White Paper

May 20, 2021
Keywords or phrases:
Octet® Instrument, 21 CFR Part 11, BLI, PQ, IQOQ,
GMP, GxP, Software Validation, Method validation,
Instrument Qualification, QC, Potency Assays,
Regulatory Compliance

Enhanced Productivity and Labor Efficiency in
Lot Release and In-Process Testing of Biologics
in GxP Laboratories
David Apiyo, Fremont, CA
Correspondence
Email: octet@sartorius.com

Abstract
Octet® systems come with distinct advantages including ease of use, throughput, low maintenance, and microfluids-free configurations, enabling them to provide GxP users with enhanced productivity and labor efficiency for lot release and in-process
testing of Biologics. Octet® instruments can be equipped with Octet® 21 CFR Part 11 Software for compliance with regulatory
requirements. Compliance is further enhanced with the availability of instrument qualification kits including IQOQ and PQ
kits that ensure the platform performs as stipulated. This whitepaper includes a few examples in which Octet® systems havebeen used under GxP compliance for different applications and shows the relative benefits over alternate technologies.

Find out more: www.sartorius.com

Introduction

Enhanced productivity: Enhances productivity byincreasing the capacity to run 20X–40X and 8X–16X more
QC potency samples testing/day than ELISA and SPR*
respectively.
Cost savings: Reduced hands-on operation allows for
10X less analyst hands-on time than ELISA, resulting in
FTE cost savings of > $40,000*.
Time savings: Robust instrument resulting in significant
maintenance cost savings and low downtime compared
to SPR; no fluidics means no clogging of samples and lessinstrument downtime. You will not need a second backup
instrument to support uninterrupted operation.
Ease of use: The Octet® platform is approximately 2X
faster than ELISA and SPR* in method validation for
ligand binding and potency assays.
Complete Octet® GxP package: Comes with all requirements for GMP compliance – IQOQ protocols and kits,user guides, performance qualification (PQ) protocols andkits, Octet® 21 CFR Part 11 Software with audit trails, software validation package and biosensor validation support.
Octet® R8 and Octet® RH16 systems come with specifically optimized performance qualification kits.
Successful use: Currently validated by and used in
multiple CROs and CDMOs world-wide.

* Please contact Sartorius for details.

A.

Sartorius’ Octet® instruments utilize BLI technology tomonitor biomolecular interactions in real time. They are an
ideal replacement for ELISA and HPLC techniques for thequantification of antibodies and recombinant proteins andare especially suitable for product potency lot release assays.
The platebased and non-fluidic format also offers GxPusers distinct advantages over comparative SPR based
techniques. Octet® systems provide higher throughput,with the flexibility to run 2 to 96 samples simultaneously,
and better sample versatility, including the ability to analyze
crude samples and more tolerance to diverse sample
matrices. In addition, ease of use, low maintenance andhigh data precision speeds time to results throughout
the drug development process. The Octet® platform isparticularly well-suited for GxP and QC laboratories.

B.

C.
Reflected Beam

Incident
White
Light

Wavelength (nm)

BLI Signal
Processing

ΔλRelative Intensity

BLI Signal
Processing
Relative Intensity

Reflected Beam
Incident
White
Light

Bio-Layer Interferometry (BLI) is an optical technique thatanalyzes the interference pattern of white light reflectedfrom two surfaces: a layer of immobilized protein on thebiosensor tip and an internal reference layer (Figure 1A).
Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that canbe measured in real time (Figure 1A and 1B). The binding
between a ligand immobilized on the biosensor surface
and an analyte in solution produces an increase in optical
thickness measured as a wavelength shift, Δ λ (Figure 1C).

Relative Intensity-

Key Considerations

Wavelength (nm)Wavelength (nm)Figure 1: Relative intensity of the light reflection pattern from the two surfaces on the biosensor. Octet® sytems with BLI technology measure thedifference in reflected light’s wavelength (Δλ) between the two surfaces.

