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June 15, 2018 (Vol. 38, No. 12)

Validating Anti-Idiotypic Binders for Trastuzumab

Avacta’s Affimer Reagents Compare Favorably to Traditional Antibodies in Regulated Bioassays

  • During drug development and clinical follow-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-idiotypic reagents to monitor these biotherapeutics during the drug development process. Affimer® reagents, a class of non-antibody-binding proteins developed by Avacta, show high target specificity and sensitivity, in addition 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 bioanalysis assay. This method incorporated an Affimer reagent, specific to the therapeutic anti-HER2/neu antibody trastuzumab (Herceptin), as the capture reagent in an ELISA. The goal of this project was aimed at replacing the currently used recombinant HER2 antibody capture reagents with an Affimer binder, as well as to qualify the Affimer performance using the criteria required for critical reagents.

  • Dynamic Assay Range

    Click Image To Enlarge +
    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%.

    A sandwich ELISA assay was established to investigate the scope of Affimer binders as critical reagents within regulatory bioanalytical assays, using an anti-trastuzumab Affimer for capture and an anti-trastuzumab antibody for detection. Following initial optimization, ELISA plates were coated with Affimer binders at 1.04 μg/mL. Samples were then diluted 1:10 into a human serum matrix, and detection was acccomplished via a biotinylated anti-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 established 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 bioanalytical 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 Control (LQC): 180 ng/mL; Middle Quality Control (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 accuracy and reproducibility both within and between assays.

  • Selectivity and Sensitivity

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    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.

    As part of the regulatory validation analysis, specificity and selectivity of the Affimer-based assay for the trastuzumab target antibody was determined in the disease state matrix, using samples of individual human donor serum from patients with metastatic breast cancer. Eight different human donor samples were analyzed, of which seven showed selectivity for the trastuzumab target within breast cancer serum with recovery of the spiked trastuzumab analyte at the LLOQ (Figure 2). The final patient sample showed previous exposure to trastuzumab. Consequently, this patient’s sample lacked selectivity.

    This selectivity assessment meets the regulatory target acceptance criteria for the assay, where 80% of samples show selectivity with sufficient bias and precision from the nominal values, and demonstrates the functional capability of the Affimer reagent, which remained highly selective for the target with no matrix effect across the range of individual patient serum samples.

  • Stability at Room Temperature

    Benchtop stability of the Affimer as a coating for the ELISA assay was assessed by storing a vial of Affimer reagent at ambient room temperature for 24 ± 4 hours prior to its use to coat ELISA plates. Comparison of the standard curve responses from both the control and Affimer reagent stored at ambient temperature were highly comparable, revealing theoretical concentrations for LQC and HQC samples with accuracy and precision well within the target specification for stability, of ±20% bias and ≤20% precision.

  • Lot-to-Lot Performance

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    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.

    A major issue with the use of anti-idiotypic reagents in bioanalysis assays is poor lot-to-lot reproducibility. Resolving this issue and maintaining assay performance can require extensive standardization between different lots. As Affimer reagents are simple to produce, the variability of recombinant protein between different production lots is minimized.

    Four separate lots of the anti-trastuzumab Affimer reagent were analyzed concurrently by comparing the reproducibility of their calibration curves for accuracy and precision (Figure 3). A high level of consistency across all four lots was demonstrated, with the estimated concentrations generated from each respective standard curve yielding an inter-assay accuracy and precision well within the target acceptance criteria for regulatory bioanalysis assay reagents.

  • Conclusion

    For this study, Covance validated the use of Affimer reagents as part of a high-performance bioanalytical assay according to regulatory standards. Affimer reagents demonstrated excellent target sensitivity, stability, and a broader dynamic assay range compared to the current antibody-based assay, with no detectable matrix effect.

    These advantages could lead to assay benefits including improved inter-assay accuracy and reproducibility between patient samples, as well as a reduction in the number of required dilutions and the number of sample repeats. Additionally, reducing the variability found in critical assay reagents through the adoption of reproducible substances, such as Affimer binders, could offer efficiency savings for drug development processes.

    A key feature of Affimer technology is the rapid development of highly specific binders without the need for affinity maturation (within just three months), allowing further reductions in project timelines and cost savings to many development programs. Taken together, these data demonstrate the validity of Affimer binders as alternative, or complementary, critical reagents to traditional antibodies for regulated pharmacokinetic assays of biotherapeutics. Finally, the results presented here highlight the scope of Affimer reagents in a regulated bioanalysis setting.

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