● The Novel System for Quantification of AggregatedAntibodies
Life Science Research Laboratory
No.2 R&D / Bioscience Division
Research and Development PlanningLife Science Research Laboratory

Kotohiro FURUKAWA
Toshinobu TOYOSHIMA
Takeshi NAGAHIRO
Koji KATAYAMA

qualitative analysis or improving quantitativity. For

1．Introduction

example, Shimadzu Corporation focused on improving

Antibody drugs have become a focus of attention

the quantitative performance of laser diffraction/

in recent years as therapeutic drugs for diseases

scattering by limiting the size range to 40 nm to 20

such as cancer, and their global market has been

µm, and then launched in 2013 the Aggregates Sizer,

expanding rapidly 1). Antibody drugs have complex

a system that enables evaluation of aggregates inprotein structures of far larger molecules in contrast

drugs. Malvern Panalytical Ltd. launched in 2018 theto small molecule drugs such as aspirin, which have

Zetasizer Ultra, a system that enables determination ofbeen commonly used, and antibodies in the drugs are

particle concentration using the multi-angle dynamic

known to be partially denatured by various stresses

light scattering method requiring multiple detectors toincluding shaking, agitation, and heat, and then to form

receive scattering light from different angles, instead

2-4). Concerns have been raised that suchof the dynamic light scattering method. However, none

aggregated antibodies may reduce therapeutic effects orof these methods have achieved accurate analysis

aggregates

5, 6)

, and

of polydisperse samples. Moreover, since, with any

the necessity of quantification of aggregated antibodiesof these methods, the particle size distribution ishas been increasingly recognized worldwide as a key

determined based on the scattering light from a sample

factor of quality management, as can be seen from thecontaining particles, quantitative performance is

description in the Food and Drug Administration (FDA)

significantly affected by the refractive index and density

cause adverse effects such as allergic reactions

7)of particles; however, an accurate determination of theseguidelines .
However, aggregated antibodies, which can range

properties of aggregated antibodies is quite difficult.

widely from a few tens of nm to several tens of µm, need

From these perspectives, quantification of aggregatedto be measured using an appropriate method according

antibodies, especially ranging from 0.1 to 2 µm, is stillto their particle size: size-exclusion chromatography

challenging.

for particles of 0.1 µm or less, and microscopy-based

We therefore started to develop a system that enables

methods such as micro flow imaging for particles of 2

quantification of particles ranging from 0.1 to 2 µm,µm or more 8). For particles ranging from 0.1 to 2 µm,

a less established area, by first separating particles

available methods are limited to qualitative analysis

according to their size and then detecting the separatedusing the dynamic light scattering method and theparticles individually. Here, we report the overview oflaser diffraction scattering method, and even the United

the system we developed and the verification results

States Pharmacopeia (USP)

9)

currently recommends

analysis using multiple methods and states that a newof quantification of aggregated antibodies in antibody
drugs.

technology is expected to be developed, indicating

We have developed the novel system with thethat no analysis techniques have been established foraim of establishing a method with high reliability

particles in such a size range.

for quantification of aggregated antibodies, and withIn recent years, analysis techniques have been

the goal of contributing to the society by providing

developed by combining quantitative analysis withthe system as a highly accurate quality management
81

TOSOH Research & Technology Review Vol.65（2021）

downstream while being pushed and aligned to one

technology for antibody drugs.

sidewall in the pinched segment by the buffer solution.
At this time, the centers of the particles were located

2．Technical Overview

on different flow streamlines with a slightly different

In the process of the development of our novel system

distance from the sidewall according to the particle

for quantification of aggregated antibodies (hereinaftersize. Such distances were amplified in the subsequent

referred to as“this system”), we first established thebroadened segment (relatively wide channel), and theparticle separation technology and particle detection

particles were then separated.

technology separately, developed the new particle

In general, the separation performance in the PFF

separation-detection technology by integrating themethod is significantly affected by the ratio of theestablished technologies, and then evaluated ourwidths of the pinched segment and broadened segment.

technology for this system by comparing it with a

Moreover, our research suggested that since ourcompetitive method. In the subsequent sections, wedevelopment only covered particles ranging from 0.1 toreport details of our technology and its development

2 µm subject to Brownian motion, the important points

process.

