1. High-resolution SPR surrogate potency assay to facilitate comparability and biosimilar studies
Veronica Fridh, GE Healthcare, Uppsala, Sweden November 2017
Presentation based on study performed by Robert Karlsson, Åsa Frostell and Veronica Fridh at R&D in Uppsala.
Abstract
Biotherapeutic drugs, such as monoclonal antibodies, are large, complex biomolecules whose structural features can impact their biological activity. Several molecular interactions contribute to antibody function and an array of potency assays is needed to measure and monitor critical quality attributes (CQA) throughout the characterization process. In this presentation, we present ways that Biacore™ assays can be simplified and used as surrogate potency assays to facilitate comparability and biosimilar studies. Specifically, we outline the use of a capture-based Biacore assay to simplify assay development. We also demonstrate how this assay can be expanded to include both antigen and receptor binding to estimate relative potency related to several CQAs of a therapeutic antibody in a single sensorgram.
Finally, based on our understanding that a dose response curve, determined by EC50 or PLA analysis accurately reflect changes in active concentration only if binding kinetics remain unchanged, we present a tool box including a) report point analysis for EC50 determination, b) sensorgram comparison functionality to determine whether antibody association and dissociation profiles are consistent with expected binding properties and c) Calibration Free Concentration Analysis as a supporting tool, in order to ensure unambiguous determination of relative potencies.

2. To cover all Critical Quality Attributes of a theraputic antibody, an array of potency assays is needed
Several interactions contribute to antibody function
EC50 a measure of potency
“Potency …the specific ability or capacity of the product, …to effect a given result.” (21 CFR 600.3) Y
Antigen 1
Antigen 2
Fcg-receptors
Complement
C1q
FcRn
Receptor 2
Receptor 1
Drug Conc
Result
EC50
100 50
A short introduction: SPR systems are widely used for characterization of antibody activities such as antigen and FC receptor binding.
In early stages of antibody development, the interactions are characterized in detail for example by epitope binning and kinetic assays.
In later stages, the focus is more on potency determinations and on assays that can ensure that the established critical quality attributes (CQA) are maintained. To cover all Critical Quality Attributes of a theraputic antibody, an array of potency assays is usually needed.
Biacore and other ligand binding assays are often referred to as a surrogate potency assay as they do not measure the biological effect directly. [In Biacore, the EC50 is obtained as a result of e.g. measuring the binding level between an antigen and a concentration series of an antibody.]
Early phase: Kinetic assays for in-depth characterization
Ligand binding assay or surrogate potency assay used to indicate biological effect
KA PP
November 2017

3. Development of a new surrogate Biacore™ potency assay
In the present study we aimed to illustrate new possibilities with Biacore for binding activity measurements.

4. Biacore™ potency assay setup for an anti-TNF-a antibody
-50
450
950
1450
1950
2450
2950
3450
3950
4450 10 70
130
190
250
310
370
430
490
550
610
670
730
Biotinylated
TNF-a
Anti-TNF-a antibody
Assay setup using Biotin CAPture Kit
Sensor Chip CAP
Anti-TNF-a antibody
Biotinylated TNF-α
Biotin CAPture reagent
Response (RU)
Biotin CAPture
reagent
User 1: 63 sensorgrams
User 2: 65 sensorgrams
As model system, we used anti-TNF alpha antibodies and their interaction to the antigen TNF alpha. To facilitate assay development we chose a capture assay format based on the Biotin CAP Kit (GE Healthcare) and biotinylated TNF-alpha (left). In that way the surface was easily prepared and can be regenerated and reused over many cycles.
The sensor surface is functionalized in two steps by injecting Biotin Capture reagent and biotinylated TNFalpha. Antibody is then injected and the cycle ends with injection of a regeneration solution.
To the right the corresponding sensorgrams are shown. Data from 2 users and in total 128 sensorgrams, were used to study assay performance.
The work was conducted on a Biacore T200.
Time (s)
Capture format minimizes assay development time
KA PP
November 2017

5. Reproducible functionalization of CAP surface
User 1, 63 cycles
3768 average
0.23 CV%
3789 max
3756 min
User 2, 65 cycles
3745 average
0.51 CV%
3783 max
3721 min
400
800
1200
1600
2000
2400
2800
3200
3600
Response (RU)
Reproducible binding of
Biotin CAPture reagent
CV < 1%
Very reproducible capture of Biotin Capture reagent with CV < 1% for both users
KA PP
November 2017

