Application Guide

August 25, 2022
Keywords or phrases:
Biologics, Parallel Line Analysis, EC50, Potency,
Drug Activity, Lot Release

A Simple Method to Determine
Relative Potency (EC50) UsingOctet BLI Technology®

Stuart Knowling, PhD, Senior Scientist
Sartorius Corporation, 47661 Fremont Blvd, Fremont, CA 94538 USA
Correspondance
Email: octet@sartorius.com

Introduction
Unlike small molecule assays where the active concentration of a drug substance can be considered to be directly inline with the total concentration prepared; biological assays have the inherent issue that the total protein concentration
determined using absorbance or a dye-binding assay often does not correlate well to the observed response.
This variation in active protein concentration is especially prevalent when considering cell line development, wherealthough total protein yield is a major factor the binding parameters of individual clones and their active concentrationis the main consideration when determining the optimal clones to progress and in a recent study, ~32% of monoclonal
antibodies were found to contain one or more additional heavy or light chains, which degrades the desired propertiesof the antibody.1 Therefore, in order to determine how much of the biologic is correctly folded and active, a bioassay(potency assay) is performed.
A potency assay is a quantitative measure of biological activity; it must represent the intended biological effect(mechanism of action) and, where possible, be related to the clinical response. Potency assays help provide assuranceof the quality and consistency of the product, and common applications include the characterization of antibodies,
vaccines, therapeutic proteins, cell and gene therapies in manufacturing, stability testing, and lot release.2
Find out more: www.sartorius.com

Classically, Bio-Layer Interferometry (BLI) assays have been used to observe the response of the analyte (insolution) binding to the ligand (attached to the biosensor). This assay format may present issues when using
kinetics and the global affinity to determine potency as the kinetic association rate constant (ka) is dependent
upon the active protein concentration, and as such, the global affinity value (KD), and a potency assay may bemore suitable. Due to the variability in a biological assay, a measurement of activity of the result of the test material
relative to a known standard is more suitable and is known as relative potency (RP). Relative potency assumes thatthe reference and test material are biologically similar and that the test material behaves like a concentration or
dilution of the standard.2 Real-time Octet® BLI assays provide several benefits over standard endpoint assays suchas ELISA. Direct, label-free assays substantially reduce assay development time compared to endpoint assays as
there is no need to choose secondary antibodies, which may cross-react with capture molecules and requirelabeling optimization due to indirect detection. Total assay and hands-on times can be substantially reduced
thanks to the Octet® BLI Discovery and Analysis Software where traditional long washing and incubation steps canbe removed, which frees up more time for additional experiments. Key advantages of real-time, label-free Octet®
BLI assays are that low-affinity interactions can be elucidated more easily due to the removal of wash steps, andkinetics and affinity of each interaction can be determined for each interaction, unlike endpoint assays from which
critical association kinetic information is missing and only affinity can be calculated.
Octet® BLI Discovery and Analysis Software now contains a new module for quickly and easily assessing relative
potency using the same assay format currently used for quantitation and kinetic assays, allowing you to measure EC50
similarity using a range of parameter logistic equations (PL), linear and semilog lines. This document introduces basic
assay design and an introduction to the new software module.

Appropriate Assay Design
When designing a relative potency assay, a sigmoidal
response (EC50) is preferred if saturation of the doseresponse is possible. Sigmoidal responses for relative
potency calculation should contain at least sevenconcentrations of the test material and span thelower asymptote, transition region, and upper
asymptote (Figure 1).

Figure 1 Using the Potency Module in Octet® BLI
Discovery and Analysis Software
Lower
Asymptote

2TransitionRegionUpper
Asymptote

Designing and setting up a relative potency assay issimple using the experiment wizard where DoseResponse and Custom Dose Response modules havebeen added in addition to Quantitation, Kinetics, andEpitope Binning modules. Dose Response assays aresimilar to quantitation or advanced quantitation assays
but the analyte concentrations of test materials are
known in advance while Custom Dose Response assays
are similar to standard kinetic assays.
Several fully customizable two and three-step basic assay
templates are included for Dose Response, and these follow
a simple naming convention (Figure 2). A two-step assay
contains a sample and detection step, whereas a three-step
assay contains an additional loading step for the biosensor.
For example, the 2_step_8CH_96W_1TestAndStandard_7conc
method is a 2-step method for an 8-channel system using a
96-well plate that contains one test material and one
standard, both with seven standard analyte concentrations(number of samples in a column plus a buffer blank).

The workflow of the assay is easily observed in the plate
definition tab and modified by clicking on the modify button.
The concentrations for the standard and the test material canbe entered manually or by highlighting the concentration
column and right-clicking to ‘Set Well Data’ (Figure 3).

Prior to performing the assay, the method can be reviewed
in the Review Experiment Tab by simply clicking or sliding
the slide bar to view each step.

Figure 2

Figure 3

Data Analysis
During data analysis the Octet® Analysis Studio software
automatically detects, assigns, and preprocesses thedata generated using the Octet® BLI discovery softwareand displays this in the Preprocessed Data tab. The Pre­

processed Data tab is a good time to perform a visualquality control check to check for any issues during theassay, such as if one channel has not loaded as well as
the others and can be excluded.

