Expert's Opinion

Qualification of an Assay

By Nicolas Fourrier, Director Biomarker and Biopharmaceutical Testing, SGS; Abhishek Saharia, Director of Cell-Based Assays & Biologics, DiscoverX; Fiona Greer, Global Director, Biopharma Services Development, Life Sciences, SGS | November 30, 2016

With applications for investigating functional comparability of an originator and biosimilar

Establishing the Quality Target Product Profile (QTPP) is the first step of biosimilar characterization, to determine the exact structure and variation of the originator molecule. Regulatory authorities then require head-to-head comparison at multiple levels to demonstrate that the proposed biosimilar is “similar or highly similar” to the reference. An integrated approach with structural and functional characterization is essential to demonstrate biosimilarity prior to clinical development, and observations made here could result in a shortened clinical process. This integrated approach to characterization requires the use of functional assays to determine pharmacological activity, providing comparative evidence to support data from structural characterization.

However, in vitro or in vivo potency assays can be expensive and time consuming, with readouts that are often far downstream from the initial drug/target interaction. Readily available cell-based assays that provide a rapid response to a drug are an attractive alternative. DiscoverX, a leader in cell-based assays for drug discovery, has developed a series of cell-based assays that rely on the native biology of target receptors to quantitatively measure drug potency. Its PathHunter technology, based on a split enzyme system, has been used to develop these mechanism of action (MOA)-based assays that are rapid and easy to use (Figure 1).

The power of these assays for potency analysis of biosimilar drugs was illustrated and qualified with an assay for the angiogenesis inhibitor, bevacizumab. Bevacizumab potency has traditionally been measured by proliferation of primary human umbilical vein endothelial cells (HUVEC), which takes 96 hours to run and is highly variable. The PathHunter bevacizumab bioassay can be performed in less than 24 hours and has high reproducibility, accuracy and precision. In addition, the PathHunter bioassay has been developed into a kit that includes cryopreserved, ready-to-assay cells, and reagents necessary to run the assay. The use of cryopreserved ready-to-assay cells further reduces assay time by eliminating the need to culture the cells prior to running the assay.

At SGS, the biomarker and biopharmaceutical testing laboratory has further qualified the PathHunter bevacizumab bioassay for use in comparability and potency testing. The assay was initially verified with VEGF165 at 12 concentrations from 0.0483–200 ng/mL with eight replicates, analyzed as four sets of duplicates. The results demonstrated a 4-parameter dose response curve with high reproducibility (Figure 2). The mean EC80 for VEGF165 was 3.93 ng/mL.

Similarly, bevacizumab activity was tested as four sets of duplicates with concentrations of drug ranging from 1.54–6000 ng/mL, using the defined EC80. As expected, the assay had a 4-parameter curve with high reproducibility (Figure 3). The mean IC50 for bevacizumab was 31.2 ng/mL, which is comparable to the ED50 of 50 ng/mL observed in a traditional HUVEC proliferation assay.

Comparison of data from a single plate when including all the rows, to the same data excluding the outer wells, demonstrated similar outputs, indicating that minimal edge effects in the assay. Analyzing the data from the same plate by pairing rows far away from each other (A-E, B-F, C-G and D-H ) reduced the overall %CV compared to pairing of rows next to each other. This suggested that the assay plate design include the use of duplicate wells that are furthest away from each other (Table 1).

Finally, to qualify the assay, two duplicate preparations of bevacizumab at 100% and 50% were prepared and tested on three different plates and two different days. Table 2 presents the data for 100% bevacizumab tested on three different plates on the same day and Table 3 compares those same three plates with a plate (D) run on a different day. In all cases, the data show good comparability with an inter-run mean IC50 of 45.6 ng/mL and an inter-day mean IC50 of 43.2 ng/mL.

Table 4 presents similar results for the 50% sample of bevacizumab. Again, there is good comparability between the plates and between the two days. The inter-day IC50 precision had a mean of 75.4 ng/mL with a standard deviation of 18.8 and CV% of 25.

The accuracy of the assay was evaluated by comparing the IC50 obtained with bevacizumab solution at 50% strength to the IC50 obtained with bevacizumab solution at 100% strength. Relative accuracy was calculated using the following formulas:

Relative potency (%) =  IC50 of 100% strength solution of bevacizumab x 100
                                       IC50 of 50% strength solution of bevacizumab

Relative accuracy (%) = Calculated relative potency x 100
                                       Theoretical relative potency

The inter-run relative potency had a mean of 57% with an SD of 7 and %CV of 12. The inter-run relative accuracy had a mean of 115% with a SD of 14 and a %CV of 12. The IC50 had acceptable intermediate precision and good relative accuracy and precision at the level of relative potency. These data support the conclusion that this assay is appropriate for the evaluation of bevacizumab potency and its biosimilars, and offers a number of advantages over the HUVEC assay. CP

• Lamerdin, J., H. Daino-Laizure, N.W. Charter, and A. Saharia Accelerating biologic and Biosimilar Drug Development 2016, BioProcess
• International, pp. 36-44.
• ICH Q6B: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
• ICH Q2(R1): Validation of Analytical Procedures: Text and Methodology
• USP guidance chapter “1032”: Development of Biological Assays
• USP guidance chapter “1033”: Bioassay Validation
• USP guidance chapter “1034”: Biological Assay Analysis

Nicolas Fourrier, Director Biomarker and Biopharmaceutical Testing, SGS
Abhishek Saharia, Director of Cell-Based Assays & Biologics, DiscoverX
Fiona Greer, Global Director, Biopharma Services Development, Life Sciences, SGS