DESC:A METHOD FOR DETECTION AND QUANTITATION OF ANTI-IL6R ANTIBODY
FIELD OF INVENTION
The present invention relates to an analytical method for detection and quantitation of antibody in a sample. Specifically, the method relates to detection of anti-IL6R antibody.
BACKGROUND OF THE INVENTION
Bioanalytical methods form the core of all clinical and non-clinical pharmacology studies during the development of a drug. Validated bioanalytical methods give critical pharmacokinetic, safety and efficacy-related information relating to the drug, adding value to regulatory review and decision making. The parameters routinely evaluated during bioanalytical method validation include information relating to specificity, selectivity and accuracy of the method, range of analyte concentration that can be detected with the method (viz. upper limit and lower limit of detection and quantitation), inter- as well as intra-assay variability, dilution effects, ability of assay to mitigate interference and parameters relating to the analyte of interest, among others. The fit-for-purpose (FFP) concept states that the level of validation should be appropriate for the intended purpose of the study. (Bioanalytical Method Validation Guidance for Industry, May 2018, Available at: https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf)
Pharmacological studies are essential regulatory pre-requisites in the development of both small molecule drugs as well as biologic products. Biologic products are therapeutic drugs engineered and produced by means of recombinant DNA technology. Therapy using biologic products has made tremendous progress, has revolutionized the treatment of many diseases where existing therapeutic measures may have been less successful. Examples of approved biologic products are adalimumab, tocilizumab, abatacept, trastuzumab and rituximab, among others.
Tocilizumab is a humanized monoclonal antibody against human interleukin-6 receptor (IL-6R) and is approved for the treatment of autoimmune disorders such as rheumatoid arthritis. It is administered in two dosage forms i.e. intravenous (IV) formulation or subcutaneous (SC) injection. For drugs approved for administering intravenously and subcutaneously, developing a single method for pharmacokinetic (PK) assessments for two routes of administration can be advantageous as it can be both time and cost effective. However, developing a single method can pose a significant bioanalytical challenge as subcutaneous studies require a highly sensitive assay to measure approximately 7-8 half lives and intravenous studies require such sensitive assays to have high dilution linearity. In addition, in tocilizumab clinical studies with healthy volunteers, where the serum IL-6 levels are in 1-10 pg/mL range and soluble IL-6R in 25-75 ng/mL range, accurate and precise quantitation of tocilizumab can be challenging due to target interference.
Therefore there is a need to develop a time saving and cost-effective method, which is at the same time, selective in detecting tocilizumab, is sensitive enough to measure upto 7-8 half lives and has high dilution linearity.
SUMMARY OF THE INVENTION
The present invention describes a method for detection and quantitation of free anti-IL6R antibody in biological matrix sample. The method can be employed for all pharmacokinetic studies of tocilizumab in healthy volunteers in a time saving and cost-effective manner, wherein the antibody is administered intravenously or subcutaneously. Particularly, the invention discloses an ELISA-based method for detection and quantitation of tocilizumab. With the claimed method, it is possible to detect free antibody as low as 50 ng/mL in the sample.
BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Specificity of the method for quantitation of tocilizumab. Specificity was evaluated by testing other IgG1 MAbs in the current assay format. High Quality Control (HQC), Upper limit of quantitation (ULOQ) and Lower limit of quantitation (LLOQ) were prepared with Tocilizumab and three other IgG1 MAbs.
Figure 2: Dilution Linearity of biosimilar DRL_TC Vs. RoActemra®. DRL_TC and RoActemra® were diluted upto 51200 fold and Dilution linearity upto 12800 has been established for both DRL_TC and RoActemra®. AQL denotes Above quantitation limit and BQL denotes Below quantitation limit.
Figure 3: Evaluation of impact of soluble IL-6 on quantitation of Tocilizumab. Serum samples were spiked with 50 ng/mL Tocilizumab (LLOQ) with varying concentrations of IL-6 from 0-1000 ng/mL and samples were analysed in the PK assay for quantification of Tocilizumab. Data is represented from three independent/different sets.