3

Dose Response Analysis
The Dose Response Analysis tab (Figure 4) allows the user
to have access to a range of powerful data fitting options

that are updated in real-time and shown in the Fit Graphwindow (Figure 4A).

Figure 4HICDAEF
Report points for use can easily be set using the sample
response window (Figure 4B) by placing the blue verticalline where desired or a specific report point time andnumber of points to be averaged can be set in the Report
Point Settings (Figure 4C). It is also possible to normalize
to the standard group across samples.
Thanks to fully customizable two- and three-step basic
assay templates, it is possible to generate multiple data
sets of test materials and standards. The grouping window(Figure 4D) allows you to group the data in a number ofdifferent ways including the default averaged data sets or
the presentation of individual replicate curves wheredesired. An important part of a relative potency assay isdetermining the similarity (EC50) of the test material(s)versus a known standard, and this can easily be set using
the ‘Set Standard Group’ option. If set, normalization of
the test materials to the standard can be performed using
the normalize to Standard Group option (Figure 4D),
which takes the minimum and maximum response fromthe standard curve and applies normalization to all thetest material curves. The scale in the Fit Graph window(Figure 4A) changes accor­dingly from nm response to ascale of 0–100.

4BG
Of critical importance in determining similarity (EC50) is themodel used for fitting the standard and test material(s).
Octet® BLI analysis software offers a range of fit parameters
including independent and global fits, which applies thesame parameter fit to all the groups but each test material
has its own EC50 value. Data can be fitted to 3, 4, and 5
parameter logistic (PL) fit equations along with linear andsemilog line fits. Pre- and post-transition baselines play akey role in determining accurate relative potency andtherefore, if these parameters are not well defined, a top
and/or bottom value can be assigned for all curves prior
to fitting (Figure 4E).
Real-time monitoring of load steps using label-free assays
is a major advantage over endpoint-based assays andallows the user to assess whether observed differences are
due to loading issues or actual variability in the assessed
samples. Responses that propagate due to differences in
the loading level are often not linear and therefore, theZ-score func­tion allows the user to set an acceptancecriterion for the loading tolerated threshold. Whenchecked, loading Z-score (Figure 4F) adds a data column
to the Dose Response Data (Figure 4G), which shows thevariability in loading. Where the loading Z-score is above
the user-defined threshold, the data set is flagged forfurther investigation.

Analyzing Similarity
Fitted data can be viewed at all times in the Fit Graphwindow (Figure 4A) and similarity results viewed in thedata table Similarity Results tab (Figure 4G), where eachtest material's fit is compared to the standard and theirsimilarity based on EC50 is shown.
A range of data views is available using the ‘Fit Graph
View’ box (Figure 4H) where test material(s) and standard
data can be shown stacked on a single graph or in pairs orindividual graphs. Stacked graph view is the default view,which shows the standard and all test materials on thesame chart. Once prepared, data can easily be exported
in numerous ways (Figure 4I) for further analysis or
custom reports can be made in the report tab.

Assessing the Relative Potency of
Human IgG Antibody Samples
Assessment of antigen binding for antibody
development is a critical step, and the ability toaccurately detect changes in binding is critical. In asetup that would be suitable for assessing twounknown antibodies against a reference standard: anOctet® R8 8-channel BLI system and AHC2 biosensors(18-5142) were used in a standard 2-step dose response
assay format. A 12-point dilution series (50–0.02 µg/mL) of human IgG (hIgG) reference samples were thentested using an anti-hIgG Fab detection step (5 µg/mL). The assay was then repeated with either a plus orminus 30% hIgG concentration series (65–0.03 µg/mL
and 35–0.01 µg/mL, respectively), and the data
analyzed using the Octet® Analysis Studio Dose
Response module (Figure 5). All curves were assessedusing a 4 PL equation and normalized against thereference standard series. Each concentration series
showed an R2 value >0.99 for each data set, andcomparison of the EC50 ratio for each series exhibiteda similarity score of 1.34 and 0.74 for the plus or minus
30% hIgG concentration series when compared to thereference standard, in good agreement with thepredicted value. Combined with the Octet® Analysis
Studio Dose Response module, the above assay format
allows users to accurately assess antigen binding ofmultiple antibodies with good accuracy.

Figure 5Conclusions
The addition of Dose Response Analysis to the Octet® BLIAnalysis Studio software allows users to rapidly assess theEC50 of samples and determine similarity compared to a
standard data set. The Octet® BLI Analysis Studio software
is fully compatible with data generated in previous ver­sions of the software allowing rapid assessment of bothpre­viously generated and new data without the need toexport data to third-party software.

Real-time, label-free relative potency assays are now simpler
than ever on the Octet® BLI systems meaning that laborious
endpoint assays with long wash and incubation steps andhigh hands-on time can become a thing of the past. Octet®
BLI relative potency assays allow the user to observe theloading levels of their proteins and assess the data according­ly, and unlike endpoint assays, kinetics of the interactions canalso be extrapolated, even for low-affinity molecules.
5References1.

2.

Bradbury A, Trinklein N, Thie H, et al. When
Monoclonal Antibodies Are Not Monospecific:
Hybridomas Frequently Express Additional Functional
Variable Regions. MAbs. 2018;10(4):539–546.
<1032> Design and Development of Biological Assays.
doi:10.31003/uspnf_m1354_01_01

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