THERAPEUTIC ANTIBODIES

Application eBook

CONTENTS
Structure Studies
Secondary structure
Secondary structure prediction for Herceptin® using CD
spectroscopy ……………………………………………………………………………… 4
Secondary structure prediction for IgG using IR spectroscopy……………………………………………………………………………………………………… 8
Orthogonal assessment of secondary structure for IgG …………… 11
Tertiary structure
Near-UV CD measurement of high-concentration IgG ……………… 16
Quaternary structure
Assessment of aggregates in antibody drugs by SEC ……………… 19

Similarity Studies
Single assessment
Orthogonal tertiary structure similarity assessment using near-UV
CD and ﬂuorescence spectroscopy …………………………………………… 25
Orthogonal assessment
4-way Orthogonal HOS similarity assessment of biosimilars using
multiple spectroscopic techniques and statistical calculations……………………………………………………………………………………………………… 30

Stability Studies
Thermal denaturation
Thermal denaturation analysis of monoclonal antibodies using
CD spectroscopy ………………………………………………………………………… 37
Thermal denaturation analysis of monoclonal antibodies using
FTIR spectroscopy ……………………………………………………………………… 41
Thermal denaturation analysis of monoclonal antibodies using
Raman spectroscopy …………………………………………………………………… 44
Heat-induced denaturation and refolding study of VHH
antibodies …………………………………………………………………………………… 48
pH & salt denaturation
pH & salt denaturation analysis of VHH antibodies using CD
spectroscopy ……………………………………………………………………………… 53

Forced Degradation Studies
Monitoring of aggregates and degradation products by SEC in
FDS of antibody drugs ………………………………………………………………… 57
Statistical similarity assessment of therapeutic antibodies in FDS
using CD spectroscopy ……………………………………………………………… 63

Introduction
The market for therapeutic antibodies has been
dramatically expanding over the past decades.
Antibody drugs exhibit therapeutic effects, such as
inhibiting the growth of malignant tumor cells andthe activity of immune cells by binding antigens
expressed in target cells with high affinity andspecificity. Such antibody drugs are expected tobe effective therapeutic agents for unmet medical
needs.

Quaternary structure
Tertiary structure
Secondary structurePrimary structure
NH3+(CH2)3 HO

The higher order structures (HOS) formed by
Peptide bondtherapeutic antibodies determines their efficacy.
Because antibodies are unstable macromolecules,
their HOS can be affected by various stimuli during
Protein higher-order structure (HOS)
the manufacturing process, resulting in reduced
drug efficacy and impurities derived from the target
substance. Therefore, HOS is considered to be a
Critical Quality Attribute (CQA) and is subject to similarity studies, stability studies, and forced degradation
studies in the R&D process from target assessment to lead optimization. In addition, it is monitored during thepost-launch manufacturing process.
N
H

N

O

CH3

n

Antibody structures are hierarchically formed as primary, secondary, tertiary, and quaternary structures, and it
is important to comprehensively evaluate each structure in the characterization of antibodies. This application
eBook presents examples of evaluations using various analytical instruments, including Circular Dichrosim(CD) spectrometers, Fourier-transform infrared (FTIR) spectrometers, Raman imaging microscopes and High
Performance Liquid Chromatography (HPLC).

R&D
TargetAssessment

Hit
Identification

Lead
Identification

Manufacture
Lead
Optimization

ClinicalStudies

GMP
Manufacturing

De novo Sequencing

Method development

Stability studies

Quality testing

Clone selection

Establishing CQA’s

Comparability studies

Product release

Epitope mapping

Forced Degradation Studies

High Order Structure

Similarity studies

R & D and manufacturing scheme for antibody drugs

STRUCTURE STUDIES | SECONDARY STRUCTURE

Secondary Structure Prediction for Herceptin®
using CD Spectroscopy

MRE

Fraction / %CD spectrometer

Herceptin®
Image from the RCSB PDB (http://www.
rcsb.org/) of PDB ID 1HZH.
SampleIntroduction

Wavelength

Measurement

Helix

Sheet

Turn

OthersSecondary structure prediction

Graphical abstract

Higher-order structure (HOS) is a critical attribute that controls the function, safety and efficacy of therapeuticproteins.1) During the development of protein therapeutics, characterization of the HOS is required in orderto achieve robust manufacturing processes, to develop stable formulations and to manufacture biosimilars
identical to the innovator.2) When investigating protein HOS, Circular Dichroism (CD) is the most well-known
technique for studying protein secondary structure. This method is an easy, time-saving method, commonlyused to monitor the effect of manufacturing, formulation and storage conditions on therapeutic proteins2).
CD spectroscopy is a classic technique for the secondary structure prediction of proteins in solution. The
secondary structure content is estimated based on CD signals in the far-UV region (170 to 250 nm), which areassociated with electronic transitions involving peptide bonds.3) The protein concentration required to detect
the minimal CD signal in this range is low: approximately 0.1 mg/mL when using a 1 mm path length cuvette.
This is a great advantage when the amount of protein is limited, which is commonly the case.
In this study, the secondary structure content of Herceptin® was analyzed using CD spectroscopy.
Keywords
Circular Dichroism (CD) spectroscopy, secondary structure prediction, BeStSel, Herceptin®, trastuzumab,
therapeutic antibody, biopharmaceutical, higher-order structure (HOS)

Experimental
Sample• Herceptin® (trastuzumab, Roche): powder dissolved in ultrapure water to 0.16 mg/mL
Measurement and analysis system
J-1500 CD spectrometer• Accurate and efficient normalization of protein
CD spectrum realized by simultaneous CD and
Abs measurements.

4

BeStSel4)• Secondary structure prediction method
developed by Dr. József Kardos at Eötvös
Loránd University.
• Highly accurate estimation of β-sheet content bydividing β-sheets into four classes.

Measurement Conditions
Bandwidth:		
D.I.T.: 			
Data pitch: 		Scanning speed:

1.0 nm2 sec.
0.1 nm50 nm/min.

Accumulation:
Optical path length:
Sample volume:

9
1 mm
200 µL

Results
CD and absorbance spectra in the far-UV region were simultaneously obtained using the J-1500 (Figs. 1a and1b). For secondary structure prediction, the measured CD spectrum is normalized using the absorbance orconcentration to generate the Mean Residue Ellipticity (MRE) spectrum or the molar CD spectrum. Here, themeasured CD spectrum was normalized using the absorbance at 205 nm to generate the MRE spectrum (Fig.
1c). 3, 5) The secondary structure content was then predicted using BeStSel (Fig. 1d).

5

a)

1.0

b)

4

0.8

2
Abs.

CD / mdeg

3

1

0.4

0
-1

0.2

-2
-3
190

200

210

220
230
240
Wavelength / nm

250

d)

4000

200

210

220
230
240
Wavelength / nm

250

Sheet

Others

260

40

2000
0
-2000

30
20
10

-4000
190

190

50

Fraction / %MRE / deg cm2 dmol-1

0.0

260

6000

c)

0.6

200

210

220
230
240
Wavelength / nm

250

260

0

Helix

Turn

Fig. 1 Spectra and predicted secondary structure contents.
a) CD, b) absorbance, c) MRE spectra in far-UV region, and d) secondary structure contents.

5Conclusions
The secondary structure of Herceptin® was easily predicted from the CD spectrum using BeStSel. CD
spectroscopy is the strongest, fastest approach to determining protein secondary structure. The simplicity ofthis technique makes it applicable for efficient screening of protein solutions under different conditions, suchas buffer composition or temperature to determine their effects on protein structural stability.

Additional Information• Application: Highly efficient CD/CPL systems for accurate simultaneous CD/Abs and CPL/FL
measurements #1.
https://www.jasco-global.com/solutions/highly-efficient-cd-cpl-systems-for-accurate-simultaneous-cd-absand-cpl-fl-measurements-1/• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/• BeStSel web server:
https://bestsel.elte.hu/index.php

References
1) S. A. Berkowitz, D. J. Houde: “Biophysical Characterization of Proteins in Developing Biopharmaceuticals(Second Edition)”. (2015), (Elsevier B. V., Amsterdam).
2) L. L. Liu, L. Wang, J. Zonderman, J. C. Rouse, H. Y. Kim: J. Pharm. Sci., 109, 3223 (2020).
3) A. Micsonai, É. Bulyáki, J. Kardos: Methods Mol. Biol. (N. Y.), 2199, 175 (2021).
4) A. Micsonai, F. Wien, L. Kernya, Y. H. Lee, Y. Goto, M. Réfrégiers, J. Kardos: Proc. Natl. Acad. Sci. U. S. A.,
112, E3095 (2015).
5) N.J. Anthis, G.M. Clore: Protein Sci., 22, 851 (2013).

About the Author
Ai Yamane is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
UV-vis, and fluorescence spectroscopy.

6Secondary Structure Estimation by CD Spectroscopy- BeStSel

BeStSel web server:
https://bestsel.elte.hu/index.php

Recently, Micsonai et al. developed the BeStSel
algorithm that accurately estimates the secondary
structure composition from the CD spectrum bytaking into account the parallel-antiparallel orientationof the β-strands and the twist of the antiparallel
β-sheets. BeStSel has the following features: 1) highestimation accuracy for a wide range of proteins
including β-structure-rich-proteins such as antibodies,2) providing eight types of secondary structure
information 3) a capability to predict the protein foldfollowing the CATH classification and 4) an open web
server.1–5)

1) Micsonai, A., Wien, F., Kernya, L., Lee, Y. H., Goto, Y., Réfrégiers, M., & Kardos, J.
: Proc. Natl. Acad. Sci. U.S.A., 112, E3095 (2015)
2) Micsonai, A., Wien, F., Bulyáki, É., Kun, J., Moussong, É., Lee, Y. H., Goto, Y.,
Réfrégiers, M., & Kardos, J.: Nucleic Acids Res., 46, W315 (2018)
3) Micsonai, A., Bulyáki, É., & Kardos, J.: Methods in molecular biology (Clifton, N.J.),
2199, 175 (2021)
4) Micsonai, A., Moussong, É., Lee, Y. H., Murvai, N., Tantos, Á., Tőke, O., Réfrégiers,
M., Wien, F., & Kardos, J.: Front. mol. biosci, 9 (2022)
5) Micsonai, A., Moussong, É., Wien, F., Boros, E., Vadászi, H., Murvai, N., Lee, Y. H.,
Molnár, T., Réfrégiers, M., Goto, Y., Tantos, Á., & Kardos, J.:Nucleic Acids Res.,50,
W90 (2022)

Seamless offline analysis - Spectra Manager BeStSel
While many academic researchers use the BeStSel web server, pharmaceutical companies
are unable to use their proprietary data on the site, and seek a development environment
where the data can be analyzed oﬄine. Addressing this need, an oﬄine BeStSel softwarein Spectra Manager™ 2.5 (Spectra Manager BeStSel) was jointly developed, and enables
seamless analysis in an oﬄine capacity. Furthermore, Spectra Manager™ 2.5 CFR BeStSel was
also developed, ensuring compatibility with GxP and satisfying computer system validation(CSV), electronic record/electronic signature (ER/ES) and data integrity (DI) for practicing
ALCOA+ principles.

BeStSel program

7

STRUCTURE STUDIES | SECONDARY STRUCTURE

Secondary Structure Prediction of IgG
using IR Spectroscopy

Amide I

Herceptin®
Image from the RCSB PDB (http://www.
rcsb.org/) of PDB ID 1HZH.

Wavenumber / cm-1

SampleMeasurement

IR SSE-4X
Fraction / %

Abs.

FTIR
with single-reflection ATR

Helix

Sheet

Turn

OthersSecondary structure prediction

Graphical abstract

Introduction
Like Circular Dichroism (CD) spectroscopy, infrared (IR) spectroscopy can be used to study protein secondary
structure. IR is also a fast measuring method, but unlike CD, proteins in both highly concentrated solutions (upto several hundred mg/mL) and the solid state can be measured.
Protein secondary structure is estimated based on absorption associated with the amide I band (1700 to 1600cm-1), which is caused by C=O stretching vibrations of the protein backbone. It is known that the amide I bandis sensitive to the β-sheet structure, which is convenient for monoclonal antibody (mAb) analysis. The higher
concentration limits of IR spectroscopy make it apt for manufacturing conditions, where the formulation mustbe high concentration and Higher-order structure (HOS) evaluation is needed. 1,2)
In this study, the secondary structure content of a highly concentrated IgG solution was analyzed using FTIR
spectroscopy.

Keywords
Fourier-transform infrared (FTIR) spectroscopy, Attenuated Total Reflectance (ATR), secondary structure
prediction, IgG, monoclonal antibody (mAb), therapeutic antibody, biopharmaceutical, higher-order
structure (HOS)

Experimental
Sample• Protein: IgG, from rabbit serum (Sigma Aldrich) 11.4 mg/mL and 116 mg/mL• Buffer: 20 mM citric acid buffer (150 mM NaCl, pH 6.0)

8

Measurement and Analysis System

FT/IR-4X FTIR spectrometer with ATR PRO 4X single-reﬂection ATR• High S/N and resolution with small body size.
• Protein solution measurement with one drop ofsample (approx. 10 µL) with ATR PRO 4X.

IR SSE-4X IR secondary structure estimation program• Secondary structure prediction program
developed by JASCO.
• Improved precision and agreement with CD
spectroscopy by enhancement of reference
model.

Measurement Conditions
Resolution:
Sample volume:

4 cm-1
10 µL

Accumulation: 100 (2 min.)N2 purge:
Yes

Results
The IR spectrum of a 116 mg/mL IgG solution was measured using the FT/IR-4X with ATR PRO 4X (Fig. 1a).
A relatively low-concentration IgG solution (11.4 mg/mL) was also measured for comparison. The secondary
structure content for both solutions was predicted using the IR SSE-4X (Fig. 1b). The results indicated a change
in the secondary structure as the concentration increased, including an increase in β-sheets and others aswell as a decrease in turns.

a)

0.12

11.4 mg/mL

50

116 mg/mL

0.10

40
Fraction / %

0.08
Abs.

b)

0.06

30
20

0.04
10

0.02
0.00
1700

1680

1660
1640
-1
Wavenumber / cm

1620

1600

0

Helix

Sheet

Turn

OthersFig. 1 IR spectrum and predicted secondary structure contents.

9Conclusions
The secondary structure of IgG was easily analyzed using IR spectroscopy, an equivalently strong techniqueto CD spectroscopy for protein secondary structure prediction. It can be applied not only to protein solutions,but also suspensions and powders, as well as highly concentrated solutions. The sample amount needed fora solution is only 10 µL when using the ATR unit. Because of its versatility, IR spectroscopy allows secondary
structure analysis of therapeutic proteins, which can be aggregated or highly concentrated.

For more information…
• Application: Analysis of the effects of low temperature on the secondary structure of VHH antibodies.
https://www.jasco-global.com/solutions/analysis-of-the-effects-of-low-temperature-on-the-secondarystructure-of-vhh-antibodies/• Application: Protein secondary structure analysis in low concentration aqueous solutions.
https://www.jasco-global.com/solutions/protein-secondary-structure-analysis-in-low-concentrationaqueous-solutions/• Product information: FTIR spectrometer.
https://www.jasco-global.com/product-spectroscopy/ftir-spectrometer/

References
1) S. A. Berkowitz, D. J. Houde: “Biophysical Characterization of Proteins in Developing Biopharmaceuticals(Second Edition)”. (2015), (Elsevier B. V., Amsterdam).
2) L. L. Liu, L. Wang, J. Zonderman, J. C. Rouse, H. Y. Kim: J. Pharm. Sci., 109, 3223 (2020).

About the Author
Ai Yamane is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
UV-vis, and fluorescence spectroscopy.

Secondary Structure Estimation by IR Spectroscopy- IR SSE Program The IR SSE program estimates the secondary structure of a sample from a spectrum obtained using
the multivariate analysis technique.
This program includes two types of standard models (standard calibration curves) created using the
PCR and PLS multivariate analysis methods, based on the infrared spectra of 28 or 17 types of proteins
contained in a database or published in the literature, respectively.
Instead of using the standard model, a custom calibration model created using the [Create SSE
Calibration Model] program can also be used to estimate the secondary structure of sample proteins.

