(W1130-02-10) Choosing the Right Bioanalytical Platform to Accommodate a Biotherapeutic Pharmacokinetic Measurement in Serum and CSF:  A Case Study

Purpose: Measurements of therapeutic antibody levels for neurodegenerative diseases are usually performed in both serum and cerebrospinal fluid (CSF). Due to the 0.1% drug partition coefficient from serum to CSF and the need of distinct sensitivity and dynamic range, two different assays are often needed to support clinical pharmacokinetic (PK) determination. The Gyrolab platform offers different Bioaffy compact discs (CDs) that allow to fine-tune the sensitivity and dynamic range of the assays while using the same critical reagents. We recently evaluated different Gyrolab Bioaffy CDs, including the recently available, high sensitivity 4000 nL CD, to fulfill the assay requirements for sensitivity and dynamic range required for drug measurement in both serum and CSF in support of a PhI program intended for a neurology indication. (1) This resulted in assays with different dynamic ranges, using the same reagents and with minimal assay development to transition from serum to CSF matrix. The use of artificial CSF as surrogate matrix for human CSF was also evaluated.

Methods: An immunoassay was developed to measure a monoclonal antibody (mAb) therapeutic concentration in human serum and CSF using the Gyrolab platform. Three Bioaffy CDs (200, 1000, and 4000 nL) were evaluated for matrix tolerance, sensitivity and dynamic range. The same capture and detection reagents were used for both serum and CSF assays targeting the anti-idiotypic regions of the therapeutic antibody. The capture reagent was an in-house generated biotinylated rabbit anti-therapeutic mAb (biotin-486), and the detection reagent was an in-house generated AlexaFluor 647-conjugated rabbit anti-therapeutic mAb (AF-30). All CDs were evaluated using a standard (std) curve ranging from 6,000 to 0.01 ng/mL, prepared by spiking the mAb therapeutic in 5% human serum pool in assay diluent. Quality controls were prepared by spiking the mAb therapeutic at upper limit of quantitation, lower limit of quantitation, high, mid, and low levels for both matrices.

Results: The signal to background ratio was calculated on each of the three Bioaffy CDs evaluated to compare the sensitivity of each of the CDs (Figure 1). The highest quantitation limit of each CD was determined by calculating the recovery of the std points in each CD, with  ±25 % relative error (%RE) passing criteria, leading to a maximum quantitation value of 666.7, 2000 and 6000 ng/ml for the Bioaffy 4000, 1000 and 200 nl CDs, respectively (Figure 1). Upon CD selection, assays for serum and CSF matrices were qualified for PK assessment. A direct correlation with CD volume capacity and sensitivity was observed, with the Bioaffy 4000 nl CD offering the highest level of sensitivity. However, an inverse correlation was observed in terms of dynamic range, with the Bioaffy 200 nl CD extending the upper range of the assay by over 20-fold over the Bioaffy 4000 nl CD. A single Bioaffy CD could not provide appropriate sensitivity and dynamic range to support drug measurements in both serum and CSF. Given its wider dynamic range at higher concentrations, the 200 nl CD was selected for PK determination in serum matrix, with a std curve range from 2.74–2000 ng/ml and an assay sensitivity of 54.8 ng/mL, which was sufficient to reach the anticipated C_trough of 70 ng/ml. The Bioaffy 4000 nl CD was selected for the PK determination in CSF, with a std curve range from 0.24–15.36 ng/ml and an assay sensitivity of 2.4 ng/mL, allowing to reach the anticipated CSF C_trough of 4 ng/ml. The assays were qualified following a pre-established acceptance criteria, with results summarized in Table 1. The use of artificial CSF as surrogate matrix for human CSF was determined to be appropriate.

Conclusion: The selection of an appropriate bioanalytical platform is integral to assay performance, sample testing, and mitigating potential risks and delays, especially when the assays require different sensitivity and dynamic range. The Gyrolab platform was chosen to develop and qualify a clinical PK assay in human serum and CSF matrices for a phase I program designed to support an indication within neurology disease space. Three distinct Gyrolab Bioaffy compact discs (CDs) were evaluated, providing flexibility in terms of dynamic range and sensitivity. This allowed to develop and qualify the assays using the same critical reagents, thereby minimizing assay development and allowing a direct, quantitative comparison of drug levels within serum and CSF compartments. Our results underscore the utility of the Gyrolab platform for pharmacokinetic assays, where sensitivity requirements significantly differ between the test matrices. Lastly, it was concluded that artificial CSF is a viable, cost-effective surrogate matrix for the development of clinical pharmacokinetic assays.

References: (1) AR Foley, G Sperinde, SK Fischer. Bioanalysis 2023. DOI: 10.4155/bio-2023-0179

Acknowledgements: The authors are employees of Genentech Inc., a member of the Roche group, and may have an equity interest in Roche. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the abstract apart from those disclosed.

Figure 1. Comparison of assay performance using three different Bioaffy CDs (4000, 1000 and 200 nL).

Table 1. Serum and CSF PK assay parameters summary.