Application Note

May 20, 2021
Keywords or phrases:
Process Development, Process Efficiencies, Quality
Control, Binding Capacity, Dynamic Binding Capacity,
Bio-Layer Interferometry, Bioprocessing, Downstream
Bioprocessing, Protein Quantitation

Enhancing Efficiency and Economics
In Process Development and Manufacturing
of Biotherapeutics
Rashi Takkar, Sriram Kumaraswamy
Correspondence
Email: octet@sartorius.com

Abstract
The high costs of discovery, development and production of therapeutic drugs necessitate the need for improved process
efficiencies and economics. Analytical tools that circumvent traditional biologics analysis processes such as the need forlabeling of reagents while allowing for real time visualization of data can help save development time and facilitate improved
efficiencies during process development. Octet® instruments are used in a wide range of applications in process development and biotherapeutics manufacturing; including in early bioprocess development applications such as cell line development and clone selection and in downstream applications such as the determination of dynamic binding capacity for affinity
purification columns. The technology’s ability to monitor binding interactions in real-time coupled with its capability to analyze samples in their crude matrix and in high throughput can help shorten development and analysis times dramatically leading to significant cost savings. In this application note, insights from Octet® instrument adopters at GlaxoSmithKline andAragen Biosciences illustrate multiple examples where the Octet® system has been used to improve process efficiencies andhighlights its advantages over HPLC and ELISA in various segments of process development.

Find out more: www.sartorius.com

Introduction
Analytical techniques that measure protein quantity andquality are used in nearly all stages of research, processdevelopment and manufacturing of biotherapeutics. UV
spectroscopy, ELISA and HPLC have been in use for
decades for protein quantitation in physiological andprocess samples, and continue to be the workhorses
despite their many limitations. To characterize thefunctional activity of proteins during biotherapeutic
development, label-free biosensor-based binding assays
are increasingly being utilized. The high cost and lengthy
times associated with drug discovery and development
have forced biopharm companies to improve the efficiency

and economics in all stages of development. These key
drivers have fueled the search for innovative analyticaltechniques that provide improved performance andspeed without increasing costs. Biopharmaceuticalcompanies have enthusiastically adopted Sartorius’
Octet® systems due to their broad utility in protein
quantitation and functional characterization combinedwith enhanced throughput, decreased sample preparation
requirements, and low cost of operation. This white paper
describes the use of Octet® instruments for protein
quantitation, particularly in the areas of processdevelopment and quality control.

Target ID &validation

Lead screening& selection

Lead optimization& characterization

Preclinicaldevelopment

Processdevelopment

QC &manufacturing

Mechanismof action –biomolecularinteractions

Screeningfor binders(hybridoma, phageor lysates)

Aﬃnitymaturation –binding kinetics

Pharmacokinetics(PK)

Cell line develop.–titer and growth
media assessment

Activity assay

ELISA assay
development

Aﬃnity/ on-rate/oﬀ-rate ranking
of clones

Fc engineering/humanization –FcRn binding

Pharmacodynamics(PD)

Immunogenicity

Chromatography
conditions
optimize – DBC,
binding, wash,
elution and CIP

Epitope mapping
Epitope binning

Mammalianclone selection
for scale-up

Quantitation
speciﬁc

Loading
concentration forsmall-scale
puriﬁcation

Figure 1: Applications of Octet® instruments in the drug research and development process.

2

Contaminanttesting – insulin,
HCP and residualProtein A

FcRn binding
assay

Binding
kinetics

Advantages over ELISA and HPLC
The principles of concentration measurement with an
Octet® system are similar to established immunoassays
such as ELISA and HPLC. However, protein quantitation
protocols on the Octet® platform provide several
advantages. The Octet® platform monitors binding ofproteins from solution to a biosensor surface in real time,
without need for labels or other detection reagents. This
real-time monitoring of binding interactions enables clear
discrimination between specific and non-specific binding
signals, which can shorten assay development times
dramatically. Octet® assays are also much faster:quantitation of a 96-well plate requires 15–30 minutes, or
60 minutes for a 384-well plate, depending on the
instrument model. Figure 2 provides a comparison ofanalysis times. Analysis of 70 samples on an Octet® R8system requires as little as 55 minutes including operator
hands-on time, whereas ELISA or HPLC assays require atleast 22 hours including several hours of analyst
involvement. Samples run on Octet® systems are also
recoverable, so that researchers may save and reuseprecious sample for other experiments. In addition, Octet®
assays are not affected by absorption interferences incolored samples or by light scattering with turbid samples,
enabling measurement of analyte concentration in crude
matrices such as cell culture supernatant, cell lysate andserum. Octet® concentration assays are complemented by
the platform’s ability to measure functional activity. For
example, titer for a monoclonal antibody (mAb) can be
determined using biosensors coated with Protein A, while
the functional activity of the mAb can be assessed in asecond assay step involving binding to its specific antigen.
In contrast, HPLC and A280 spectroscopy can determine
only the total protein concentration of a sample, andseparate assays must be used to measure biological activity.