IR SSE-4X Secondary Structure Estimation program

10

STRUCTURE STUDIES | SECONDARY STRUCTURE

Orthogonal Assessment of Secondary Structure
for IgG

ORTHOGONALASSESSMENT

Fraction / %FTIRwith singlereflection ATR

Therapeutic antibody(e.g. highly concentrated IgG solution)
SampleIntroduction

Helix Sheet Turn Others Helix Sheet Turn Others

CD spectrometer

CD Spectroscopy
Measurement

IR Spectroscopy

Secondary Structure Estimation

Graphical abstract

The market for therapeutic antibodies has
dramatically expanded over the past decades since
their first approval in 1986. As a result of this growth,therapeutics antibodies are a large part of new drugs
approved in recent years.1) However, the physicalcharacteristics of antibodies may change dependingon their production and storage environments.
These physical characteristics are referred to as
critical quality attributes (CQAs) and may affect thesafety and potency of antibody drugs.2) Therefore,
there is a need to assess the impact of production
and storage conditions on the CQAs of antibodies,and this assessment should ideally be conductedat their prescribed concentration. Most therapeutic
antibody drugs are prescribed at a relatively highconcentration of up to 200 mg/mL3); however,proteins are known to behave differently at highconcentration compared to lower concentrations.3)

Main CQAs
Primary structure
Higher-Order
Structure
Glycosylation

Main technique
Orthogonal
CD
IR
DSC
Fluorescence
NMR

Product Related
Variant
Process Related
Variant
Fig. 1 CQAs of therapeutic antibodies.7)

The higher-order structure (HOS) of antibodies has an important influence on the CQAs because it directly
affects physiological activity.4) Circular Dichroism (CD) and infrared (IR) spectroscopy are the most popular
techniques for secondary structure evaluation of proteins. CD spectroscopy is used to obtain secondary
structural information for relatively low-concentration protein solutions with a maximum concentration of about
10 mg/mL. IR spectroscopy is used for secondary structure prediction for protein solutions with concentrationsof up to 100 mg/mL, as well as protein suspensions or powders.
When assessing the physical properties of therapeutic antibodies, the importance of orthogonal assessment
has been stressed.5) In this approach, each physical characteristic is assessed by two or more techniques thatare based on different principles. Orthogonal assessment is expected to improve the efficiency and lower thecost of clinical trials during biosimilar development.6)
Here, the secondary structure of IgG, the most common antibody used in antibody drugs, was assessed using
CD and IR spectroscopy as orthogonal methods.
Keywords
Circular Dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, Attenuated Total
Reflectance (ATR), secondary structure prediction, BeStSel, IgG, monoclonal antibody (mAb), therapeutic
antibody, biopharmaceutical, higher-order structure (HOS), Critical Quality Attribute (CQA), orthogonalassessment
11Experimental
Sample• Protein: IgG, from rabbit serum (Sigma Aldrich)
5.8 mg/mL, 11.4 mg/mL, 17.5 mg/mL and 23.1 mg/mL• Buffer: 20 mM citric acid buffer (150 mM NaCl, pH 6.0)
Measurement and analysis system
J-1500 CD spectrometer• Accurate and efficient normalization of protein
CD spectrum obtained by simultaneous CD and
Abs measurements.
0.01 mm path length demountable cuvette

BeStSel8)• Very short path length enabling CD
measurement of high-concentration protein
solutions.
• Easy cleaning simply by wiping off the samples
using lab tissues soaked with ultrapure water.

• Secondary structure prediction method
developed by Dr. József Kardos at Eötvös
Loránd University.
• Highly accurate estimation of β-sheet content bydividing β-sheets into four classes.

FT/IR-4X FTIR spectrometer with ATR PRO 4X single-reﬂection ATR• High S/N and resolution with small body size.
• Protein solution measurement with one drop ofsample (approx. 10 µL) with ATR PRO 4X.

IR SSE-4X IR secondary structure estimation program• Secondary structure prediction program
developed by JASCO.
• Improved precision and agreement with CD
spectroscopy by enhancement of reference
model

12

Measurement Conditions<Far-UV CD measurement>
Bandwidth:
1.0 nm
D.I.T.:
2 sec.
Data pitch:
0.1 nmScanning speed:
50 nm/min.

Accumulation:
Optical path length:
Sample volume:4
0.01 mm
1.8 µL

Accumulation:
N2 purge:

100 (2 min.)Yes<IR measurement>
Resolution:
Sample volume:

4 cm-1
10 µL

Results
CD and absorbance spectra in the far-UV region were simultaneously obtained using the J-1500 (Figs. 2aand 2b). The CD signal and absorbance both increased with increasing sample concentration. For secondary
structure prediction, the measured CD spectrum is normalized using the absorbance or concentration to
generate the Mean Residue Ellipticity (MRE) spectrum or the molar CD spectrum. Here, the measured CD
spectra were normalized using the absorbance at 205 nm to generate the MRE spectra (Fig. 2c).9,10) The shapeof the MRE spectra did not change despite the difference in concentration.
IR spectra were measured using the FT/IR-4X and ATR PRO 4X (Fig. 2d). Although the absorbance increased
with increasing sample concentration, the amide I band (1700 to 1600 cm-1) did not show significant changes
in shape with concentration.

a)15

11.4 mg/mL

10

5.8 mg/mL3

17.5 mg/mL

511.4 mg/mL
17.5 mg/mL

2.5

23.1 mg/mL

0

Abs.

CD / mdeg

3.5b)

5.8 mg/mL

23.1 mg/mL

2
1.5

-5

1

-10

0.5

-15

0

-20

200

220

240

260

200

c)

5.8 mg/mL

4000

17.5 mg/mL

2000

5.8 mg/mL

011.4 mg/mL

Amide I

0.020

23.1 mg/mL

260

0.030
0.025

11.4 mg/mL

240

17.5 mg/mL
23.1 mg/mL

Abs.

MRE / deg cm2 dmol-1

d)

6000

220
Wavelength / nm

Wavelength / nm

0.015

-2000
0.010

-4000

0.005

-6000
-8000
200

220

240

260

0.000
1700

1600

Wavelength / nm

1500

1400

1300

1200

Wavenumber / cm-1

Fig. 2 CD and absorbance spectra in far-UV region and IR spectra of IgG solutionsa) CD, b) absorbance, c) MRE spectra in far-UV region, and d) IR spectra.

13

a)

50

X-ray crystallographyb)

Sheet 43.2 %Others 40.9 %Fraction / %

40
30
20

Turn 12.6 %100

Helix 3.2 %
5

10
15
20
Concentration / mg mL-1

25

5

10
15
20
-1
Concentration / mg mL

25

Fig. 3 Secondary structure prediction.
Secondary structure contents obtained using a) CD spectroscopy and b) IR spectrum.

Table 1. What should be checked for proper use of CD and IR spectroscopy
Item

CD (far-UV)IR

Buffer and additives

Do not show excessive absorbance in the bare minimum
wavelength for SSE (200 to 250 nm).

Do not show absorbance in amide I band (1700 to 1600cm-1).

Sample state• Solution (up to several tens mg/mL)• Only transparent, suspension not accepted.

• Solution (up to several hundreds mg/mL)• Suspension• PowderThe secondary structure content was predicted from MRE spectra using BeStSel (Fig. 3a) and from the amide
I band using the IR SSE-4X (Fig. 3b). The secondary structure content for IgG (PDB ID: 1HZH) obtained by X-ray
crystallography is shown in Fig. 3 for comparison.11) In this concentration range, the secondary structure content
did not show a significant change in either the CD or IR secondary structure prediction. This agrees with theobservation that the shape of the MRE (Fig. 2c) and IR (Fig. 2d) spectra did not depend on concentration. Also,
the secondary structure contents obtained by CD and IR spectroscopy, as well as X-ray crystallography, arein good agreement.

Conclusions
The agreement between the CD and IR spectroscopy confirms the usefulness of these two techniques fororthogonal assessment of protein secondary structure. As shown in Table 1, the most appropriate choice oftechnique depends on the buffer and sample used. For instance, the amide I band in the IR spectrum derived
from C=O stretching in proteins may overlap with the absorbance bands for certain buffers and additives.
When this happens, accurate baseline subtraction is difficult, resulting in poor precision for secondary
structure prediction. On the other hand, in the CD spectrum, the baseline can be accurately subtracted aslong as the solvent does not show excessive absorbance. Therefore, for certain types of buffers and additives,
CD is the better choice. However, CD measurements can only be performed for transparent solutions withconcentrations of up to a few tens of mg/mL, even when using a very short path length. In contrast, IR
measurements can be performed on protein solutions with concentrations of more than 100 mg/mL, as wellas protein suspensions and powders. Accurate prediction of secondary structure can be achieved only when
the appropriate technique is selected depending on the solvent type and sample state.
Both CD and IR spectroscopy are simple methods for HOS assessment. The present results confirm theapplicability of both techniques in the development and quality control processes for therapeutic antibodies
and biosimilars.

14

Additional Information• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/• Product information: FTIR spectrometer.
https://www.jasco-global.com/product-spectroscopy/ftir-spectrometer/• BeStSel web server:
https://bestsel.elte.hu/index.php

References
1) R. M. Lu, Y. C. Hwang, I. J. Liu, C. C. Lee, H. Z. Tsai, H. J. Li, H. C. Wu: J. Biomed. Sci., 27:1, (2020). doi: 10.1186/
s12929-019-0592-z.
2) European Medical Agency: “ICH Guideline Q11 on Development and Manufacture of Drug Substances(Chemical Entities and Biotechnological/Biological Entities)” < https://www.ema.europa.eu/en/documents/
scientific-guideline/ich-guideline-q11-development-manufacture-drug-substances-chemical-entitiesbiotechnological/biological-entities_en.pdf>, (accessed 2022.07.28)
3) S. Uchiyama, E. Krayukhina, M. Noda: Pharmacology, 74, 12 (2014).
4) Y. Tokunaga, K. Takeuchi: Int. J. Mol. Sci., 22, 46 (2020).
5) World Health Organization: “Guidelines on the Quality, Safety, and Efficacy of Biotherapeutic Protein Products
Prepared by Recombinant DNA Technology”, < https://cdn.who.int/media/docs/default-source/biologicals/
biotherapeutics/trs_987_annex4.pdf?sfvrsn=d4ba378a_5&download=true>, (accessed 2022.07.28)
6) U.S. Food and Drug Administration: “Development of Therapeutic Protein Biosimilars: Comparative
Analytical Assessment and Other Quality-Related Considerations”, <https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/development-therapeutic-protein-biosimilars-comparativeanalytical-assessment-and-other-quality>, (accessed 2022.07.28)
7) N. Nupur, S. Joshi, D. Gulliarme, A. S. Rathore: Front. Bioeng. Biotechnol, 10:832059, (2022).
8) A. Micsonai, F. Wien, L. Kernya, Y. H. Lee, Y. Goto, M. Réfrégiers, J. Kardos: Proc. Natl. Acad. Sci. U. S. A.,
112, E3095 (2015).
9) N.J. Anthis, G.M. Clore: Protein Sci., 22, 851 (2013).
10) A. Micsonai, É. Bulyáki, J. Kardos: Methods Mol. Biol. (N. Y.), 2199, 175 (2021).
11) E. O. Saphire, P. W. Parren, R. Pantophlet, M. B. Zwick, G. M. Morris, P. M. Rudd, R. A. Dwek, R. L. Stanfield, D.
R. Burton, I. A. Wilson: Science, 293, 1155 (2001).

About the Author
Ai Yamane is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
UV-vis, and fluorescence spectroscopy.

15

STRUCTURE STUDIES | TERTIARY STRUCTURE

Near-UV CD Measurement of High-Concentrated
IgG
CD spectrometer

Normalized CD

Near-UV CD spectra

Therapeutic antibody(e.g. highly concentrated IgGsolution)

0.1 mm and 0.5 mm
path length cuvette

SampleMeasurement

S-S
Trp
Phe

Tyr

Wavelength / nm

Graphical abstract

Introduction
Beyond secondary structure, Circular Dichroism(CD) spectroscopy is an excellent method to probe
the tertiary structure of proteins. As previously
mentioned, therapeutic antibody drugs are oftenprescribed at high concentration of up to 200
mg/mL (Fig. 1). Our previous example paired CD
and infrared (IR) spectroscopy to bridge the low
to high concentration gap for secondary structureevaluation. To evaluate the proteins physicalcharacteristics and Critical Quality Attributes (CQAs)
1)
on CD we can employ the near UV regions.
The near UV region of the CD spectrum is related
to the tertiary structure of the antibody studied. The
concentrations which can be studied in this region
are allowed to be much higher because of thesmaller molar extinction coefficient.

100 to 200 mg/mL
29 %1 to 9 mg/mL
19 %50 to 99 mg/mL
16 %10 to 49 mg/mL
36 %Fig. 1 Prescribed concentration of therapeutic antibodies.3)

Here, near-UV CD spectra of IgG solutions with concentrations of more than 150 mg/mL were successfully
obtained using a short path length cuvette. This allows one to study the possible effects of low and highconcentrations of antibody on physiological activity.2,3)

Keywords
Circular Dichroism (CD) spectroscopy, IgG, monoclonal antibody (mAb), therapeutic antibodies, higher-order
structure (HOS), tertiary structure, critical quality attributes (CQAs), high-concentrated protein, short path
length cuvette

Experimental
Sample• Protein: IgG, from rabbit serum (Sigma Aldrich)
5.8, 11.4, 17.5, 23.1, 43.3, 61.7, 76.8, 114.9, and 156.2 mg/mL• Buffer: 20 mM citric acid buffer (150 mM NaCl, pH 6.0)
16

Measurement and analysis system
J-1500 CD spectrometer• Accurate and efficient normalization of protein
CD spectrum obtained by simultaneous CD and
Abs measurements.
• High-concentration protein measurement using
short path length cuvette.

Measurement Conditions
Bandwidth:
D.I.T.:
Data pitch:
Scanning speed:
Accumulation:

1.0 nm4 sec.
0.1 nm20 nm/min.
4 (9 for 156.2 mg/mL)

Optical path length:
1 mm (5.8 to 11.4 mg/mL)
0.5 mm (17.5 to 23.1 mg/mL)
0.1 mm (43.3 mg/mL or higher)
Sample volume:
280 µL (5.8 to 11.4 mg/mL)
220 µL (17.5 to 23.1 mg/mL)
22 µL (43.3 mg/mL or higher)

Results
CD and absorbance spectra in the near-UV region were simultaneously obtained using the J-1500. IgG with awide range of concentration was measured using an appropriate path length for each concentration. CD andabsorbance spectra normalized based on the path length are shown in Figs. 2a and 2b.
V

b)

10
5

S-S

0
-5

Trp

Tyr

-10

Normalized Abs.

Normalized CD / mdeg

a)

Phe

-15
-20

3

5.8 mg/mL

2.5

11.4 mg/mL2

23.1 mg/mL

1.5

61.7 mg/mL

17.5 mg/mL
43.3 mg/mL
76.8 mg/mL
114.9 mg/mL

1

156.2 mg/mL

0.5

250

270

290

310

0

320

250

270

c)

0.4

d)

0.2

Normalized CD / mdeg

0

-0.2

-0.4

290

310

10

5

0

-5

5.8 mg/mL
260

270

280
Wavelength / nm

320

Wavelength / nmNormalized CD / mdeg

Wavelength / nm

290

300

156.2 mg/mL
-10

260

270

280

290

300

Wavelength / nm

Fig. 2 CD and absorbance spectra in near-UV regiona) Normalized CD, and b) normalized absorbance spectra in near-UV region. c) and d) show the regions between 255 and 305 nm
for 5.8 mg/mL and 156.2 mg/mL in (a), respectively.

17

Both the CD signal and the absorbance increased with increasing concentration. In the near-UV region, there
are minor CD peaks between 255 and 305 nm derived from disulfide bonds and aromatic amino acids. Thesesignals were clearly detected at both the minimum and maximum concentrations (Figs. 2c and 2d).

Conclusions
Selecting an appropriate path length allowed CD measurements of highly concentrated protein solutions(> 150 mg/mL) in the near-UV region, which has not been achieved before.

Additional Information• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/

References
1) R. M. Lu, Y. C. Hwang, I. J. Liu, C. C. Lee, H. Z. Tsai, H. J. Li, H. C. Wu: J. Biomed. Sci., 27:1, (2020). doi: 10.1186/
s12929-019-0592-z.
2) European Medical Agency: “ICH Guideline Q11 on Development and Manufacture of Drug Substances(Chemical Entities and Biotechnological/Biological Entities)” < https://www.ema.europa.eu/en/documents/
scientific-guideline/ich-guideline-q11-development-manufacture-drug-substances-chemical-entitiesbiotechnological/biological-entities_en.pdf>, (accessed 2022.07.28)
3) S. Uchiyama, E. Krayukhina, M. Noda: Pharmacology, Japan, 74, 12 (2014).
4) Y. Tokunaga, K. Takeuchi: Int. J. Mol. Sci., 22, 46 (2020).

About the Author
Ai Yamane is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
UV-vis, and fluorescence spectroscopy.

