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ArticleOpalescence Arising from Network Assembly in Antibody Solution
Yoshitaka Nakauchi, Suguru Nishinami, Yusuke Murakami, Toshihiko Ogura, Hideaki Kano,and Kentaro Shiraki*
Cite This: Mol. Pharmaceutics 2022, 19, 1160−1167

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ABSTRACT: Opalescence of therapeutic antibody solutions is
one of the concerns in drug formulation. However, the mechanistic
insights into the opalescence of antibody solutions remain unclear.
Here, we investigated the assembly states of antibody molecules asa function of antibody concentration. The solutions of bovine
gamma globulin and human immunoglobulin G at around 100
mg/mL showed the formation of submicron-scale network
assemblies. The network assembly resulted in the appearance of
opalescence with a transparent blue color without the precipitatesof antibodies. Furthermore, the addition of trehalose and arginine,previously known to act as protein stabilizers and protein
aggregation suppressors, was able to suppress the opalescencearising from the network assembly. These results will provide animportant information for evaluating and improving protein formulations.
KEYWORDS: opalescence, network assembly, liquid−liquid phase separation, antibody, additive■

INTRODUCTION
The property of opalescence has also been reported inproteins, where it refers to the cloudiness or turbidity of theprotein solution.1 In general, opalescence occurs via two
optical mechanisms, namely Rayleigh scattering and Mie
scattering.2 Rayleigh scattering is the elastic scattering of lightby small particles, such as protein monomers and oligomers,
having a diameter much smaller than the wavelength ofincident light. In contrast, Mie scattering refers to the elastic
scattering of light by large particles, like protein amorphousaggregates, with a diameter similar to or larger than the
wavelength of the incident light. According to European
Pharmacopoeia, opalescence is quantiﬁed in terms of anephelometric turbidity unit, which denotes the amount of
scattered light detected at a scattering angle of 90°.3
Therapeutic antibody formulations often display an
opalescence appearance, despite their great target speciﬁcity
and eﬃciency.1,4 Clinically, subcutaneous (SC) route ispreferred for the administration of biologics owing to low
cost, ease of application, and lower risk of complications.
However, SC administration imposes a limitation of volume
and the dosage is several mg kg−1, requiring the antibodies tobe concentrated,5,6 which strongly promotes opalescence andviscosity of antibody formulations.7,8 Recently, the development of syringe and the improvement of formulation form
have been done to overcome these challenges to facilitate thedelivery of large amounts of drug via SC route.9 The
opalescence of antibody formulations is considered to possibly© 2022 American Chemical Society

lead to the loss of structural stability and biological function.
Besides this, it hinders the development of a placebo
formulation for clinical studies.1,10 Several studies havereported that antibody concentration and solution conditions,
including solution pH, ionic strength, and temperature,signiﬁcantly aﬀect the opalescence of antibody formulations.11,12 The molecular mechanism responsible for thisopalescence is mainly associated with the ability of antibodies
to form reversible liquid droplets via liquid−liquid phaseseparation (LLPS). This characteristic of antibodies iscontributed to their large and ﬂexible structure and thepresence of noncovalent protein−protein interactions, including electrostatic4,13,14 and dipole interactions.15,16 Various
studies have reported the existence of oligomers17,18 and theprecursors of droplets and oligomers12,19−21 in antibody
solution. However, the mechanism responsible for the
opalescence of antibody solutions without precipitation
remains to be established.
Additives are utilized as an eﬀective strategy to control andunderstand the interactions in protein solutions. Various types
Received: December 3, 2021
Revised: February 16, 2022
Accepted: March 1, 2022
Published: March 11, 2022