Protein quantitation of 70 complex samples
Octet® systems
Sample andinstrument prep
30 minSample analysis
10 min60 min total assay time
40 min operator hands-on time

Sample run time
20 minHPLC
Sample andinstrument prep
4 hr

Sample injection and run time
18 hr

Sample analysis
50 min23 hr total assay time
5 hr operator hands-on time

ELISA
Bind capture
antigen to plate
Overnight

Sample andinstrument prep
1 hr

Sample run time
4 hr

Sample analysis
30 min21.5 hr total assay time
5.5 hr operator hands-on time

Figure 2: Comparison of protein quantitation in complex matrices using Octet® systems and alternative methods.

3

Bio-Layer Interferometry
Technology (BLI)

Biosensors

Standard

BLI technology monitors and analyzes the interference
pattern generated from the reflection of white light fromtwo different surfaces: a layer of immobilized protein on thebiosensor tip and an internal reference layer (Figure 3). Any
increase or decrease in the number of binding molecules tothe biosensor surface produces a change in optical
thickness that causes a shift in the interference pattern.
Unbound molecules in complex matrices and changes in
the refractive index of the surrounding medium haveminimal effect on the interference pattern. BLI technology
simplifies protein quantitation by enabling specificmeasurement in complex samples. The one-step Dip andRead assay format uses native proteins, without need forlabels or secondary reagents.

Bound molecule

0.8
0.6
0.4
0.2

Unbound molecules
have no eﬀect

Wavelength (nm)Figure 3: Bio-Layer Interferometry is an optical analytical techniquethat analyzes the interference pattern of white light reflected from two
surfaces. Changes in the number of molecules bound to the biosensor
causes a shift in the interference pattern that is measured in real time.

Binding Rate (nm)

Biocompatible
surfaceUnknown

1.0

BLI signal processing
Relative Intensity

Incident
white
light

Reference0

Accurate determination of biologically relevant protein
concentrations is essential to several areas in thebiopharmaceutical industry including research,
bioprocessing, quality control and manufacturing. The
Octet® platform uses a simple Dip and Read approach for
rapid analysis of samples in 96 and 384-well microplate
formats. The concentration of the target protein or
antibody in a sample is determined via a direct binding or
sandwich assay. Biosensors coated with a capture
molecule, called the ligand, are dipped into solutions
containing the analyte in a highly parallel, automatedmethod to measure binding interactions. In a typicalquantitation assay, a standard curve is generated using
known amounts of the protein analyte, and unknown
sample concentrations are interpolated from the standard
curve (Figure 4). Concentration can be calculated from theinitial binding rate of the interaction which is based on theinitial slope or from the binding rate at equilibrium.

4120

Binding Rate (nm)Concentration Measurement

Time (sec)Concentration

Figure 4: A typical quantitation assay setup. Biosensors dip into
standards run in duplicate to obtain binding rate (nm) vs. time (sec)curves. The initial slope of the interaction is used to create thestandard curve of the binding rate vs. concentration. The concentrationof an unknown sample is then interpolated from the standard curve.

Quantitation Applications for
Drug Development
Research and Early Bioprocess Development
Pe-adapted
Host Cells

The Octet® platform is a useful tool for cost-effective protein expression screening in research and early bioprocessdevelopment with several significant benefits.