18

STRUCTURE STUDIES | QUATERNARY STRUCTURE

Assessment of Aggregates in Therapeutic
Antibodies by SEC
Separation by SEC

Aggregates &Multimers

Intensity

Packing particle
in SEC column

Therapeutic antibody

Monomer

Fragments

Retention time

UV chromatogram

Inert LC-UV system

SampleMeasurement

Graphical abstract

Introduction
Since the first monoclonal antibody was approved by the U.S. Food and Drug Administration (FDA) in 1986,
the market for therapeutic proteins has expanded rapidly, and antibody drugs have become a predominanttreatment modality for various diseases over the past 25 years.1) The development of biopharmaceuticals, suchas therapeutic antibodies, requires the evaluation of physicochemical, chemical, biological or microbiologicalproperties that should be within the appropriate range to ensure their efficacy and safety. These propertiesare called Critical Quality Attributes (CQAs) and are analyzed by various analytical methods.2–4)
During the manufacturing process, transportation and storage therapeutic antibodies may form dimer, trimer
or higher order aggregates due to mechanical stress or environmental factors.5, 6) These aggregates need
to be evaluated as one of the CQAs because they may cause a decrease in drug efficacy and side effects.
In 2014, the FDA provided guidance on the evaluation of the immunogenicity of therapeutic protein products(the ability of a drug to act as an antigen, produce anti-drug antibodies in the body and provoke an immune
response). For protein aggregates contained in biopharmaceuticals, it is recommended to assess the validityof each analysis result by comparing the results using multiple methods with different measurement principles(orthogonal assessment).7)
Moreover, evaluation of physicochemical properties is considered to be important to show that the subsequent
products (biosimilars) have similar quality, safety and efficacy to preceding biopharmaceuticals (innovators).
Among the physicochemical properties, higher-order structure (HOS) is an essential element of characterization,
which should include similarity assessment of primary to tertiary structure and quaternary structure, including
aggregates, using appropriate analytical techniques, and again the Orthogonal Assessment for one property
is recommended.8, 9)Aggregates are classified by their size into < 100 nm, 100 nm to 1 µm, 1 µm to 100 µm, and > 100 µm categories,
and analytical methods are employed according to each size.10, 11) Size Exclusion Chromatography (SEC) is
often used as one of the qualitative and quantitative evaluation methods for aggregates of 50 nm or less.12, 13)
In this article, antibody drugs with IgG-derived sequences in addition to IgG from human serum were measured
using SEC to separate and detect monomer, multimers, and aggregates. The measured antibody drugs were
innovator rituximab (MabThera®) and biosimilar (RIABNI™) for the treatment of non-Hodgkin lymphoma, andinnovator trastuzumab (Herceptin®) for the treatment of breast cancer.
Keywords
High Performance Liquid Chromatography (HPLC), Size Exclusion Chromatography (SEC), TSKgel G3000SWXL,UV detector, IgG, monoclonal antibody (mAb), MabThera®, RIABNI™, rituximab, Herceptin®, trastuzumab,
therapeutic antibody, biosimilar, biopharmaceutical, higher-order structure (HOS), quaternary structure, Critical
Quality Attribute (CQA), aggregate
19Experimental
Sample• IgG, from human serum (Merck)• MabThera® (rituximab, Roche) 100 mg/10 mL• RIABNI™ (rituximab, AMGEN) 100 mg/10 mL• Herceptin® (trastuzumab, Roche) 150 mg powder for concentrate for solution for infusion
Measurement and analysis system
LC-4000 HPLC system<System configuration>
Pump:
PU-4080i (with DG-4000-04)
Autosampler: AS-4050i (with TC-4000-1)
Column oven: CO-4060
UV detector: UV-4075 (with inert flow cell unit)
Chromatography data system:
ChromNAV Ver. 2(with GPC calculation program)

Measurement Conditions
Column:Flow rate:
Wavelength:Standard:

TSKgel G3000SWXL(7.8 mmI.D. × 300 mmL, 5 µm)
0.8 mL/min.
220 nmProtein standard mix
15–600kDa (Merck)
30 mg/mL in water

Eluent:

0.2 mol/L sodium phosphate buffer(pH 6.7)
Column Temp.:
25 ºC
Injection volume:
10 µL

Results
Fig. 1 shows UV chromatograms of a blank (ultrapure water) and a protein standard sample. The column
used in this experiment has an exclusion limit with globular proteins of 800 kDa and a measurement range ofapproximately 10 to 500 kDa. As shown in Fig. 2, a molecular weight calibration curve was created from theretention times and molecular weights of the four components, except for p-aminobenzoic acid, that eluted
after the permeation limit (around 13.8 minutes). Based on this calibration curve, the molecular weight of eachsample was calculated.
Fig. 3 shows a UV chromatogram of IgG derived from human serum. Table 1 shows the calculation resultsof the peak top molecular weight (Mp) of IgG derived from human serum and the area ratio of respective
sections.

20

Standard

LogM = 5.21796 + 0.398763*tR- 0.0504095*tR^2 + 1.06239e-003*tR^3

42000002

LogM

Intensity / µV

6.0

3

Blank

3000005.0

1100000

5
4.0

0
0.0

4.0

8.0

12.0

16.0

20.0

7.0

8.0

9.0

Retention time / min.

Fig. 1 UV chromatograms of a blank and a protein standard
1: Thyrogrobulin (670 kDa), 2: γ-globulins (150 kDa), 3:
Ovalbumin (44.3 kDa), 4: Ribonuclease A (13.7 kDa), 5:
p-amino-benzoic acid (0.137 kDa)a) 150000

10.0

11.0

b)

4 (monomer)

Calibration curve
Baseline
Section range

30000

3

5.2
20000

4
1

10000

4.8

2
4.4

0

0
0.0

4.0

5.6

LogM

1

2

Intensity / µV

100000

50000

13.0

Fig. 2 Molecular weight calibration curve

3

Intensity / µV

12.0

Retention time / min.

8.0

12.0

16.0

20.0

6.0

Retention time / min.

8.0

10.0

12.0

4.0
14.0

Retention time / min.

Fig. 3 UV chromatogram of IgG derived from human seruma) Full scale, and b) enlarged view near the baseline at 6-14 min. (shown with molecular weight calibration curve)
Pretreatment: 1 mg of IgG derived human serum was dissolved in 1 mL of ultrapure water.

Table 1. Calculation results of peak top molecular weights and area ratios of IgG derived from human serum
converted based on the results of standard sample
Peak No.

Section range [min.]Baseline range [min.]tR [min.]

Mp

Section area

Area %

1

6.873–7.120

6.000–18.000

7.043

78893811988

2

7.120–8.107

6.000–18.000

7.987

535189234438

0.16
3.04

3

8.107–9.243

6.000–18.000

8.703

371957116056615.04

49.243–13.573

6.000–18.000

10.1171559036308812

81.76

Similarly, Figs 4 to 6 and Tables 2 to 4 show the UV chromatograms and calculation results of molecular weightfor MabThera®, RIABNI™ and Herceptin®, respectively. The peaks that elute after the permeation limit in Figs 4
to 6 are considered to be the peaks derived from the additives contained in each antibody drug.
Comparison of the Mp of the monomer peaks (peak 3) in Tables 1 to 4 revealed that the molecular weight ofeach antibody drug was about 10 kDa smaller than that of IgG. The molecular weights of MabThera®, RIABNI™
and Herceptin® were calculated to be 145 kDa (published value: 145 kDa), 146 kDa (published value: 145 kDa)
and 149 kDa (published value: 148 kDa), respectively. All the molecular weights were close to the publishedvalues.14–16)
In addition, judging from their molecular weights (27 to 28 kDa), peak 4 eluted between 12 to 13 minutes ineach sample except for IgG, which is thought to correspond to one light chain constituting each antibody drug
eluted as a fragment peak.
21

a)

3 (monomer)

600000b)Calibration curve
Baseline

200000Intensity / µV

400000

2

5.2

8000
3

2

4.8

1

4000

4

1

5.6

Section range

4.4

4
0

0
0.0

4.0

8.0

12.0

16.0

4.0
6.0

20.0

LogM

Intensity / µV

12000

8.0

10.0

12.0

14.0

Retention time / min.

Retention time / min.

Fig. 4 UV chromatogram of MabThera®
a) Full scale, and b) enlarged view near the baseline at 6-14 min (shown with molecular weight calibration curve)
Pretreatment: 100 mg/10 mL of MabThera® was diluted with ultrapure water to 1 mg/mL.

Table 2. Calculation results of peak top molecular weights and area ratios of MabThera®
converted based on the results of standard sample
Peak No.

Section range [min.]1a)Baseline range [min.]8.100–9.043

6.000–18.000

29.043–9.510

39.510–12.117

4

12.117–13.570

Mp

Section area

Area %

8.743

3638866.000–18.000

9.257

270669

28743

0.21

6.000–18.000

10.22014527113226663

98.80

6.000–18.000

12.407

27215

27055

0.20

1050560.78

b)

3 (monomer)

800000tR [min.]

Calibration curve
Baseline

12000

Section range

Intensity / µV

4000002200000

5.2

8000
1

4000

3

2

4.8

4

1

4
0

0
0.0

4.0

8.0

12.0

16.0

4.0
6.0

20.0

8.0

10.0

12.0

Retention time / min.

Retention time / min.

Fig. 5 UV chromatogram of RIABNI™
a) Full scale, and b) enlarged view near the baseline at 6-14 min (shown with molecular weight calibration curve)
Pretreatment: 100 mg/10 mL of RIBNI™ was diluted with ultrapure water to 1 mg/mL.

Table 3. Calculation results of peak top molecular weights and area ratios of RIBANI™
converted based on the results of standard sample
Peak No.

Section range [min]122

Baseline range [min]8.027–9.043

6.000–18.000

29.043–9.507

3

9.507–12.093

4

12.093–13.630

tR [min]

4.4

Mp

Section area
125379Area %

8.737

3652230.73

6.000–18.000

9.183

282810

36310

0.21

6.000–18.000

10.217145605

17083631

98.86

6.000–18.000

12.403

27291

34582

0.20

14.0

LogM

Intensity / µV

600000

5.6

a)

b)

3 (monomer)

6000

Calibration curve
Baseline
Section range

Intensity / µV

4000
4000002200000

5.2
2

3

4.8

1

2000

4

1

4.4
4
0

0
0.0

4.0

5.6

LogM

Intensity / µV

600000

8.0

12.0

16.0

4.0
6.0

20.0

8.0

10.0

12.0

14.0

Retention time / min.

Retention time / min.

Fig. 6 UV chromatogram of Herceptin®
a) Full scale, and b) enlarged view near the baseline at 6-14 min. (shown with molecular weight calibration curve)
Pretreatment: 1 mg of Herceptin® was dissolved in 1 mL of ultrapure water.

Table 4. Calculation results of peak top molecular weights and area ratios of Herceptin®
converted based on the results of standard sample
Peak No.

Section range [min]1

Baseline range [min]6.057–8.050

6.000–18.000

28.050–9.387

3

9.387–12.107

412.107–13.373

tR [min]

Mp

Section area
3800

Area %

6.737

873721

0.03

6.000–18.000

8.690

374673

70616

0.48

6.000–18.000

10.18314897414738698

99.45

6.000–18.000

12.37

28058

7586

0.05

Table 5. Comparison of area ratios of HMWS, monomer, and LMWS for each sample
MW classification

HMWS

Monomer

LMWS

SampleMabThera®

0.99 %

98.80 %

0.20 %RIABNI™

0.94 %

98.86 %

0.20 %

Herceptin®

0.51 %

99.45 %

0.05 %

Table 5 shows the results of comparing the area ratios of monomer, High Molecular Weight Species (HMWS)
containing aggregates that elute earlier than the monomer and Low Molecular Weight Species (LMWS) thatelute later than the monomer in each sample. These results show that the innovator and biosimilar of rituximab(MabThera® and RIABNI™) exhibit very similar area ratios. Comparison with the data reported by Goyon etal. revealed that HMWS of MabThera® was 0.99% (literature value: 0.7%) and LMWS was 0.20% (literature
value: 0.3%), both of which were close to their literature values. On the other hand, in the case of innovator
trastuzumab (Herceptin®), the comparison value of LMWS is not available, but the HMWS was 0.51% (literature
value: 0.4%), which is also very close to the literature value.17)

Conclusions
Through this experiment, it was confirmed that the measurement method using SEC can successfully separate
and detect monomer and aggregates containing multimers in antibody drugs. In addition, it was also confirmedthat this method is a powerful tool for evaluating the similarity of HOS, which is an important item of CQAs.

23

Additional Information• Application: Analysis of Aggregates in Antibodies by SEC.
https://www.jasco-global.com/solutions/analysis-of-aggregates-in-antibodies-by-sec/• Product information: HPLC.
https://www.jasco-global.com/product-chromatography/integrated-hplc-system/

References
1) R. M. Lu, Y. C. Hwang, I. J. Liu, C. C. Lee, H. Z. Tsai, H. J. Li, H. C. Wu: J. Biomed. Sci., 27:1, (2020).
2) European Medicines Agency: “ICH Topic Q6B Specifications: Test Procedures and Acceptance Criteria
for Biotechnological/Biological products” <https://www.ema.europa.eu/en/documents/scientific-guideline/
ich-q-6-b-test-procedures-acceptance-criteria-biotechnological/biological-products-step-5_en.pdf>,(accessed 2022. 5. 25).
3) European Medicines Agency: “ICH guideline Q8 (R2) on pharmaceutical development” <https://www.
ema.europa.eu/en/documents/scientific-guideline/international-conference-harmonisation-technicalrequirements-registration-pharmaceuticals-human-use_en-11.pdf>, (accessed 2022. 5. 25).
4) European Medicines Agency: “ICH guideline Q11 on development and manufacture of drug substances(chemical entities and biotechnological/biological entities)” < https://www.ema.europa.eu/en/documents/
scientific-guideline/ich-guideline-q11-development-manufacture-drug-substances-chemical-entitiesbiotechnological/biological-entities_en.pdf>, (accessed 2022. 5. 25).
5) L. Zang: Am. Pharm. Rev., 21 (2), (2018). <https://www.americanpharmaceuticalreview.com/FeaturedArticles/348833-Factors-Influencing-Biotherapeutic-Monoclonal-Antibody-Aggregation/>, (accessed 2022.
5. 25).
6) H. C. Mahler, W. Friess, U. Grauschope, S. Kiese: J. Pharm. Sci., 98 (9), 2909 (2009).
7) U.S. Food and Drug Administration: “Guidance for Industry: Immunogenicity Assessment for TherapeuticProtein Products” <https://www.fda.gov/media/85017/download>, (accessed 2022. 5. 25).
8) European Medicines Agency: “Guideline on similar biological medicinal products containing biotechnologyderived proteins as active substance: quality issues (revision 1)” <https://www.ema.europa.eu/en/documents/
scientific-guideline/guideline-similar-biological-medicinal-products-containing-biotechnology-derivedproteins-active_en-0.pdf>, (accessed 2022. 5. 25).
9) U.S. Food and Drug Administration: “Guidance for Industry: Scientific Considerations in Demonstrating
Biosimilarity to a Reference Product” <https://www.fda.gov/media/82647/download>, (accessed 2022. 5.
25).
10) L. O. Narhi, J. Schmit, K. B. Peters, D. Sharma: J. Pharm. Sci., 101 (2), 493 (2012).
11) S. Uchiyama: Am. Pharm. Rev., 22 (4), 18 (2018).
12) S. Fekete, A. Beck, J. L. Veuthey, D. Guillarme: J. Pharm. Biomed. Anal., 101, 161 (2014).
13) B. Bobály, S. F. Souverain, A. Beck, J. L. Veuthey, D. Guillarme, S. Fekete: J. Pharm. Biomed. Anal., 147,
493 (2018).
14) European Medicines Agency: “Assessment report MabThera” <https://www.ema.europa.eu/en/
documents/variation-report/mabthera-h-c-165-ii-149-epar-assessment-report-variation_en.pdf>, (accessed
2022. 5. 25).
15) AMGEN: “RIABNI™ (rituximab-arrx) Safety Data Sheet” <https://www.msds.amgen.com/-/media/Themes/
Amgen/msds-amgen-com/msds-amgen-com/documents/riabni-sds-20201218.pdf>, (accessed 2022. 5.
25).
16) Australian Government Department of Health: “AUSTRALIAN PRODUCT INFORMATION Herceptin(trastuzumab)
powderfor
injection”<https://www.ebs.tga.gov.au/ebs/picmi/picmirepository.nsf/
pdf?OpenAgent&id=CP-2011-PI-02054-3>, (accessed 2022. 5. 25).
17) A. Goyon, V. D’atri, O. Coals, S. Fekete, A. Beck, D. Guillarme: J. Chromatogr. B, 1065-1066, 35 (2017).

About the Author
Satoe Iijima is a chromatography application scientist at JASCO Corporation (Tokyo, Japan) specializing in LC,
LC/MS, SFC, and SFC/MS.

24

SIMILARITY STUDIES | SINGLE ASSESSMENT

Orthogonal Tertiary Structure Similarity Assessment
using Near-UV CD and Fluorescence Spectroscopy

Therapeutic antibody
SampleNear-UV CD
FluorescenceInt.

CD / mdeg.

Orthogonal assessment

Wavelength / nm

Near-UV CD spectra
Measurement

Wavelength / nm

Fluorescence spectra

Graphical abstract

Introduction
In recent years, there has been a rapid increase in the development, manufacturing and marketing of antibody
drugs. Antibody drugs are expected to become therapeutics for malignant tumors and unmet medical needs
because of their high specificity in targeting only antigens expressed on target cells.
ICH Q5E1) for biopharmaceuticals, including antibody drugs, specifies that the similarity of the higher-order
structure (HOS) should be evaluated before and after changes in the manufacturing process. Circular
Dichroism (CD) spectroscopy is widely used to evaluate the quality of antibody drugs because it can easily
obtain information on the secondary and tertiary structure of antibodies.
Along with innovator antibody drugs, many biosimilar drugs with the same amino acid sequence as theinnovator, but produced using different expressing cells, are now also being used. With the accelerated
development of such biosimilars, there has been a growing focus on how to compare the HOS of innovatorsand biosimilars, and guidelines have been issued by the FDA,2) EMA3) and other institutions around theworld.4,5) These guidelines stress orthogonal assessment of the HOS of innovators and biosimilars using
multiple methods based on different principles, increasing the importance of using multiple analytical
instruments to measure a single critical quality characteristic.
The J-1500 CD spectrometer, in combination with the FMO-522 fluorescence monochromator and theFDT-538 fluorescence detector, can measure near-UV CD spectra and fluorescence spectra that provide
information on protein tertiary structure, making it easy to perform orthogonal assessments. Here, we report
the results of an orthogonal assessment of Herceptin® and MabThera® antibody drugs with different targets
and formulation conditions, by measurement of near-UV CD and fluorescence spectra.
Keywords
Circular Dichroism (CD) spectroscopy, fluorescence spectroscopy, Herceptin®, MabThera®, trastuzumab,
rituximab, therapeutic antibodies, biopharmaceutical, higher-order structure (HOS), tertiary structure,orthogonal assessment, inner-filter effects (IFE)25

Experimental
Sample• Trastuzumab
Herceptin®(Roche): powder dissolved in ultrapure water to 10 mg/mL
Additives: trehalose hydrate 4.7 mg/mL, L-histidine hydrochloride hydrate 0.11 mg/mL,
L-histidine 2.6 x 10-3 mg, polysorbate 2.1 x 10-2 mg/mL• Rituximab
MabThera® (Roche) 100 mg/10 mL
Additives: sodium citrate dihydrate 7.4 mg/mL, sodium chloride 9.0 mg/mL,
polysorbate 80 0.7 mg/mL

Measurement and analysis system

J-1500 CD spectrophotometerwith FMO-522/FDT-538 ﬂuorescence monochromator/detector• Successive measurement for CD andfluorescence spectrum• Orthogonal assessment
for protein tertiary structure2
Fluorescence

1

4
Transmitted light3
Incident light

Measurement overview(1. FMO-522 monochromator, 2. FDT-538 detector, 3. Sample, 4. CD/UV detector)

26

Excitation
light intensity

Excitation
light intensitya)b)Fluorescence

1.5

Excitation

1.0

Excitation

Fluorescencemonochromator

320

340

360

380

400

Wavelength / nm

Int.