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of small additives, particularly sugars,22,23 osmolytes,24,25 andkosmotropic salts,26,27 have been reported to enhance thestability and solubility of proteins. Besides this, additives, likeurea,28 guanidine hydrochloride,29 and chaotropic salts,30 are
known to suppress protein aggregation. Among these, argininehydrochloride (ArgHCl) is the most versatile additive that has
been widely used in protein solution for suppressing protein
aggregation,31,32 enzyme inactivation,33 and nonspeciﬁc surface
adsorption.34,35 In particular, ArgHCl is known to improve theviscosity,36 opalescence,37 and LLPS38,39 of highly concentrated antibody solutions. Recently, allantoin, hydantoin, andcaﬀeine, having a diﬀerent chemical backbone from arginine,
have also been proposed as possible substitutes of ArgHCl for
certain applications.40−43
Here, we reported the nanoscale network assembly ofantibodies at high concentrations with the aim to propose a
uniﬁed mechanism for the opalescence of antibody formulation
depending on the antibody concentrations. We investigated
two polyclonal antibodies, bovine gamma globulin (BGG) andhuman immunoglobulin G (HGG), which also contain a large
number of antibody molecules with diﬀerent subclasses,
speciﬁcity, and diﬀerent biophysical properties, typicallyisoelectric points. Spectroscopic techniques, namely coherent
anti-Stokes Raman scattering (CARS) and scanning electronassisted dielectric microscopy (SE-ADM), were utilized toinvestigate the labile structure of antibody solutions at highconcentrations. Further, we investigated the eﬀects of trehaloseand ArgHCl on the opalescence of antibody solution to
elucidate their utility as additives and establish the underlying
mechanisms.

Article1000 mM additives were transferred to a quartz cell with a 1
mm path length, and measurements were performed at 25 °C.
Microscopic Imaging. The morphology of the microscaleassemblies of antibodies was observed using bright-ﬁeld or
ﬂuorescence microscopy. To prepare stained BGG samples,the primary amine groups of lysine residues present on thesurface of BGG were chemically modiﬁed using FITC. BGG
solutions at 9−230 mg/mL concentrations were prepared bymixing FITC-labeled BGG with unlabeled BGG in 10 mM
MOPS buﬀer (pH 7.0) at a ratio of less than 1% labeled BGG.
The samples were transferred to a 96-well microplate (productnumber: 3474, Corning Inc., NY, USA) and observed using aphase contrast inverted ﬂuorescence microscope (BZ-X710,
Keyence Inc., Osaka, Japan). The FITC-labeled BGG
molecules were detected using excitation and emission maxima
of 470 and 525 nm, respectively. Notably, no signiﬁcantdiﬀerences were observed between the bright-ﬁeld microscopic
images of FITC-labeled and unlabeled BGG molecules.
CARS Imaging. Label-free antibodies forming microscaleassemblies were observed using a multiplex CARS spectroscopic imaging system.45 The main laser source was a custommade, dual-ﬁber output synchronized laser source (OPERA
HP, Leukos, Limoges, France). In the laser housing, the master
laser output, with a wavelength of 1064 nm, temporal durationof 50 ps, and repetition rate of 1 MHz, was divided into two.
One output was introduced into the large-mode area photonic
crystal ﬁber and used as the pump beam, ω1, which wascollimated by a collimator lens (F810APC-1064, Thorlabs,
Newton, NJ, USA). The other output was injected into a
photonic crystal ﬁber to obtain ultra-broad-band supercontinuum radiation ranging from visible to near-infrared(NIR). The NIR spectral components of the SC were selectedusing an optical ﬁlter (IR80, Kenko-optics, Tokyo, Japan) andwere used as the Stokes beam (ω2). The SC was collimated by
an oﬀ-axis parabolic mirror (RC04APC-P01, Thorlabs) to
avoid chromatic dispersion. The two beams were superimposed by a notch ﬁlter (NF03-532/1064E-25, Semrock,Rochester, NY, USA) and guided into a modiﬁed inverted
microscope (ECLIPSE Ti with modiﬁcation, Nikon, Tokyo,
Japan) in collinear geometry. Two laser pulses were tightly
focused on the sample through a microscope objective (CFI
Plan Apo 60× NA 1.27, water-immersion, Nikon). The
samples were placed on a piezoelectric stage (Nano-LP200,
Mad City Labs, Madison, WI, USA) for three-dimensional (x,y, and z) position selection. The full scanning range of the xyzpiezo stage was 200 × 200 × 200 μm3. The second-harmonic
generation (SHG), third-harmonic generation (THG), andultra-multiplex CARS signals were collected using a second
objective lens (Plan S Fluor 40× NA 0.6, Nikon) and spectrally
separated by a dichroic mirror. The CARS signal transmitted
through the dichroic mirror was detected using a spectrometer(LS785, Princeton Instruments, Trenton, NJ, USA) equippedwith a CCD camera (BLAZE 100HR, Princeton Instruments).
The spectral coverage and spectral resolution of the CARS
signal were ∼3500 and 8 cm−1, respectively. The SHG and
THG signals reﬂected by the dichroic mirror were detectedusing a spectrometer (SpectraPro300i, Princeton Instruments)
equipped with a CCD camera (PIXIS 100 B, Princeton
Instruments). The exposure time at each spatial position was
50 ms for signal detection. The image size in pixels andmicrons were 101 × 101 pixels and 50 × 50 μm2, respectively,
and the step size was 0.5 μm.