-

Octet® platform advantages
Antibody and protein concentrations can be determinedin crude matrices, such as cell lysates or hybridoma
supernatants, saving time and resources when processing
a large number of samples.
Octet® assays have a dynamic range of greater than twoorders of magnitude, enabling a single quantitation assay
to be utilized across all development stages – from earlycell culture to production bioreactors.
Octet® systems perform rapid quantitation with minimaluser involvement. 96 samples are analyzed in as little as
20 minutes, and 384 samples in 70 minutes. With
additional plate handling automation, Octet® RH16
systems can process more than 1200 samples per day.
Samples are analyzed in a non-destructive method andare fully recoverable, which is advantageous when working
with low sample volumes and precious samples.
Octet® systems are easy to learn and operate. Multiple
analysts can operate the instrument with minimal training,allowing rapid integration of these systems into laboratory
workflows.
Early clone selection
In clone selection, thousands of hybridoma or phage clones
are screened to determine positive binding clones and theirprotein secretion levels. Titer measurements are used to
select high-producing clones and to normalize thefunctional activity of these clones in crude matrices.
Integration of an Octet® system into the antibody discovery
workflow affords increased screening throughput. With
Octet® RH16 and Octet® RH96 instruments, automatedplate handling can also be added to achieve even higher
throughput. Octet® quantitation assays are also used todetermine loading levels of chromatography columns forsmall-scale purification.
Cell line development
Harvest samples are screened on Octet® systems to selecthigh-expressing clones during various scale-up procedures
involving 96-, 48-, 24- and 6-well plates, fed batch shakeflasks, and bioreactors (Figure 5). Octet® assays also areused to determine protein levels during media development for fed-batch and bioreactor processes (Figure 5).
This is performed by comparing protein secretion levels fol-

VectorTransfections
96-well Plates
Range: 1–300 µg/mL
Throughput: 1000s clones

24-, 12- and 6-well Plates

Octet® system

Range: 1–500 µg/mL
Throughput: 200–500 clones

Octet® system
T-flasks
Range: 1–500 µg/mL
Throughput: 100–150 clones

Octet® system
Shake Flasks
Range: 1–700 µg/mL
Throughput: 50–100 clones

Octet® system

Fed-batch Shake Flasks
Range: 1–700 µg/mL
Throughput: 12–20 clones

Octet® system

1–3 L Seed Bioreactors
Range: 1–4 g/L
Throughput: 2–5 clones

Figure 5: Protein titer assessment and growth media optimization
using the Octet® system at different stages of cell line development.

5lowing variations in feeding regimes, strategies and concentrations. Data acquisition and subsequent data analysis
can be performed rapidly for hundreds of samples, bypassing traditional processing bottlenecks. Please see Sartorius
Application Note “Fc-Fusion Protein Quantitation in Cell
Culture Supernatants” for more information.

Downstream Process Development
Efficient development of manufacturing processes for antibodies and recombinant proteins is a critical need for biopharmaceutical companies. Increasingly stringent
regulatory requirements targeting better understanding
and control of manufacturing processes are expected to
impact product quality and performance. The Octet® platform can quickly determine the impact of multiple process
variables at different stages of the purification process, andhelp identify optimal conditions that provide protein product with the desired yield, binding specificity and potency(Figure 6). Pre-configured reagents and protocols are available for rapid quantitation of protein products, host cell
proteins (HCP), and residual Protein A levels during purification processes.

--

Octet® platform advantages
One Octet® instrument can be used to measure protein
titer, host cell proteins and residual Protein A contaminantlevels.
Octet® assays are faster to develop and run than ELISA
and HPLC assays.
Octet® assays can be automated with robotic and liquidhandling systems for complete, walk-away screening.

Cell culture
harvest---Optimizing binding, wash
and elution conditions
Protein binding/activity
Residual Protein A
HCP (general & specific)
Protein recovery

Binding, wash and elution conditions
Numerous chromatography binding and elution conditions
are tested during optimization studies, including different
buffer compositions, salt, pH, operating temperature andsample injection volume. High-throughput tools, such asmini columns and 96-well filter plates, often are used toscreen these process variables. The impact of different conditions on product titer and quality can be analyzed rapidly
and effectively on Octet® systems, speeding identificationof optimal chromatography conditions (Figure 6).
Contaminant testing
Downstream purification processes must remove host cell
proteins, residual Protein A and residual DNA impurities.
According to guidance from regulatory authorities, host cell

Polishing chromatography(at least 2 steps)