Int.

1.0

0.5

0.0
300

Fluorescence

1.5

Fluorescencemonochromator

0.5

0.0
300

320

340

360

380

400

Wavelength / nm

Fig. 1 Effect of cuvette shape on IFE reductiona) 1 mg/mL Herceptin®, optical path length: 10 mm, b) 10 mg/mL Herceptin®, optical path length: 1 mm

Measurement Conditions<Near-UV/CD measurement>
Bandwidth:
D.I.T.:
Accumulation:

1.0 nm4 sec.
9

Scanning speed:
Data pitch:
Optical path length:

20 nm/min.
0.1 nm
1 mm

Accumulation:
Data pitch:
Optical path length:

4
1 nm
1 mm

<Fluorescence measurement>
Excitation bandwidth: 2.0 nm
Excitation wavelength: 280 nm
Emission bandwidth: 10 nm
D.I.T.:
1 sec.

Results

Confirmation and correction of inner filter effects
To measure the near-UV CD spectra of antibodies, an optical path length of 10 mm and a concentrationof about 1 mg/mL are considered optimal. When measuring fluorescence spectra under these conditions,the excitation light is absorbed by the sample and does not reach the center of the cuvette, resulting in asignificant decrease in fluorescence spectral intensity (Fig. 1a).
This phenomenon is called the Inner-Filter Effect (IFE). In this report, to suppress the IFE, the optical path
length and sample concentration were set to 1 mm and 10 mg/mL, respectively (Fig. 1b), thereby reducing the
IFE and achieving both near-UV CD and fluorescence spectrum measurement conditions.
Fig. 2 shows the concentration dependence of the fluorescence spectral intensity for Herceptin®. Fig. 2ashows the measured fluorescence spectrum, where it can be seen that a good signal-to-noise ratio was
obtained. On the other hand, Fig. 2b shows the fluorescence intensity at 340 nm plotted against concentrationand indicates that the linearity between the concentration and fluorescence intensity is impaired. This isthought to be due to the IFE, which could not be eliminated using the 1 mm optical path length cuvette alone.
Therefore, IFE correction6) was performed using Equation (1).

27

1.0
0.9

0.4

2.0

0.7

1.5

0.6

Int.

Int.@340 nm

0.6

c)

2.0

0.8

0.8

Int.

2.5

b)

Int.@340 nm

a)

1.0

1.0

0.5

d)

1.5

1.0

0.4
0.2

0.5

0.3

0.0
300

0.2
350
Wavelength / nm

5-1
Concentration / mgmL

400

10

0.5

0.0
300

350
Wavelength / nm

5-1
Concentration / mgmL

400

10

Fig. 2 Confirmation of IFE correction under near-UV CD and fluorescence spectral measurement conditions.
a) and b): Fluorescence spectra and intensity at 340 nm of Herceptin® c) and d): IFE-corrected fluorescence spectraand intensity at 340 nm of Herceptin® 1 mg/mL (gray), 5 mg/mL (red), and 10 mg/mL (blue)

2

a)

3.5

0

3.0

-1

2.5

-2

Int.

CD / mdeg

1

4.0

-3

Herceptin®
MabThera®

2.0
1.5

-4
-5

b)

Phe

Tyr

1.0

Trp
Herceptin®
MabThera®

-6
260

280

300

320

0.5
340

0.0
300

Wavelength / nm

320

340

360

380

400

Wavelength / nm

Fig. 3 Orthogonal assessment of tertiary structure of Herceptin® and MabThera®
a ) Near-UV/CD spectrum, b) Fluorescence spectrum

Fcorr = Fobs • 10 Absex+ Absem
2
Fcorr: Fluorescent intensity after correlation
Fobs: Measured fluorescent intensity
Absex, Absem: Absorbance at excitation and emission wavelength
Absex is the absorbance at 280 nm, which is the excitation wavelength when measuring the fluorescence
spectrum, and therefore can be obtained from the absorption spectrum acquired by the CD spectrometer at
the same time as the near-UV CD spectrum. Since Absem is the absorbance at the fluorescence wavelength,an absorption spectrum in the wavelength range of the fluorescence spectrum (300 to 400 nm) is required.
However, since the measurement range for the near-UV CD spectrum is 250 to 340 nm, a separate measurementof the 300 to 400 nm absorption spectrum is required. In the case of proteins, the absorbance at 300 to 400
nm is extremely small, so Absem can be approximated as 0, and the information necessary for IFE correction
can be obtained only by measuring the near-UV CD spectrum. The results of IFE correction of fluorescence
spectra using this method show that the fluorescence intensity at 340 nm has a highly linear dependence on
the concentration, with a correlation coefficient of 0.999 (Figs. 2c and 2d). Thus, the fluorescence spectrum
can be corrected and a high-quality fluorescence spectrum can be obtained together with the CD spectrum.

28Comparison of tertiary structures of antibody drugs
Near-UV/CD and fluorescence spectra of Herceptin® and MabThera® are shown in Fig. 3. The near-UV CD
spectrum is normalized by the absorbance at 280 nm, and the fluorescence spectrum is normalized by theabsorbance at 280 nm after IFE correction is performed, as for the near-UV CD spectrum.
In the near-UV CD spectra, there are significant differences in the spectral shape at 295 nm, 280 nm and near
260 nm for the two antibody drugs. These differences indicate different environments around the side chainsof amino acids for tryptophan, tyrosine and phenylalanine, respectively, indicating that the two antibody drugs
form different tertiary structures (Fig. 3a). The fluorescence spectra also show differences in peak position andintensity for the two antibody drugs (Fig. 3b). This reflects the difference in polarity around tryptophan, and aswith the near-UV CD spectrum, indicates that the two antibody drugs form different tertiary structures. These
results confirm the differences in the tertiary structure of Herceptin® and MabThera® by using two different
measurement principles.

Conclusions
The J-1500 CD/fluorescence measurement system has made it possible to detect differences in the tertiary
structure of Herceptin® and MabThera®, using orthogonal methods based on different principles. This system
can detect even slight differences in tertiary structure with high sensitivity, making it effective for development
and quality control of antibody drugs.

Additional Information• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/

References
1) ICH Q5E, Comparability of Biotechnological/ Biological Products Subject to Changes in their Manufacturing
Process (2004)
2) U.S. Food and Drug Administration: “Development of Therapeutic Protein Biosimilars: Comparative Analytical
Assessment and Other Quality-Related Considerations”, <https://www.fda.gov/regulatoryinformation/
search-fda-guidance-documents/development-therapeutic-protein-biosimilars-comparativeanalyticalassessment-and-other-quality>, (accessed 2022.07.28)
3) European Medicines Agency: “Guideline on similar biological medicinal products containing biotechnologyderived proteins as active substance: quality issues (revision 1)” <https://www.ema.europa.eu/en/
documents/scientific-guideline/guideline-similar-biological-medicinal-products-containingbiotechnologyderived-proteins-active_en-0.pdf>, (accessed 2022. 5. 25).
4) Department of Biotechnology, Government of India: "Guidelines on similar biologics: Regulatory requirements
for marketing authorization in India" (2012)5) Center for drug evaluation of China National Medical Product Administration: "Technical guideline fordevelopment and evaluation of biosimilars" (2015).
6) J. R. Lakowicz: Principles of Fluorescence Spectroscopy, (2006), (Springer, New York)

About the Author
Taiji Oyama is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
IR, Raman, UV-vis, and fluorescence spectroscopy.

29

SIMILARITY STUDIES | ORTHOGONAL ASSESSMENT

4-way Orthogonal HOS Similarity Assessment of
Biosimilar Using Multi Spectroscopic Techniqueand Statistical Calculation
Rituximab
MabThera®
(innovator)

JWQHOS-531

Far-UV CD
Near-UV CD

RIABNI™
(biosimilar)

Mean MeanRIABNI™

ORTHOGONALASSESSMENT?

MabThera®

Raman
SampleFTIR

Measurement

-2SD

2SD

-2SD

2SD

Distance

Spectral similarity test

Graphical abstract

Introduction
The market for therapeutic antibodies has been dramatically expanding over the past decades. Antibody
drugs exhibit therapeutic effects, such as inhibiting the growth of malignant tumor cells and the activity of
immune cells by binding antigens expressed in target cells with high affinity and specificity. Such antibody
drugs are expected to be effective therapeutic agents for unmet medical needs. Many antibody drugs arebeing used worldwide, and along with leading antibody drugs called innovators, many biosimilars, which havethe same amino acid sequence as the innovators but are produced using different expression cells, are also
beginning to be used.
Biosimilars tend to be thought of as having the same function and structure as the innovators because
they have the same amino acid sequence; however, antibodies are subjected to various stimuli and posttranslational modifications during the production process, which may result in a significant loss of function.
The main factors are thermal stimulation and fragmentation by proteases; therefore, measuring changes inantibody structure due to stimulation or modification is an essential step in R&D and quality control of antibody
drugs. Guidelines issued by the U.S. Food and Drug Administration (FDA), European Medicine Agency (EMA)
and other regulatory agencies indicate the importance of measuring Critical Quality Attributes (CQAs) oftherapeutic antibodies by analytical methods in the quality assessment of biosimilars.1–4)
In addition, these guidelines state that a CQA should be evaluated using multiple analytical methods withdifferent principles, i.e., orthogonal methods, rather than using a single analytical method.

Innovator

Higher Order Structure

Biosimilar
ClinicalSecondary structure

PK/PD
FT-IR

FarUV/CDFL

PreclinicalFunctional
Characterization

Physicochemical
Characterization
Fig. 1 The importance of physicochemical characterization in biosimilar development

30

NearUV/CD

Raman
Tertiary structure

Orthogonal Assessment

As shown in Fig. 1, it is recommended to use far-UV CD and FTIR for the evaluation of secondary structure,and near-UV CD, Raman, fluorescence, or two or more of these methods for the analysis of tertiary structure.
Furthermore, there is the requirement for an objective assessment of structural similarity described in ICHQ5E. The FDA's guidance places the most important Tier 1 method on objective and statistical evaluation ofanalytical similarity between biosimilars and their innovators. There is a growing demand for a process toobjectively determine the similarity of spectra.5,6)
Here we report the results of a higher-order structure (HOS) similarity assessment of Herceptin® (the innovatorof Trastuzumab), MabThera® (the innovator of Rituximab) and RIABNI™ (a biosimilar to MabThera®) using the
HTCD Plus high-throughput CD spectrometer, FT/IR-4X FTIR spectrometer, NRS-4500 Raman microscope andthe qHOS program.
Keywords
Circular Dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, Attenuated Total
Reflectance (ATR), Raman spectroscopy, similarity assessment, Herceptin®, MabThera®, trastuzumab, rituximab,
therapeutic antibody, biosimilar, biopharmaceutical, higher-order structure (HOS), secondary structure, tertiary
structure, orthogonal assessment, high-throughput screening

Experimental
Sample• Trastuzumab
Herceptin® (Roche): powder dissolved in ultrapure water to 10 mg/mL
Additives: trehalose hydrate 4.7 mg/mL, L-histidine hydrochloride hydrate 0.11 mg/mL, L-histidine 7.4 x 10-2
mg/mL, polysorbate 2.1 x 10-2 mg/mL• Rituximab
MabThera® (innovator, Roche) and RIABNI™ (biosimilar, AMGEN) 100 mg/10 mL
Additives: sodium citrate dihydrate 7.4 mg/mL, sodium chloride 9.0 mg/mL, sodium hydroxide 9.0 mg/mL,
polysorbate 80 0.7 mg/mL
Measurement and analysis system
HTCD Plus high-throughput CD measurement system• Fully automated measurement of up to 192
samples.
• Pre-registered flush method for protein or DNA/RNA samples can be selected to eliminate
sample carry-over.

FT/IR-4X FTIR spectrometer with ATR PRO 4X single-reﬂection ATR• High S/N and resolution with small body size.
• One drop (approx. 10 µL) ATR measurement

31

NRS-4500 Raman spectrometer• Microanalysis• Chemical imaging

JWQHOS-531 qHOS
• Statistical HOS similarity test program developed
by JASCO.
• High-sensitive detection of spectral difference bynoise-weighted Euclidean distance.
• Support for spectra obtained by multiple
instruments.

Measurement Conditions<Far-UV/CD measurement>
Optical path length:
Bandwidth:

0.2 mm
1.0 nmAuto washing:
Scanning speed:On
20 nm/min<Near-UV/CD measurement>
Optical path length: 0.5 mm
Bandwidth:
1.0 nmAuto washing:
Scanning speed:On
50 nm/min<IR measurement>
Detector:
ATR crystal:

Resolution:
Accumulation:

4 cm-1
128

Exposure:
Number of scans:

45 sec.
32

TGS
Diamond<Raman measurement>
Laser:
532 nmGrating:
900 gr./mm
Analysis conditions
Distance calculation method:
Euclidean distance
Weighting:
Noise

Statistical testing:Significance level:

Welch's t-test*
0.05*Welch’s t-test tests for significant differences in spectral distances, taking into account the variances in bothstandard and sample spectral distances. Data for multiple standard and unknown samples are needed. Thismethod is used for comparing the HOS of innovators and biosimilars, the HOS for antibody drugs before andafter manufacturing process changes and for lot inspections.

32

Results
First, to confirm that this system can discriminate between the tertiary structures of antibody drugs with different
targets and formulation conditions, we determined the similarity of the tertiary structures of MabThera®, theinnovator of Rituximab, and Herceptin®, the innovator of Trastuzumab (Fig. 2). The shapes of the near-UV/
CD spectra of MabThera® and Herceptin® differ significantly (Fig. 2a). The Cotton effect at around 260,
280 and 290 nm reflects the intermolecular interactions and environment of aromatic amino acids, such as
phenylalanine, tyrosine and tryptophan, respectively, indicating that the tertiary structures of these antibody
drugs are different. MabThera® and Herceptin® have similar amino acid sequences and the difference is onlyin the Fv region, but the near UV/CD spectra can sensitively detect the difference in their tertiary structures.
Similarly, the average distances between MabThera® and Herceptin® calculated from the CD spectra showa large difference (Fig. 2b). The p-value obtained from the t-test is below the significance level of 0.05,
indicating that Herceptin® has a different tertiary structure to MabThera®. These results show that the system
can clearly distinguish differences in the tertiary structure of different antibody drugs.
Near-UV/CD

a)

b)

0

Mean

Herceptin®

PheCD / mdeg

Mean

-2

-2SD

2SD

0.6

0.8

MabThera®
Trp-4Herceptin®
MabThera®

Tyr
260

280

300

320

0.0

0.2

0.4

1.0

Distance

340

Wavelength / nm

Result of t-test
t-value = 49
p-value = 0.0

Similarity failed

Fig. 2 Similarity assessment of the tertiary structure of MabThera® and Herceptin®, antibody drugs with different targeting andformulation conditions.

Far-UV/CDa)

MabThera®
RIABNI™
2SD

0.002

d2Int./dλCD / mdeg

2

ATR FT-IR

b)

0

0.000
-0.002
MabThera®
RIABNI™
2SD-0.004

-2
200

210

220

c)
RIABNI™

240

250

Result of t-test

Wavelength / nm

t-value = 1.08
p-value = 0.15

Mean Mean

Similarity passed

1700

0.02

2SD

2SD

0.04
Distance

0.06

0.08

0.10

1660
1640
1620
-1
Wavenumber / cm

-2SD

-2SD

0.00

1600

Result of t-test
t-value = -1.3
p-value = 0.106

Similarity passed

Mean Mean

MabThera®
-2SD

1680

d)
RIABNI™

-2SD

MabThera®

0.00

230

2SD

2SD

0.01
Distance

Fig. 3 Orthogonal similarity assessment for the secondary structure of MabThera® and RIABNI™

33

Near-UV/CD

CD / mdeg

0

Phe

0.000

Tyr

-2.5
Trp

-5.0

260

280

MabThera®
RIABNI™
2SD

300

320

340

Mean Mean

-2SD

0.00

Result of t-test

2SD

-2SD
0.02

0.06

1600

1400

0.08

0.10

1200

Raman shift / cm-1

1000

800

Result of t-test
t-value = -1.3
p-value = 0.106

Mean Meand)

Similarity passed

RIABNI™

MabThera®

Distance

Tyr
Phe

-2SD
2SD

0.04

Trp
MabThera®
RIABNI™
2SD

1800

t-value = 1.098
p-value = 0.15

Similarity passed

RIABNI™

MabThera®

-0.002-0.004

Wavelength / nm

c)

Raman

b) 0.002
d2Int./dλ

a)

2SD

-2SD

2SD

0.00

0.006
Distance

Fig. 4 Orthogonal similarity assessment for the tertiary structure of MabThera® and RIABNI™

Fig. 3 shows the results of orthogonal similarity assessment of the secondary structure. Both MabThera® and
RIABNI™ Far-UV/CD spectra show a negative Cotton effect at 217 nm, which is characteristic of β-sheets (Fig.
3a). IR spectra around 1700 to 1600 cm-1 are derived from amide I, and the spectral shape is known to change
reflecting the secondary structure (Fig. 3b). The secondary differential spectrum of amide I shows a negative
peak around 1640 cm-1, which is characteristic of β-sheets, indicating that the structure is rich in β-sheets, as
indicated by the CD spectrum. Furthermore, both the CD and IR spectra of MabThera® and RIABNI™ show
good agreement in spectral shape.
The distributions of the distances between MabThera® and RIABNI™ are close to each other (Figs. 3c and 3d).
The p-value is larger than the significance level of 0.05.
Fig. 4 shows the results of orthogonal similarity assessment of the tertiary structure. Although the near-UV/CD
spectra of MabThera® and Herceptin® show significant differences despite most of the amino acid sequences
being identical, the near-UV/CD spectra of MabThera® and its biosimilar, RIABNI™, show very good agreement(Fig. 4a).
The same is true for the Raman spectra: the Raman second derivative spectra at 1800 to 700 cm-1 including
tryptophan (around 1550 cm-1), phenylalanine (around 1000 cm-1) and tyrosine (around 900 cm-1) are identicalfor MabThera® and RIABNI™. The entire spectrum below 1600 cm-1 is in agreement with those for MabThera®
and RIABNI™ (Fig. 4b).
The distribution of the distances between
MabThera® and RIABNI™ are close to each other(Figs. 4c and 4d). The p-value is larger than thesignificance level of 0.05.
The similarity of the biosimilar and innovator
HOS was confirmed in the secondary and tertiary
structures.