6. Reproducible capture of biotinylated TNF-a
User 1, 63 cycles
39.4 average
0.77 CV%
40.1 max
38.8 min
User 2, 65 cycles
36.4 average
1.12 CV%
37.5 max
35.7 min 30 33 36 39 42 45
Response (RU)
CV ~ 1%
0.5% BSA added to TNF-a
Very reproducible capture of biotinylated TNF-alpha with CV close to 1% for both users. Important.
For this specific assay we found that addition of 0.5% BSA to biotinylated TNF alpha improved reproducibility. (It is not a general recommendation but something to investigate during assay development).
KA PP
November 2017

7. Acceptable baseline stability
Change in baseline
User 1, 63 cycles
41482 average
41532 max
41447 min
User 2, 65 cycles
41520 average
41594 max
41470 min
40000
40500
41000
41500
42000
42500
43000
Response (RU)
No significant changes in baseline level beween cycles indicate optimized regeneration
Small and negligible drop in baseline level indicates unchanged surface properties
No significant changes in baseline level beween cycles (increase, decrease or jumps), indicates optimized regeneration
KA PP
November 2017

8. Determination of EC50 from the Biacore™ dose-response curve
200
400
600
800
0.01
0.1 1 10
100
1000
Antibody concentratation (µg/mL)
Potency
report point
200
400
600
800
100
300
Response (RU)
Time (s)
Antibody
0.05–120 µg/mL X
EC50
2.6 µg/mL
Biotinylated
TNF-a
Let me remind you about the Biacore response.
We are here looking at the antibody response (binding level) upon interaction with TNF alpha, taken just after the end of the injection.
If the response is plotted versus the concentration we obtain dose response curves. EC50 values are using a 4 parameter logistic model.
KA PP
November 2017

9. Excellent repeatability of antibody binding in wide concentration range
User 1
User 2
Antibody conc. n=3 (g/mL)
Average response (RU)
Stdev (RU)
CV(%)
0.02
-0.9
0.12
-13.3
0.05
2.4
0.00
0.0
0.16
11.1
0.15
1.4
0.49
31.1
0.26
0.8
1.48
65.2
0.35
0.5
4.44
105.7
0.56
13.33
144.9
0.86
0.6
40.00
174.0
1.22
0.7
120.00
190.0
0.10
0.1
360.00
199.4
1.14
Antibody conc. n=4 (g/mL)
Average response (RU)
Stdev (RU)
CV(%)
0.02
-1.0
0.15
14.6
0.07
2.4
0.13
5.5
0.22
11.7
0.08
0.7
0.66
32.9
0.4
1.98
66.4
0.25
5.93
104.8
0.46
17.78
139.7
0.2
53.33
164.4
0.54
0.3
160.00
179.2 -
A look at the assay performance in terms of binding level responses, shows very good repeatability over a broad range of concentrations, here from 0.05 to 360 µg/mL
For best performance, samples were analyzed using a ”low to high concentration” run order. This means that standards and samples are run in a single series and not in separate series. (At each concentration the run order was randomized)
Different concentration ranges, partly overlapping (blue)
Run order: samples and standards in one series, low to high concentration
KA PP
November 2017

10. Assay ready to be validated
Biacore™ potency assay is linear with respect to concentration and capable of detecting stressed induced change
Linearity
Stressed antibody sample
-50 50
100
150
200
0,001
0,01
0,1 1 10
1000
Response (RU)
Antibody concentration µg/mL
-50 50
100
150
200
250
0,01
0,1 1 10
1000
Response (RU)
Antibody conc (µg/ml)
Reference
EC µg/mL
1 h stress
EC µg/mL
Relative potency for stressed sample derived from EC50 ratio: 3.69/8.38=43.8%
130%
70%
Sample relative conc. (%)
EC50 (µg/mL)
Relative potency (%)
Reference, 100%
4.23
100.0
130
3.30
128.1 70
5.90
71.7
The linearity of the assay was investigated following deliberately change in antibody concentration (130% and 70%, resp) compared to the reference sample (100%). Higher concentrations shifted the dose-response curves to the left and lower concentrations shifted the dose response curve to the right. Thus, relative potency values are linear with respect to changes in concentration.
The assay was further able to detect stress induced changes in another antibody. The relative potency dropped by around 40% following 1 hour of heat-treatment at 60°C.
Together all these results demonstrate that our assay is fit for purpose. We have shown that the assay is repeatable, that dose response curves have a good shape and EC50 values are meaningful. This, the assay is ready to be validated.
Assay ready to be validated
KA PP
November 2017

11. Assessing several Critical Quality Attributes in one Biacore™ SPR assay
Now the Biacore assay can be extended to look at several CQA in a single sensorgram.

