DESC:FIELD OF THE INVENTION
The present invention relates to a method for determining anti drug antibodies in a biological sample using double antigen bridging assay.
BACK GROUND OF THE INVENTION
Biopharmaceuticals have emerged as an attractive therapeutic molecule for treatment of various diseases. Most of biopharmaceuticals are protein based drug molecules, which are complex in nature and have high molecular weight. When administered to a patient, then may lead to several undesirable immune responses. One such common drug induced undesirable adverse response is the production of anti drug antibodies (ADA).
Production of anti drug antibody molecules may depend upon the immunogenicity of the drug molecule. Further, such generated anti drug antibody molecules may bind to the administered drug and neutralize the drug’s activity which in turn may affect pharmacokinetic and pharamcodynamic properties of its administered drug. Hence, it is one of the recommendations of Food and Drug Administration (FDA) to measure anti drug antibodies raised against the administered drug. Further, the FDA guidelines clearly states that the immunogenicity assay for detection of anti drug antibodies should have sufficient sensitivity and specificity. The method should also detect different isotypes of immunoglobulins including IgM and IgG in a biological sample.
In ADA assays, “sensitivity” is defined as the lowest concentration of anti drug antibody that is detected by the assay. Further, the sufficient sensitivity of the ADA assay may vary from product to product and are greatly influenced by selection of reagents and controls employed in a particular ADA assay format.
A number of different assay formats are available for measuring anti drug antibodies in a sample such as, but are not limited to, direct binding enzyme-linked immunosorbent assay (ELISA), bridging ELISA, radioimmunoprecipitation assays (RIPA), Surface Plasmon Resonance (SPR), and bridging electrochemiluminescence assays. Each assay has its advantages and disadvantages based on rapidity of throughput, sensitivity, and availability of reagents. The major differences between each of these assays are the number and vigor of washes, and availability/exposure of binding sites on antigen (epitope) which can have direct correlation on assay sensitivity.
Among the assay formats, double antigen bridging assays are highly preferable, since such the method, once optimized, can be applied to immunogenicity testing and is dependent only on the antigen specificity of ADAs and independent of host species in which the ADAs were generated. Various parameters involved in this assay play an important role in detection of ADA and the sensitivity of the assay. These parameters include concentration of coating antigen, concentration of detection antigen and the number of wash steps employed to the assay. Further, the concentration of coating antigen is known to be prone for “hook effect”, wherein a concentration other than optimal concentration of the coating antigen leads to false negative results. Lesser (or) higher coating concentration of the antigen leads to insufficient availability of binding sites on antigen to ADA as those binding sites might be covered with the antigen in case of excess coating of antigen and lesser binding sites are available in case of coating with lesser antigen. Hence, it is necessary to optimize the coating density of an antigen employed in double antigen bridging assays.
Further, the concentration of primary detection is also important to achieve greater sensitivity of the assay. Optimization of these parameters may vary from product to product.
Apart from above stated parameters which may interfere in the assay sensitivity, interferences present in a biological matrix may also impact the performance of the assay, which in turn affect the sensitivity of the assay. Pre-treatment of samples such as protein A/G treatment, acid treatment are known to overcome such interferences. However, such interferences may vary depending up on the indication and nature of the administered drug.
Hence, it is essential to develop an optimized immunoassay method for measuring anti drug antibodies raised against therapeutic protein molecules.
The primary object of the invention is to develop a sensitive and optimized assay format for detecting anti drug antibodies in a biological sample. Once, the format is optimized can be used in any host cells.
SUMMARY OF THE INVENTION
The present invention discloses a method for detecting anti drug antibodies raised against a therapeutic protein molecule by employing a double antigen bridging assay, wherein the ADA is captured by both biotinylated antigen and digoxigenylated antigen. Further, the concentration of coating antigen is optimized in a way such that, it prevents the hook effect, which in turn helps in achieving greater sensitivity by reducing the possibility of false negative data that poses risk to clinical subjects / patients.
In addition, the developed method detects different isotypes of immunoglobulins including but not limited to IgM anti drug antibodies and/or IgG anti drug antibodies.
The assay also exhibits tolerance to serum interferences. Hence, the method demonstrates a sufficient sensitivity in a neat serum.
The disclosed method is sensitive enough to detect as low as 4 ng/mL of IgG ADA and 250 ng/mL of IgM ADA.
DESCRIPTION OF THE INVENTION
Various embodiments of the disclosed invention provide a method for detecting antidrug antibodies in a biological sample.