34

Table 1. Raman shift and assignments to tertiary structure for protein
Raman shift

Assignment-11555 cm

Tryptophan

1000 cm-1

Phenyl alanine

850 cm-1

Tyrosine-1

750 cmTryptophan

Conclusions
By combining the latest HTCD Plus, FT/IR-4X and NRS-4500 with qHOS, we were able to comprehensively
assess the HOS similarity of biosimilars against innovators in an orthogonal manner. This system can be used
not only to evaluate the similarity of biosimilars, but also to easily and accurately determine the changes in the
HOS of antibody drugs caused by post-translational modifications and external stimuli.

Additional Information• Application: Orthogonal HOS Similarity Assessment of Biosimilar Using Multi Spectroscopic Techniqueand Statistical Calculation.
https://www.jasco-global.com/solutions/orthogonal-hos-similarity-assessment-of-biosimilar-using-multispectroscopic-technique-and-statistical-calculation/• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/• Product information: FTIR spectrometer.
https://www.jasco-global.com/product-spectroscopy/ftir-spectrometer/• Product information: Raman imaging microscope.
https://www.jasco-global.com/product-spectroscopy/raman/

References
1) U.S. Food and Drug Administration: “Development of Therapeutic Protein Biosimilars: Comparative Analytical
Assessment and Other Quality-Related Considerations”, <https://www.fda.gov/regulatoryinformation/
search-fda-guidance-documents/development-therapeutic-protein-biosimilars-comparativeanalyticalassessment-and-other-quality>, (accessed 2022.07.28)
2) European Medicines Agency: “Guideline on similar biological medicinal products containing biotechnologyderived proteins as active substance: quality issues (revision 1)” <https://www.ema.europa.eu/en/documents/
scientific-guideline/guideline-similar-biological-medicinal-products-containingbiotechnology-derivedproteins-active_en-0.pdf>, (accessed 2022. 5. 25).
3) Department of Biotechnology, Government of India: "Guidelines on similar biologics: Regulatory requirements
for marketing authorization in India" (2012)
4) Center for drug evaluation of China National Medical Product Administration: "Technical guideline fordevelopment and evaluation of biosimilars" (2015).
5) International Conference on Harmonisation (ICH) of Technical Requirements for Registration of
Pharmaceuticals for Human Use. ICH AUTHORIZED MANUFACTURING CHANGES FOR THERAPEUTIC
MABS 833 Harmonised Tripartite Guideline Comparability of Biotechnological/ Biological Products Subject
to Changes in their Manufacturing Process Q5E. (2004)
6) Y. Tsong, X. Dong, and M. Shen: J. Biopharm. Stat., 27(2), 197 (2017)

About the Author
Taiji Oyama is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
IR, Raman, UV-vis, and fluorescence spectroscopy.

35

Spectral Similarity Test - qHOS Program
The qHOS program converts the spectral difference into a parameter called the "Euclidean distance".
By weighting the Euclidean distance to account for noise and spectral variation, differences between
spectra can be detected with high sensitivity ("weighted Euclidean distance"). The qHOS program
allows statistical significance testing to determine whether the differences between spectra are due
to noise, sample preparation errors or are in fact chemically meaningful. Three types of statistical tests
can be selected according to the purpose: Student’s t-test, Welch’s t-test and Equivalence test (TOST).

Features of each testing method
Assay

Features

Student's t-test

This method tests for significant differences in spectral distances, taking into account the variance in standard sample spectral
distances.
Data for multiple standard samples and a single unknown sample are used. This test is used for quality assessment of
manufactured antibody drugs.

Welch's t-test

This method tests for significant differences in spectral distances, taking into account the variances in both standard and sample
spectral distances.
Data for multiple standard and unknown samples are used.
This test is used for comparing the HOS of innovators and biosimilars, the HOS for antibody drugs before and after
manufacturing process changes, and for lot inspections.

Equivalence test
(TOST)

This method tests for significant differences in spectral distances, taking into account the variances in both standard and sample
spectral distances. In this method, a range (equivalence margin) is set in which the distance between the spectrum of eachstandard sample and the average spectrum of standard samples, and the distance between the spectrum of each unknown
sample and the average spectrum of unknown samples, are equivalent.
Data for multiple standard and unknown samples are used.
This test is based on the guidelines for similarity tests published by the FDA.

Calculation of distance between standard andsample spectra

1

Average spectrum forstandard

Sample spectrum
1.0

(

1.0

0.5
0.0
-0.5
210

220

230

240

250

260

Wavelength / nm

2

-

0.5
0.0
-0.5
210

220

230

240

250

260

Wavelength / nm

There is a small difference in the distance between
the standard and the sample

2

)=

0.6
0.4

0.2

0.0
210

2

×

3

1.0

1.0

0.8

0.8

0.6
0.4
0.2
210

220

230

240

Wavelength / nm

250

260

=

220

230

−

240

2
250

Wavelength / nm

Samples

-2SD

260

2

2SD

-2SD

2SD

passedDistance

There is a large difference in the distance between
the standard and the sample
Confidence interval

0.4

Significant

Mean

difference

0.2
0.0
210

220

230

240

Wavelength / nm

250

260

Samples2SD-2SD

Similarity test

Standard
Weighted Euclidean
distance:

2SD

-2SD
0

Confidence =interval

Procedure for spectral similarity assessment.

36

No difference

Similarity test

Standard
0

−

0.6

Mean

Confidence interval

1.0
0.8

Weighting function−

Evaluate difference in distance between
standard and sample

failedDistance

+

2

+

2

: t-value at significance level: Number of sample: Number of standard

STABILITY STUDIES | THERMAL DENATURATION

Thermal Denaturation Analysis of Monoclonal
Antibodies by CD Spectroscopy

Fraction / %CD spectrometer

Monoclonal antibody (mAb)
SamplePeltier cell holder
Temperature / °C

Measurement

Denaturation analysis

Graphical abstract

Introduction
Antibodies, which are proteins, form their structure and exert their activity through a complex series of noncovalent bonds, and may lose their activity due to various external stimuli. Therefore, the evaluation of structural
stability is extremely important in the development and formulation of candidate antibodies. Thermodynamic
stability of antibodies is most frequently evaluated by applying heat stress, and Differential Scanning Calorimetry(DSC) is used to directly measure the calorimetric changes that accompany the transition from the natural state
to the denatured state.1) On the other hand, DSC does not provide detailed information on conformationalchanges induced by heat. CD spectroscopy is an easy and rapid method for obtaining information on thesecondary structure and other higher-order structures of proteins in solution and can be used to evaluate thestructure of proteins under perturbations, such as heat. Therefore, CD spectroscopy is widely used as themethod that can estimate the specific conformational changes of proteins induced by heat.2)
Recently, Andras et al. developed the BeStSel algorithm, which can accurately estimate the proportion of
secondary structure in a protein based on the shape of its CD spectrum. BeStSel has the following features:1) high accuracy in analyzing β-sheet littered proteins such as antibodies, 2) 8 types of secondary structure
categories and 3) high accuracy in classifying CATH.3,4) Therefore, we have employed BeStSel to trace
the structural changes of antibodies induced by thermal stress in detail using temperature dependent CD
spectroscopy.

Keywords
Circular Dichroism (CD) spectroscopy, secondary structure prediction, BeStSel, Herceptin®, trastuzumab, IgG,
monoclonal antibody (mAb), therapeutic antibody, biopharmaceutical, secondary structure, denaturation, heat

Experimental
Sample• Herceptin®(trastuzumab, Roche): powder dissolved in ultrapure water to 0.025 mg/mL• IgG, from human serum (Sigma Aldrich): powder dissolved in ultrapure water to 0.025 mg/mL

37

Measurement and analysis system
J-1500 CD spectrometer• Accurate and efficient normalization of protein
CD spectrum realized by simultaneous CD and
Abs measurements.

PTC-510 single-position Peltier cell holder• Temperature range of 0 to 100 °C with ameasurement probe that can be placed insideor adjacent to the cell and a magnetic stirrer toeliminate thermal gradients.

BeStSel• Secondary structure prediction method
developed by Dr. József Kardos at Eötvös
Loránd University.
• Highly accurate estimation of β-sheet content bydividing β-sheets into four classes.

Measurement Conditions
Bandwidth:
D.I.T.:
Data pitch:
Scanning speed:

1.0 nm2 sec.
0.1 nm50 nm/min.

Accumulation:
Optical path length:
Sample volume:
Temperature range:

45 mm
300 µL
30 to 95 °C

Results
The thermal denaturation spectra of Herceptin® and human IgG are shown (Figs. 1a and 1b). Both antibodies
showed a spectral shape with a minimum around 217 nm, which is characteristic of the β-sheet at 30 °C, but
the peak disappeared as the temperature increased.
In order to analyze these structural changes in detail, secondary structure prediction was performed on thesetemperature change data using BeStSel to determine the change in the ratio of each secondary structurewith temperature. The secondary structure ratio of others, β-strands and random coils at room temperature
was consistent with that for human IgG (PDB ID 1IGT) obtained from the X-ray crystal structure (data notshown). The ratio of others increased while the ratio of β-strand decreased with heating for both Herceptin®
and human IgG (Figs. 1c and 1d). The Tm values for Herceptin® and human IgG estimated from these curves
were consistent with previously reported values.5,6)
38

Herceptin®

b)

MRE / deg cm2 dmol-1

4000
2000

30 °C

0
-2000
95 °C

-4000
-6000
200

c)

210

220
230
Wavelength / nm

240

2000
30 °C
0
-2000

d)

220
230
Wavelength / nm

240

250

Others52.5

Fraction / %Fraction / %

210

55.0

45
40
35

50.0
47.5
45.0

30
25

95 °C

-4000
-6000
200

250

β-strand50

4000

MRE / deg cm2 dmol-1a)Human IgG

42.5

40

60
Temperature / °C

40.0

80

40

60
Temperature / °C

80

Fig. 1 Melting spectra of antibody
Thermal denaturation spectra for a) Herceptin®, and b) human IgG. c) β-strand, d) others fraction of Herceptin® (red)and human IgG (blue)

Distorted
helix

a) 5

Left-twisted
β-strand

b)

d) 24

2

4
3
2

1

0

18
16

20
18
16
14

14

1

50
75
Temperature / °C

Fraction / %

3

22
20
Fraction / %Fraction / %Fraction / %

c) 22

6

Right-twisted
β-strand5

4

0

Relaxed
β-strand

50
75
Temperature / °C

12

12
50
75
Temperature / °C

10

50
75
Temperature / °C

Fig. 2 Results of secondary structure prediction using BeStSel
Predicted secondary structure fraction of a) distorted Helix, b) left-twisted, c) relaxed, and d) right-twisted β-strand of Herceptin®
(red) and human IgG (blue).

Fig. 2 shows the changes in the secondary structure ratios of the helix and the β-strand as a function oftemperature. A large difference was observed between Herceptin® and human IgG. This result indicates thatthe combination of temperature variation spectroscopy and BeStSel can provide detailed information on the
structure of antibodies.

39

Conclusions
By combining BeStSel with temperature change measurements by CD spectroscopy, we were able to precisely
trace the thermally-induced conformational changes of antibodies.

Additional Information• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/

References
1) Daisuke Ama, Jun Hasegawa, Susum Uchiyama and Kiichi Fukui:. Netsu sokutei (Japan), 38(1), 9, (2011)2) Norma J Greenfield: Nat. Protoc, 1(6), 2527 (2006)
3) A. Micsonai, F. Wien, L. Kernya, Y. H. Lee, Y. Goto, M. Réfrégiers, J. Kardos: Proc. Natl. Acad. Sci. U. S. A.,
112, E3095 (2015).
4) A. Micsonai, É. Bulyáki, J. Kardos: Methods Mol. Biol. (N. Y.), 2199, 175 (2021).
5) Leina Moro Perez, Azalia de la Caridad Rodrıguez Taño2, Lazaro Roberto Martın Marquez, Jose Alberto
Gomez Perez, Aisel Valle Garay Rances Blanco Santana: PLoS One, 14(5), e0215442 (2019)6) Jason Moggridge, Kyle Biggar, Neal Dawson and Kenneth B. Storey: Technol Cancer Res Treat., 16(6), 997(2017)

About the Author
Taiji Oyama is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
IR, Raman, UV-vis, and fluorescence spectroscopy.

40

STABILITY STUDIES | THERMAL DENATURATION

Thermal Denaturation Analysis of Monoclonal
Antibodies by IR Spectroscopy
Intermolecularβ-sheetIntermoluecular β-sheet

FTIR with single-reflection ATR

d2A/dλAggregationby heatIntramolecularβ-sheetIntramolecularβ-sheetWavenumber / cm-1

IR spectrum
Measurement

SampleGraphical abstract

Introduction
In recent years, therapeutic antibodies have been actively developed, manufactured and marketed. Antibody
drugs are often used as therapeutic agents to inhibit the proliferation of malignant tumor cells or the activityof immune cells and are highly anticipated for diseases for which complete treatment is not yet available.
Active ingredients of therapeutic antibodies are proteins, which have the potential to undergo aggregation
when exposed to various external stimuli. Thus, efforts have been made to minimize aggregation in R&D andmanufacturing processes of antibody drugs.1)
Infrared (IR) spectroscopy is widely used to analyze the secondary structure of proteins in trace amounts andin a short time. It is used to analyze the ratio of the secondary structure because the shape of the amide I peakvaries depending on the structure of the main chain of the protein (i.e. the secondary structure). In addition, it
has recently been shown that Fourier-transform infrared spectrometer (FTIR) can detect intermolecular β-sheet
formation associated with protein aggregation and is used as a tool for detecting aggregates and elucidating
the mechanism of aggregation.2,3) In this study, we report on the ATR-FTIR detection of aggregation inducedby sample heating of MabThera®, a commercially available antibody drug.
Keywords
Fourier-transform infrared (FTIR) spectroscopy, Attenuated Total Reflectance (ATR), MabThera®, rituximab,
therapeutic antibody, biopharmaceutical, secondary structure, aggregation, denaturation, heat, intermolecularβ-sheetExperimental
Sample• MabThera® (rituximab, Roche) 100 mg/mL• Additives: sodium citrate dihydrate 7.4 mg/mL, sodium chloride 9.0 mg/mL, sodium hydroxide 9.0 mg/mL,
polysorbate 80 0.7 mg/mL

41

FT/IR-4X FTIR spectrometer with ATR PRO 4X single-reﬂection ATR• High S/N and resolution with small body size.
• Protein solution measurement with one drop ofsample (approx. 10 µL) with ATR PRO 4X.
• Easy measurement of suspensions and powder
samples that contain aggregates as well as
solutions

Measurement Conditions
Resolution:
Sample volume:

4 cm-1
10 µL

Accumulation: 100 (2 min.)N2 purge:
Yes

Results
Fig. 1 shows IR spectra measured after MabThera® was heated at various temperatures for 10 min., and thenreturned to room temperature. All spectra are subtracted from the buffer spectrum. Fig. 1a shows that the
spectral shapes for amide I and amide II, which reflect the higher-order structure (HOS) of the protein, change
as the heating temperature increases. Fig. 1b shows the second-derivative spectra at each temperature.
Fig. 1c shows the change in the intensity of the 1640 and 1620 cm-1 peaks as a function of temperature. As
the heating temperature increases, the intensity of the 1640 cm-1 peak derived from intramolecular β-sheets
becomes weaker, while the intensity of the 1620 cm-1 peak derived from intermolecular β-sheets becomesstronger. This behavior is consistent with previous reports showing the formation of intermolecular β-sheetsof antibodies by aggregation.

Conclusions
It was found that FTIR combined with ATR can be used to measure aqueous antibody solutions containing
aggregates, and easily detect structural changes in trace amounts. This system has proven to be a useful
tool for the evaluation of antibody drug aggregates and the elucidation of their molecular mechanisms in thescreening and formulation of antibody candidates.

c)d2A/dλd2A/dλb)

Normalized

a)

1700

1600
Wavenumber / cm-1
25 °C

1500
60 °C

1660

1640
1620
-1
Wavenumber / cm

70 °C

80 °C

1600

40

60
Temperature / °C

1640 cm-1

Fig. 1 Thermal denaturation analysis of MabThera® using FTIR.
a) Normalized IR absorbance spectra, b) second-derivative spectra, and c) transition of inter- and intramolecular β-sheets.