were to set a flow rate condition at which particles
could flow promptly through the pinched segment［1］Particle Separation Technology

and to achieve a design of the microchannel with

［1-1］Verification of Particle Separation Performance

seamless streamlines between the pinched segment andPinched-Flow Fractionation

10, 11)(hereinafter referred

to as“the PFF method”), which hydrodynamicallyseparates particles according to their size, was adopted
as a particle separation technology (Fig.1). In the PFF

InletsSample solution

Pinchedsegment

Broadened segment

method, a microdevice, on which microchannels witha height and width of micrometers were formed, wasused for separation. First, sample solution containing
particles and buffer solution not containing particles
were introduced from two inlets into the pinchedsegment (relatively narrow channel). When the flowrate of the buffer solution was set to be enough larger

Buffer solution

Fig.１

than that of the sample solution, the particles flowed(a)Pinchedsegment

Broadened
segment

0.2 µmPrinciple of Pinched-Flow Fractionation

0.5 µmObservation
area

50 µm
0.2 µm(b)

0.5 µmObservation
area

Fig.２

82

Separation of particles by Pinched-Flow Fractionation

50 µm

東ソー研究・技術報告

broadened segment. The overview of particle separation

第 65 巻（2021）［1-3］Particle Separation Experimentby the PFF method is shown in Fig.2. In the PFF

As fluorescent polystyrene beads, Fluorescentmethod, the microchannel is generally broadened 90°

Polymer Particles (Green) (Bangs Laboratories,

to both sides at the junction of the pinched segment andInc.; absorbance maximum, 480 nm; fluorescence10); however, with this structure, amaximum, 520 nm) was adopted for particles of 0.2 µm,

complete separation of fluorescent particles of 0.2 µm

and Fluoresbrite BB (Polysciences, Inc; absorbanceand 0.5 µm (Polysciences Inc.) in the sample solution

maximum, 360 nm; fluorescence maximum, 407 nm)

could not be observed under a fluorescence microscope.

for particles of 0.5 µm. As suspension for the particles,

On the other hand, a complete separation of fluorescentphosphate buffer solution containing 0.05% (v/v)

particles of 0.2 µm and 0.5 µm has been achieved byTween 20 was adopted. Before experiment, foreign

establishing the structure of the microdevice, whereparticulate matters were removed from the buffer

the channel is designed not to be broadened toward

solution using a syringe filter with a pore size of 0.45

the sidewall on the sample solution inlet side, or theµm (Merck Millipore Ltd.), and fluorescent particles ofsidewall to which particles are aligned.

0.2 µm and 0.5 µm were diluted with the buffer solution,broadened segment

The separation experiment described above wasrespectively, to 3.3 ng/mL and 7.5 ng/mL. The flow rate

conducted through the following processes.

was adjusted with a syringe pump (KD Scientific Inc.)［1-2］Fabrication of Microdevice

while introducing sample solution and buffer solution

A microdevice used for verification of particle

into the microchannel. Fluorescence images wereseparation performance was fabricated by making aobtained separately at the corresponding wavelengths,

mold and then pouring the resin into the mold andusing an inverted microscope IX71 (Olympus

curing it. First, the procedure of fabricating a mold is

Corporation) by irradiating the mercury lamp excitation

noted; Photoresist SU-8 3005 (Microchem Corp.) waslight to the observation area and then shooting video

dropped on a 4-inch bare silicon wafer (Philtech Inc.),of the target area for 2 seconds using a digital CMOS

and a photoresist thin film was formed using a spin

camera ORCA-FLASH (Hamamatsu Photonics K.K.).

coater (Mikasa Co., Ltd.). At that time, cyclopentanone(Tokyo Chemical Industry Co., Ltd.) was added to［2］Particle Detection Technology

dilute SU-8 3005 according to the desired thickness［2-1］Overview of Particle Detectionof the thin film to be formed. Subsequently, a desired

The Coulter method 12), which electrically measures

microchannel pattern on a chrome mask was transferredindividual particle, was adopted as particle detection

to the photoresist thin film using a mask aligner (Ushio

technology (Fig.3). The Coulter method is a technologyInc.) and was then developed using SU-8 Developerthat enables electrical measurement of particles on a(Microchem Corp.) to fabricate a desired mold.

device in which a pair of electrodes is placed across a

Next, uncured SYLGARD SILICONE ELASTOMER

fine hole called an aperture. In the method, the slight

KIT (Dow Corning Toray Co., Ltd.) was poured into

decrease in the electrical current when particles pass

the fabricated mold and was then heated at 80ºC forthrough the aperture is used. The decrease range of thetwo hours to fabricate Polydimethylsiloxane (PDMS)

electrical current value (when particles pass throughwith the microchannel pattern transferred. The cured

the aperture) and the decreased signal pulse count

PDMS was unmolded and was shaped to a desired size

are dependent on, respectively, each particle size andby cutting it with a knife, and then the inlets and outlets

particles concentration.

