White Paper

October 10, 2022
Keywords or phrases:
Octet® Ligand Binding, Kinetics, GxP, GLP

Instrument Comparability Assessment:
Kinetics Precision Assessment in Ligand
Binding Assays on the GxP-Compliant
Octet RED96e and Octet R8 Instruments®

®

Bob Dass, Senior Scientist and David Apiyo, Manager of BioA Applications
Sartorius Corporation, 47661 Fremont Blvd, Fremont, CA 94538 USA
Correspondence
Email: Octet@Sartorius.com

Introduction
To ensure the quality of pharmaceutical and other biological products that support clinical trials and post market assay
activities, it is important to identify the specific critical processes and quality attributes early in development and to put inplace methods for their evaluation throughout the life cycle of product development and release. The International
Conference on Harmonization (ICH) Q2(R1)1 publication provides guidance on the validation of analytical technologies
and helps form the basis of key quality attributes and parameters that need to be assessed for pharmaceuticals intended
for use in humans. The ICH Q2(R1) publication details multiple parameters that need to be established and assessed during
the validation of analytical procedures that are included as part of the registration process during applications forpharmaceutical drugs and biologics in general.
These parameters often need to be established during assay method development and qualification and prior to method
validation. They include, but are not limited to, assessing specificity, accuracy and precision and are dependent on the typeof assay to be validated. For example, while quantitation assays require that the method in use should exhibit linearity,

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accuracy and limits of quantitation among other parameters, there is a bigger emphasis on specificity and precision whenit comes to ligand binding assays where affinity constants are the main output parameter in evaluation (FDA Bioanalyticalmethod validation).
While this whitepaper does not discuss these specific requirements in detail, it nevertheless focuses on comparability
assessment of Octet® RED96e and Octet® R8 instruments—the Bio-Layer Interferometry (BLI) analytical platforms
commonly used in establishing supporting data and in some cases lot-release methods for pharmaceutical products
and other biologics through ligand binding assays.

Bio-Layer Interferometry (BLI) Technology
Bio-Layer Interferometry (BLI) analyzes the interference
pattern of white light reflected from two surfaces: a layerof immobilized protein on the biosensor tip and aninternal reference layer. Any change in the number ofmolecules bound to the biosensor tip causes a shift in
the interference pattern that can be measured in realtime. The binding between a ligand immobilized on thebiosensor surface and an analyte in solution produces anincrease in optical thickness measured as a wavelength
shift, Δ λ (Figure 1). In BLI, biomolecular interactions
between two binding partners is converted into response
signals in real-time allowing users to design methods

that can help in rapidly optimizing binding assays ofvarious biological molecules. BLI is a label-freetechnology that allows users to determine associationand dissociation rate constants in kinetic characterization
experiments; key determinants in affinity constantderivation and information not available with end-point
analysis techniques such as ELISA. In addition, BLI canbe used for protein quantitation determination, hence it
can be applied at various stages of drug development
allowing users to circumvent limitations of ELISA and
HPLC platforms.

Figure 1
Relative Intensity of Light Reflection From the Two Biosensor Surfaces
Incident white light

1.0
Δλ

BLI signal processing

Biocompatible surface

Bound molecule

Relative Intensity

Reflected beam
0.8

0.6

0.4

0.2

Unbound molecule
Wavelength (nm)Note. BLI is an optical technique that measures interference patterns of white light reflected from two surfaces on the tip of a biosensor.
Interactions between biological molecules at the tip of the biosensor causes a shift in interference patterns and is measured in real-time.

2

Octet RED96e Versus Octet R8®

®

Octet® systems utilize a standard microplate format thatenables high-throughput, automated binding analysis
directly from 96- and/or 384-well plates and great
flexibility in assay design. The RED96e and the R8 areboth 8-channel instruments capable of analyzing up to
8 samples in tandem and are compatible with 96-well
micro-titer sample plates.
The RED96e system was developed to operate reliably ina regulated environment and came with features that
included a sample plate cover and a wide range ofsample temperature control (15–40 °C) allowing the

instrument to run un-attended for up to 12 hours. The R8
on the other hand was introduced into the market as partof the modular Octet® R series that are available inconfigurations of 2, 4 or 8 channels (Figure 2). The
R-series is designed to provide Octet® users with theflexibility of having a field upgrade performed of theirlower through-put instruments (R2 and R4) to the
8-channel Octet® R8 (R2→R4, R2→R8 or R4→R8) as
their through-put needs increase. The R8 maintains
identical features to RED96e and comes with GxPcompatible products as illustrated below.