Figure 4: Evaluation of impact of soluble IL-6R on quantitation of Tocilizumab. Serum samples were spiked with 50 ng/mL Tocilizumab (LLOQ) and 2.5µg/mL Tocilizumab (ULOQ) with varying concentrations of sIL-6R from 0-300 ng/mL and samples were analysed in the PK assay for quantification of Tocilizumab. Values highlighted in Bold indicate %RE more than ±25%. Data is represented from four independent preparations/sets
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person having ordinary skill in the art to which the invention pertains.
The term “sample” herein refers to a discrete material of biological origin that can be sampled and processed in a reproducible manner. Examples are blood, serum, plasma, urine, feces, cerebrospinal fluid, saliva, sputum, and various discrete tissues.
The term “blank” herein refers to sample of a biological matrix to which no analytes have been added, that is used to assess the specificity of the bioanalytical method.
A term “calibration standard” refers to a biological matrix to which a known amount of analyte has been added. Calibration standards are used to construct calibration curves from which the concentrations of analytes in quality control samples and in unknown study samples are determined.
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 “control sample” refers to a sample with a known quantity of analyte that is used to monitor the performance of a bioanalytical method and to assess the integrity and validity of the results of the unknown samples analyzed in an individual run.
The term “dilution linearity” as used herein refers to the extent to which the sample’s dose response is linear and in the desired assay range.
The term “free anti-IL6R antibody” refers to anti-IL6R antibody that is not bound to its ligand.
The term “lower limit of quantification” or “LLOQ” refers to the lowest amount of an analyte in a sample that can be quantitatively determined with acceptable precision and accuracy.
The term “quantitation range” refers to the range of concentrations, including ULOQ and LLOQ that can be reliably and reproducibly quantified with accuracy and precision through the use of a concentration-response relationship.
The term “recovery” refers to the extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method.
The term "reference product" refers to a currently or previously marketed recombinant protein, also described as the "originator product" or "branded product" serving as a comparator in the studies.
Ther term “sample” refers to a generic term encompassing controls, blanks, unknowns, and processed samples.
The term “sensitivity” refers to the lowest analyte concentration that can be measured with acceptable accuracy and precision (i.e., LLOQ).
The term “standard curve” refers to the relationship between the experimental response values and the analytical concentrations (also called a calibration curve).
The term “system suitability” refers to determination of instrument performance (e.g., sensitivity and chromatographic retention) by analysis of a set of reference standards conducted prior to the analytical run.
The term “upper limit of quantitation” or “ULOQ” refers to the highest amount of an analyte in a sample that can be quantitatively determined with precision and accuracy.
The present invention is advantageous for pharmacokinetic studies of drugs approved for being administered intravenously and subcutaneously, as developing a single platform method proves to be time saving and cost effective. The method is sensitive enough to measure approximately 7-8 half lives of the drug and has high dilution linearity. In addition, the method allows accurate and precise quantitation of tocilizumab with reduced target interference during clinical studies with healthy volunteers, where the serum IL-6 levels are in 1-10 pg/mL range and soluble IL-6R in 25-75 ng/mL range. With the claimed method, it is possible to detect the free antibody as low as 50 ng/mL in the biological matrix with reduced target interference.
In an embodiment, the invention discloses a method for detecting and quantifying free anti-IL6R antibody in a sample wherein the method comprises:
a) adding capture antibody to a substrate, wherein the capture antibody is in monovalent Fab format,
b) incubating the substrate for more than 12 hours with the capture antibody thereby coating the substrate with the capture antibody,
c) adding the sample onto the coated substrate of step b),
d) adding a diluted conjugated antibody,
e) incubating for more than 60 minutes in shaking condition, and
f) detecting and quantifying anti-IL6R antibody by colorimetry or fluorimetry;
wherein the lower limit of quantitation of anti-IL6R antibody is about 50 ng/mL and the method is able to tolerate upto 1000 ng/mL of IL-6 in the sample.
In an embodiment, the anti-IL6R antibody is tocilizumab.
In another embodiment, concentration of the capture antibody is preferably 0.20-0.60 µg/mL.
In another embodiment, the conjugated antibody is diluted in PBS containing 5% BSA.
In an embodiment, the conjugated antibody is HRP conjugated antibody.