2

Octet® Instrument Qualification

Figure 2: Sartorius’ Octet® systems meet a wide range of throughput
needs. The higher throughput Octet® RH16 and RH96 systems analyze
either 16 or 96 samples at a time, respectively (top). The lower throughput
Octet® R Series systems analyze either 2, 4 or 8 samples at the same time
respectively (bottom).

Analytical Instruments in the
GxP Environment
Analytical methods that provide reliable and accurate data
are necessary to ensure quality standards are met during
the production and release of drug products. In order toensure data accuracy, it is critical that the performance of
the applied analytical instruments, the computer systems
used, and the developed methods for data acquisition
meet required specifications.
Several official regulatory guidelines including Good
Laboratory Practice (GLP), Good Manufacturing Practice(GMP) and ISO Standards exist and provide guidance on
meeting compliance.

---

Sartorius, an ISO 9001 certified company, offers Octet 21
CFR Part 11 Software and full GxP products and services for
Octet® instruments (Figure 2). These include:
Octet® CFR Software and Sartorius FB server features forsecure, traceable electronic record keeping that enable
compliant data acquisition and data analysis in laboratories
working under GxP and 21 CFR Part 11 regulations.
IQOQ/PQ packages that ensure that Octet® systems
are qualified and operate as intended and that theperformance meets specifications.
Customer-run software validation support
Biosensor validation support services
Technical support assistance

Instrument qualification is critical to meeting complianceguidelines in any analytical laboratory developing, qualifying
and validating assays in the GxP environment. Sartorius
offers a rigorous installation qualification and operational
qualificaton (IQOQ) kit that includes a user guide, reagents,
and documentation required for instrument installation andqualification. In addition, a comprehensive performance
qualification (PQ) kit is also available that can be used for
performance qualification and periodic system checks.
The IQOQ and PQ kits can be purchased separately at thetime of instrument installation or any other time for regularsystem performance checks to ensure calibration andmaintenance of the instrument.

Installation Qualification andOperational Qualification (IQOQ)
The IQOQ Kit provides a checklist of instrument components
and documented verification that the Octet® instrument,
accessories and computer system as supplied and installed,
comply with Sartorius’ specifications. It also provides a comprehensive and documented verification that the Octet®
instrument operates as intended. It verifies that various critical operational parameters such as the optical system,alignment of instrument, plate, temperature and sample
plate shake speed meet the stipulated specifications. The
IQOQ Kit can be added at time of instrument purchase orat any other time during the lifespan of the Octet® instrument. Sartorius recommends the IQOQ procedure to berepeated after an instrument move or repair and following
a software upgrade.

Performance Qualification (PQ)®

The PQ Kit is used to verify that the Octet® instrument isfully functional after installation. System functionality is
tested for both quantitation and kinetics analysis. The kit
contains biological samples, reagents and biosensors foreach analyses and is used to demonstrate that consistentand reproducible results are obtained within productspecifications.
The qualification process for kinetics applications is two-fold:
1 . Assess instrument performance for ligand loading onto
the biosensor surface with stipulated loading acceptance criteria
2 . Assess instrument performance for analyte binding to theimmobilized ligand with stipulated acceptance criteria.

3

The qualification process for quantitation applications
asseses performance of known concentrations of ProstateSpecific Antigen (PSA) used in the generation of a standard
reference curve. Assay performance is evaluated against
stipulated percent acceptance variation statistics.

Octet® CFR Software andSoftware Validation Services
Software validation and the use of 21 CFR Part 11-compliant
software are regulatory requirements prior to using any
analytical instruments in a GxP environment. Octet® CFR
Software includes features such as controlled user accessand electronic signatures. In addition, the Sartorius FB
Server module manages the information recorded during
user sessions. Software validation provides users with theconfidence that data integrity and accuracy are always
maintained. The Software Validation services provide asingle source for meeting all software compliance needs,
including demonstration that the Octet® Software iscompliant with the 21 CFR part 11 final rule. This validation
package includes an instruction manual that provides
guidelines on how to verify functions in the FB Server
monitor and Octet® Analysis Studio Software. In addition,
it provides example data sets for the comparison of
Octet® software-generated data to other software suchas Microsoft® Excel® and GRAPHPAD PRISM™.