Aﬃnity chromatography
Optimizing dynamic binding
capacity of column resins
Detect protein breakthroughin flow-through in crude
matrixDynamic binding capacity (DBC)of chromatography columns
Affinity chromatography often is the first major purification
procedure performed on harvested cell culture samples indownstream bioprocessing. The dynamic binding capacity(DBC) of an affinity chromatography column is defined as
the amount of protein that will bind to the column resin
under a defined condition. DBC is determined by continuously loading a sample containing a known concentrationof target protein and monitoring this protein in the flowthrough fractions. Quick determination of DBC using HPLCor A280 spectroscopy is hampered by the presence of large
amounts of host cell proteins in the flow-through fractions.
Specific detection of the protein of interest among contaminants is straightforward with Octet® systems, reducing thetime required to optimize purification conditions (Figure 6).

----

Optimizing dynamic binding
capacity of resins
Bind and elute mode: determine
protein breakthrough point
Flow-through mode: determine
final fraction when target
protein detectable
Optimizing binding, elution,
and wash conditions
Protein recovery
Protein binding/activity
Residual protein A
HCP

Figure 6: Use of Octet® systems in the downstream purification process of proteins and antibodies.

6

Viral
ﬁltration

Ultraﬁltration
diaﬁltration--

Optimize different
formulations based on:
Protein concentration
required for stability
Protein activity/binding

proteins in a drug substance should be below detectablelevels using a highly sensitive analytical method, and as arule this level should not exceed 100 ppm. The type of assay
required for HCP determinations depends on the phase ofclinical studies for which the material is produced. For earlier clinical phases, a generic assay may be sufficient. However, a process-specific HCP assay generally is required forphase 3 and later studies. Leached Protein A is another contaminant of concern in process development. The elution of
antibodies during Protein A chromatography requires
acidic conditions, which in turn can accelerate leaching ofProtein A from the column. Residual Protein A levels should
not exceed 10 ppm in the final drug product.

Customer Highlight: GlaxoSmithKline
The analytical lab at GlaxoSmithKline incorporated ageneric HCP assay on the Octet® QK384 system to streamline their workflow in process development. The automatedOctet® HCP assay required minimal analyst intervention
and provided more accurate and precise results than their

Activity assay

manual ELISA assay (Table 1). Hands-on time for preparation and processing of 1–3 assay plates was reduced to
30 minutes from the previous 2.5 hours with manual ELISA,
and antibody consumption decreased by 40%.
More information on the development of the HCP assay on
Octet® systems can be found in Technical Note, Host Cell
Protein Dectection on the Sartorius website.
Process development assays for residual Protein A andproduct titer can be fully automated on Octet® RH16 systems using external liquid handling platforms. The Octet®
assay for leached Protein A is highly sensitive with a LLOQ
of 0.20 ppm, has >2.5 logs of dynamic range, and is fasterthan competing methods. A residual Protein A assay on the
Octet® RH16 system can be completed in 1 hour and45 minutes per plate with minimal analyst involvement,
compared to a minimum of 3.5 hours for ELISA (including
significant analyst hands-on time). For more information on
the Octet® residual Protein A quantitation protocol, seeTechnical Note, Dip and Read Residual Protein A Detection
Kit on the Sartorius website.

Quantitation assay

Streptavidin
FabBiotinLigand — binds FabAntibody

Fc

Protein A — binds Fc

Figure 7: An activity assay can be developed on the Octet® platform by immobilizing a specific biotinylated ligand on the biosensor and thendetecting binding of an analyte, Fab or protein. In the quantitation assay, mAb titer is determined using Protein A-loaded biosensors, which
does not measure mAb activity towards its target.

7

Quality Control
Octet® systems provide robust and highly reproducible
assays for protein concentration and functional activity, andare suitable for operation in quality control and manufacturing environments. Protein activity and various kinetic assays
are used to support in-process testing, drug potency, lotto-lot variability and stability studies.

-

Octet® platform advantages
Octet® systems are designed for GLP/GMP
environments, and provide 21 CFR Part 11 compliancetools.
Octet® assays provide detailed information about thebinding behavior of protein products, and reveal subtle
differences in binding activity between production lots.