12. Example 1 Dual potency assay for evaluation of two CQA in one sensorgram
Biacore™ assay extended with injection of FcγRI
Two potency assessments obtained in a single experiment
Anti-TNF-a antibody
[conc series]
FcgRI [fixed conc]
TNF-α
capture
Anti-TNF-a antibody conc (µg/ml)
EC50
FcγRI
binding
TNF-α RI
In the first example, antibody binding is followed by FcgRI binding. Here the binding levels for the receptor increase with increasing antibody concentration (or response). This data allows EC50 values to be determined for both antigen and Fc gamma-receptor binding.
In this way it becomes easy to define an array of potency data to provide a comprehensive potency profile of the antibody. Similar assays can be developed for bi-specific antibodies or other combinations of molecular mechanisms.
The dual potency assay may even be extended to include additional binding events provided the antibody do not dissociate from the antigen. In such a scenario, receptors that dissociate rapidly can be injected first, folllowed by injections of high affinity receptors.
[This type of assay is difficult with ELISA as it may require dual labels and as the second binding may be unstable.]
Sequential binding events
KA PP
November 2017

13. Example 2 Dual potency assay for evaluation of two CQA in one sensorgram
Biacore™ assay extended with injection of TNF-α receptor
Two potency assessments obtained in a single experiment
IC50
Anti-TNF-a antibody conc (µg/ml)
TNF-α receptor
binding
TNF-α
EC50
TNF-α
[capture
Anti-TNF-a antibody
[conc series]
TNFα receptor
[fixed conc] RI
In the second example, the assay was extended with an injection of the TNF-alpha receptor, natural binding partner of TNF-alpha.
The anti-TNF-a antibody and TNF-α receptor compete for binding to captured TNF-αlpha. Here we demonstrate that receptor binding is reduced or eliminated when the antibody binds to TNF-alpha. This allows us to prepare two plots; one for the antigen and one for the receptor and both EC50 and IC50 values can be determined.
Competing binding events
KA PP
November 2017

14. What do dose-response curves tell us? New ways to compare data
Let’s take a step back and look closer at what dose response curves tell us.

15. A shift in EC50 can be linked to changes in both concentration and kinetic parameters
Simulation 1 (fast kinetics)
Simulation 2 (slower kinetics)
What does it mean for Biacore™ assay, ELISA and other dose/response measurements? 10 20 30 40 50 60 70 80 90
100
110
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
Response
Concentration (M)
Reference 10 20 30 40 50 60 70 80 90
100
110
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
Response
Concentration (M)
ka&kd *0,5 10 20 30 40 50 60 70 80 90
100
110
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
Response
Concentration (M)
kd*0,5 10 20 30 40 50 60 70 80 90
100
110
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
Response
Concentration (M)
ka*0,5 10 20 30 40 50 60 70 80 90
100
110
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
Response
Concentration (M)
conc * 1.3 10 20 30 40 50 60 70 80 90
100
110
1E-12
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06 10 20 30 40 50 60 70 80 90
100
110
1E-12
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
Reference
conc * 1.3
ka*0,5
ka&kd *0,5 10 20 30 40 50 60 70 80 90
100
110
1E-12
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
Reference
EC50 values are not only linked to
concentration data.
The same EC50 values can be obtained for samples with different kinetic properties.
Differences in dissociation rates may
be missed for stable binders.
kd*0,5
Reference = Anti-TNFa antibody –TNFa interaction
ka 1.6*106 M-1s-1 ; kd 8.5*10-5 s-1
Concentration (M)
Use the animation and read the texts that appear.
[Background: Simulation of data performed using a 1:1 binding model with six input parameters (interaction and dissociation time, concentration, binding capacity (100 RU), association rate constant and dissociation rate constant). Case study 1: 100s interaction time, Case study 2: 180 interaction time, 300s dissociation time.]
Reference = Interferon a-2a -IFNAR2 interaction
ka 1.2*107 M-1s-1 ; kd 2.5*10-2 s-1
Changes in conc, ka and kd shift EC50
Changes in conc and ka shift EC50
Changes in kd alone do not shift EC50
KA PP
November 2017

16. Dose-response curves may not tell the whole story
Dose-response curves may not tell the whole story. Biacore™ software enable new ways to compare data Y
Sensor Chip CAP
Anti-TNF-a antibody
Biotin-TNF-α
Dose-response curves
EC50 or PLA
Report point data
Calibration-free concentration analysis (CFCA)
Active concentration analysis
Based on our simulations it is clear that dose response curves accurately reflect changes in active concentration only if binding kinetics remain unchanged.
Here we present a Biacore toolbox in order to ensure unambiquous determination of relative potencies.
We have shown that capture based methods can be used for potency assays, thereby simplifying assay development.
From the sensorgrams, report points are used for PLA analysis or for calculation of EC50
By adding a dissociation phase and including replicates, the Biacore software functionality Sensorgram comparison can be used to study changes in the dissociation constant, not revealed by the EC0 curve (as shown by the simulations).
Calibration free concentration analysis, CFCA, is a binding assay that can be used to determine relative active concentrations directly and without interference from kinetic properties
Let us have a closer look at these methods.
Replicates
Extended dissociation phase
Sensorgram comparison
Kinetic binding profiles
KA PP
November 2017