42

1620 cm-1

80Additional Information• Product information: FTIR spectrometer.
https://www.jasco-global.com/product-spectroscopy/ftir-spectrometer/

References
1) Y. Yunyu and L. Zang: Am. Pharm. Rev., 21 (2), (2018). <https://www.americanpharmaceuticalreview.com/
Featured-Articles/348833-Factors-Influencing-Biotherapeutic-Monoclonal-Antibody-Aggregation/>,(accessed 2022. 5. 25).
2) Garrett Baird, Chris Farrell, Jason Cheung, Andrew Semple, Jeffery Blue & Patrick L. Ahl: Protein J., 39(4),318 (2020)
3) Bhavana Shivu, Sangita Seshadri, Jie Li, Keith A Oberg, Vladimir N Uversky & Anthony L Fink:, Biochemistry,
52(31), 5176 (2013)

About the Author
Taiji Oyama is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
IR, Raman, UV-vis, and fluorescence spectroscopy.

43

STABILITY STUDIES | THERMAL DENATURATION

Thermal Denaturation Analysis of Monoclonal
Antibodies by Raman Spectroscopy
Inter molecularβ-sheetRaman spectrometer
80 °C
Aggregates

Int.

Aggregationby heat

50 °C
Non heatedIntra molecularβ-sheet-1

Wavenumber / cm

Raman spectrum
Measurement

SampleGraphical abstract

Introduction
Antibody drugs are currently employed to inhibit the
proliferation of malignant tumor cells or the activityof immune cells, and are prime research candidates
for treatment of conditions which currently have no
medical routes to treatment. Antibody therapeutics
have several factors involving environment,
storage condition and manufacturing that can spur
detrimental behavior, such as aggregation when
exposed to various external stimuli. To develop andprovide conditions which prevent the aggregationof antibody drugs, there is a large amount of R&D
being conducted to identify when, how and why
aggregation occurs during the manufacturing
processes.1–3)

Table 1. Various Raman measurement system
Micro Raman

Fiber Raman

NRS-5500/7500

NRS-4500

RMP-500• Microanalysis• Chemical imaging• High sensitivity• In-situ measurement• Process monitoring• High flexibility

Raman spectroscopy can analyze the secondary and tertiary structures of proteins in solid or aqueous solution
nondestructively and without pretreatment. Because of its wide applicability from microscopic measurement
to process monitoring using fibers, Raman spectroscopy has attracted much attention as a method to evaluate
physical and scientific properties and to monitor the quality of therapeutic antibodies.4)
Here we report a Raman spectroscopic evaluation of tertiary structural changes in MabThera®, a commerciallyavailable antibody drug, due to aggregation.

Keywords
Raman spectroscopy, MabThera®, rituximab, therapeutic antibody, biopharmaceutical, higher-order structure(HOS), secondary structure, tertiary structure, aggregation, denaturation, heat44

Experimental
Sample• MabThera® (rituximab, Roche) 100 mg/mL• Additives: Sodium citrate dihydrate 7.4 mg/mL, sodium chloride 9.0 mg/mL, sodium hydroxide 9.0 mg/mL,
polysorbate 80 0.7 mg/mL

NRS-4500 Raman spectrometer• Desktop micro Raman• High sensitivity• Multi laser excitation• Quick Raman imaging• Full automated and Class 1 laser safety

MSD-462 Micro sampling disc*• Sample volume: 10 µL• Optical path length: 1 mm
* Accessory for J-1500 CD spectrometer

Measurement Conditions
Laser:
Exposure:Slit:

532 nm70 sec.
Φ 100

Accumulation: 20
Grating:
900 gr./mm
Objective:
LMPFLN 50x

Results
Fig. 1 shows Raman spectra of MabThera® without heating, MabThera® heated at 50 °C for 10 min. and theaggregates generated by heating at 80 °C for 10 min. Except for the spectrum of the aggregates, the spectraof the buffer were subtracted. In each spectrum, peaks characteristic of the secondary and tertiary structuresof the protein were clearly observed, as shown in Table 2.

45

Amide I

Trp

Amide IIIPhe

Tyr Trp

Table 2. Raman shift and assignments.5)
Int.

80 °C Aggregates

Raman shift
-1

50 °C
Non heated
1800

1600

1400
1200
1000
Wavenumber / cm-1

800

600

a)

1650 cm

Amide I

1555 cm-1

Tryptophan

1300 cm-1

Amide III-1

Fig. 1 Raman spectra of MabThera® and its aggregates
Blue: Non heated, Orange: Heated at 50 °C for 10 min, Gray: aggregates
generated by heating at 80 °C for 10 min. Each spectrum was normalized
by the amide I band.

b)Assignment

1000 cm

Phenylalanine

850 cm-1

Tyrosine

750 cm-1

Tryptophanc)I850/I830 = 1.6

Int.

Int.

d2A/dλ

I850/I830 = 0.91

80 °C
Aggregates
50 °C

I850/I830 = 0.91
Non heated
1680
1660
Raman shift / cm-1

1600

1550
Raman shift / cm-1

1550

900

850
Raman shift / cm-1

Fig. 2 Enlarged view of a) second-derivative Raman spectra and b,c) Raman spectra
Blue: Non heated, Orange: Heated at 50 °C for 10 min, Gray: aggregates generated by heating at 80 °C for 10 min. Each spectrum
was normalized by the amide I band.

The second-derivative bands for amide I are consistent for the unheated and 50 °C heated samples, buta peak shift is observed in the aggregates, indicating that the secondary structure has changed (Fig. 2a).
The band at 1550 cm-1 derived from tryptophan also shows a change in the band shape of the aggregatescompared to the unheated and 50 °C heated samples, indicating a change in the tertiary structure (Fig. 2b).
Similar changes are observed for the triplet bands derived from tyrosine (Fig. 2c). Curve fitting was applied tothese bands, and the intensity ratio of I850/I830 increased in the aggregates, indicating that the phenolic -OH
group of tyrosine changed from a donor to an acceptor during aggregation process.5)

Conclusions
Raman spectroscopy has proven to be a useful tool for easily evaluating the secondary and tertiary structuresof therapeutic antibodies in aqueous solution, aggregates and other forms. JASCO offers a variety of Raman
spectrometers, including microscopic and fiber type systems, to meet the needs of various applications, fromdevelopment to quality monitoring of antibody drugs.

Additional Information• Product information: Raman spectrometer.
https://www.jasco-global.com/product-spectroscopy/raman/

46

References
1) Y. Yunyu and L. Zang: Am. Pharm. Rev., 21 (2), (2018). <https://www.americanpharmaceuticalreview.com/
Featured-Articles/348833-Factors-Influencing-Biotherapeutic-Monoclonal-Antibody-Aggregation/>,(accessed 2022. 5. 25).
2) Wei Bingchua, Woon Nicholas, Dai Lu, Fish Raphael, et al.: MAbs, 14(1), e2007564 (2022)
3) Ilokugbe Ettah and Lorna Ashton: Antibodies, 7(3), 24 (2018)
4) Bethan S. McAvan, Leo A. Bowsher, Thomas Powell, John F. O’Hara, Mariangela Spitali, Royston Goodacre,and Andrew J. Doig: Anal. Chem., 92(15), 10381 (2020)
5) Nikki Kuhar, Sanchita Sil, Taru Verma and Siva Umapathy: RSC Adv., 8, 25888 (2018)

About the Author
Taiji Oyama is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
IR, Raman, UV-vis, and fluorescence spectroscopy.

47

STABILITY STUDIES | THERMAL DENATURATION

Heat-Induced Denaturation and Refolding Studyof VHH Antibody
VHH antibody

Fraction / %CDR3

CD / mdeg

Camelid
antibody

CD spectromer

CDR1
CDR2

CH2 CH2

6-position Peltier
cell changer

CH3 CH3

SampleTemperature / °C

Temperature / °C

Denatuation curve

Secondary structure prediction

Measurement

Denaturation analysis

Graphical abstract

Introduction

The coronavirus S glycoproteins sit on the surface of the virus particle and facilitate receptor binding andmembrane fusion, which leads to entry of the virus into host cells (Fig. 1).1) Therefore, the S proteins are themain target for therapeutic drug design by inhibiting binding.
VHH antibodies are small, single-domain antibodies corresponding to the variable region of a heavy chain
camelid antibody (see “Sample” in the graphical abstract above). They are highly stable with respect to heat,
have high affinity and binding specificity, a low molecular weight which increases mass production efficiency
and can be easily modified.2) Because of these features, VHH antibodies have been pursued as therapeuticmodalities.2) Since the structure of antibodies may change and cause a loss in their activity depending on theformulation conditions and storage environment, it is necessary to evaluate their stability in order to ensure
quality when they are applied to therapeutic uses. In particular, thermal denaturation is highly reversible
depending on the protein, increasing the need to determine the denaturation temperature and refolding
ability in the stability evaluation of antibody drugs.
Here, we evaluate the thermal stability and refolding ability of three different types of VHH antibodies using a
CD spectrometer. The secondary structure contents were estimated using the BeStSel3,4) program to further
correlate the denaturation temperatures with structural changes to the VHH antibody framework.
Spike Protein
VHH antibody

RBD

S1

FP

S1

HR1
S2
RNA

HR2
TM
CP

SARS-CoV-2

Spike Protein

CH3 CH3

IgG

Fig. 1 Schematics of the spike protein and VHH antibody structures.

Keywords
Circular Dichroism (CD) spectroscopy, secondary structure prediction, BeStSel, VHH antibody, therapeutic
antibody, biopharmaceutical, secondary structure, denaturation, refolding, heat, SARS-CoV-2
48

Experimental
SampleDenaturation study• Protein: anti-SARS-Cov-2 VHH antibody (three types with different sequences)No. 1 (0.4 mg/mL), No. 2 (0.28 mg/mL), No. 3 (0.2 mg/mL)• Buffer: 20 mM PBS
Refolding study• Protein: anti-SARS-Cov-2 VHH antibody (three types with different sequences)No. 1 (0.1 mg/mL), No. 2 (0.1 mg/mL), No. 3 (0.1 mg/mL)• Buffer: 20 mM PBS
Measurement and analysis system
J-1500 CD spectrometer• Accurate and efficient normalization of protein
CD spectrum obtained by simultaneous CD and
Abs measurements.
MPTC-513 automatic 6-position Peltier cell changer• Automated measurements of 6 samples while
maintaining or ramping their temperature.
• Temperature range of 0 to 100 °C.
JWTDA-519 denatured protein analysis program• Tm calculation based on temperature vs. CD
sigmoid curve.

BeStSel• Secondary structure prediction method
developed by Dr. József Kardos at Eötvös
Loránd University.
• Highly accurate estimation of β-sheet content bydividing β-sheets into four classes.

49

Measurement Conditions
Denaturation study
The denaturation curve for the VHH antibody was obtained by heating the sample from 25 to
90 °C while measuring the CD value at 217 nm to track the amount of β-sheet structure. The
onset temperature (To), denaturation temperature (Tm) and end temperature (Te) were calculated
from the obtained denaturation curves.
Wavelength:
D.I.T.:
Temp. ramp rate:

217 nm2 sec.
1 °C/min.

Bandwidth:
Temp. interval:
Temp. range:

1 nm
1 °C
25 to 90 °C

Refolding study
The sample was heated from 25 °C to the end temperature (Te) obtained from the denaturation
curves, and then cooled back to 25 °C. CD spectra were measured at the start temperature,
onset (To), denaturation temperature (Tm), and end temperature (Te). Secondary structure
analysis was performed on the obtained CD spectra using BeStSel and the refolding ability wascalculated from the amount of change in the β-sheet structure at 25 °C before and after heating.
Bandwidth:
D.I.T.:
Data pitch:

1 nm
4 sec.
0.1 nmScanning speed:
Accumulation:
Optical path length:

50 nm/min.
9
1 mm

Results
Denaturation study
Fig. 2 shows the denaturation curve based on the CD value at 217 nm, which reflects changes in the β-sheet
structure. Table 1 shows the onset (To), denaturation temperature (Tm) and end temperature (Te) obtained
from the denaturation curve. Comparing the denaturation temperatures, the thermal stability of the samples
decreases in the following order: No. 3, 1 and 2.

-8

a)

-12

-16

CD / mdeg

CD / mdeg

-14

-18
-20
Fitting curve

-22
-24

b)

-10
-11

-5

-12

-6

-13
-14

40

-7
-8

Fitting curve

-15

No. 1
30

Fitting curve

-9

No. 2
50
60
70
Temperature / °C

80

90

-16

30

40

No. 3
50
60
70
Temperature / °C

80

90

-10

Fig. 2 Temperature vs. CD denaturation curves.
Anti-SARS-Cov-2 VHH antibody a) No. 1 (0.4 mg/mL), b) No. 2 (0.28 mg/mL), and c) No. 3 (0.2 mg/mL).

50

c)

-4

CD / mdeg

-10

-3

-9

30

40

50
60
70
Temperature / °C

80

90

Table 1. Onset temperature (To), denaturation temperature (Tm), and end temperature (Te).

Sample

57

65

75

57

63

75

No. 3

65

73

80

50

Cooling

40

3
2

25

57

65

30
20

0

75

25

40

4
3
2

57

63

20

40

4
3
2

57

63

65

73

Temperature / °C

80

73

Temperature / °C

80

25

57

63

40
30
20

0

75

25

57

63

75

Temperature / °C
60
50

10

0

75

10

5

65

65

50

15

20

57

Temperature / °C

20

25

25

Temperature / °C

30

0

0

75

10

0

75

10

1

65

5

Fraction / %5
Fraction / %Fraction / %

50

57

60

Temperature / °C

6

25

25

15

25

20

Temperature / °C

30

0

75

40
30

10

20

Temperature / °C

0

0

75

10

1

No. 3

65

Fraction / %5
Fraction / %Fraction / %

50

25

10

Temperature / °C

6

0

57

Others505Temperature / °C

No. 2

60

15

10

1

Turn

Fraction / %4

20

Fraction / %Heating

Sheet

Fraction / %

Helix

5

0

Te / °C

No. 2

Fraction / %Fraction / %No. 1

Tm / °C

No. 1

Fraction / %6

To / °C

40
30
20
10

25

65

73

Temperature / °C

80

0

25

65

73

80

Temperature / °C

Fig. 3 Changes in estimated secondary structure content as a function of temperature. Red bars show changes in heating process,while blue bars show changes in cooling process.

Refolding study
In order to reduce the effect of aggregation, the sample was heated to the end temperature (Te) determinedfrom the previously obtained denaturation curves and cooled immediately after the sample became denatured.
Fig. 3 shows the secondary structure analysis result of the obtained CD spectra using BeStSel.

51

Conclusions
For all samples, when the temperature increases, the β-sheet content decreases, and the proportion of turnand other structures increases. In addition, the proportion of α-helix structures increases with increasing
temperature for sample No. 1 only. The thermal reversibility study indicates that the β-sheet, turn, and otherstructures in all three samples are reversible, as well as the α-helix structure of No. 1. While the framework of
VHH antibodies is considered to be conserved, the structure of the Complementarity Determining Regions(CDR) that bind to the antigens is variable. The framework is primarily composed of β-sheet structures, andany changes in structure can potentially affect the structure of the CDR. Focusing on the percentage of theβ-sheet secondary structure, the effective refolding before and after heating was calculated for samples No. 1,
2 and 3 and were 97.3 %, 95.0 % and 93.5 %, respectively. Therefore the ability of the samples to refold after
heating and cooling assumes the following order, No. 1, 2 and 3, further supporting the melting temperature
data shown in Table 1.

Additional Information• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/• BeStSel web server:
https://bestsel.elte.hu/index.php

References
1) L. Duan, Q. Zheng, H. Zhang, Y. Niu, Y. Lou, H. Wang: Front. Immunol., 11:576622, (2020).
2) R. J. Hoey, H. Eom, J. R. Horn: Exp. Biol. Med., 244, 1568 (2019).
3) A. Micsonai, F. Wien, L. Kernya, Y. H. Lee, Y. Goto, M. Réfrégiers, J. Kardos: Proc. Natl. Acad. Sci. U. S. A.,
112, E3095 (2015).
4) A. Micsonai, F. Wien, E. Bulyaki, J. Kun, E. Moussong, Y.H. Lee, Y. Goto, M. Refregiers, J. Kardos: Nucleic
Acids Res., 46, W315 (2018).

About the Author
Satoko Suzuki is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
CPL, UV-vis, and fluorescence spectroscopy.