were drilled using a puncher. After surface treatment

With the Coulter method, particles can be measured

was applied to the unmolded PDMS and a glass slide

individually and accurately even if their inclusion rate

(Matsunami Glass Ind., Ltd.) using an oxygen plasma

is low; however, there is an issue that the measurable

generator (Meiwafosis Co., Ltd.), the treated PDMS

range of particle size (dynamic range) is relatively

and glass slide were bonded together to fabricate asmall. Specifically, in this method, the dynamic range of

microdevice.

particles is said to be 2% to 60% of the aperture diameter83

TOSOH Research & Technology Review Vol.65（2021）

Power source

Pump suction

Ａ
AmmeterCurrent+

Electrodes

Particlesize-dependent

Aperture

Time

Fig.３

Principle of Coulter methodin general. Moreover, particles larger than the apertureµm (width) × 0.4 µm (height) × 10 µm (length) eachdiameter needs to be eliminated before measurement

for small particles, 2 µm (width) × 0.8 µm (height)

using a filter with a pore size smaller than the aperture× 10 µm (length) each for medium particles, and 3.5

diameter. This is because the aperture may beµm (width) × 4.5 µm (height) × 20 µm (length) eachclogged by such particles, if any, and then subsequent

for large particles. A platinum electrode was inserted

measurements are prevented.

into each outlet at the downstream of the aperture, andBased on the issue above, we subsequently

DC power supply and a current amplifiers (current

established the Coulter method in a microdevice into voltage converter; NF Corporation) were placed

which multiple apertures with the size of the particles

between the two platinum electrodes to form a circuit.

to be separated are placed, to develop a system that

The output of the current amplifiers (current to voltage

can individually detect particles ranging from 0.1 to 2

converter) was connected to an A/D convertor, to whichµm, a less established area. In this section, we reporta PC was connected to obtain electrical signal using a

the process of establishing the Coulter method in the

self-made software set up with LabVIEW.

fabricated microdevice.
［2-2］Development of Particle Detection Channel
Three sizes of apertures were used to detect
particles ranging from 0.1 to 2 µm: for detection of［3］Particle Separation-Detection Technology［3-1］Integration of Particle Separation Technology
and Particle Detection Technology

small particles, medium particles, and large particles.

A schematic overview of our developed microdevice

A schematic overview of the Coulter method is shown

where particle separation technology and particle

in Fig.4. The adopted structure was as follows: thedetection technology are integrated is shown in Fig.5.

channel in which the separated particles flow (particle

With this system, particles ranging from 0.1 to 2 µm

collection channel) branched off downstream into two

can be evaluated individually by first separating thechannels with a fine structure (aperture) at each branch

particles according to their size using the PFF method

point, each of which communicated with a downstream

and then introducing the separated particles into theoutlet. In this structure, each particle collection channel

three particle collection channels with the apertures of

had two apertures. The size of the two apertures was 1

the corresponding particle size. The detection resultsA

To outlet
Aperture

Separated particles

Fig.４

84

Aperture

A schematic overview of the microfluidic Coulter method

東ソー研究・技術報告第 65 巻（2021）

of a sample containing mixed particles of 0.1, 0.2, 0.5,

were diluted with phosphate buffer solution containing

1, 2 µm are shown in Fig.6. Signal pulses of particles of

0.05% (v/v) Tween 20 so that the final concentration

0.1 and 0.2 µm were detected at the apertures for small

could be 5 µg/mL.

particles as shown in Fig.6 (a), signal pulses of particles［3-2］Comparative Evaluation between Our Methodof 0.2 and 0.5 µm at the apertures for medium particles

and the Laser Diffraction/Scattering Method

as shown in Fig.6 (b), signal pulses of particles of 0.5, 1

A comparative evaluation was conducted by

and 2 µm at the apertures for large particles as shown

comparing our developed method shown in Fig.7

in Fig.6 (c) and (d). These detection results of particles

with another technology, Aggregates Sizer (Shimadzu

ranging from 0.1 to 2 µm obtained at the three sizes ofCorporation), using a sample containing mixed particles

apertures are integrated into a histogram (Fig.7). As canof 0.1, 0.2, 0.5, 1, 2 µm. As can be seen from thebe seen from the five peaks formed in the histogram,histogram in Fig.8, the peaks of 0.2 µm and 2 µm were

the particles were separated according to their size. As

lost and a peak size of 0.1 µm was increased, indicatinga sample containing mixed particles, Duke Standards

that an accurate measurement of samples containing(Thermo Fisher Scientific K.K.) were adopted, which

mixed particles was difficult.

