Specification
DESC:INTRODUCTION
The present invention relates to the field of quantification of biotherapeutic drugs. More particularly, the invention relates to a method of detection and quantification of free TNF-a targeting biotherapeutic drug in serum sample. The invention is particularly important for pharmacokinetic studies of a given TNF-a targeting biotherapeutic.
BACKGROUND
The analysis of plasma or serum samples for the presence and levels of a given therapeutic protein after its administration inside the body forms the core of pharmacokinetic (PK) studies. PK assessments enable safe and effective management of a given drug molecule and are hence recommended by regulatory bodies during clinical trials.
During a PK study, the absorption, distribution, metabolism and elimination of the drug molecule over a course of time within the body is measured. Parameters such as bioavailability, biologic half-life, renal clearance and toxic plasma concentrations of the drug are generally established from the results of these studies. It also help in designing the dosage regimen of the drug.
As the parameters chosen (e.g. number of samples, sample processing) for a PK study specifically influence the performance and sensitivity of the assay the methodology adopted needs to be carefully designed in each case.
For example, it is necessary that sufficient number of samples are collected over several time points to arrive at high accuracy values in comprehending pharmacokinetics of a particular drug. Also, serum sample is preferred over blood plasma for any detection or analytical assay, as, during processing of plasma to serum, a large number of plasma components that might interfere in the assay are removed and/or reduced during the processing step. Given this, using serum does not entirely mitigate the problem of interference, as serum has its own interfering factors. Also, the binding of capture and detection reagents with the desired ligand is subject to interference from multiple non-specific and undesired specific factors. Hence interference mitigation remains a challenge to be addressed even when serum samples are employed.
Enzyme-linked immunosorbent assay (ELISA) is one of the preferred methods for detection/measurement of the therapeutic molecule in a biological matrix (serum/blood). There are different formats available for ELISA and it basically involves coating an appropriate ligand on to a solid phase, allowing the analyte of choice to bind to the solid phase, and detection of the said analyte by complementary agents. The assay also includes blocking and intermittent washing steps. The washing steps, buffer condition, dilution factors and type of coating and detection reagents used, among other parameters, are optimized so as to enable removal of non-specific binding/interactions and thereby enable detection of the analyte of interest with superior specificity. In case of therapeutic antibodies, the assay design can also help in the quantification of free, bound or unbound forms of the drug. Hence, it is critical to design a specific methodology (comprising individual steps, reagents used, dilution factor etc.) from case-to-case, in a manner that stabilizes the interaction of the detection agent with the analyte, with minimal background noise.
Biotherapeutic drugs are made by means of recombinant DNA technology and include growth factors, hormones and antibodies. Biotherapeutic-based therapy has made tremendous progress, predominantly in the field of oncology and rheumatology. Rheumatological ailments (e.g. rheumatoid arthritis (RA), psoriatic arthritis (PsA), Crohn’s disease (CD)) are often characterized by an elevated presence of TNF-a in the circulating blood. TNF-a targeting biotherapeutic drugs bind specifically and with high affinity to the soluble and transmembrane forms of tumor necrosis factor-a (TNF-a), thereby inhibiting the binding of TNF-a to its receptors and consequent disease manifestation. Examples of currently approved TNF-a targeting biotherapeutics are adalimumab, infliximab, etanercept, certolizumab and infliximab.
In the case of adalimumab, based on the PK profile data available in literature (Abbvie-Humira Prescribing Information, US) and sensitivity calculation approaches, a sensitivity of around 50 ng/mL is required. In addition, while detecting free adalimumab, there is also a need to mitigate interference due to TNF-a binding factors (other than adalimumab) that could be present in the serum. Currently reported studies that achieves a desirable sensitivity is the assay use means such as electrochemiluminescence (ECL) and mass spectrometry based PK determination. These methods have superior tolerance and sensitivity, but are costly and time-consuming. ELISA based assays have a larger dynamic range, are cost effective and comparatively less time consuming. Available literature on ELISA-based PK studies of adalimumab within the desirable sensitivity range uses TNF-a as capture ligand, which understandably are subject to interference from TNF-a-binding agents in the serum other than the analyte (drug) of interest.