■

EXPERIMENTAL SECTION
Materials. BGG (lot number: SLBZ2175; Sigma-Aldrich
Co., St. Louis, MO, USA) at 99% purity and HGG (lotnumber: 180820-0256; Equitech-Bio, Inc., Kerrville, TX, USA)at 97% purity were obtained. 3-(N-morpholino)propanesulfonic acid (MOPS), sodium dihydrogen phosphate,sodium chloride (NaCl), L-arginine hydrochloride (ArgHCl),ﬂuorescein-4-isothiocyanate (FITC), and trehalose dihydrate
were obtained from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). All the chemicals were of reagent grade.
Preparation of Antibody Stock Solutions. The
lyophilized powders of proteins BGG and HGG were dissolvedin pure water to prepare antibody stock solutions (>230 mg/mL). Then, these proteins were dialyzed against pure water or
buﬀer solutions overnight at 4 °C to remove the small
impurities. The concentration of the antibodies was measured
at an absorbance of 280 nm against the appropriate blank using
an ultraviolet−visible spectrometer (ND-1000; NanoDrop
Technologies, Inc., Wilmington, DE, USA). The extinction
coeﬃcients (E1%
280nm) of BGG and HGG were 13.8 and 13.7,
respectively.44
Turbidity Measurement. To evaluate the opalescenceappearance of the protein solutions, two turbidity measurements were conducted as follows. Each sample solution wasirradiated with laser light, and the transmitted light at 550 nm
was detected at an angle of 180° using an ultraviolet−visible
spectrophotometer (V630; Japan Spectroscopic Co., Ltd.,Tokyo, Japan), while the scattered light at 510 nm was
detected at a scattering angle of 90° using a spectroﬂuorometer(FP-6500; Japan Spectroscopic Co., Ltd., Tokyo, Japan). The
sample solutions containing 0−230 mg/mL antibody and 0−1161

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ArticleFigure 1. Opalescence arising from the microscale assembly of antibodies. (a) Visual appearance of BGG solutions at diﬀerent concentrations. The
white horizontal line is a white paper placed on the other side of the test tubes. (b) Quantitative measurement of light transmittance for 0−230 mg/
mL BGG solutions in the absence or presence of NaCl. The values were obtained in a single experiment. (c) Bright-ﬁeld (upper) and ﬂuorescence(lower) micrographs for FITC-labeled BGG solutions. (d) CARS at 2939 cm−1and (e) THG images for unlabeled BGG solution at 230 mg/mL.
All the samples were prepared in 10 mM MOPS buﬀer (pH 7.0). Scale bars represent 50 μm.

■

SE-ADM Imaging. A structure consisting of nanoscale
networks of antibodies in solutions was observed using a highresolution SE-ADM imaging system.46−48 This system involved
a thermal ﬁeld emission scanning electron microscope(SU5000, Hitachi High-Tech Co., Japan) at room temperature. A 1.0 μL aliquot of a sample solution containing 9−230
mg/mL ﬂuorescently stained BGG and 10 mM MOPS buﬀer(pH 7.0) was transferred to a sample holder mounted onto theSEM stage. The detector terminal was connected to apreampliﬁer under the holder.46 The electrical signal fromthe preampliﬁer was fed into an analog-to-digital converter
after low-pass ﬁltering, as previously described.47,48 The SEADM images of 1280 × 1020 pixels were captured at amagniﬁcation of 20,000−50,000 at a scanning time of 40 s,
working distance of 7 mm, electron beam acceleration voltageof 3.5 kV, and current of 10 pA.
The SE-ADM signals from SU5000 were transferred to apersonal computer (Intel Core i7, 3.8 GHz, Windows 10). The
high-resolution SE-ADM images from the low-pass ﬁlter andthe scanning signals were then processed using MATLAB
R2020a software with an image-processing toolbox (MathWorks Inc., Natick, MA).46−48 The SE-ADM images wereﬁltered using a two-dimensional (2D) Gaussian ﬁlter (GF)with a kernel size of 7 × 7 pixels and a radius of 1.2σ, where σis the half-width of the Gaussian function. Backgroundsubtraction was performed by subtracting the SE-ADM images
from the ﬁltered images using a broad GF (400 × 400 pixels,200σ).

