Method For Measuring Free Antibody In Sample

DESC:FIELD OF THE INVENTION The present invention relates to a method for measuring free (unbound) antibody present in a sample. BACKGROUND OF THE INVENTION Analysis of plasma or serum samples for presence and levels of a given drug 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 (ADME) 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 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 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. While detecting free adalimumab, there is also a need to mitigate interference due to binding factors other than the originally intended ligand (non-specific binding). 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. Hence, there is a need for developing a simple, cost effective method for assaying vedolizumab present in samples within a desired sensitivity level and target tolerance as well as optimal interference mitigation. SUMMARY OF THE INVENTION Consequently, the primary object of this invention is to provides a simple, sensitive and costeffective method for detecting and measuring vedolizumab in a sample, which is able to remove matrix interference substantially. Another objective of the method is to enable and qualify the said method with superior lower limit of quantitation. Accordingly, present invention discloses an ELISA-based method that can measure vedolizumab in the sample at a concentration as low as 100 ng/mL of the antibody in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Representative standard curve showing concentration of vedolizumab vs. absorbance at 450-630 nm using claimed method DETAILED DESCRIPTION OF THE INVENTION In an embodiment, the invention discloses a method for quantifying unbound vedolizumab present in a sample, wherein the method comprises: a) diluting the sample with an assay diluent; b) contacting the sample with a solid substrate coated with a first ligand of the antibody; c) removing the unbound material in the sample by washing the solid substrate with 0.1% phosphate buffered saline with Tween 20 (PBST) solution; d) contacting the solid substrate of step c) with a second ligand of the antibody wherein, the second ligand is conjugated with a detection reagent; e) measuring the output from of the detection reagent of step d), thereby quantifying vedolizumab in the sample; wherein, the method can measure vedolizumab in the sample at a concentration as low as 100 ng/mL and wherein the method is interspersed with wash cycles using a buffer containing 0.1% PBST. In an embodiment, the first ligand is a monoclonal antibody, a F(ab’) fragment or F(ab’)2 fragment, preferably a F(ab’) fragment. In another embodiment, the second ligand is a monoclonal antibody, a F(ab’) fragment or F(ab’)2 fragment, preferably a monoclonal antibody. In another embodiment, the first ligand is coated onto the solid substrate at the concentration of 1.4 µg/mL. In yet another embodiment, the second ligand is contacted at a concentration of at least 40 ng/mL or 50 ng/mL or 60 ng/mL or 70 ng/mL. In yet another embodiment, the sample is diluted at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold or at least 50 fold. In a further embodiment, the detection reagent is horse radish peroxidase.

The method uses 3,3',5,5'-Tetramethylbenzidine (TMB) as a substrate of horse radish peroxidase for the detection of the second order complex, thereby detecting vedolizumab. However, the second ligand of step d can be conjugated with any commercially available suitable labelling agent (such as but not limited to ruthenium, iodine etc.,) as can be understood by a person skilled in the art and shall be detected using appropriate substrate using the principle of enzyme linked immunosorbent assay. The upper and lower limits of detection of vedolizumab in samples, possible by this method are 2000 ng/mL and 50 ng/mL respectively. Sensitivity of the assay has been validated using commercially available vedolizumab ENTIVYO®. Further, the method mitigates interference due to non-specific agents binding to a4ß7 integrin. Definitions: The term “capture antibody” refers to antibody that binds and retains analyte from the sample. The term “conjugated antibody” refers to antibody that is linked to a label and can be used for the indirect detection of the analyte (when bound to the analyte). Conjugated antibodies are often linked to a variety of colorimetric or fluorimetric probes. The term “free antibody” refers to antibody in the solution that is not yet bound to the corresponding ligand. The term “F(ab’) fragment” refers to monovalent antigen-binding fragment of the antibody. The F(ab’) fragment, with a molecular weight of approximately 45 kDa, consists of one constant region and one variable region of each of the heavy and light chain. The term “F(ab’)2 fragment” refers to a fragment of antibody that consists of two monovalent antigen binding regions that are linked together by disulfide bonds with a molecular weight of approximately 110 kDa. “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. The term “sample” refers to a generic term encompassing controls, blanks, unknowns, and processed samples. “Sensitivity” of the assay is defined as the lowest concentration of standard sample drug preparation which consistently provides signal in the assay.