* VHH antibody was kindly provided by RePHAGEN https://rephagen.com/

52

Denatured Protein Analysis ProgramThis program evaluates a single protein substance in solution that is in equilibrium at a certain
temperature, and enables calculation of the thermodynamic parameters (Tm, ΔH and ΔS) of its proteinfrom the data with variable temperatures.
𝑁𝑁𝑁𝑁

𝐾𝐾𝐾𝐾𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷

ln 𝐾𝐾𝐾𝐾𝐷𝐷𝐷𝐷 = −∆𝐻𝐻𝐻𝐻 1 ∆𝑆𝑆𝑆𝑆� +𝑅𝑅𝑅𝑅 𝑇𝑇𝑇𝑇 𝑅𝑅𝑅𝑅

KD is the ratio of [N] and [D], and is calculated by using the experimental data, which is the measured
CD values θi as a function of temperature Ti. This program approximates θi by the following equation,and calculates the KD.
�𝜃𝜃𝜃𝜃(𝑇𝑇𝑇𝑇)=

𝜃𝜃𝜃𝜃𝑁𝑁𝑁𝑁 𝑇𝑇𝑇𝑇 + 𝐾𝐾𝐾𝐾𝐷𝐷𝐷𝐷 (𝑇𝑇𝑇𝑇)𝜃𝜃𝜃𝜃𝑁𝑁𝑁𝑁 𝑇𝑇𝑇𝑇
1 + 𝐾𝐾𝐾𝐾𝐷𝐷𝐷𝐷 (𝑇𝑇𝑇𝑇)
𝑁𝑁𝑁𝑁𝜃𝜃𝜃𝜃𝑛𝑛𝑛𝑛 𝑇𝑇𝑇𝑇 = � 𝑎𝑎𝑎𝑎𝑖𝑖𝑖𝑖,𝑛𝑛𝑛𝑛 𝑇𝑇𝑇𝑇
𝑖𝑖𝑖𝑖=1𝑁𝑁𝑁𝑁−𝑖𝑖𝑖𝑖

𝑁𝑁𝑁𝑁

𝐾𝐾𝐾𝐾𝐷𝐷𝐷𝐷 𝑇𝑇𝑇𝑇 = � 𝐴𝐴𝐴𝐴𝑖𝑖𝑖𝑖,𝐷𝐷𝐷𝐷 𝑇𝑇𝑇𝑇 𝑁𝑁𝑁𝑁−𝑖𝑖𝑖𝑖
𝑖𝑖𝑖𝑖=1𝑁𝑁𝑁𝑁 = 2JWTDA-519 denatured protein analysis program

53

STABILITY STUDIES | PH & SALT DENATURATION

pH and Salt-Induced Denaturation Study of VHH
Antibody using CD Spectroscopy
HTCD Plus
high-throughput CD
measurement system

VHH antibody
CDR1
CDR3

CDR2

t-value

Camelid
antibody

CH2 CH2

NaCl / mM

pH

CH3 CH3

6-position Peltier cell changer
SampleMeasurement

Spectral similarity test
Denaturation analysis

Graphical abstract

Introduction
Since their first approval in 1986, therapeutic antibodies have dramatically expanded their market over the past
decades.1) Antibodies are known to lose their native structure due to changes in the surrounding environment,such as temperature, pH and salt concentration, ultimately resulting in the loss of their function. Therefore, thekey to achieving a robust life cycle of therapeutic antibodies is to understand the effects of formulation andstorage conditions on their structure.2)
Circular Dichroism (CD) spectroscopy is a classic technique for monitoring the structural stability of biologicalmacromolecules, such as proteins and nucleic acids. It is especially well-known for providing fast secondary
structure prediction of proteins under homogeneous conditions.
In this study, the CD spectral difference between denatured VHH and native-state VHH was statisticallyanalyzed using the qHOS program. To assess the effects of pH and salt concentration, the VHH was measured
under 63 different solvent conditions using an HTCD Plus, high-throughput CD measurement system, thatprovides fully automated sample loading to flow washing. The denaturation temperature (Tm) for VHH under
the different solvent conditions was obtained from the CD denaturation curves. The spectral difference andthe Tm showed a high correlation, suggesting that pre-screening of the pH and salt concentration can be
performed before time-consuming thermal screening.
Keywords
Circular Dichroism (CD) spectroscopy, similarity assessment, VHH antibody, therapeutic antibody,
biopharmaceutical, denaturation, pH, salt concentration, heat, high-throughput screening, SARS-CoV-2

Experimental
Sample• Protein: anti-SARS-Cov-2 VHH antibody 0.5 mg/mL• Buffer*: 50 mM citric acid for pH 3, 4, 5, and 6, 50 mM phosphoric acid for pH 7 and 8, and 50 mM CHAPS
for pH 9, 10, and 11.
*NaCl concentration varied as follows: 10, 100, 200, 400, 600, 800, and 1000 mM.

54

Measurement and analysis system

J-1500 CD spectrometer• Accurate and efficient normalization of protein
CD spectrum obtained by simultaneous CD and
Abs measurements.

HTCD Plus high-throughput CD measurement system• Fully automated measurement of up to 192
samples.
• Pre-registered flush method for protein or DNA/RNA samples can be selected to eliminate
sample carry-over.

MPTC-513 automatic 6-position Peltier cell changer• Automated measurements of 6 samples while
maintaining or ramping their temperature.

JWQHOS-531 qHOS
• Statistical HOS similarity test program developed
by JASCO.
• High-sensitive detection of spectral difference bynoise-weighted Euclidean distance.
• Support for spectra obtained by multiple
instruments.

Measurement Conditions
pH and salt induced denaturation
Bandwidth:
D.I.T.:
Data pitch:

1 nm
2 sec.
0.1 nmScanning speed:
Accumulation:
Optical path length:

100 nm/min.
4
0.2 mm55

Thermal denaturation
Bandwidth:
D.I.T.:
Temp. interval:

1 nm
2 sec.
1 °C

Temp. ramp rate:
Optical path length:
Wavelength:

1 °C/min.
1 mm
236 nmSpectral Distance Test
• Reference: 10 samples of 0.5 mg/mL anti-SARS-Cov-2 VHH antibody at pH 7 and 200 mM NaCl were
measured as a representative native state to create a reference set.
• Subjects: 0.5 mg/mL VHH was prepared at different pHs using the buffers described in the “Sample”
section.
See page 36 for more details of the spectral similarity test using the qHOS program.

Results
pH- and salt-induced denaturation
CD spectra of VHH for 200 mM NaCl and different pH values are shown in Fig. 1a. The CD spectra for pH 3
and pH 4 display a negative peak at 217 nm, reflecting the β-sheet structure, and a peak at 227 nm, which isconsidered to be due to β-sheet interactions. These peaks deviate from the native VHH CD spectrum for pH 7.
CD spectra of VHH were measured while changing the NaCl concentration from 10 to 1000 mM and the pHfrom 3 to 11. The CD spectrum changes from the native state (200 mM NaCl and pH 7) were quantified using
the qHOS program. Fig. 1b shows the relationship among the VHH antibody pH, the NaCl concentration andthe t-value. The t-value is small in the pH range from 6 to 9, and the structure is not significantly different fromthat of the reference sample at pH 7 and an NaCl concentration of 200 mM. On the other hand, at pH 5 andlower, or pH 10 and higher, the t-value is large, indicating that the structure has changed. It can also be seenthat the structure changes as the NaCl concentration increases.
Thermal denaturation
Using the 6-position Peltier cell changer, which automatically rotates 6 cells while changing the temperature,
the denaturation temperature (Tm) was obtained for each combination of pH and NaCl concentration. Tm wascalculated from the thermal denaturation curves obtained by monitoring the CD signal at 236 nm. Fig. 2
shows the relationship between the t-value shown in Fig. 1a and the Tm for each solvent condition.

a)

4

pH 7

1

t-value

CD / mdeg.

2

b)

pH 8
pH 9
pH 10
pH 11

pH 3
pH 4
pH 5
pH 6

3

0
-1
-2
-3

210

220

230

240

Wavelength / nm

250

260

140
120
100
80
60
40
20
0

1000
800
600
4

400

6

8
pH

10

NaCl / mM

200
12

0

Fig. 1 Screening of pH and NaCl concentration.
a) CD spectra of VHH antibody for 200 mM NaCl and different pH. b) relationship between pH and NaCl concentration of VHH
antibodies and t-value.

56

120
100

t-value

80
60
40
20
0
45

50

55

Tm / °C

60

65

70

Fig. 2 Relation between denaturation temperature and t-value.

Conclusions
The combination of the HTCD Plus, an automated CD measurement system and the qHOS program isa breakthrough approach to statistically analyzing the effects of solvent conditions. This approach isespecially convenient for understanding the robust conditions for preparation, synthesis and storage ofbiopharmaceuticals.
In the thermal denaturation study, there was a high correlation between the t-value and the Tm. This implies thatthe spectral difference test is effective for primary screening of a large number of buffers before conducting
a time-consuming thermal stability evaluation.

Additional Information• Application: Structure Evaluation of G-quadruplex aptamers Using High-Throughput CD Measurement
System and Principal Component Analysis.
https://www.jasco-global.com/solutions/structure-evaluation-of-g-quadruplex-aptamers-using-highthroughput-cd-measurement-system-and-principal-component-analysis/• News: JASCO releases new CD accessory HTCD Plus.
https://www.jasco-global.com/news/jasco-releases-new-cd-accessory-htcd-plus/• Product information: CD spectrophotometer.
https://www.jasco-global.com/product-spectroscopy/circular-dichroism/

References
1) R. M. Lu, Y. C. Hwang, I. J. Liu, C. C. Lee, H. Z. Tsai, H. J. Li, H. C. Wu: J. Biomed. Sci., 27:1, (2020).
2) L. L. Liu, L. Wang, J. Zonderman, J. C. Rouse, H. Y. Kim: J. Pharm. Sci., 109, 3223 (2020).

About the Author
Satoko Suzuki is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
CPL, UV-vis, and fluorescence spectroscopy.

* VHH antibody was kindly provided by RePHAGEN https://rephagen.com/* This application was developed under the guidance of Professor Kohei Tsumoto of the
University of Tokyo.
57

FORCED DEGRADATION STUDIES (FDS)

Monitoring of Aggregates and Degradation
Products by SEC in FDS of Antibody Drugs
Separation by SEC

Heat
Freeze-thaw

Glycation(Lys)

Agitation

Reduction
Oxidation(Met, Trp)

Packing particle
in SEC column

Aggregates &Multimers

Intensity

Deamidation
(Asn
Asp)

Aggregates

Monomer

Fragments

pHLight
irradiation

Major degradiation
pathway

Sample (Forced degradation study FDS)

Retention time

Inert LC-UV system

Measurement

UV chromatogram

Graphical abstract

Introduction
Since the first monoclonal antibody (mAb) was approved by the U.S. Food and Drug Administration (FDA) in
1986, the market for therapeutic proteins has expanded rapidly, and antibody drugs have become predominanttreatment modalities for various diseases over the past 25 years.1) The stability of a drug molecule is very
important because it affects the safety and efficacy of the drug. In products with higher-order structures (HOS),
such as antibody drugs, the maintenance of molecular structure and biological activity depends on noncovalent bonds, as well as covalent bonds of the molecule and is affected by various environmental factors.2)
In the guidelines of the FDA and International Council for Harmonization of Technical Requirements for
Pharmaceuticals for Human Use (ICH), the need for stability test data is described to understand how thequality of drug substances and drug products changes over time in different environments 3, 4). Therefore, aForced Degradation Study (FDS) to purposely deteriorate and degrade the drugs is conducted at each stage
from research and development through marketing approval of antibody drugs. FDS is an effective method
for clarifying the degradation pathways and structures of degradation products of drug substances and drug
products and guaranteeing stability. Also, it is useful for the development of manufacturing processes andselection of storage conditions and containers.5, 6) Common test conditions for FDS include heating, freezing
and thawing, agitation, pH, light irradiation, oxidation, reduction and glycation.7, ) Since one of the degradation
pathways that occurs under many of these conditions is aggregates that are deeply related to immunogenicity,
Size Exclusion Chromatography (SEC) that can successfully separate and detect monomer and aggregates,
including multimers, is used as an analytical method.5, 7)
In this article, we performed various forced degradation tests on MabThera® (rituximab), which is a therapeutic
drug for non-Hodgkin's lymphoma, and measured untreated and forcibly degraded MabThera® by SEC in order
to compare the changes in the amount of monomer and aggregates, including multimers and degradation
products.
Keywords
High Performance Liquid Chromatography (HPLC), Size Exclusion Chromatography (SEC), TSKgel G3000SWXL,UV detector, Forced Degradation Study (FDS), MabThera®, rituximab, therapeutic antibody, biopharmaceutical,
aggregation, degradation

58

Experimental
Sample• MabThera® (rituximab, Roche) 100 mg/10 mL
Additives: sodium citrate dihydrate 7.4 mg/mL, sodium chloride 9.0 mg/mL, sodium hydroxide 9.0 mg/mL,
polysorbate 80 0.7 mg/mL
Measurement and analysis system
LC-4000 HPLC system<System configuration>
Pump:
PU-4080i
Autosampler: AS-4050i (with TC-4000-1)
Column oven: CO-4060
UV detector: UV-4075 (with inert flow cell unit)
Chromatography data system:
ChromNAV Ver. 2(with GPC calculation program)

Measurement Conditions
Column:Flow rate:
Wavelength:Standard:

TSKgel G3000SWXL(7.8 mmI.D. x 300 mmL, 5 µm)
0.8 mL/min
220 nmProtein standard mix
15-600kDa (Merck)
30 mg/mL in water

Eluent:0.2 M sodium phosphatebuffer (pH 6.7)
Column Temp.:
25 °C
Injection volume:
10 µL

Results
Fig. 1 shows UV chromatograms of a blank (ultrapure water) and a protein standard sample. The column
used in this experiment has an exclusion limit with globular proteins of 800 kDa and a measurement range ofapproximately 10 to 500 kDa. As shown in Fig. 2, a molecular weight calibration curve was created from theretention times and molecular weights of the four components, except for p-aminobenzoic acid, that eluted
after the permeation limit (around 13.8 min). Based on this calibration curve, the molecular weight of eachsample was calculated.

300000Standard

LogM = 5.21796 + 0.398763*tR- 0.0504095*tR^2 + 1.06239e-003*tR^3

42000002

LogM

Intensity / µV

6.0

3

Blank

5.0

1

100000

5
4.0

0
0.0

4.0

8.0

12.0

16.0

20.0

Retention time / min.

Fig. 1 UV chromatograms of a blank and a protein standard
1: Thyrogrobulin (670 kDa), 2: γ-globulins (150 kDa), 3: Ovalbumin(44.3 kDa), 4: Ribonuclease A (13.7 kDa), 5: p-amino-benzoic acid
(0.137 kDa)

7.0

8.0

9.0

10.0

11.0

12.0

13.0

Retention time / min.

Fig. 2 Molecular weight calibration curve59Fig. 3 shows the UV chromatogram of MabThera® without degradation treatment. Figs. 4 to 7 show UVchromatograms of MabThera® that had been oxidized, glycated, deamidated and heated, respectively. In
addition, Figs. 8 to 10 show the UV chromatograms of light-irradiated MabThera® with the irradiation timechanged in three steps of 1, 2 and 5 minutes. The peaks around 14 minutes in Figs. 3 to 10 are the peaks thateluted after the permeation limit of the column. Although detailed calculation results of molecular weight are
not shown here, the monomer peaks were identified from each peaks top molecular weight in Figs. 3 to 10.
In Figs 3 to 10, the peaks of each chromatograms were categorized into monomer, High Molecular Weight
Species (HMWS), including aggregates that elute earlier than monomer, and Low Molecular Weight Species(LMWS), including degradation products that elute later than monomer. Table 1 shows the comparison resultsof each category ratio (area %) to the total peak area. From these results, the ratio of HMWS of MabThera®
that had been oxidized, glycated, deamidated, heated and light-irradiated (1 minute) increased by about 0.2–
0.6% and that of LMWS increased by about 0.1–0.4% as compared to untreated MabThera®. Regarding lightirradiation, it was found that the ratios of the monomer gradually decreased and those of the HMWS and
LMWS increased significantly as the irradiation time increased.

Calibration curve

Monomer

a)

b)

12000

Intensity / µV

400000200000

5.6

Monomer

5.2

8000

4.8

HMWS

LMWS

4000

4.4
0

0
0.0

4.0

8.0

12.0

16.0

4.0
6.0

20.0

8.0

10.0

12.0

14.0

Retention time / min.

Retention time / min.

Fig. 3 UV chromatogram of untreated MabThera®
a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with molecular weight calibration curve)
Pretreatment: 100 mg/10 mL of MabThera® was diluted with ultrapure water to 1 mg/mL.

6000

Intensity / µV

Intensity / µV

400000

Monomera)200000

Monomer
HMWS

4000

LMWS
2000

0

0
0.0

Oxidized
Untreated

b)

4.0

8.0

12.0

Retention time / min.

16.0

20.0

6.0

8.0

10.0

12.0

Retention time / min.

Fig. 4 UV chromatogram of oxidized MabThera®
a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: One µL of 30% hydrogen peroxide solution was added (final concentration: 0.3% hydrogen peroxide) to 100 µL of
MabThera® 100 mg/10 mL solution, and the solution was left at room temperature for 3 hours. Then, the solution was dialyzed
against 10 mM sodium phosphate buffer (6 mM disodium hydrogen phosphate-4 mM potassium dihydrogen phosphate, pH 7.0)
using a dialysis cellulose tube (Visking tubing, manufactured by Serva), and finally diluted 10 times with ultrapure water.

60

14.0

LogM

Intensity / µV

600000a)400000

Monomerb)

6000

Glycated
Untreated

Intensity / µV

Intensity / µV

Monomer
300000200000

HMWS

4000

LMWS
2000

100000
0

0
0.0

4.0

8.0

12.0

16.0

20.0

6.0

8.0

10.0

Retention time / min.

12.0

14.0

Retention time / min.