RESULTS
Opalescence of Antibody Solutions Depending on
the Concentration. The opalescence behavior of BGG
solutions was investigated at room temperature and physiological pH. The gamma globulin is a mixture of total IgGs insera, and most of their isoelectric points are in the pH range of
7−9, with some having acidic pI. In a previous study, it has
been suggested that antibody solutions show opalescence wellat pH near the pI of proteins.12 Microscopic images for thevisual appearance of BGG solutions at diﬀerent concentrations
are shown in Figure 1a. The test tube ﬁlled with buﬀer isclearly visible in these images. An increase in BGG
concentration induced changes in the visual appearance of
BGG solutions. BGG solutions prepared at 46 and 230 mg/mL
concentrations without NaCl appeared cloudy. In comparison
to this, BGG solution at 115 mg/mL concentration was alsoslightly turbid in comparison to buﬀer solution; however, thesample solution was clear in this case. The overnightincubation of the opalescent solutions of BGG at 46, 115,
and 230 mg/mL at room temperature resulted in the
precipitation of only the 46 mg/mL solution. Following this,nonprecipitating opalescent solutions of 115 and 230 mg/mL
concentrations were centrifuged at 18,800g for 20 min, and aliquid−liquid phase separation (LLPS) was observed only for
230 mg/mL solution, with more protein-rich phase (lower
phase) than protein-poor phase (upper phase). As shown in
Figure 1b, the quantity of transmitted light decreased with anincrease in the concentration of BGG from 0 to 100 mg/mL
without NaCl. In contrast, the transmittance of light increased
at BGG concentrations of 115−145 mg/mL. Addition of NaCl
suppressed the turbidities of BGG solutions.
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ArticleFigure 2. Opalescence arising from the nanoscale network assembly of antibodies. (a) Visual appearance of BGG solutions at diﬀerent
concentrations. Each sample was irradiated with laser light at 532 nm from a certain position (indicated by black triangle arrow). (b) Quantitative
measurement of the light scattering intensity for 0−230 mg/mL BGG solutions, in the absence or presence of NaCl, monitored at a scattering of
90°and wavelength at 510 nm. The values were obtained in a single experiment. (c) SE-ADM image for 115 mg/mL BGG solutions. The black areashows the presence of BGG, and the pixel intensity corresponds to 0. Scale bar represents 1 μm. (d) Mesh size of the network assembly observed in
115 mg/mL BGG solution. The SE-ADM image is an expansion of the area surrounded by the white dotted line shown in (c). Pixel intensity shows
the density of BGG molecules present on the white dotted line in the enlarged image. All the samples were prepared in 10 mM MOPS buﬀer (pH7.0).