“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. EXAMPLES Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. During development of an ELISA based pharmacokinetic assay for detecting vedolizumab present in patient’s sample, various parameters were optimized to achieve high 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. Wash program that uses phosphate buffered saline solution with 0.1% PBST contributes to enhanced sensitivity and helps to obviate non-specific interference. Example 1: Measurement of vedolizumab Normal human pooled serum samples spiked with various concentrations of vedolizumab were diluted at least 10 fold in 5%BSA in PBST and incubated overnight at 2-80C in microtitre wells coated with monovalent human recombinant F(ab’) (anti-vedolizumab) that recognizes free vedolizumab at a concentration of 1.50 µg/mL in 1X PBS. Wells were washed with 0.1% PBST (3-5X) followed by addition of secondary antibody. Secondary antibody was human antivedolizumab monoclonal antibody (IgG1) conjugated with horse radish peroxidase (HRP) at a concentration of 40-70 ng/mL. Samples was incubated with secondary antibody for 60 mins under shaking conditions. Wells were washed followed by addition of TMB substrate (3,3',5,5'- Tetramethylbenzidine). Reaction was terminated and wells were read at 450-630 nm. The addition of substrate (TMB) gives color that is measured at 450 nm with a correction wavelength of 630 nm measured in dual wavelength combination as Lm1–Lm2. The intensity of the color is directly proportional to the amount of vedolizumab protein present in the samples. A representative standard curve for vedolizumab may be found in Figure 1. The lowest limit of quantitation of the analyte (vedolizumab) was found to be 100 ng/mL, while highest limit being 3600 ng/mL.

Example 2: Concentration of coating & detection reagents Ten standard concentrations points were run with coating concentration 1.500 µg/mL and various concentrations of detection reagent (40 to 70 ng/mL). Standards were prepared by spiking Entyvio® in pooled normal human serum. Results are tabulated in Table 1. Based on the results, it could be concluded that the combination of 1.500 µg/mL coating reagent concentration with any of the detection reagent concentrations assessed (40 ng/mL, 50 ng/mL, 60 ng/mL or 70 ng/mL) achieved signal gradation across the standard and high S/N ratio recovery within acceptable range. Table 1: Assessment of influence of concentration of coating & detection reagents Example 3: Assessment of matrix interference Matrix interference refers to interference in the drug quantification due to the presence of endogenous matrix components. This was analyzed by spiking various concentrations of vedolizumab in Pooled Normal Human Serum (PNHS) or assay diluent (5% BSA in 1X PBS) at 10 fold sample dilution. Vedolizumab was measured by method as described in Example 1. The results are shown in Table 3. Buffer curve refers to vedolizumab spiked in the assay diluent Coating reagent conc.- 1.5ug/mL Detection reagent conc.- 40.000ng/mL Detection reagent conc.- 50.000ng/mL Sample Conc. (µg/mL) Mean OD Value Mean conc. (µg/mL) %CV %RE S/N Mean OD Value Mean conc. (µg/mL) %CV %RE S/N STD01 10.000 2.132 11.132 1 11 355 2.533 11.182 0 12 362 STD02 5.000 1.802 4.411 2 -12 300 2.130 4.457 1 -11 304 STD03 3.500 1.700 3.655 1 4 283 1.978 3.547 0 1 283 STD04 2.500 1.476 2.568 1 3 246 1.737 2.591 0 4 248 STD05 1.000 0.797 0.997 0 0 133 0.945 1.013 1 1 135 STD06 0.500 0.413 0.497 0 -1 69 0.484 0.494 5 -1 69 STD07 0.250 0.181 0.234 2 -7 30 0.201 0.220 1 -12 29 STD08 0.100 0.081 0.114 2 14 14 0.094 0.113 1 13 13 STD09 0.050 0.040 0.059 2 19 7 0.046 0.059 5 18 7 STD10 0.030 0.024 0.034 5 14 4 0.028 0.037 0 23 4 MB 0.000 0.006 NA 0 NA NA 0.007 NA 3 NA NA Detection- 60.000 ng/mL Detection- 70.000 ng/mL Sample Conc. (µg/mL) Mean OD Value Mean conc. (µg/mL) %CV %RE S/N Mean OD Value Mean conc. (µg/mL) %CV %RE S/N STD01 10.000 2.828 11.675 1 17 404 3.014 10.716 2 7 377 STD02 5.000 2.385 4.338 1 -13 341 2.655 4.754 1 -5 332 STD03 3.500 2.241 3.555 2 2 320 2.444 3.538 1 1 306 STD04 2.500 1.986 2.619 0 5 284 2.147 2.521 0 1 268 STD05 1.000 1.073 1.009 2 1 153 1.196 1.003 4 0 150 STD06 0.500 0.534 0.488 3 -2 76 0.629 0.508 9 2 79 STD07 0.250 0.229 0.229 2 -8 33 0.252 0.224 2 -11 32 STD08 0.100 0.102 0.114 0 14 15 0.116 0.115 2 15 15 STD09 0.050 0.055 0.065 3 31* 8 0.057 0.060 6 19 7 STD10 0.030 0.030 0.035 2 18 4 0.035 0.036 4 18 4 MB 0.000 0.007 NA 1 NA NA 0.008 NA 5 NA NA