Figure 2 and Table 1
The Modular Octet® R Series: R2, R4 and R8

Octet® R2

Octet® R4

Octet® R8

Number of spectrometers

2

4

8

Maximum simultaneous reads

2

4

8

Temperature control

15–40 ⁰C

15–40 ⁰C

15–40 ⁰C

Evaporation control

No

No

Yes

Upgradeability

Yes

Yes

No

GxP package availability

No

No

Yes

Note. Lower read heads can be upgraded to a higher configuration at user site

Octet R8 GxP Compliance®

GxP users are often looking for analytical instruments thatoffer simplicity, innovation and speed, and have small lab
footprints with user-friendly software that allows forautomation. Other key considerations include robust
instruments that experience minimal downtime while atthe same time allowing for ease of use; properties thatare desirable in QC environments. For QC assays, thetechnology also needs to demonstrate methods thatare stability indicating. The Octet® R8 is designed in aconfiguration that provides simplicity and ease of use,
and yet allows for enhanced productivity by increasing
the capacity to test QC and other samples.

Sartorius’s Octet® R8 GxP package comes with all
requirements for GMP compliance including IQOQ
protocols and kits, user guides, performance
qualification (PQ) protocols and kits, 21 CFR Part 11
software with audit trails, software validation package
and biosensor validation support.

3Octet Comparability Studies: Octet RED96e vs Octet R8®

®

It should be noted that the first step in the developmentof a ligand binding assay using label-free technologies
such as the BLI is the selection of an appropriate assay
format. This includes the selection of the solid-surface orbiosensor chemistry in the case of the Octet® platform;
the determination of the molecule to be immobilized
onto the biosensor and the optimization and subsequentqualification of assay condition such as buffers, pH, ligand
immobilization concentrations, etc. This white paper isintended to showcase a comparison between the®

performance of a pre-developed and qualified ligand
binding assay on the Octet® RED96e and its R8
counterpart. While a typically qualified ligand binding
assay would need to assess multiple qualification
parameters including specificity, accuracy, robustness
and precision among others, here, we focus only on
precision assessment by comparing parameters that
include equilibrium dissociation and other rate constants
determined by multiple analysts on both instruments, toshowcase comparability.

Materials and Methods
Materials
 Instruments: Octet® R8 and Octet® RED96e instrumentwith Octet® BLI Discovery and Analysis Studio Software.
 Samples: Biotinylated Anti HER2 Antibody (R&D systems;
Cat. No. BAF1129) and HER2 receptor protein (ACRO
Biosystems; Cat. No. HE2-H5212).
 Biosensors: High Precision Streptavidin 2.0 (SAX 2.0; Cat.
No. 18-0047)
 Sample plates: 96-well, black, flat bottom microplate,
Greiner Bio-One Part No. 655209
 Buffers: 20 mM Tris-Triton, pH 7.4 (Sartorius)
Methods
To provide information on the comparability of the binding
kinetics data between Octet® R8 and Octet® RED96e abinding kinetics assay was performed by 3 analysts on both
systems using the same reagents and a pre-established
Octet® kinetics method. Prior to the start of the assay, theSAX2.0 biosensors were hydrated (pre-wet) for at least
10 minutes in the assay buffer (20 mM Tris-Triton buffer,

4

pH 7.4). The assay included an initial baseline (biosensors
equilibration) step where the hydrated biosensors weredipped in assay buffer for 120 seconds followed by a dip in
the biotinylated anti-HER2 antibody @ 1 μg/mL for
400 seconds (ligand load step). A second baseline step
where the biosensors were dipped in assay buffer for
180 seconds to remove any non-specifically boundsubstances in the ligand loading step was performed before
the ligand-bound biosensors were dipped for 180 seconds
in HER2 analyte protein samples. Samples were tested atconcentrations ranging from 57.1 nM–0 nM (2-fold dilution)
during the association step. A final dissociation step of1800 seconds in assay buffer was run to establish analyte
off-rates.
Figures 3–5 show overlayed replicates of binding curves
obtained by 3 different analysts on the two instruments;
RED96e (A) and R8 (B) and fitted to generate precisioncomparison data as captured in Tables 2–4 (full dissociation
steps not shown).

Figure 3
HER2 to Anti-HER2 Binding Analysis Obtained by Analyst 1
A. Octet® RED96e
0.25
57.1 nM

0.20

28.6 nM
14.3 nM

0.10

7.1 nMnm

0.15

3.6 nM

0.05

1.8 nM
0.9 nM

0
0

50

100

150

200

250

300

350 400
Time (s)

450

500

550

600

650

700

750

B. Octet® R8
0.25
57.1 nM

0.20

28.6 nM
14.3 nM

0.10

7.1 nMnm

0.15

3.6 nM

0.05

1.8 nM
0.9 nM

0
0

50

100

150

200

250

300

350 400
Time (s)