To identify the reason for the cloudy appearance of BGG
solutions, we investigated the formation of microscaleassemblies with increasing BGG concentrations. Figure 1cshows the bright-ﬁeld and ﬂuorescence images of BGG
solutions containing FITC-labeled BGG. BGG solutions at 9
and 46 mg/mL showed the presence of droplet-like assemblythat was found to be spherical at the BGG concentration of 9
mg/mL, whereas the droplets at 46 mg/mL concentration
were spherical and multiphased. In the case of 115 mg/mL
BGG solution, no microscale assemblies were observed, while
the BGG solution at 230 mg/mL BGG concentration showed
the presence of protein-poor droplets. A multiplex CARS
microscope was also used to observe the BGG droplets, andlabel-free images were acquired for the assembly of
biomolecules. Figure 1d shows the CARS images for unlabeled
BGG solution at 230 mg/mL. In the CARS image, the peakintensities for BGG (at 2939 cm−1 for CH3 symmetricstretching vibration) derived outside the droplets were foundto be much larger than the intensities recorded inside. It
should be noted that the inside of the droplet has a noticeable
CH2 stretching vibration (at 2850 cm−1) compared to theoutside. Perhaps, BGG solution contains aliphatic moleculesthat cannot be removed by dialysis. In general, the THG
process gives a signal when there is a diﬀerence in therefractive index. Thus, the boundary, interface, and assemblies
with submicrometer scale should be THG-active. As shown in
Figure 1e, THG activity was observed in the case of proteinpoor BGG droplets. In contrast, the SHG process that gives

signals in the case of noncentrosymmetric structures was notobserved in the present study, indicating the absence ofnoncentrosymmetric structures such as the amyloid49 or
crystal-like ordered structures in the BGG solutions (datanot shown).
Nanoscale Network Assembly of Highly Concentrated Antibodies. We further investigated the submicronscale assembly states of the antibody solutions using SE-ADM.
Figure 2a shows the irradiation of 4 and 230 mg/mL BGG
solutions with green laser, resulting in the spreading of lightthroughout the solution. Interestingly, a strong light scattering
was observed only in the path of laser beam in the case of 115
mg/mL BGG solution. Figure 2b shows the quantitative values
for scattered light intensity in BGG solutions recorded at ascattering angle of 90°. Addition of 10 and 50 mM NaCl
suppressed the scattering of light, indicating that electrostaticinteraction was responsible for the opalescence. Next, SEADM was used to investigate the mechanism responsible for
opalescence in antibody solutions without microscale assemblies. SE-ADM allows the direct observation of nanoscale
objects in solution, subjecting samples to very little stress.
Figure 2c shows the SE-ADM images for the nanoscale
network assembly of BGG at 115 mg/mL, characterized by thepresence of almost equal volumes of the diluted and condensed
phases of BGG molecules. The network assembly showedmesh size of hundreds of nanometers, while the width of BGG
condensates comprising the network structure was found to betens of nanometers (Figure 2d).
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ArticleFigure 3. Size and density of network assembly depending on antibody concentrations. (a) SE-ADM images of HGG solutions at diﬀerent
concentrations. The black area shows the presence of BGG, and the pixel intensity corresponds to 0. Scale bars represent 500 nm. (b) Histogramsfor the two-dimensional pixel intensity of SE-ADM images shown in (a). Solid lines represent the Gaussian distribution curves calculated for eachhistogram. (c) Quantitative measurement of light transmittance and scattered light intensity for 0−150 mg/mL HGG solutions as a function of
antibody concentrations. The determined values were the averages of triplicate experiments. All the samples were prepared in 20 mM Na phosphatebuﬀer (pH 7.0).

Ultracentrifugation of the Network Assembly. Next,
the change in the opalescence of antibody solutions arising due
to the application of mechanical stress on the network
assembly was studied using ultracentrifugation. Figure 4

The formation of nanoscale network assembly was also
investigated in HGG solutions. Unlike BGG solutions, HGG
did not exhibit any precipitation or LLPS in water even whensubjected to centrifugation at 18,800g for 20 min. Figure 3ashows the SE-ADM images for HGG solutions at 60−150 mg/
mL. As expected, HGG solutions showed the formation of
network assemblies in a concentration-dependent manner.
Similar to BGG, the mesh size of the HGG network assembly
was also measured to be around hundreds of nanometers, andit changed as a function of HGG concentration. The mesh size
at 60 and 100 mg/mL appeared to be larger than 150 mg/mL,
consistent with the light scattering intensity. The statisticalanalysis for the mesh size of network assemblies is shown in
Figure 3b. The histogram analysis of the two-dimensional pixel
intensity for each SE-ADM image showed that the density ofantibodies in network assemblies increased as a function of
antibody concentration. The maximum distribution of antibodies in the concentrated and diluted phases was attained at
100 mg/mL concentration. As shown in Figure 3c, theopalescence in HGG solutions was found to be concentrationdependent. The light transmittance decreased until 100 mg/
mL HGG concentration; however, it increased with further
increase in HGG concentrations. These results indicated thatthe network assemblies were formed at high concentrations of
HGG solution, in the range of 60 and 150 mg/mL, even in thepresence of slight diﬀerences in the mesh size.