at varying concentrations. Matrix curve refers to vedolizumab spiked in the PNHS at varying concentrations. % Difference refers to percentage difference between OD values of buffer curve and matrix curve. Based on the results, it was found that the method is free of matrix interference at 10 fold dilution of sample. Concentration (µg/mL) Buffer curve Mean OD Value Matrix curve Mean OD Value Average OD value of buffer and matrix curve % Difference 10.000 3.051 3.084 3.068 -1 5.000 2.899 2.909 2.904 0 3.500 2.755 2.820 2.788 -2 2.500 2.559 2.611 2.585 -2 1.000 1.695 1.789 1.742 -5 0.500 1.023 1.086 1.055 -6 0.250 0.553 0.550 0.552 1 0.100 0.219 0.255 0.237 -15 0.030 0.066 0.085 0.076 -25 MB 0.018 0.016 0.017 NA Table 3: Assessment of matrix interference ,CLAIMS:CLAIMS: We claim: 1. A method for quantifying unbound vedolizumab present in a sample, wherein the method comprises: a) diluting the sample with an assay diluent; b) contacting the sample with a solid substrate coated with a first ligand of the antibody; c) removing the unbound material in the sample by washing the solid substrate with 0.1% phosphate buffered saline with Tween 20 (PBST) solution; d) contacting the solid substrate of step c) with a second ligand of the antibody wherein, the second ligand is conjugated with a detection reagent; and e) measuring the output from of the detection reagent of step d), thereby quantifying vedolizumab in the sample; wherein, the method can measure vedolizumab in the sample at a concentration as low as 100 ng/mL and wherein the method is interspersed with wash cycles using a buffer containing 0.1% PBST. 2. The method as claimed in claim 1 wherein, the first ligand is a monoclonal antibody, a F(ab’) fragment or F(ab’)2 fragment. 3. The method as claimed in claim 1 wherein, the first ligand is coated onto the solid substrate at the concentration of 1.4 µg/mL. 4. The method as claimed in claim 1 wherein, the second ligand is contacted at a concentration of at least 40 ng/mL or 50 ng/mL or 60 ng/mL or 70 ng/mL.