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.