Figure 4. Ultracentrifugation of antibody solution displaying the
network assembly.

shows the visual appearance of the HGG solutions containing
the network assembly before and after ultracentrifugation.
Before ultracentrifugation, the HGG solution was characterized
by an opalescent appearance with a transparent blue color.
Further, the ultracentrifugation of HGG solution at 120,000rpm resulted in the formation of clean/opalescent phaseseparation in a nonequilibrium manner. Ultracentrifugation of
the solution for a suﬃciently long time (360 min) divided theopalescent solution into two clear phases with an interface.
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Articleeﬀects on the opalescence arising from the network assembly.
This might be attributed to the preferential interaction oftrehalose,51,52 whereas cation−pi, pi−pi, and electrostaticinteractions might be involved in the interaction of ArgHCl
with antibody molecules.53−55 The study also evaluated the
combined eﬀects of these additives on the opalescence ofantibody solutions. Figure 5c shows the scattered lightintensity for 80 mg/mL HGG solutions in the presence of
300 mM trehalose and ArgHCl. The mixture of trehalose andArgHCl was found to be slightly more eﬀective in suppressing
opalescence as compared to the use of individual additives.

contain any protein. This might be attributed to the removal ofwater molecules present in the network assembly by ultracentrifugation. In particular, ultracentrifugation of protein
solutions for several hours results in the formation of glass-likeprotein condensates.50
100 mg/mL of HGG solution with 50 mM MOPS buﬀer(pH 7.0) was ultracentrifuged at 120,000 rpm and 25 °C for
the indicated time periods.
Eﬀects of Additives on the Opalescence Arising fromthe Network Assembly. Development of stable andbiologically active biopharmaceuticals demands the preventionof opalescence of antibody solutions. Trehalose is widely
utilized as a protein stabilizer, while ArgHCl has been
employed to suppress protein aggregation. The present study
investigated the eﬀects of trehalose and ArgHCl on the
opalescence arising from the network assembly. Figure 5a■

DISCUSSION
This paper investigated the size and morphology of antibody
solutions at high concentrations using ﬂuorescence microscopy, CARS, and SE-ADM. The results showed that there wasa relationship between the opalescence appearance and theformation of nanoscale network assembly or oligomer ofantibodies. The formation mechanism of nanoscale network
assemblies is considered as follows. In BGG solutions, thenetwork assembly was observed at the concentrations lying
between two critical concentrations, which were related to theantibody solubility (Figure 1c). For HGG solutions, theopalescence without precipitation was observed at higher HGG
concentrations, which was similar to the opalescence observedfor BGG solution containing small amounts of NaCl. The
opalescence without precipitation has been previously reported
in the case of therapeutic monoclonal antibody formulations,where LLPS was induced particularly at low temperature andlow ionic strength, or in the presence of molecular crowders
such as polyethylene glycol.12,19,38 The formation of a polymerrich phase in the metastable region of LLPS, existing between
binodal and spinodal curves, involves the initiation of
nucleation as a local phenomenon. In the unstable region
that arises due to spinodal decomposition, small ﬂuctuations in
the polymer density are observed in the long-range order.
These ﬂuctuations further result in the formation of areas
without sharp interface instead of droplets, which probably
represents the network assembly of antibodies. Notably, thesize of the network assembly of antibodies was in the range oftens to hundreds of nanometers. This might be driven by the
charge distribution and dipole moment on the surface of theantibodies, involving Fab−Fab and Fab−Fc interactions.56−60
The formation of a network probably requires charge
asymmetry and local interactions.
Trehalose and ArgHCl successfully suppressed the opalescence of antibody solutions. In the presence of trehalose, thenetwork structure of antibodies appeared to favor morecompact structures such as monomers and oligomers.
Trehalose is a protein stabilizer that increases the surface
tension of water, away from the protein−water interface, andenhances protein hydration.51,52 On the other hand, thesuppression eﬀect of ArgHCl on the opalescence was higher
than that of NaCl and trehalose. ArgHCl interacts directly withthe surface of proteins via cation−pi and pi−pi interactions
mediated by the guanidinium group of the side chain andelectrostatic interactions between its charged backbone and theguanidinium group.53−55 Thus, the eﬀects of trehalose andArgHCl on opalescence were mutually independent, further
supporting the enhanced combinatorial eﬀect of ArgHCl andtrehalose observed in the present study.

