Application Note

HTRF profiling services on SpectraMax iD5 Multi-Mode Microplate Reader

  • Obtain consistent compound profiling results with the HTRF optimized SpectraMax iD5 Multi-Mode Microplate Reader
  • Analyze data quickly with preconfigured protocols in SoftMax Pro Software
  • Access a wide range of acclaimed HTRF assays and services with Cisbio's US Custom Solutions Laboratory team

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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:

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].

Log Competitor

Log Competitor one

Log Competitor two

Log Competitor three

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.

CD one

CD Two

Cd three

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.

Natalia Castro | Assay Development Scientist | Cisbio

Arturo Gonzalez Moya | Custom Solutions Manager | Cisbio

Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices

简介

Cisbio 定制解决方案实验室团队与 Molecular Devices 合作,使用 Molecular Devices 读 板机生成相关数据,促进 Cisbio US 完成专 家分析服务活动。SpectraMax®iD5 多功能 读板机通过 HTRF 认证,配置增强型 TRF 检测模块和针对 HTRF 检测优化的滤光片。 使用 SoftMax®Pro 7.0.3 及更高版本软件中 预先配置的 HTRF 程序模板,包括优化的 读数设置和数据分析,便于获得更快的分 析结果。

试验细节

使用三种商用药物完成一项分析服务,这 些药物均用于 Cisbio 的 Fcγ 受体结合分析 和 FcRn。在 SpectraMax iD5 读板机上测 量结果。

Cisbio 建立的对于使用的每种药物/抗体

化合物剂量反应:化合物连续稀释 8 个浓 度点的剂量响应曲线,每个浓度下三个平 行孔。

下图展示了两 (2) 名用户在两 (2) 天内完成的 剂量反应。数据来自一个样品在所有 Fcγ 受 体和 FcRn 的测定 ( SpectraMax iD5 读板 机 ),没有异常值,浓度梯度底部约束曲线模 型 [ng / mL]

Sample#
Biosimilar
Trade Name
Mechanism of Action
1
Bevacizumab
Avastin
Anti-VEGFA mAb
2
Rituximab
Rituxan
CD20 (ADCC)
3
Etanercept
Enbrel
Anti-TNFa mAb

下图显示了所有样品的 Log IC50 和 CV % 值,样品的读数由不同的用户在 Molecular Devices 的 SpectraMax iD5 读板机上运行不同天检测获得

Log Competitor

Log Competitor one

Log Competitor two

Log Competitor three

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.

CD one

CD Two

Cd three

下表显示来自七 (7) 个受体,两 (2) 个用户的 Log IC50 [ng / mL] 和 CV % 原始值,以及来自于 SpectraMax iD5 读板机的读数

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%

结论

由于与 Molecular Devices 和 SpectraMax iD5 多功能读板机的合作,Cisbio 现在可提供定制的 HTRF 分析服务,作为一种可靠,快速和可重 现药物研发的方法,这个技术适用于使用抗体亲和力检测技术的药物研发,以及对市场上现有的相关药物进行生物相似性研究的潜力。

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