With drug development costs increasing, it is now critical for the biopharmaceutical industry to focus its efforts on identifying new technologies and methods that can predict the efficacy and safety of potential therapies as early as possible in the drug development process. For this reason, the industry is placing great emphasis on the development of predictive biomarkers with assays that can provide evidence of efficacy and safety early in preclinical development, thus providing greater assurance that lead candidates have a high probability of success.
Blood-borne biomarkers are considered desirable primarily due to the relative ease of sampling and the opportunity for serial monitoring. Immunoassays provide sensitive and specific technologies for studying protein biomarkers and a number of platforms are available, including enzyme-linked immunosorbent assay (ELISA), bead-based approaches such as xMAP®, and electrochemiluminescence (ECL).
While each platform has its specific application niche, they also need relatively large sample volumes, require long run times, and have limited dynamic ranges. Other immunoassay technologies are now available that can address these issues, defining their own analytical niche.
The Gyrolab® immunoassay platform (Gyros) is an automated, CD-based microfluidics technology that delivers high accuracy and precision for the measurement of monoclonal antibodies, drugs, proteins, and biomarkers from nanoliters of biological fluids (Figure 1). The attractiveness of the Gyrolab platform is due to its relatively short set-up and run times, built-in automation, low sample volume requirements, and broad dynamic range. EMD Millipore recently introduced services on the Gyrolab platform for use in biomarker and immunogenicity testing in its GLP-compliant laboratory.
Cytokine Biomarker Assays
Cytokines are promising biomarker candidates for predicting disease activity and response to therapy because of their prominent role in inflammation, immune response, and repair. However, the use of cytokines as biomarkers poses a few challenges, including a short serum half-life, very low (pg/mL) or undetectable serum baseline levels, potentially high disease- or drug-induced levels, lack of tissue- and toxicity-specific expression, and differential endogenous levels. Therefore, a highly sensitive cytokine biomarker assay is needed that can measure extremely low levels of proteins in biological matrices and has a large dynamic range.
A cytokine biomarker assay for the quantitation of interleukin-8 (IL-8) in cynomolgus monkey serum was developed and validated using the Gyrolab platform. Optimal parameters for quantitation were first determined, including capture and detection antibody concentrations, buffers, and matrix effects.
Recombinant IL-8 was then spiked into charcoal-stripped (protein-depleted) serum at predetermined levels and quantitated by immunoassay. The assay was validated by measuring the sensitivity, accuracy, precision, and selectivity of recombinant IL-8 in cynomolgus monkey serum (Figure 2).
The IL-8 immunoassay performed well over a wide range of analyte concentrations, ranging from 20 pg/mL to 10,000 pg/mL, and was selective for both recombinant and endogenous IL-8 as measured by spike, recovery, and parallelism, respectively. Drug treatment and disease state can result in the expression of cytokines at very high levels, and these samples must be diluted to the validated range of the assay.
To simulate theses conditions, high levels of IL-8 were spiked into serum and diluted to 1:2, 1:4, 1:8, 1:16, and 1:32. Nearly 100% recovery was observed for all dilutions, demonstrating dilutional linearity.
Finally, assay stability was measured. IL-8 remained stable over short-term (4 hr and 24 hr) stability testing in matrix at 15–30ºC and 2–8ºC; samples also remained stable (nearly almost 100% recovery) throughout six freeze-thaw cycles. Long-term stability will also be evaluated for up to one year. These data demonstrate that this cytokine biomarker assay is highly sensitive, can measure extremely low levels of proteins in biological matrices, and has a large dynamic range. In addition to IL-8, cytokine assays have recently been validated for IL-1β, IL-6, IL-10, IL-12, IFNγ, and TNFα.
With novel biologic drugs entering the market, it has become paramount to have well-defined and easily monitored safety profiles, including an evaluation of immunogenic response to the drug. Generation of antidrug antibodies in a patient can have a profound impact on the pharmacokinetics, pharmacodynamics, efficacy, and safety of a therapeutic.
The type and severity of the impact of an antidrug antibody reaction is dictated by the isotype of the antibodies generated; i.e., generation of IgE can indicate an allergic response. Therefore, it becomes important to understand the type of response that is generated in patients by determining the isotypes of the antibodies that are generated; e.g., a hypersensitivity reaction would be associated with elevated IgE levels.
An isotyping method was developed on the Gyrolab platform that is sensitive, specific, and flexible. The flexibility is derived from the method being generic to the isotypes of interest, and a sample pretreatment step can be employed to isolate therapeutic specific antibodies. This allows the opportunity to have one set of assays that can be applied to multiple therapeutics and reduce validation time.
To assess sensitivity, a curve of an antibody of known isotype (IgG/A/E/M) was prepared and analyzed. The sensitivity for any of the isotype systems (anti-IgG/A/E/M) was 15.6–125 ng/mL, which exceeds the required 250–500 ng/mL for clinical and nonclinical studies. Each assay was then challenged with each of the other isotypes to determine cross-reactivity.
The isotype specificity of each system was acceptable with no more than 0.4% cross-reactivity for any isotype in any system; thus, each system has a very high level of specificity between the analyte and the detection pairs. Matrix samples (bovine serum, canine serum, rhesus monkey serum, cynomolgus monkey serum, human serum, and buffer blank) were then assessed in each system (Figure 3).
Species specificity is mainly limited to primate antibodies (human and monkey) for most systems. The anti-IgA and anti-IgG systems detected both cynomolgus and rhesus monkey antibodies to ~80–90% of human serum levels. The anti-IgM system detected both cynomolgus and rhesus monkey antibodies to ~50% and canine antibodies to ~20% of what was detected in human serum. The IgE assay was only able to detect rhesus monkey antibodies to ~40% of what was detected in human serum. Bovine antibodies were not detected with any of the systems.
In summary, the Gyrolab platform is a suitable platform for assays that require very low sample volumes, high sensitivity, and a broad dynamic range. EMD Millipore leveraged this platform for the development of highly sensitive cytokine biomarker and immunogenicity assays in support of regulated studies for predicting the efficacy and safety of new compounds. Rapid development of these assays not only saves time and money; the low sample volumes required make it an ideal platform for the analysis of samples from small mammal models and help conserve precious clinical samples.