17. Sensorgram comparison can be included in Biacore™ potency assays to detect differences directly
Prolonging the dissociation phase for one of the potency concentrations adds kinetic information
A new sample that meets acceptance criteria appears between limits
Potency
report point
Upper limit
Lower limit 40 80
120
-100
100
200
300
400
Time (s)
Response (RU) X X
Reference
variability
window
Sample with similar potency response (X) as reference
appears outside the comparison window and receives a similarity score of < 100 %.
Sample with similar potency response (X) as reference
appears inside the comparison window and receives a similarity score of 100 %
Sensorgram Comparison is a simple tool that can identify deviations in kinetic behavior without the need to determine kinetic parameters.
A comparison window is defined, intended to capture assay or product variability. It can be established by combining data from several files and will use the average curve +/- user defined SD window, or simply min and max sensorgrams.
If the new sample falls outside the window a judgement can be made using similarity scores and by inspection of data in overlay plots. Small deviations from the window may still be acceptable.
If a new sample falls inside the window it indicates unchanged kinetic parameters and potency/relative potency values from dose response curves become reliable.
Different kinetic profiles
Similar kinetic profiles
KA PP
November 2017

18. Relative CFCA results(%)
Calibration free concentration analysis, CFCA, determines relative concentrations directly without interference from kinetic properties
Relative CFCA results revealed that paratope and FC activity are affected in the same way by stress
Different CFCA assay set-ups can be used to verify functional parts of stressed antibody 20 40 60 80
100
120
TNF-a
SuRe
Relative CFCA results(%)
Reference
1h stress
2h stress
3h stress
Sensor Chip PEG
TNF-α
Anti-TNF-a antibody: Paratope Y
Sensor Chip PEG
Anti-TNF-a antibody: Fc Y
MabSelect™ SuRe™
Calibration free concentration analysis, CFCA, is a binding assay that can be used to determine relative concentrations directly and without interference from kinetic properties.
With CFCA we can fit binding data over a range of dilutions to derive the active concentration.
In the current study we designed two different CFCA assays by
Immobilization of TNFalpha
Immobilization of MabSelect Sure , a variant of protein A
to Sensor Chip PEG in order to study changes in active concentration related to the paratope and FC activity stressed anti-TNFalpha antibodies, respectively.
Relative CFCA results revealed that paratope and FC activity of stressed anti-TNFalpha antibody are equaly affected by stress.
KA PP
November 2017

19. Potential for CFCA to support or replace EC50 data CFCA: Calibration-free concentration analysis
Relative potencies from Biacore™ dose-response curve based on capture of biotinylated TNFα
Relative CFCA results correlate perfectly with EC50
y = 1.02x-2.89
R² = - 20 40 60 80
100
120
Relative CFCA results (%)
Relative EC50 values (%)
TNF-a binding
CFCA vs EC50
y = 1.01x-1.72
R² =
MabSelect SuRe™ binding
Reference
1h stress
2h stress
3h stress
Anti-TNFα antibody
EC50
µg/mL
Relative potency (%)
Reference
3.6
100.0
1h stress
8.4
42.9
2 h stress
27.3
13.2
3 h stress
77.4
4.7
As shown earlier, exposing the antibody to heat stress lead to an increase in EC50 values and therefore a drop in relative potency.
Interestingly, is we plot the relative CFCA results against the relative EC50 values determined by our dose-reposponse curve, we can see a perfect correlation.
This finding suggests that CFCA may replace dose response curves to provide relative potency data.
Again such data will reflect potency if kinetic properties are unchanged.
So let us now summarize:
KA PP
November 2017

20. Summary and conclusions
A capture-based Biacore™ assay to estimate potencies for several CQA in a single experiment was developed
the assay show high reproducibility and can be developed for bi-specific antibodies or other molecular combinations
Sensorgram comparison and CFCA complement Biacore™ dose response curves to ensure unambiguous relative potency data
as a shift in EC50 can occur due to changes in concentration and in kinetic parameters
KA PP
November 2017

21. References
Poster
High-resolution SPR-based surrogate potency assay to facilitate comparability and biosimilar studies
KA PO
Publication
R. Karlsson, V. Fridh, Å. Frostell Journal of Pharmaceutical Analysis (submitted)
KA PP
November 2017

22. GE, GE monogram, imagination at work, Biacore, and MabSelect SuRe are trademarks of General Electric Company. © 2017 General Electric Company. First published November All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. For local office contact information, visit gelifesciences.com/contact gelifesciences.com GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden
This is a general slide on trademarks and goods and services
KA PP
November 2017

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