Customer Highlight:
Aragen Biosciences
Aragen Biosciences created a stable and scalable CHO cellline, purification platform and manufacturing process for aparticular product in a GMP environment. They developed
an Octet® assay to compare the activity and quality of a new
product lot (Lot 2) with a reference lot (Lot 1) throughout
their bioprocess and manufacturing processes. The assay

involved loading a biotinylated ligand on Streptavidin Biosensors, and measuring binding interaction of the ligand
with the protein analyte. As seen in Figure 8, Lot 2 contained a large second peak that was absent in the Lot 1 reference material. The second peak in Lot 2 exhibited aslower on-rate and much faster off-rate, indicative of aless-active fraction (Figure 9). Octet® system activity data
results were confirmed with a cell-based assay, and Aragen
was able to modify their production conditions to significantly reduce this second peak fraction.

--

Octet® quantitation assays provide a direct measure of
the biological activity of the analyte(s) (Figure 7).
Octet® assays can be easily transferred to manufacturing
operations.

Activity assays
An activity assay is generally utilized during process development, QC and manufacturing to compare various prepared lots of the drug molecule, as well as its stability.
Activity assays are critical because they differentiate active
protein from inactive or clipped variants, as those species
will not bind the ligand. Active protein concentration can be
determined using a binding assay on the Octet® platformby immobilizing a specific ligand against the target analyte
onto the biosensor, and then measuring its binding interaction with the analyte as shown in Figure 7.

Benefits of automated Octet® CHO HCP assay compared to manual ELISA
Benefit

Details

Precision

Liquid handling robot reduces pipetting variation inherent in manual pipetting

Reliability

Method performed exactly the same each time

Streamlined process

Worklist drives robotic method and creates sample plate importation files
Robotic method automatically creates and executes Octet® method file

Walk away

No analyst intervention needed to complete method after instrument loaded and diluent volumes are checked

Washing steps

No washing steps needed and plate washer integration not required

Analysts involvement

Automated Octet ~30 minutes for 1–3 assay plates
Manual ELISA ~2.5 hours per assay plate

Throughput

3 assay plates can be run in ~5 hours
38 samples/plate in duplicate wells > 108 samples in 3 plates

Antibody consumed

Re-use of coating antibody can significantly reduce consumption over multiple assay plates®

Table 1: Benefits of automated Octet® CHO HCP assay compared to manual ELISA summarized by GSK.

8

Lot 1High Speciﬁc Binding Activity

Lot 2
Low Speciﬁc Binding Activity
Peak 1

Peak 2Time (min)Time (min)Peak 1

Peak 23035

30

Presence of large
second peakcorrelated with
reduced speciﬁc
binding activity

35

Figure 8: HPLC spectra of Lot 1 and Lot 2 of a drug molecule. Lot 2 was made by Aragen Biosciences and had an
additional peak (Peak 2) compared to the reference lot (Lot 1) provided by their customer. Data provided courtesyof Aragen Biosciences.

Peak 1Time (min)Peak 230

35

Fitting View

A lower on-rate and anincreased oﬀ-rate wasclearly indicative of aless-active fraction

0.8
0.6
0.4
0.2
0

400

800
1200
Time (sec)

1600

Binding Rate (nm)Binding Rate (nm)1.00Fitting View

0.7

1.2

0.6
0.5
0.4
0.3
0.2
0.1
0

0

400

800
1200
Time (sec)

1600

Figure 9: The Octet® binding kinetics or functional assay demonstrated that Peak 1 was the active fraction. Peak 2 was theless-active fraction, with a lower on-rate and a much faster off-rate in a binding experiment. Data provided courtesy of
Aragen Biosciences.

9

Conclusion
Octet® systems deliver comprehensive characterization ofbiotherapeutics, as well as rapid and reproducible determination of protein concentrations during different stages ofthe development process. Titer and functional activity
assays on Octet® systems are useful for a broad array of
applications in target identification, lead selection, processdevelopment, formulation development, quality control,and manufacturing. In early stages of drug development,
Octet® systems provide the high throughput needed toscreen through large libraries of candidate drug molecules.

10

In later stages of process development and manufacturing,
Octet® systems provide the required reliability, robustness
and measurement accuracy. The broad utility of this single
platform makes the Octet® instrument unique in its ability
to deliver high value across a wide range of applicationneeds in biopharmaceutical discovery, development andmanufacturing processes.

11Germany
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37079 Goettingen
Phone +49 551 308 0

 F
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www.sartorius.com/octet-support

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