DESC:FIELD OF THE INVENTION
The present invention relates to the field of quantification of biotherapeutic drugs. More particularly, the invention relates to a method for the detection and quantification of free recombinant fusion protein in serum sample. The invention is particularly important for pharmacokinetic studies of a given protein biotherapeutic.
BACKGROUND OF THE INVENTION
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 helps 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
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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. Some of the currently approved biotherapeutics for treatment of rheumatological ailments include adalimumab, tocilizumab and abatacept.
There is a need for developing a simple, cost effective method for assaying abatacept in serum samples within a desired sensitivity level and target tolerance as well as optimal interference mitigation.
Consequently, the primary object of this invention is to provide a simple, sensitive and cost effective method for detecting and measuring a fusion protein biotherapeutic drug in serum samples, which is able to mitigate interference substantially. Another objective is to enable and qualify the said method with high target tolerance at its physiological levels in healthy and diseased states.
SUMMARY OF THE INVENTION
The present invention discloses an ELISA based method for detecting a fusion protein biotherapeutic drug in a serum sample. The method employs use of antibodies or antibody fragments specific to the said fusion protein 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,
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interference mitigation, desirable sensitivity and target tolerance when compared to existing methods is the art.
Examples are directed towards abatacept, a CTLA4-Ig fusion protein biotherapeutic drug. The method possesses 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 THE DRAWINGS
Figure 1 illustrates absorbance read-outs at varying concentration ranges of the sample with mean concentration values and associated attributes that fall within the acceptance criteria of the assay
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method for detecting and quantifying fusion protein biotherapeutic drug in a serum, wherein the serum sample has or is suspected to have interference factors, the method comprising:
a) diluting the serum sample in a suitable assay diluent
b) immobilizing antibody against the fusion protein onto a solid support
c) contacting the diluted sample of step a) with the solid support, such that the fusion protein present in the sample is bound to the immobilized antibody of step b) to form a first order complex
d) diluting a labelled antibody against the fusion protein in a suitable assay diluent
e) contacting the bound fusion protein of step c) with the diluted antibody of step d), to form a second order complex
f) colorimetrically detecting the complex of step g, using standard protocols of enzyme linked immunosorbent assay such that the signal-to-noise ratio detected is above 3 at 450-630nm
g) interspersing steps a) to f) with wash cycles;
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wherein, the suitable assay diluent of step a) and step d) are the same and that the method reduces interference substantially.
In a further embodiment, the labelled antibody of step e is a horse radish peroxidase labelled antibody.
In yet another embodiment, the fusion protein biotherapeutic drug is abatacept or etanercept.
The invention describes a method for detecting the level of fusion protein biotherapeutic in serum samples, wherein the sample dilution factor, assay diluent buffer, contact antibodies and wash program are selected so as to reduce non-specific interferences from the sample, leading to enhanced sensitivity and substantially high target tolerance.
The method uses 3,3',5,5'-Tetramethylbenzidine (TMB) as a substrate of horse radish peroxidase for the detection of the second order complex. However, the labeled antibody of step d) can be labeled with any commercially available suitable labelling agent (such as but not limited to ruthenium, iodine etc.,) and shall be detected using appropriate substrate using the principle of enzyme linked immunosorbent assay.
Definitions
The term ‘sample’ or ‘biological matrix’ as used herein the invention refers to blood or serum sample isolated from the blood of healthy volunteers or patients who had been administered with fusion protein biotherapeutic. The said sample has or is suspected to have non-specific interference. A ‘standard sample’ as used here in the invention, is a sample spiked with known amount of fusion protein to mimic the physiological condition of treated patient’s sample and/or to evaluate sensitivity of the assay.
“Blank Sample” is an unspiked sample (plasma from patients or from healthy volunteers) in which no fusion protein has been added.
“Patients” as used here in the invention, refers to patients suffering from rheumatological ailments.
“Sensitivity” of the assay is defined as the lowest concentration of standard sample drug preparation which consistently provides signal in the assay.
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“Lower limit of quantification (LLOQ)” is the lowest concentration of analyte that has been demonstrated to be measurable with acceptable levels of accuracy and precision.
“Upper level of quantification (ULOQ)” is the highest concentration of analyte that has been demonstrated to be measurable with stated levels of accuracy and precision.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.
EXAMPLES
During development of an ELISA based pharmacokinetic assay for detecting fusion protein biotherapeutic drug present in patient’s sample, various parameters were optimized to achieve good sensitivity and interference mitigation. One such critical parameter was the optimal dilution of the test sample such that the signal-to-noise ratio is enhanced substantially. Further, blocking agent, wash program and parameters of assay diluent have been optimized to achieve acceptable levels of sensitivity of the assay. Specific examples described below relate to abatacept – an approved fusion protein antagonist for the treatment of rheumatological ailments.
Example 1: Sample Preparation
Serum samples were obtained from volunteers who had been administered with abatacept. Standard samples are human serum samples obtained from healthy individuals that has been externally spiked with abatacept.
Various concentrations of fusion protein were spiked to prepare the standard sample. Some of the standard samples were not spiked with abatacept so as to maintain the blank sample. Further, the spiked and unspiked samples were diluted in a suitable assay diluent buffer.
Example 2: Detection of fusion protein biotherapeutic drug
Individual wells of 96 micro titer plate were coated with recombinant anti-abatacept antibody. This is the coating antibody, which is diluted in 1X phosphate buffered saline (pH 7.4) and the plate was sealed and incubated overnight at 2-8 ºC. After incubation, the plate was washed to remove uncoated anti-abatacept antibody. Followed by this, plates were blocked with 5% BSA. After incubation, blocking buffer was discarded and the plates were washed to remove remnants of blocking agent. Following this, sample, standard sample and blanks are prepared
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as in example 1 and were added to each well and incubated under shaking conditions at 25 ºC.
After incubation, the plate was washed to remove unbound material from the wells.
Subsequently, horse radish peroxidase tagged recombinant anti-abatacept antibody was added
to each well and the mixture was incubated in the dark at 25 ºC. Followed by incubation, plates
were washed. Then, 3,3’,5,5’-tetramethylbenzidine (TMB) was added to the plates and the
mixture was incubated for 30 mins at 25 º C under shaking conditions. Stop solution was added
to stop the reaction and absorbance was measured at 450 nm using a micro plate reader. During
each wash step, the plate was washed with 0.1 % PBST, five times. ,CLAIMS:We Claim:
1. A method for detecting and quantifying fusion protein biotherapeutic drug in a serum,
wherein the serum sample has or is suspected to have interference factors, the method
comprising the steps of;
a) diluting the serum sample in a suitable assay diluent
b) immobilizing antibody against the fusion protein onto a solid support
c) contacting the diluted sample of step a) with the solid support, such that the fusion
protein present in the sample is bound to the immobilized antibody of step b) to form a
first order complex
d) diluting a labelled antibody against the fusion protein in a suitable assay diluent
e) contacting the bound fusion protein of step c) with the diluted antibody of step d), to
form a second order complex
f) colorimetrically detecting the complex of step g, using standard protocols of enzyme
linked immunosorbent assay such that the signal-to-noise ratio detected is above 3 at
450-630nm
g) interspersing steps a) to f) with wash cycles;
wherein, the suitable assay diluent of step a) and step d) are the same and that the method
reduces interference substantially.
2. The method as claimed in claim 1, wherein the labelled antibody of step e is a horse
radish peroxidase labelled antibody.
3. The method as claimed in claim 1, wherein the fusion protein biotherapeutic drug is
abatacept or etanercept.