Fig. 5 UV chromatogram of glycated MabThera®
a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: Fifty-four mg of glucose was added to 100 µL of MabThera® 100 mg/10 mL solution and dissolved (final concentration:
2.5 M glucose), and then left in a constant-temperature oven (SCF-Sro) at 40 °C for 1 week. Then, the solution was dialyzed against
10 mM sodium phosphate buffer (6 mM disodium hydrogen phosphate-4 mM potassium dihydrogen phosphate, pH 7.0) using a
dialysis cellulose tube (Visking tubing, manufactured by Serva), and finally diluted 10 times with ultrapure water.

a)400000

Monomerb)

6000

Deamidated
Untreated
Monomer

Intensity / µV

Intensity / µV

300000200000

HMWS

4000

LMWS
2000

100000
0

0
0.0

4.0

8.0

12.0

16.0

20.0

6.0

8.0

Retention time / min.

10.0

12.0

14.0

Retention time / min.

Fig. 6 UV chromatogram of deamidated MabThera®
a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: 100 µL of MabThera® 100 mg/10 mL solution was dialyzed against 10 mM sodium phosphate buffer (6 mM disodiumhydrogen phosphate-4 mM potassium dihydrogen phosphate, pH was adjusted to 8.5 by 5 M sodium hydroxide solution) using a
dialysis cellulose tube (Visking tubing, manufactured by Serva), and then left in a constant-temperature oven (SCF-Sro) at 40 ºC for
1 week. Then, the solution was dialyzed against 10 mM sodium phosphate buffer (6 mM disodium hydrogen phosphate-4 mM
potassium dihydrogen phosphate, pH 7.0) again, and finally diluted 10 times with ultrapure water.

a)

Monomer

HeatedUntreated

b)

6000

HMWS
Intensity / µV

Intensity / µV

600000400000200000

Monomer

4000
LMWS

2000

0

0
0.0

4.0

8.0

12.0

Retention time / min.

16.0

20.0

6.0

8.0

10.0

12.0

14.0

Retention time / min.

Fig. 7 UV chromatogram of heated MabThera®
a) Full scale and b) enlarged view near the baseline at 6-14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: 100 µL of MabThera® 100 mg/10 mL solution was left in a constant-temperature oven (SCF-Sro) at 40 ºC for 8 days
and then diluted 10 times with ultrapure water.

6140000a)

600000b)

MonomerLight-irradiated (1 min.)Untreated

Intensity / µV

Intensity / µV

30000

Monomer

400000200000

20000

10000

HMWS
LMWS

0

0

0.0

4.0

8.0

12.0

16.0

20.0

6.0

8.0

Retention time / min.

10.0

12.0

14.0

Retention time / min.

Fig. 8 UV chromatogram of light-irradiated MabThera® (irradiation time: 1 min)a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: 100 µL of MabThera® 100 mg/10 mL solution was irradiated with light (energy: 16.3 mW, center wavelength: 247 nm)
for 1 minute using a light irradiation system (MM-3, manufactured by Bunkoukeiki).

a)

b)

40000

MonomerLight-irradiated (2 min.)Untreated

30000

400000Intensity / µV

Intensity / µV

600000200000

0
0.0

4.0

8.0

12.0

16.0

Monomer
HMWS

20000

10000

LMWS

0

20.0

6.0

Retention time / min.

8.0

10.0

12.0

14.0

Retention time / min.

Fig. 9 UV chromatogram of light-irradiated MabThera® (irradiation time: 2 min)a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: 100 µL of MabThera® 100 mg/10 mL solution was irradiated with light (energy: 16.3 mW, center wavelength: 247 nm)
for 2 min. using a light irradiation system (MM-3, manufactured by Bunkoukeiki).

a)
Monomer

400000

0
4.0

8.0

12.0

Retention time / min.

16.0

Light-irradiated (5 min.)Untreated

HMWS

30000

200000

0.0

b)

40000

Intensity / µV

Intensity / µV

600000

20.0

Monomer

20000

LMWS

10000

0
6.0

8.0

10.0

12.0

14.0

Retention time / min.

Fig. 10 UV chromatogram of light-irradiated MabThera® (irradiation time: 5 min)a) Full scale and b) enlarged view near the baseline at 6–14 min. (shown with chromatogram of untreated MabThera® )Pretreatment: 100 µL of MabThera® 100 mg/10 mL solution was irradiated with light (energy: 16.3 mW, center wavelength: 247 nm)
for 5 min. using a light irradiation system (MM-3, manufactured by Bunkoukeiki).

62

Table 1. Comparison of ratios of HMWS, monomers, and LMWS in untreated and forcibly degraded MabThera®
			Type of treatments

MW classification

HMWS

Monomer

LMWS

Untreated

1.0 %98.8 %

0.2 %Oxidized

1.5 %97.9 %

0.6 %Glycated

1.2 %98.3 %

0.5 %Deamidated

1.3 %

98.2 %

0.5 %Heated

1.6 %98.1 %

0.3 %Light-irritated (1 min)1.5 %98.3 %

0.2 %Light-irritated (2 min)

6.7 %93.0 %

0.3 %Light-irritated (5 min)21.0 %

74.4 %

4.6 %Conclusions
In this experiment, it was confirmed that aggregates and degradation products in antibody drugs generated
under various FDS test conditions can be well separated from monomers and detected by using SEC, and
SEC is a powerful method for monitoring aggregates in the FDS.

Additional Information…
• Application: Analysis of Aggregates in Antibodies by SEC.
https://www.jasco-global.com/solutions/analysis-of-aggregates-in-antibodies-by-sec/• Product information: HPLC.
https://www.jasco-global.com/product-chromatography/integrated-hplc-system/

References
1) R. M. Lu, Y. C. Hwang, I. J. Liu, C. C. Lee, H. Z. Tsai, H. J. Li, H. C. Wu: J. Biomed. Sci., 27:1, (2020).
2) European Medicines Agency: “ICH Topic Q5C Quality of Biotechnological Products: Stability Testing
Biotechnological/Biological Products” <https://www.ema.europa.eu/en/documents/scientific-guideline/
ich-topic-q-5-c-quality-biotechnological-products-stability-testing-biotechnological/biological-products_
en.pdf>, (accessed 2022. 5. 30).
3) European Medicines Agency: “ICH Topic Q1A (R2) Stability Testing of new Drug Substances and Products”<https://www.ema.europa.eu/en/documents/scientific-guideline/ich-q-1-r2-stability-testing-new-drugsubstances-products-step-5_en.pdf>, (accessed 2022. 5. 30).
4) U.S. Food and Drug Administration: “Guidance for Industry: INDs for Phase 2 and Phase 3 Studies” <https://www.fda.gov/media/70822/download>, (accessed 2022. 5. 30).
5) C. Nowak, J. K. Cheung, S. M. Dellatore, A. Katiyar, R. Bhat, J. Sun, G. Ponniah, A. Neill, B. Mason, A. Beck,
H. Liu: mAbs, 9 (8), 1217 (2017).
6) Blessy M, R. D. Patal, P.N. Prajapati, Y. K. Agrawal: J. Pharm. Anal., 4 (3), 159 (2014).
7) J. Halley, Y. R. Chou, C. Cicchino, M. Huang, V. Sharma, N.C. Tan, S. Thakkar, L. L. Zhou, W. A. Azzam, S.
Cornen, M. Gauden, Z. Gu, S. Kar, A. C. Lazar, P. Mehndiratta, J. Smith, Z. Sosic, P. Weisbach, E. S. E. Stokes:
J. Pharm. Sci., 109, 6 (2020).
8) A. Hawe, M. Wiggenhorn, M. V. D. Weert, J. H. O. Garbe, H.C. Mahler, W. Jiskoot: J. Pharm. Sci., 101 (3), 895(2012).

About the Author
Satoe Iijima is a chromatography application scientist at JASCO Corporation (Tokyo, Japan) specializing in LC,
LC/MS, SFC, and SFC/MS.

63

FORCED DEGRADATION STUDIES (FDS)

Statistical Similarity Assessment of Antibody Drugs
in FDS by Circular Dichroism (CD) and qHOS
Deamidation
(Asn
Asp)

Heat

Glycation(Lys)

Agitation

Reduction
Oxidation(Met, Trp)HTCD Plus
high-throughput CD measurement system

Freeze-thaw

Photooxidation

Aggregates

Reference(MabThera®)
No treatment

pH
Light
irradiationJWQHOS-531

Major degradiation
pathway

Sample (Forced degradation study FDS)

Mean
-2SD

2SD

Mean
-2SD

2SD

Distance

Measurement

Spectral similarity test

Graphical abstract

Introduction
In recent years, therapeutic antibodies have been actively developed, manufactured and marketed. Antibody
drugs are widely used as therapeutic agents to inhibit the proliferation of malignant tumor cells and the activityof immune cells and are also highly anticipated for diseases for which complete treatment is not yet available.
Antibodies generally exhibit high affinity and highly specific activity due to their higher-order structure (HOS)
driven by covalent and non-covalent bond formation. In the production of therapeutic antibodies, there areseveral processes, such as cell cultivation, purification and virus removal, which may lead to various stimuli,
modifications and degradations that may significantly impair the function of the drugs. Therefore, Forced
Degradation Study (FDS) is frequently conducted at each stage from the selection of candidate antibodies topost-approval.1,2) ICH Q5C provides guidelines for stability testing of biologics and describes the importanceof testing under accelerated and severe conditions.3) The main stimuli for forced degradation testing include
heating, freeze-thawing, agitation, pH, light exposure, oxidation/reduction and glycation. Rapid detection ofchanges in physicochemical properties of antibodies due to these stimuli is critical for R&D and quality control
of antibody drugs.
Circular Dichroism (CD) spectroscopy is a simple method to obtain information on the HOS of antibodies
and to identify minute changes in HOS to modification or degradation as described above. Therefore, it isrecognized as a method to evaluate HOS in ICH Q6B and is widely used as one of the measurement methods
to control antibody quality.4) With the implementation of these guidelines, there is a growing demand for amethod that can objectively and sensitively evaluate CD spectra and identify significant differences between
them. The qHOS program, developed to meet these demands, is capable of detecting small-differences inspectra with high sensitivity based on statistical tests.
Here, we report the results of statistical determination of higher-order conformational changes for rituximab
caused by forced degradation testing using the HTCD Plus, high-throughput CD measurement system, in
combination with the qHOS program.
Keywords
Circular Dichroism (CD) spectroscopy, similarity assessment, Forced Degradation Study (FDS), MabThera®,
RIABNI™, rituximab, therapeutic antibody, biopharmaceutical, aggregation, degradation, high-throughput
secreening

64

Experimental
SampleMabThera® (rituximab innovator, Roche) and RIABNI™ (rituximab biosimilar, AMGEN) 100 mg/10mL
Additives: sodium citrate dihydrate 7.4 mg/mL, sodium chloride 9.0 mg/mL, sodium hydroxide 9.0 mg/mL,
polysorbate 80 0.7 mg/mL
Met and Trp oxidation1)

Preparation• Photo-oxidation
10 mg/mL MabThera® solution was irradiated with 16.3 mWof light energy and a central wavelength of 247 nm for 1
minute at room temperature using a light irradiation system.
(MM-3, manufactured by Bunkoukeiki) before measurement.
• Reduction
Tris (2-carboxyethyl) phosphine was added to a 10 mg/
mL MabThera® solution to a final concentration of 0.001%
and left at room temperature for 30 minutes before
measurement.

Hisoxidation5, 6)

Disulfide bond
reduction7)

Met oxidation1)

Fig. 1 Types of post-translational modifications andaffected domain of antibodies by oxidation andreduction

Measurement and analysis system
HTCD Plus high-throughput CD measurement system• Fully automated measurement of up to 192
samples.
• Pre-registered flush method for protein or DNA/RNA samples can be selected to eliminate
sample carry-over.

JWQHOS-531 qHOS
• Statistical HOS similarity test program developed
by JASCO.
• High-sensitive detection of spectral difference bynoise-weighted Euclidean distance.
• Support for spectra obtained by multiple
instruments.

Measurement Conditions
Bandwidth:
D.I.T.:
Accumulation:

1 nm
4 sec.
4

Scanning speed:
Data pitch:
Optical path length:

20 nm/min.
0.1 nm
0.5 mm65

Analysis conditions
Smoothing:
Weighting:Statistical testing:Significance level:

Distance calculation method:
Euclidean distance
Number of samples:
9
Concentration correction:
Absorbance(far-UV: Abs200)(near-UV: 0.5 Abs280 )Savitzky-Golay
Noise
Welch's t-test
0.05

See page 36 for more details of the spectral similarity test using the qHOS program.

Results
Through the near-UV CD spectra the tertiary structure of RIABNI™ was determined to be identical to that of
MabThera® (Fig. 2). The near-UV CD spectrum of the biosimilar RIABNI™ was in excellent agreement with thatof the innovator MabThera®, and the distances calculated from the spectra were also small. The t-test results
showed that the p-values were above the significance level of 0.05, objectively confirming that RIABNI™ has
the same tertiary structure as MabThera®.
The spectra and identity test results for MabThera® after photo-oxidation and reduction treatments areshown in Fig. 3. The spectra after the photo-oxidation and reduction treatments do not visually appear to besignificantly different from the spectrum of the untreated MabThera®, which was used as the reference for
each. However, when the spectra are converted to distances from each other, the distributions of distances
between the reference and post-stimulation treatments are far apart.
Furthermore, a Welch's t-test based on these distances statistically determined that the stimulus-treateddata are significantly different from the untreated data. These demonstrate that the treatments have caused
significant structural and spectral changes, which would otherwise go unnoticed if visually inspected.

Conclusions
Using HTCD Plus and the qHOS program, we were able to determine, based on statistical tests, that while
the tertiary structures of MabThera® and RIABNI™, antibody drugs with the same amino acid sequence andformulation conditions, are identical, there are significant differences in tertiary structure when MabThera® is
exposed to stimuli, such as photo-oxidation and reduction. Based on statistical tests, we found that the tertiary
structures of MabThera® and RIABNI™ are identical. Thus, by combining HTCD Plus and qHOS, we were able
to objectively evaluate significant differences that could not be determined visually, and this system proved
to be useful in the evaluation of HOS in FDS.

2

a)

b)

Result of t-test
Mean

0
RIABNI™
CD / mdeg

t-value = -0.32
p-value = 0.62

-2SD

-2
Reference(MabThera®)

-4

2SD

Similarity passed

Mean
-2SD

2SD

-6
260

280

300

Wavelength / nm

320

340

0.00

0.02

0.04

0.06

0.08

0.10

Distance

Fig. 2 Results of spectral similarity test for MabThera® (red) and RIABNI™ (blue).
a) and b): mean spectra of MabThera® (red), RIABNI™ (blue), standard deviation for MabThera® (gray).

66

1

a)

0
CD / mdeg

Result of t-test

b)
Mean

Photo
oxidation-2SD-1
Reference(MabThera®)
No treatment-2

t-value = 4.2
p-value = 0.012
2SD

Similarity failed

Mean
-2SD

2SD

-3
-4

260

280

300

320

0.00

340

0.02

0.04

0.06

0.08

0.10

Distance

Wavelength / nm

Result of t-test

1

c)

d)

CD / mdeg

0t-value = 23
p-value = 0.0

Mean

Reduction-2SD-1
Reference(MabThera®)
No treatment-2

Similarity failed

2SD

Mean
-2SD

2SD

-3
-4

260

280

300

Wavelength / nm

320

340

0.00

0.02

0.04

0.06

0.08

0.10

Distance

Fig. 3 Results of spectral similarity test for non-treated (red) and stimulated (blue) MabThera®.
a) and c): mean spectra of MabThera (red), stimulated sample (blue), standard deviation for MabThera (gray). b) and d): distances andtest results for MabThera (red) and stimulated sample (blue).

References
1) C. Nowak, J. K. Cheung, S. M. Dellatore, A. Katiyar, R. Bhat, J. Sun, G. Ponniah, A. Neill, B. Mason, A. Beck,
H. Liu: mAbs, 9 (8), 1217 (2017).
2) J. Halley, Y. R. Chou, C. Cicchino, M. Huang, V. Sharma, N.C. Tan, S. Thakkar, L. L. Zhou, W. A. Azzam, S.
Cornen, M. Gauden, Z. Gu, S. Kar, A. C. Lazar, P. Mehndiratta, J. Smith, Z. Sosic, P. Weisbach, E. S. E. Stokes:
J. Pharm. Sci., 109, 6 (2020).
3) International Conference on Harmonisation (ICH) of Technical Requirements for registration of
Pharmaceuticals for Human Use. ICH AUTHORIZED MANUFACTURING CHANGES FOR THERAPEUTIC
MABS 833 Harmonised Tripartite Guideline Comparability of Biotechnological/ Biological Products Subject
to Changes in their Manufacturing Process Q5C. 1995
4) International Conference on Harmonisation (ICH) of Technical Requirements for registration of
Pharmaceuticals for Human Use. ICH AUTHORIZED MANUFACTURING CHANGES FOR THERAPEUTIC
MABS 833 Harmonised Tripartite Guideline Comparability of Biotechnological/ Biological Products Subject
to Changes in their Manufacturing Process Q6B. 1999
5) Amano, M., Kobayashi, N., Yabuta, M., Uchiyama, S. and Fukui, K.: Anal. Chem., 86(15), 7536 (2014).
6) Miyahara, Y., Shintani, K., Hayashihara-Kakuhou, K., Zukawa, T., Morita, Y., Nakazawa, T., Yoshida T., Ohkubo,
T. and Uchiyama, S.: Sci. Rep., 10, 6333 (2020).
7) Liu, H., Gaza-Bulseco, G. and Chumsae, C.: J. Am. Soc. Mass Spectrom., 20(12), 2258 (2009).

About the Author
Taiji Oyama is a spectroscopy application scientist at JASCO Corporation (Tokyo, Japan) specializing in CD,
IR, Raman, UV-vis, and fluorescence spectroscopy.

67

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