High-Precision Biomolecular Analysis
BLI is a widely adopted technique in biopharmaceuticaldevelopment and manufacturing. The throughput andflexibility of the Octet® platform has helped accelerate
almost every stage of the drug development workflow(Figure 3) in leading and start-up biotherapeutic companies,
enabling informed decisions to be made earlier.
The comprehensive tool set for compliance make Octet®
systems ideal in regulated quality control (QC) labs forconcentration and impurity analysis in both upstream anddownstream processes. In addition, Octet® systems havebecome a solution platform for kinetic and potency analysis
of drug-target and drug-Fc receptor(s) (FcγRI, FcγRIIa,
FcRIII and FcRn) interactions, and for stability analysis
by assessing changes in activity in stressed and forced
degradation samples. The high-throughput capability andflexibility of the platform enables rapid completion of assay
method development through design of experiments(DOE) essential in identifying and controlling critical quality
attributes (CQAs) that ensure consistent and reliable
quality of biopharmaceutical products.

Discovery
research

Lead screening
and selection

Lead optimizationand characterization

Preclinicaldevelopment

Processdevelopment

QC andmanufacturing

Elucidate protein
structure-function
relationshipHybridoma or
phage display
library screening

Hit validation

Immunogenicity
testing

Cell line
development

Bioreactor
monitoring

Mode of action
studiesOn-rate/oﬀ-raterankingAﬃnitymaturation

PK/PD studies

Cell culture
optimization

Product potency
assay

ELISA assay
development

Epitope binningFc engineering /humanization

Chromatography
optimization

Contaminanttesting

Dynamic binding
capacity (DBC)
determination

Fc receptor
binding assays

Kinetics application
Quantitative application

Figure 3: Select applications areas of Octet® systems in biotherapeutics development.

4

Biosensor Validation Services

Concentration Determination

Sartorius provides GxP users with biosensor support
during the method validation process. Customers are able
to order multiple lots of a biosensor for assay method
qualification and validation, and can reserve one of thelots for future purchase.

The Octet® platform provides a convenient and reliable
analytical method for measuring antibody and protein concentrations. The simple Dip and Read approach enables
streamlined workflows and rapid quantitation of 96 samples
in as little as 5 minutes on the Octet® RH96 system or 80
samples in < 30 minutes for the Octet® R8 system. Samples
can be analyzed in cell culture supernatant or in complex
media, thus removing the need for purification.

The service is available for the following biosensor types:
Protein A (ProA), Streptavidin (SA), High Precision Streptavidin(SAX), Ni-NTA (NTA) and Anti-Mouse Capture (AMC).
Customers can order up to five different biosensor lots,
up to 20 trays from each lot for evaluation, and reserve up
to 40 additional trays for future purchase.

High Precision Streptavidin 2.0 (SAX2)
Biosensors
Sartorius provides GxP users with High Precision
Streptavidin 2.0 (SAX2) Biosensors developed and qualified
for applications in downstream drug discovery and regulated
environments that have stringent assay precision requirements. SAX2 is QC-tested for the biotinylated molecule tomeet precisioncontrolled coefficient of variation (CV) specification of ≤ 4% within lot and CV range of 20% across lots.
SAX2 is suitable for both kinetics and quantitation assays
when used with biotinylated ligands.

In a typical quantitation assay, biosensors coated with capture molecules are dipped into samples in the sample plate
and the on-rate is measured in real time. The measured
on-rate is then used to determine the concentration of thetarget protein. This is done by comparing the binding signals obtained from a set of known analyte concentrations
which are used to generate a standard curve with the signals from the unknown samples. Commonly used biosensors for antibody and protein concentration determination
include Protein A- and Protein G-coated Biosensors andAnti-Human IgG (AHQ) and Anti-Mouse IgG (AMQ) Biosensors for antibody or Fc based protein molecules. AntiHIS (HIS2), Anti-GST (GST) and High Precision Streptavidin
Biosensors (SAX) are used for quantitation of recombinant
proteins. Octet® instruments measure the unique binding
properties of interacting pairs of molecules; hence the calibration with external standards used with techniques like
OD 280 measurements are not needed. The instrument’s
performance is verified through well-characterized materials that can be used to confirm the system is running within
set specifications. Since the binding properties of each specific interaction pair is unique, a stan dard or reference
curve using the purified version of the analyte in matching
matrix is required. As the association curve of a binding
event is used to quantify the analyte in the sample, only theamount of biomolecule that really binds to the immobilized
ligand on the biosensor is taken into consideration.