In one embodiment, the claimed invention discloses a method for detecting anti drug antibodies raised against a drug comprising:
a). coating of optimized concentration of biotinylated drug on a micro titer plate
b). addition of the biological sample containing antidrug antibodies and incubating for a first period of time
c). addition of digoxigenylated drug and incubating for a second period of time and,
d). addition of anti digoxigenin horseraddish Peroxidase conjugate to detect the said digoxigenylated drug and in turn the bound anti drug antibody.
In another embodiment of the invention, the drug is a therapeutic protein molecule.
In yet another embodiment of the invention, the therapeutic molecule includes GCSF, PEG GCSF, erythropoietin, darbepoetin or the like.
In another embodiment of the invention, the biological sample in the method is plasma or serum.
In yet another embodiment of the invention, the concentration of the biotinylated drug is from about 20 ng/mL to 40 ng/mL.
In yet another embodiment of the invention, the concentration of digoxigenylated drug is selected from the range of about 25 ng/mL to 200 ng/mL.
In another embodiment of the invention, the first period of time is a sufficient time which allows binding of anti drug antibody present in a biological sample to biotinylated drug.
In another embodiment of the invention, the second period of time, is a sufficient time which allows binding of digoxigenylated drug to the ADA captured by biotinylated drug.
In yet another embodiment of the invention, the method is performed at room temperature.
In yet another embodiment of the invention, the sample is incubated during first and second period of time for 1 hour at room temperature.
In another embodiment of the invention, the anti drug antibody including but not limited to IgG or IgM.
In another embodiment of the invention, biological sample is diluted from 1:10 to 1:20 prior to the subjecting of sample to said assay.
In another embodiment of the invention, the method does not require any in-process pretreatment step for detecting anti drug antibodies.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples, being provided only for purposes of illustration.
These examples should not be construed as limiting the scope of the invention in any manner.
EXAMPLES
During development of ADA assay, various parameters were optimized in an immunoassay format to detect anti drug antibodies present in a biological sample, wherein the method employed both biotinylated antigen and digoxigenylated antigen. Further, the biotinylated antigen employed in coating of a microtiter plate. The concentration of coating antigen was optimized in a way that it results in achieving better sensitivity.
The method was optimized for detection anti PEG GCSF antibodies (which is an IgG antibody) and anti PEG antibodies (which is an IgM antibody; purchased from ANP technologies) and anti-GCSF antibodies as well.
In addition, the present assay method was optimized for detection of anti darbepoetin antibodies in a biological sample.
Example 1: Assay method for detection of anti PEG GCSF and anti PEG antibodies and evaluating the sensitivity of the assay
Prior to the performance of the assay for detection of anti PEG GCSF antibodies, antibodies against PEG GCSF were raised in rabbit and were purified using PEG GCSF coupled NHS activated sepharose beads and the purified anti PEG GCSF antibodies were used as positive control in detection of anti PEG GCSF antibody assay. Biotinylated and digoxigenylated PEG GCSF molecules were prepared by conjugating with commercially available PEG GCSF molecule and such conjugation procedures were known in the art.
Step 1:
Samples were prepared by the following process, wherein serum samples obtained from healthy volunteers were spiked with various concentrations of in-house generated and purified anti PEG GCSF antibodies to mimic physiological conditions of PEG GCSF administered neutropenia patients. Such spiked samples were considered as positive controls (PC 1) in this assay. Some of the serum samples were not spiked with anti PEG GCSF antibodies to maintain negative quality control (NQC) in the assay. Further to maintain negative control, serum sample obtained from healthy individual (not pooled serum) was employed in the assay. In these experiments pooled serum which was collected from individuals and pooled, was obtained from authorized blood banks.
Some of the serum samples were also spiked with anti PEG antibodies purchased from ANP technologies, which were considered as positive controls (PC 2) in detection of anti PEG antibodies in the ADA assay.
Step II
To analyze the effect of varying concentrations of biotinylated PEG GCSF and digoxigenylated PEG GCSF on the performance of the assay, various concentrations of biotinylated PEG GCSF and digoxigenylated PEG ranging from100 ng/mL to 12.5 ng/mL were prepared by diluting in assay buffer (1% bovine serum albumin in phosphate buffer saline (PBS) solution containing pH 7.2-7.4).
First the assay was optimized with the positive control samples containing anti GCSF antibodies obtained from AbD serotec. Further, the optimized assay was employed in detection of anti PEG and anti PEG GCSF antibodies.