EXAMPLES
The invention will now be described in greater detail by reference to the following examples which further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example I
Plates are coated with monovalent Fab of human anti-Tocilizumab antibody (HCA252, Bio-Rad) to capture Tocilizumab present in human serum samples followed by detection with HRP conjugated Human Anti-Tocilizumab Antibody (HCA253P). The addition of substrate 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 Tocilizumab present in the serum samples. The calibration (standard) range of the method is 3.500 – 0.033 µg/mL and the quantification range is 2.500 – 0.050 µg/mL in neat serum.
Example II
Different drugs (viz., Adalimumab, Trastuzumab, Bevacizumab and Tocilizumab) were spiked at ULOQ (2.5 µg/mL) , LLOQ (0.05 µg/mL) and HQC (1.875 µg/mL) level in normal pooled human serum and were checked for quantification using method described in Example I.
As shown in Figure 1 signal was obtained only with Tocilizumab and not with other IgG1 MAbs. IgG1 MAbs are shown in X-axis and Mean Optical density (OD) values are shown in Y-axis.Samples spiked with Adalimumab, Trastuzumab and Bevacizumab are below quantification limit and did not give any signal in the assay. And Tocilizumab samples are recovered as per acceptance criteria. Thus, method is specific for anti-IL6R antibody.
Example III
A high spiked tocilizumab sample of 1600 µg/mL was prepared in pooled normal human serum using DRL_TC and RoActemra® and subjected to five dilutions in such a way that concentration of one point after dilution falls above the ULOQ in standard curve, three dilutions within quantification range of the standard curve and one point below the LLOQ point. The dilutions used were 1:400, 800, 1600, 6400, 12800 and 51200. The minimum required dilution (MRD) of the standards, quality controls, blank and samples in serum was 1:10. MRD step was performed in assay diluent and additional dilutions were performed in 10% (v/v) blank matrix. Five sets of each dilution fold were prepared for both drugs.
Dilutional linearity of samples were within acceptance criteria for DRL_TC and RoActemra®. Thus, sample dilution shows linearity up to 1:12800 post MRD. Hence, samples (1600 µg/mL) can be diluted maximum up to 1:12800 in addition to MRD.
Example IV
Target control samples were prepared in pooled normal human serum containing 0-1000 ng/mL concentrations of soluble IL-6 and 0-300 ng/mL sIL-6R. No drug was spiked into the target control samples. Samples were stored for at least 12 hours at -70±10°C prior to the analysis. Target control samples without drug and with drug at LLOQ level were analyzed at MRD.
All target tolerance samples without drug were below LLOQ and with drug were recovered within ±25% upto 1000 ng/mL of IL-6 for 3 out of 4 sets and upto 50 ng/mL of IL-6R for all 4 set and all samples passed for %CV criteria. Hence, method is able to tolerate upto 1000 ng/mL of IL-6 and 50 ng/mL of IL-6R in the assay.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments and examples are therefore to be considered in all respects illustrative rather than limiting the invention described herein.

,CLAIMS:WE CLAIM:
1. A method for detecting and quantifying free anti-IL6R antibody in a sample wherein the method comprises:
g) adding capture antibody to a substrate, wherein the capture antibody is in monovalent Fab format,
h) incubating the substrate for more than 12 hours with the capture antibody thereby coating the substrate with the capture antibody,
i) adding the sample onto the coated substrate of step b),
j) adding a diluted conjugated antibody,
k) incubating for more than 60 minutes in shaking condition, and
l) detecting and quantifying anti-IL6R antibody by colorimetry or fluorimetry;
wherein the lower limit of quantitation of anti-IL6R antibody is about 50 ng/mL and the method is able to tolerate upto 1000 ng/mL of IL-6 in the sample.
2. The method as claimed in claim 1 wherein, the anti-IL6R antibody is tocilizumab.
3. The method as claimed in claim 1 wherein, concentration of the capture antibody is preferably 0.20-0.60 µg/mL.
4. The method as claimed in claim 1 wherein, the conjugated antibody is diluted in PBS containing 5% BSA.
5. The method as claimed in claim 1 wherein, the conjugated antibody is HRP conjugated antibody.