepresentative MRM Conditions

MRM Chromatogram

Precision and Accuracy (QC sample)

Figure 3. Representative result of LCMS Bioanalysis by nSMOL method -Trastuzumab analysis-

Figure 4. Assay verification between validated data and new dataset using Rituximab bioanalysis in plasma. Signature peptide:

ASGYTFTSYNMHWVK (CDR1, H-chain), MRM transition: m/z 598.05>817.45.

0 50 100 150 200 Conc.0

1

2

3

4

5

6

7

Area ratio

0 50 100 150 200 Conc.0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Area ratio

a) Validated calibration data b) Calibration data using new reagent kit

Quantitation range in human plasma: 0.244 to 250 ug/mLAveraged accuracy: 100.8 %

Quantitation range in human plasma: 0.244 to 250 ug/mLAveraged accuracy: 100.3 %

Enhancement of

antibody signature

peptide-derived

ion intensity by

FG Beads Trypsin

DARTTM

No significant change was observed between individual and 10-plex data in nSMOL quantitation

because nSMOL targets all IgG molecules with high-selectivity chemistry.

Figure 1. Fab Selective Proteolysis of monoclonal antibodies by nSMOL.

Regulated LC/MS/MS bioanalysis of therapeutic antibodies based on nano-surface and molecular-orientation limited (nSMOL)

proteolysis method using a new reagent kitChristopher Gilles4 Jerry Byrne II4, Gurmil Gendeh4, Toshiya Matsubara1, Noriko Iwamoto2, Nozomi Maeshima1, Deepti Bhandarkar3, Masateru Oguri1, Rashi Kochhar3, Takashi Shimada2, and Ichiro Hirano1

1 Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan. 2 Technology Research Laboratory, Shimadzu Corporation, Tokyo, Japan. 3 Shimadzu Analytical India Pvt. Ltd., Mumbai, India. 4. Shimadzu Scientific Instruments, Columbia, MD

Figure 2. Standard protocols of nSMOL workflow.