Natalia Castro | Assay Development Scientist | Cisbio
Arturo Gonzalez Moya | Custom Solutions Manager | Cisbio
Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices
Introduction
Cisbio’s Custom Solutions Laboratory team has worked in collaboration with Molecular Devices to generate relevant data promoting the expert profiling service activity completed by Cisbio US utilizing current Molecular Devices readers.
The SpectraMax® iD5 Multi-Mode Microplate Reader is HTRF-certified, with an enhanced TRF detection module and filters optimized for HTRF detection. Preconfigured HTRF protocols in SoftMax® Pro Software 7.0.3 and higher include optimized reader settings, as well as data analysis, for faster results.
Experimental details
A profiling service was completed using three commercial drugs all on Cisbio’s Fcγ receptor binding assays and FcRn. The SpectraMax iD5 reader was used for measurement of the results. For each drug/antibody used, Cisbio established:
- Dose-response curves in triplicate with eight (8) concentrations
- Control 1: Labeled cells expressing the receptor of interest only (min)
- Control 2: Labeled cells expressing the receptor of interest and IgG-red only (max)
Compound dose response: 8-point dose response curve of serially diluted compound performed in triplicate.
| Sample# | Biosimilar | Trade Name | Mechanism of Action |
|---|---|---|---|
| 1 | Bevacizumab | Avastin | Anti-VEGFA mAb |
| 2 | Rituximab | Rituxan | CD20 (ADCC) |
| 3 | Etanercept | Enbrel | Anti-TNFa mAb |
The following graphs contain the dose responses from two (2) users on two (2) different days. Data includes one single sample in all Fcγ receptors and FcRn assay (SpectraMax iD5 reader), with no outliers, bottom constrained and concentration [ng/mL].
The following graphs show Log IC50 and CV% values for all samples. Samples were run over multiple days with multiple users with readings from Molecular Devices SpectraMax iD5 Reader.
The following tables represent the Log IC50 [ng/mL] and CV% raw values from seven (7) receptors, two (2) users, and readings from the SpectraMax iD5 reader.
| CD16a (Fcγlla V158) | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 4.516 | 4.285 | 4.29 |
| User 1 – Run 2 | 4.544 | 4.317 | 4.235 |
| User 2 – Run 1 | 4.521 | 4.327 | 4.174 |
| User 2 – Run 2 | 4.528 | 4.279 | 4.158 |
| AVG | 4.527 | 4.302 | 4.214 |
| STDV | 0.011 | 0.02 | 0.052 |
| CV% | 0.2% | 0.5% | 1.2% |
| CD16a (Fcγlla F158) | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 5.137 | 5.045 | 4.875 |
| User 1 – Run 2 | 5.112 | 4.922 | 4.848 |
| User 2 – Run 1 | 5.13 | 4.919 | 4.748 |
| User 2 – Run 2 | 5.134 | 4.906 | 4.685 |
| AVG | 5.128 | 4.948 | 4.789 |
| STDV | 0.01 | 0.056 | 0.076 |
| CV% | 0.2% | 1.1% | 1.6% |
| CD32a (Fcγlla H131) | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 4.445 | 4.556 | 4.598 |
| User 1 – Run 2 | 4.606 | 4.373 | 4.647 |
| User 2 – Run 1 | 4.679 | 4.511 | 4.528 |
| User 2 – Run 2 | 4.524 | 4.536 | 4.609 |
| AVG | 4.564 | 4.494 | 4.596 |
| STDV | 0.088 | 0.072 | 0.043 |
| CV% | 1.9% | 1.6% | .09% |
| CD32a (Fcγlla R131) | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 4.57 | 4.671 | 4.654 |
| User 1 – Run 2 | 4.576 | 4.58 | 4.691 |
| User 2 – Run 1 | 4.649 | 4.693 | 4.742 |
| User 2 – Run 2 | 4.656 | 4.695 | 4.662 |
| AVG | 4.613 | 4.660 | 4.687 |
| STDV | 0.04 | 0.047 | 0.034 |
| CV% | 0.9% | 1% | 0.7% |
| CD32b (Fcγllb) | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 5.184 | 5.353 | 5.499 |
| User 1 – Run 2 | 5.258 | 5.494 | 5.442 |
| User 2 – Run 1 | 5.389 | 5.526 | 5.545 |
| User 2 – Run 2 | 5.447 | 5.593 | 5.646 |
| AVG | 5.32 | 5.492 | 5.533 |
| STDV | 0.104 | 0.088 | 0.075 |
| CV% | 2% | 1.6% | 1.4% |
| CD64 (Fcγl) | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 2.751 | 2.23 | 2.937 |
| User 1 – Run 2 | 2.744 | 2.537 | 2.824 |
| User 2 – Run 1 | 2.778 | 2.605 | 3.028 |
| User 2 – Run 2 | 2.759 | 2.494 | 2.712 |
| AVG | 2.758 | 2.467 | 2.875 |
| STDV | 0.0127 | 0.1422 | 0.1188 |
| CV% | 0.5% | 5.8% | 4.1% |
| FcRn | |||
|---|---|---|---|
| Avastin (Bevacizumab) | Rituxan (Rituximab) | Enbrel (Etanercept) | |
| User 1 – Run 1 | 6.977 | 6.812 | 6.958 |
| User 1 – Run 2 | 7.022 | 7.046 | 7.019 |
| User 2 – Run 1 | 6.976 | 6.742 | 6.875 |
| User 2 – Run 2 | 6.812 | 6.833 | 7.191 |
| AVG | 6.947 | 6.858 | 7.011 |
| STDV | 0.08 | 0.114 | 0.116 |
| CV% | 1.2% | 1.7% | 1.7% |
Conclusion
Due to the collaboration with Molecular Devices and the SpectraMax iD5 Multi-Mode Microplate Reader, Cisbio can now offer custom HTRF profiling services as a reliable, fast and reproducible approach to drug discovery using technology suitable for antibody affinity and the potential of biosimilarity studies using relevant drugs available on the market.