Sample solution

Aperture A
Aperture B

Aperture C

Buffer solution

Fig.５

（b）

0.1

－0.1
－0.2

0.1 µm

－0.3

2.5

0.2 µm2
1.5
10.2 µm

－0.4
－0.5

3.5
3

0

Voltage (V)Voltage (V)（a）

A schematic overview of the developed microdevice

0.5 µm

0.5

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0

0.1

0

0.02

0.04

0.06

0.08

（c）

（d）

0.22

Voltage (V)Voltage (V)

0.12

0.14

0.16

0.18

0.2

0.97

0.98

0.99

1

0.4
0.2

0.2
0.18

0.5 µm

0.16
0.14

0
－0.2
－0.4
－0.6

0.12
0.1

0.1

Time (sec)Time (sec)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

2.0 µm－0.8

1.0 µm
0.16

0.18

0.2

－1
0.9

0.91

0.92

0.93

Fig.６

0.94

0.95

0.96

Time (sec)Time (sec)Electrical signal detected from each aperture

85

7

7

6

6

5

5

Concentration(µg/mL)

Concentration(µg/mL)TOSOH Research & Technology Review Vol.65（2021）

4
3
2

4
3
2

1
0

1

0.05

0.1

0.2

0.5

1

2

0

5

0.05

0.1

2
1

0.05 0.1 0.2

0.5

1

2

5

(c)

3

Concentration(µg/mL)

Concentration(µg/mL)

Concentration(µg/mL)

(b)

3

0

2
1
0

0.05 0.1 0.2

Particle size (µm)

Fig.９

1

2

5

Resolution of PS standard particles (0.1, 0.2, 0.5, 1.0,
2.0 µm) by Laser Diffraction

Fig.８

Resolution of PS standard particles (0.1, 0.2, 0.5, 1.0,
2.0 µm) by developed method

(a)

0.5

Diameter of particles (µm)

Diameter of particles (µm)

Fig.７

0.2

0.5

1

2

5

3
2
1
0

0.05 0.1 0.2

Particle size (µm)

0.5

1

2

5

Particle size (µm)

Time course of protein aggregates (a) 0 min (b) 10 min (c) 120 min

3．Detection of Aggregated Antibodies in
Antibody Drugs

microdevice in which the particle separation technology
using the PFF method and the particle detection
technology using Coulter method are integrated. WithThe following three types of samples were preparedthis system, aggregated antibodies in commerciallyusing a commercially available antibody drug:(a) aavailable antibody drugs can be detected and theircontrol sample (the drug to which no treatment wasaggregation process over time can be evaluated,

applied), (b) a sample of the drug that was heated atproviding the possibility of using this system for

60℃ for 30 minutes and then left at room temperature

quality management of antibody drugs. This system isfor 10 minutes, (c) a sample of the drug that was heatedexpected to contribute not only to improving the quality

at 60℃ for 30 minutes and then left at room temperatureof antibody drugs because it enables quantification offor 120 minutes. Each of three samples was measured

a small amount of aggregates in antibody drugs, butusing our developed technology. As can be seen

also to improving the performance of antibodies usedfrom the histograms in Fig.9, while no particles were

for antibody drugs because it enables verification of thedetected from the control sample, the peak of particles

stability of antibodies in antibody drugs by applying athat was suspected to be aggregates were detected fromstress (e.g. thermal degradation test).

the heat-treated samples, and peak top position was
shifted over time to the larger side of the particle size.

4．Conclusion

5．Acknowledgments
We express our sincere thanks to Prof. Minoru Seki,
Graduate School of Engineering, Chiba University for

As described above, we have achieved the novel

his technical guidance and advice for our development,

system that enables quantification of particles ranging

and to his laboratory staffs advising us and supporting

0.1 to 2 µm, a less established area, by developing theour development.

86東ソー研究・技術報告第 65 巻（2021）

We also thank Mr. Kazuki Iijima, Asahi Kasei
Medical Co., Ltd. for his technical assistance with ourdevelopment.

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