Genetic Engineering & Biotechnology News

JUN15 2018

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14 | JUNE 15, 2018 | Genetic Engineering & Biotechnology News | GENengnews.com Amanda Nicholl During drug development and clinical fol- low-up, regulatory bioanalysis of therapeutic proteins requires the use of critical reagents that can specifically identify and accurately quantify each biotherapeutic within patient samples. Antibodies currently represent the "gold standard" of affinity reagents, and while traditional antibodies have been refined to the point where they are specific, sensitive, and reasonably reliable, they can be limited by their development speed, complexity to produce on an industrial scale, and lot-to-lot variation in assay performance. With an increasing number of antibody- based therapeutics entering the clinic, there is increased demand for validated, anti-idio- typic reagents to monitor these biotherapeu- tics during the drug development process. Affimer ® reagents, a class of non-antibody- binding proteins developed by Avacta, show high target specificity and sensitivity, in ad- dition to being rapidly developed and easily produced with minimum variation between lots—thus meeting the key requirements for critical reagents in pharmacokinetic assays. The following study was performed by Covance Laboratories to qualify the use of Affimer reagents in a key regulatory bio- analysis assay. This method incorporated an Affimer reagent, specific to the therapeutic anti-HER2/neu antibody trastuzumab (Her- ceptin), as the capture reagent in an ELISA. The goal of this project was aimed at replac- ing the currently used recombinant HER2 antibody capture reagents with an Affimer binder, as well as to qualify the Affimer per- formance using the criteria required for criti- cal reagents. Dynamic Assay Range A sandwich ELISA assay was established to investigate the scope of Affimer binders as critical reagents within regulatory bioanalyti- cal assays, using an anti-trastuzumab Affimer for capture and an anti-trastuzumab antibody for detection. Following initial optimization, ELISA plates were coated with Affimer bind- ers at 1.04 μg/mL. Samples were then diluted 1:10 into a human serum matrix, and detec- tion was acccomplished via a biotinylated an- ti-trastuzumab monoclonal antibody at 1 μg/ mL, with further incubation using the enzyme conjugate streptavidin horseradish peroxidase at a 1:2000 dilution. When a concentration curve was estab- lished for the assay, the use of the Affimer protein as a capture reagent allowed a broad dynamic assay range for the quantification of trastuzumab, from 60 to 2000 ng/mL (Figure 1). This assay range is more than double the range of the current antibody- based method that is used in regulatory bio- analytical assays by Covance and that spans from 40 to 750 ng/mL. Accuracy and Precision When the the bias and precision values of the Affimer-based assay were determined, the individual quality control concentrations— Lower Limit of Quantification (LLOQ): 60 ng/mL; Upper Limit of Quantification (ULOQ): 2000 ng/mL; Lower Quality Con- trol (LQC): 180 ng/mL; Middle Quality Con- trol (MQC): 1000 ng/mL; and Higher Quality Control (HQC): 1400 ng/mL—were reverse calculated from the standard curve. Both the bias and precision values were well within the acceptable regulatory limits of the assay, at ±10.8% bias and ≤12.4% coefficient of variance (CV) for intra-assay values (n = 6) and ±4.2% bias and ≤19.2% CV for the inter-assay values. These results demonstrate the absence of matrix effects in the Affimer-based assay—improving ac- curacy and reproducibility both within and between assays. Selectivity and Sensitivity As part of the regulatory validation anal- ysis, specificity and selectivity of the Affimer- based assay for the trastuzumab target an- tibody was determined in the disease state Avacta's Affimer Reagents Compare Favorably to Traditional Antibodies in Regulated Bioassays Validating Anti-Idiotypic Binders for Trastuzumab Drug Discovery Tutorial Figure 2. Selectivity of the anti-trastuzumab Affimer reagent in the disease state matrix. Trastuzumab target antibody was spiked into serum samples from metastatic breast cancer patients at the LLOQ and analyzed for analyte recovery. Seven of eight human samples were analyzed, and analyte recovery was within the validated assay parameters. Figure 1. Use of the anti-trastuzumab Affimer binder as a capture reagent allows a broad dynamic assay range for the detection of trastuzumab. Standard curve profiles of six independent runs are highly comparable with cumulative recoveries across concentrations within ±7.5% of nominal values and precision (CV) across the standard curve points of ≤10.8%. Figure 3. Affimer reagents show a high level of lot-to-lot reproducibility in their performance as critical anti-idiotypic reagents in the bioanalysis of trastuzumab.

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