DESC:FIELD OF INVENTION
The present invention relates to an analytical method for detection and quantitation
of fusion protein in a sample.
BACKGROUND OF THE INVENTION
Bioanalytical studies form an important part of the clinical and non-clinical
pharmacology studies during drug development. Validated bioanalytical studies
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-ValidationGuidance-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.
Abatacept is a biologic drug that belongs to the class of T-cell co-stimulation
modulators and is used for the treatment of autoimmune disorders such as
rheumatoid arthritis (RA). 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
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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.
Therefore there is a need to develop a time saving and cost-effective method, which
is at the same time, selective in detecting Abatacept, is sensitive enough to measure
upto 7-8 half lives and has high dilution linearity.
SUMMARY OF THE INVENTION
Accordingly, present invention describes an ELISA based method for detection and
quantitation of CTLA-4 Fc fusion protein in a sample. The method employs use of
antibodies specific to the said fusion protein 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 costeffectiveness, interference mitigation and desirable sensitivity when compared to
existing methods is the art. With the claimed method, it is possible to detect free
fusion protein as low as 5 ng/mL in the sample.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Representative Standard curve showing concentration of abatacept vs.
absorbance at 450-630 nm using the method
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 “capture antibody” refers to antibody that binds and retains analyte from
the sample.
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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 “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 using a concentration-response relationship.
The 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 “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
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is sensitive enough to measure approximately 7-8 half lives of the drug and has high
dilution linearity. With the claimed method, it is possible to detect the free fusion
protein as low as 5 ng/mL in the biological matrix.
In an embodiment, the invention discloses a method for detecting and quantifying
CTLA-4 Fc fusion protein in a sample wherein the method comprises:
a) diluting the sample with an assay diluent;
b) contacting the diluted sample of step a) with a solid substrate coated with a
capture antibody, wherein the capture antibody is in monoclonal format of
IgG2a subclass;
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 detection antibody under
shaking condition wherein, the detection antibody is conjugated with a
detection reagent;
e) measuring the output from of the detection reagent of step d), thereby
quantifying abatacept in the sample; wherein,
the method can measure CTLA-4 Fc in the sample at a concentration as low as
5 ng/mL.
In an embodiment, the CTLA-4 Fc fusion protein is abatacept.
In another embodiment, concentration of the capture antibody is preferably 0.5 to 1
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.
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Example I
Normal human pooled serum samples spiked with various concentrations of
abatacept were diluted at least 10 folds in 5%BSA in PBST and incubated overnight
at 2-8
0C in microtitre wells coated with coating antibody (Anti human monoclonal
IgG2a antibody) raised against CTLA-4 to capture free CTLA-4 Fc fusion protein
present in the samples at a concentration of 0.50 µg/mL in 1X PBS. Wells were
washed with 0.1% PBST (3-5X) followed by addition of secondary antibody.
Secondary antibody was HRP conjugated Human Anti-Abatacept Antibody.
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
abatacept protein present in the samples.
A representative standard curve for abatacept may be found in Figure 1. The lower
limit of quantitation of the analyte (abatacept) using present method was found to
be 5 ng/mL, while upper limit was 80 ng/mL.
Example II
A high spiked abatacept sample of 2.5 mg/mL was prepared in pooled normal
human serum 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 1:10000, 35714, 71429, 208333 and 714286 (DL1, DL-2, DL-3, DL-4 and DL-5 respectively). 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. Data is shown in Table 1.
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Dilutional linearity of samples were within acceptance criteria. Sample dilution
displayed linearity up to 1:208333 post MRD. Hence, it could be concluded that
samples (2.5 mg/mL) can be diluted maximum up to 1:208333 in addition to
MRD.
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.
Table 1: Dilution Linearity of abatacept ,CLAIMS:CLAIMS:
We claim:
1. A method for detecting and quantifying CTLA-4 Fc fusion protein in a sample
wherein the method comprises:
a) diluting the sample with an assay diluent;
b) contacting the diluted sample of step a) with a solid substrate coated with a
capture antibody, wherein the capture antibody is in monoclonal format of
IgG2a subclass;
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 detection antibody under
shaking condition wherein, the detection antibody is conjugated with a
detection reagent;
e) measuring the output from of the detection reagent of step d), thereby
quantifying abatacept in the sample; wherein,
the method can measure CTLA-4 Fc in the sample at a concentration as low as
5 ng/mL.
2. The method as claimed in claim 1 wherein, the CTLA-4 Fc fusion protein
is abatacept.
3. The method as claimed in claim 1 wherein, concentration of the capture
antibody is 0.5 to 1 µg/mL.
4. The method as claimed in claim 1 wherein, the conjugated antibody is
diluted in PBS containing 5% BSA.