5

In quantitation assays, precision, linearity and accuracy are
key parameters that must be demonstrated in order for theassay to be validated for use in manufacturing. In addition,
the limit of quantitation (LOQ) should be determined toincrease confidence in the assay’s performance. As an
example, the Octet® system was used to determine method
precision and accuracy in the quantitation of recombinant
insulin.1 High Precision Streptavidin Biosensors (SAX) were
coated with anti-insulin antibody and used to bind purified
insulin samples. Known insulin sample concentrations wereprepared in Sartorius’ 1X Kinetic Buffer at concentrations
ranging from 0–50 μg/mL and used in binding assays(Figure 4A) to generate a standard/reference curve(Figure 4B). Three samples (25, 6.25 and 1.56 μg/mL) of
insulin were also spiked in 1X Kinetics Buffer and treated as
unknown samples. All samples were run in triplicate. Data wasanalyzed using the initial slope binding rate analysis modein Octet® Analysis Studio Software. The standard samples
exhibited concentration % CVs of < 3% (Table 1), while theunknown samples exhibited concentration % CVs well
below 5% indicating excellent precision. Dose recovery for the
unknown samples was found to be within 90–110%, indicating
excellent method accuracy (Table 2). A similar approach canbe used for other recombinant proteins.

A.

B.
0.20

4.0

Binding Rate (nm)Binding Rate (nm)

0.16

3.0

2.0

1.0

0

0.12

0.08

Standard
Fitting Curve

0.04

0

20

40

60
Time (sec)

80

0

100

0

10

20

30
40
Concentration (µg/mL)

50

60

Figure 4: Binding of known concentrations of recombinant insulin (A) used to generate a standard/reference curve (B) for the quantitation ofrecombinant insulin. SAX Biosensors loaded with anti-insulin were used for the studies.

Table 1: Insulin quantitation. Standard samples exhibit excellent precisionat < 3% CV.

Table 2: Insulin quantitation. Unknown samples, n=3, exhibit excellent
precision and accuracy.

Conc.
μg/mL

Binding rate,
nm/s

Conc. avg,
μg/mL

Conc.
% CV

Conc.
μg/mL

Bindingrate, nm/s

Conc. avg,
μg/mL

Conc.
% CV

%
Recovery

50

1.02

50

2.39

25

0.5182

25.9

1.41

104

25

0.5

25

1.88

6.25

0.064

6.4

0.1638102

12.5

0.1872

12.5

1.24

1.56

0.0066

1.7

1.4

106

6.25

0.0611

6.2

1.48

3.13

0.01893.1

0.34761.56

0.0058

1.6

2.83

0.78

0.00120.8

0.283100.00010

0

6High-precision Ligand Binding andPotency Assays

Stability and Forced Degradation
Studies

Ligand binding kinetics assays are increasingly finding use
as batch lot release methods; especially in potency assays
where an assessment of product stability and function canbe made. In these binding kinetics studies, the interaction isoften assessed through the measurement of either theaffinity of the analyte/drug product to a receptor or ligand
immobilized on the bisensor surface or by monitoring the
drug product‘s binding response signals (Req) as a functionof concentration and comparing it to a control reference
product for relative potency assesments2. Sartorius offers
ready-to-use biosensors such as Ni-NTA and FAB2G
Biosensors for the immobilization of different panels of
Fc gamma receptors that can in turn be used to bind drug
products for potency assesment.3

Octet® platforms can also be used for developing stabilityindicating methods, and are suitable for measuring anddistinguishing between fully-functional drug products andthose whose binding activities have been affected bydegradation. In one study, the Octet® platform was used to
distinguish between native and deactivated antigen andshowed reduced binding activity of the deactivated antigen(ref), hence proving the assay was stability indicating.
In another study4 (Figure 6), the affinity of an IgG1 to an Fc
gamma receptor IIIa molecule was shown to decrease withincreasing percent deglycosylation, further indicating that
Octet® systems are suitable for use in developing stabilityindicating methods.