Figure 5. Eﬀects of additives on opalescence arising from the network
assembly. (a) SE-ADM images for 80 mg/mL HGG solution, in thepresence of 300 mM trehalose or ArgHCl. Scale bars indicate 500 nm.
(b) Quantitative measurement of light scattering intensity for 80 mg/
mL HGG solution in the presence or absence of additives (i.e.,trehalose, ArgHCl, or NaCl). (c) Ratio of intensity for 80 mg/mL
HGG solution with 300 mM trehalose and ArgHCl; the intensity of
HGG solution without additives corresponds to 1. The determinedvalues were the averages of triplicate experiments. All the samples
were prepared in 10 mM MOPS buﬀer (pH 7.0).

shows the SE-ADM images for the 80 mg/mL HGG solution
in the presence of trehalose and ArgHCl. In the absence ofadditives, the network assemblies were observed in HGG
solution at concentrations ∼80 mg/mL (Figure 3a). However,the HGG network assembly was found to be broken anddissolved in the presence of 300 mM ArgHCl. Further, theaddition of 300 mM trehalose suppressed the formation of
network structural assembly and resulted in the formation of
non-networked oligomers. These additives displayed aconcentration-dependent suppression of opalescence in theorder, ArgHCl > NaCl > trehalose (Figure 5b). These results
suggested that trehalose and ArgHCl induced independent1165

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CONCLUSIONSThis paper identiﬁed the submicron network-like phaseseparation of antibodies, which suggests a mechanistic insight
into the opalescence of antibody solution as a function of its
concentration. Arginine and trehalose successfully suppressed
the opalescence of the antibody solutions by aﬀecting the
network assembly. These results will provide a fundamental
information for the development of liquid biopharmaceuticals
and for understanding the protein nature.

■

AUTHOR INFORMATION

Corresponding Author

Kentaro Shiraki − Faculty of Pure and Applied Sciences,
University of Tsukuba, Tsukuba 305-8573, Japan;
orcid.org/0000-0003-3438-4076; Email: shiraki@bk.tsukuba.ac.jp

Authors

Yoshitaka Nakauchi − Faculty of Pure and Applied Sciences,
University of Tsukuba, Tsukuba 305-8573, Japan
Suguru Nishinami − Faculty of Pure and Applied Sciences,
University of Tsukuba, Tsukuba 305-8573, Japan
Yusuke Murakami − Ph.D. Program in Humanics, Universityof Tsukuba, Tsukuba 305-8577, Japan; International
Institute for Integrative Sleep Medicine (WPI-IIIS),
University of Tsukuba, Tsukuba 305-8575, Japan
Toshihiko Ogura − Health and Medical Research Institute,
National Institute of Advanced Industrial Science andTechnology (AIST), Tsukuba 305-8566, Japan;
orcid.org/0000-0001-8953-6562
Hideaki Kano − Department of Chemistry, Kyusyu University,
Fukuoka-shi 819-0395, Japan

Complete contact information is available at:https://pubs.acs.org/10.1021/acs.molpharmaceut.1c00929
Funding

This work was partly supported by JSPS KAKENHI (grant
number 18H02383).
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
We are grateful to Dr. Atsushi Hirano (AIST, Japan) and Dr.
Shunsuke Tomita (AIST, Japan) for valuable discussion andintroducing us as collaborators.

■

ABBREVIATIONS
ArgHCl, arginine hydrochloride; HGG, human immunoglobulin G; BGG, bovine gamma globulin; SE-ADM, scanningelectron-assisted dielectric microscopy; CARS, coherent antiStokes Raman scattering; LLPS, liquid−liquid phase separation; GLPC, glass-like protein condensates■

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