Hence, there is a need for developing a simple, cost effective method for assaying adalimumab in serum samples within a desired sensitivity level and target tolerance as well as optimal interference mitigation. Further, the logic adopted for the said assay for adalimumab could also be extended to tailor suitable methodologies for other TNF-a-targeting biotherapeutic drugs.
Consequently, the primary object of this invention is to provide a simple, sensitive and cost effective method for detecting and measuring TNF-a-targeting biotherapeutic drug in serum samples, which is able to remove interferences substantially. Another objective is to enable and qualify the said method with high target tolerance for TNF-a at it’s physiological levels in healthy and diseased states.
SUMMARY OF THE INVENTION
The present invention discloses an ELISA based method for detecting TNF-a-targeting biotherapeutic drug in a serum sample having or suspected of having interference due to TNF-a binding agents. The method employs the use of antibodies or antibody fragments specific to the said TNF-a-targeting biotherapeutic drug as coating and detection reagents, wherein the methodology in entirety significantly reduces non-specific interactions and/or the interferences in the assay. In addition, the method combines cost-effectiveness, interference mitigation, desirable sensitivity and target tolerance when compared to existing methods is the art.
Examples are directed towards adalimumab, a TNF-a-targeting biotherapeutic drug. Through the disclosed method, it is possible to detect and quantitate adalimumab as low as ~50 ng/ml in serum samples. Also, the upper limit of quantitation is 3000 ng/ml, thus highlighting the dynamic range of the method. The target tolerance of the method is 5 ng/ml of TNF-a, which is well within the physiological levels of TNF-a in healthy as well as diseased states.
Further, the method possess acceptable accuracy and precision and qualifies for its robustness and consistency as per the validation requirements of regulatory agencies.
In addition, by optimizing the dilution factor of the sample, working concentrations of detection reagent and by use of cost-effective detection modes in an ELISA format, the disclosed method provides a cost-effective assay methodology that also mitigates interference substantially.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 illustrates a comparison between two different dilution conditions of the serum sample viz, 1:10 and 1:25 wherein the samples were diluted in a suitable assay diluent buffer and experiment performed as described in Example 2.
Figure 2 illustrates sensitivity and range of the ELISA based assay wherein OD values of samples with concentration range from 0.05 µg/mL to 3.000 µg/mL is captured in triplicate
Figure 3 illustrates target tolerance of the assay wherein % RE and % CV were within the acceptance range in the presence of TNF-a up to a concentration of 5 ng/mL. Sample without analyte were found to be 3 and overall OD profile of the curve is desirable in the case of 1:10 dilution condition when compared to the dilution of 1:25. Hence dilution factor of not above 1:10 was optimized for the ELISA based assay of adalimumab.
,CLAIMS:WE CLAIM:
1. A method for detecting and quantifying TNF-a binding biotherapeutic drug in a serum sample comprising:
a) immobilizing an unlabelled anti-drug antibody to a solid phase
b) contacting diluted serum sample with the solid phase of step a) to form a first order complex
c) blocking with a buffer devoid of non-ionic surfactant
d) contacting the first order complex of step b) with diluted labelled anti-drug antibody to form a second order complex
e) detecting the second order complex of step d), using standard protocols of enzyme linked immunosorbent assay
f) interspersing steps a) to e) with wash cycles using a buffer containing 0.1% nonionic surfactant;
wherein interference due to TNF-a binding agents other than the drug is mitigated substantially.
2. The method according to claim 1 wherein diluent used for serum sample and labelled anti-drug antibody are the same.
3. The method according to claim 1 wherein the TNF-a binding biotherapeutic drug is detected to a level of sensitivity of up to 50 ng/mL and at target tolerance towards TNF-a of at least 5 ng/mL
4. The method according to claim 1 wherein, the labelled antibody of step d is a horse radish peroxidase labelled antibody.
5. The method according to claim 1 wherein, the TNF-a binding biotherapeutic drug is infliximab, adalimumab, golimumab, certolizumab or etanercept.
6. The method according to claim 1 wherein, the nonionic surfactant is polyoxyethylene sorbitan monolaurate.