Association

Dissociation

1.6E-6

1.4E-6

KD (M)In all cases, reproducibility measured through precision andaccuracy are key metrics. Octet® systems are highly suitable
for these assays as they decrease method development
time significantly compared to SPR and ELISA techniques.
An overlay of replicate data (Figure 5) for the binding of an
FcRn molecule to an IgG, with the IgG immobilized onto
Anti-Human Fab-CH1 (FAB2G) Biosensors shows excellent
reproducibility.

1.2E-6

1.0E-6.60
R2 = 0.9883

Binding Rate (nm).50
8.0E-7

.40
.30

0

20

% Deglycosylation

40

60

Figure 6: Affinity analysis of the binding of an IgG1 to an Fc gamma
receptor as a function of deglycosylation.

.20
.10
0
0

20

40

60
Time (sec)

80

100

Figure 5: Overlay of several replicates of FcRn-IgG interactions on the
Octet® platform using FAB2G Biosensors. Fitting of data traces was doneusing a 1:1 model with global fitting and a 5-second dissociation step (fitlines in magenta).

Technical Support
Sartorius offers comprehensive technical and service
support for Octet® system users in the GxP environment.
Field Applications Scientists and Engineers are trained toprovide guidance in various aspects of compliance, including
assay development and qualification. Performance certification (PC) that includes instrument preventive maintenanceand the necessary documentation is also available to GxPusers, and is recommended every 6 months.

7Summary

Table 3: Sartorius Octet® platform GxP product offerings and typical
GxP/QC applications.

Sartorius offers Octet® system users complete qualfication,
validation and support solutions that ensure compliance inthe regulatory space, and allow rapid development,
optimization and validation of assay methods for various
applications in GLP and QC laboratories. A summary of
product offerings and assay recommendations are listed
in Table 3.

References
1.

Steve Turner et al., Sartorius Application Note:
Customized Quantitation of Recombinant TherapeuticProteins Using High Precison Streptavidin (SAX)
Biosensors.
2. Nathan Oien et al., KBI Potency webinar: From
Screening to QC: Development considerations for
Octet® methods.
3. Michael Sadik et al., Bioprocessing Online, Accelerate
Biopharmaceutical Development with Novel Analytical
Techniques, April 28th, 2017.
4. Renee Tobias et al; Sartorius Application Note: Analysis
of FcRn-Antibody Interaction on the Octet® Platform.

Germany
Sartorius Lab Instruments GmbH & Co. KG
Otto-Brenner-Strasse 20
37079 Goettingen
Phone +49 551 308 0F or further contacts, visit
www.sartorius.com/octet-support

Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
For Research Use Only.
WP-41-0293 Rev F

Item

Description

IQOQ Kit andservice

Includes items required for the verificationand documentation of instrumentcomponents and for the qualifcationof instrument operational features

PQ KitIncludes biosensors and reagents required
to verify that the instrument performancemeets specifications in binding andquantitation applications

Octet® CFR
Software

21 CFR Part 11 data acquisition and analysis
software that ensures compliance with 21 CFRPart 11 regulations

Software validation
supportSartorius offers user support during validationof Octet® Software

Biosensors forvalidation support

Sartorius will work with customers tosequester multiple lots of biosensors (fora select group of biosensors) during assay
method qualification and method validation
processes

Key recommended
Octet® platform
GxP applications

Lot release, in-process testing and investigational
new drug (IND) testing assays, potency assays,
ligand binding, stability, detection limit and titerdetermination methods

USA
Sartorius Corporation
565 Johnson Avenue
Bohemia, NY 11716
Phone +1 888 OCTET 75
Or +1 650 322 1360