450

500

550

600

650

700

750

5

Figure 4
HER2 to Anti-HER2 Binding Analysis Obtained by Analyst 2
A. Octet® RED96e
0.25
57.1 nM
0.20

28.6 nM0.15
nm14.3 nM
7.1 nM

0.10

3.6 nM
1.8 nM
0.9 nM

0.05
0
0

50

100

150

200

250

300

350 400
Time (s)

450

500

550

600

650

700

750

B. Octet® R8
0.25
57.1 nM

0.20

28.6 nM0.15
nm14.3 nM

0.10

7.1 nM
3.6 nM

0.05

1.8 nM
0.9 nM

0
0

6

50

100

150

200

250

300

350 400
Time (s)

450

500

550

600

650

700

750

Figure 5
HER2 to Anti-HER2 Binding Analysis Obtained by Analyst 3
A. Octet® RED96e
0.25
0.20

57.1 nM
28.6 nMnm

0.1514.3 nM
0.10

7.1 nM
3.6 nM

0.05

1.8 nM
0.9 nM

0
0

50

100

150

200

250

300

350 400
Time (s)

450

500

550

600

650

700

750

B. Octet® R8
0.25
57.1 nM

0.15

28.6 nMnm

0.2014.3 nM
0.10

7.1 nM
3.6 nM
1.8 nM

0.05

0.9 nM

0
0

50

100

150

200

250

300

350 400
Time (s)

450

500

550

600

650

700

750

7

Table 2
Intra-Analyst Variation (% CV) of the Measure Binding Kinetics Parameters of Affinity (KD), Association Rate (ka) andDissociation Rate (kd) on the Octet® RED96e
KD (M)

kd (s-1)

ka (M-1s-1)

Mean

STDEV

% CV

Mean

STDEV

% CV

Mean

STDEV

% CV

Analyst 1

2.75E-10

4.68E-11

17.00

4.31E+05

1.65E+04

3.82

1.18E-04

1.56E-05

13.22

Analyst 2

3.61E-10

2.44E-11

6.75

4.55E+05

1.98E+03

0.44

1.64E-04

1.04E-05

6.32

Analyst 3

3.04E-10

5.59E-12

1.84

4.37E+05

2.01E+04

4.59

1.33E-04

8.49E-06

6.38

Table 3Intra-Analyst Variation (% CV) of the Measure Binding Kinetics Parameters of Affinity (KD), Association Rate (ka) andDissociation Rate (kd) on the Octet® R8
KD (M)ka (M-1s-1)

kd (s-1)

Mean

STDEV

% CV

Mean

STDEV

% CV

Mean

STDEV

% CV

Analyst 1

2.69E-10

2.64E-11

9.81

4.16E+05

1.42E+04

3.41

1.12E-04

1.485E-05

13.23

Analyst 2

3.67E-10

2.76E-11

7.51

4.46E+05

3.42E+04

7.66

1.63E-04

2.828E-07

0.17

Analyst 3

4.39E-10

4.38E-11

10.00

4.39E+05

1.34E+04

3.05

1.92E-04

1.344E-05

6.99

Table 4
Inter-Analyst Variation (% CV) of the Measure Binding Kinetics Parameters of Affinity (KD), Association Rate (ka) andDissociation Rate (kd) on the Octet® R8 and Octet® RED96e
KD (M)ka (M-1s-1)

kd (s-1)

Mean

STDEV

% CV

Mean

STDEV

% CV

Mean

STDEV

% CV

Octet R8

4.03E-10

5.05E-11

12.53

4.34E+05

1.53E+04

3.53

2.051E-05

1.78E-04

11.54

Octet RED96e

3.33E-10

4.09E-11

12.29

4.46E+05

1.24E+04

2.79

2.23E-05

1.49E-04

15.01

®

®

8

Benefits of the Octet® Platform in
GMP Applications

Examples of Approved Drugs Using
the Octet® Instrument

Octet® Bio-Layer Interferometry (BLI) systems offer anadvanced, fast, robust and fluidics-free approach forscreening and characterizing molecular interactions suchas protein-protein and protein-small molecule analysis.
The Octet® platform enables a huge variety of applications
performed at various stages of biologics development—from early selection to validation to manufacturing andquality control, e.g. concentration determination of theactive analyte, ligand binding, or contaminant testing for
lot release and in-process testing. It is estimated that for atypical ligand binding method, an Octet® system will be 2X
faster for method development than traditional ELISA. The
sample plate format coupled with real-time analysis andthe high-throughput readout allows for a more rapid
assessment of different assay conditions, that in-turn help
speed up assay optimization. Benefits include:

Octet® data was used as part supporting data for the assess­ment of functional activity of pembrolizumab (Keytruda;
EMEA approved in 2015). Keytruda as monotherapy isindicated for the treatment of advanced (unresectable ormetastatic) melanoma in adults. The active substance ofKeytruda is pembrolizumab which is a humanized mono­clonal antibody that binds to human PD-1 and blocks theinteraction between PD-1 receptor and its ligands. It is an
IgG4 monoclonal antibody with Class-II mechanism ofaction (binding to cell-bound antigen not involving Fc
effector function). The Octet® together with ELISA wereused for invitro binding studies of pembrolizumab.2

1. Parallel processing of samples—reduced assay
development time.
2. Faster analysis across many samples allows for faster
decision making and is especially useful in assay
method development when multiple conditions must
be investigated for method qualification.
3. One-step direct binding assay—minimal sample prep,
automated assay without manual steps. Assay setup iseasy and fast; complete walkaway while experiment isrunning and minimal manual intervention reduces therisk of human errors.
4. Few moving parts that require maintenance—QC
environment ready. Octet® instruments require very low
maintenance. There are no microfluidics that need to
be guarded against clogs, contamination, and leaking.
5. Off the shelf biosensors such as Protein A (Pro A),
Protein G (ProG), Anti-human Capture (AHC2) andAnti-mouse Capture (AMC) are available for direct
capture of ligands. High precision streptavidin (SAX andSAX2.0) biosensors deliver enhanced precision for
GMP applications.
6. Comprehensive screening and characterization toolacross a diverse range of applications—antibody andprotein quantitation, Fc-receptor binding, contaminanttesting and general product characterization.

The Anti-PD-L1 monoclonal antibody Tecentriq(atezolizumab) was approved by EMEA in 2017 and later by
the FDA in 2018. It is used in the treatment of patients withlocally advanced or metastatic urothelial carcinoma whohave disease progression during or following platinumcontaining chemotherapy and other cancers. Octet®
instruments were used for supporting data in the binding
characterization of the drug to its receptor PD-L1 among
other studies.
In addition to these approved drugs, there are multiple
instances where the Octet® has been used along withother techniques to provide supporting data for a various
drug applications. For example, Theramex Ireland Limited
applied for marketing authorization at the EMA for Livogiva
in 2019 with a positive opinion of granting the sameprovided in 2020. Livogiva is a biosimilar to Forsteo andis used for the treatment of osteoporosis. The active
gradient is Teriparatide, PTH (1-34) the biologically active
34-amino acid N-terminal fragment of the 84-amino acidnative parathyroid hormone, PTH (1-84). The Octet® wasused to verify biosimilarity between the two drugs infunctional binding assay.3
STADA Arzneimittel AG filled an application for marketing
authorization at the EMA for Oyavas in early 2020 with apositive response obtained in January 2021. Oyavas has
been developed as a similar biological medicinal product(biosimilar) to the reference medicinal product Avastin,which contains bevacizumab as the active substance.
Bevacizumab is a recombinant humanized monoclonal
antibody, which specifically binds to human vascularendothelial growth factor (VEGF), preventing its inter­action with VEGF receptors (VEGFRs) on the surface ofendothelial cells. In this case, the Octet® was used for
confirming specificity of the biosimilar through functionalbinding assessment with irrelevant antigens.4 Many otherexamples of drugs developed with the Octet® BLI used forsupporting characterization data exist in literature.
9Conclusion

References

A Kinetics Ligand binding comparability assay using
HER2 receptor binding to anti-HER2 as a model systemand focused on the key ICH guideline on precision
analysis for such studies indicates that there areinsignificant differences in performance between thetwo instruments. The CVs of < 20% observed for thevarious parameters studied are similar to what would be
expected within the same instrument for the same
analyst for a high affinity binding study implying that
there are no detectable differences between the Octet®
R8 and the RED96e. Since the data shows that the
Octet® R8 and the RED96e produce similar results whenusing identical binding pairs and since there are nofeature differences between the two instruments,
Octet® users currently possessing the RED96e and whoneed to replace the instrument with a newer version or
who need additional instruments due to emerging
needs should feel confident that methods developed on
the RED96e can be successfully transferred onto Octet®
R8 without any impact on performance. Moreover, data
derived from both instruments should be applicable forregulatory filing to support BLA, NDA or any otherproduct related regulatory application.

1. ICH Q2R1 Validation of Analytical Procedures:
Text and Methodology
2. https://www.ema.europa.eu/en/documents/assessmentreport/keytruda-epar-public-assessment-report_en.pdf
3. LIVOGIVA, INN-teriparatide (europa.eu)
4. Oyavas - INN-bevacizumab (europa.eu)

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Phone +49 551 308 0
For additional information,
visit www.sartorius.com

Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
For Research Use Only. 4082 Rev B.
Status 10 | 2022

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Phone +1 888 OCTET 75
Or +1 650 322 1360