96 well micro titer plate was coated with 100 µl/well of 5 µg/mL of streptavidin and plate was washed with 0.05% PBST (Phosphate Buffer Saline solution with Tween-wash buffer) and wells were blocked by adding 250 µl/well of 1% BSA (blocking buffer) and were incubated at room temperature for 1 hour. Various concentrations of the prepared biotinylated PEG GCSF was added to each well and incubated at room temperature for 1 hour with shaking at 300 rpm. All the wells were washed with wash buffer. 100 µl of 100 ng/mL of positive control containing anti GCSF antibody was added to each well and was incubated for 1 hour at room temperature with shaking at 300 rpm. After incubation, wells were washed with wash buffer for three times. 100 µl of varying concentrations of digoxigenylated PEG GCSF was added to each well and was incubated for 1 hour at room temperature with shaking at 300 rpm. After incubation, all the wells were washed with wash buffer for three times followed by 100 µl of anti digoxigenin horseradish peroxidase (which was diluted in 1% BSA in the ratio of 1: 2500) was added to all the wells and was incubated for 1 hour at room temperature with shaking at 300rpm. After incubation with secondary detection reagent, wells were washed with wash buffer for three times and 100 µl of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate was added to each well and was incubated for 15 minutes at room temperature. 100 µl of stop solution (1N H2SO4) was added to stop the reaction and absorbance at 450 nm was measured using a micro plate reader.
Results of the assay are given below in Table 1 and 2. Data was analyzed in terms of mean absorbance/optical density (Mean OD) and P/N ratio (positive control/negative quality control). P/N ratio is also known as S/N ratio (Signal to noise ratio).
Table 1: Mean absorbance of positive control sample (100 ng/mL of anti GCSF antibody) in presence of varying concentrations of biotinylated and digoxigenylated PEG GCSF.
Biotinylated PEG GCSF Concentration (ng/mL) Digoxigenylated PEG GCSF (ng/mL)
100 50 25 12.5
Mean OD % CV Mean OD % CV Mean OD % CV Mean OD % CV
100 0.570 1 0.539 2 0.492 3 0.386 2
Blank 0.115 8 0.068 3 0.056 1 0.059 16
50 0.668 1 0.563 4 0.573 1 0.414 2
Blank 0.114 13 0.068 6 0.061 2 0.055 12
25 0.704 2 0.678 3 0.625 2 0.506 1
Blank 0.113 6 0.070 3 0.057 2 0.050 2
12.5 0.555 0 0.525 0 0.487 1 0.422 2
Blank 0.150 10 0.094 4 0.067 4 0.663 10

Table 2: S/ N ratios of 100 ng/mL of anti GCSF antibody in presence of various concentrations of biotinylated and digoxigenylated PEG GCSF
Biotinylated PEG GCSF Concentration (ng/mL) Digoxigenylated PEG GCSF (ng/mL)
100 50 25 12.5
100 4.9 7.9 8.8 6.6
50 5.8 8.2 9.5 7.5
25 6.2 9.6 10.9 10.0
12.5 3.7 5.6 7.3 6.7
Blank (NQC) 1.0 1.0 1.0 1.0

From the above results, it is evident that, as decreasing the concentration of biotinylated PEG GCSF and digoxigenylated PEG GCSF mean absorbance and S/N ratios are increased. However, maximum S/N ratio and mean absorbance values are observed in presence of 25 ng/mL of biotinylated PEG GCSF and 25 ng/mL of digoxigenylated PEG GCSF. After 25 ng/mL of both biotinylated and digoxigenylated PEG GCSF, there is a decrease in absorbance and S/N ratio. Hence, optimal concentrations of 25 ng/mL of biotinylated and digoxigenylated PEG GCSF are selected for further assay process.
Step III
To assess the sensitivity of the assay using above optimized assay format in detection of anti PEG GCSF antibody, 1000 ng/mL to 0.0610 ng/mL of rabbit anti PEG GCSF was diluted in pooled human serum. Further, the samples were diluted to 1: 10 ratio in assay buffer (1% BSA in PBS).
96 well micro titer plate was coated with 100 µl/well of 5 µg/well of streptavidin and plate was washed with 0.05% PBST (wash buffer) and wells were blocked by adding 250 µl/well of 1% BSA (blocking buffer) and were incubated at room temperature for 1 hour. 25 ng/mL of biotinylated PEG GCSF was added to each well and incubated at room temperature for 1 hour with shaking at 300 rpm. All the wells were washed with wash buffer. 100 µl of positive controls containing various concentrations of anti PEG GCSF antibody was added to each well and was incubated for 1 hour at room temperature with shaking at 300 rpm. After incubation, wells were washed with wash buffer for three times. 100 µl of 25 ng/mL of digoxigenylated PEG GCSF was added to each well and was incubated for 1 hour at room temperature with shaking at 300 rpm. After incubation, all the wells were washed with wash buffer for three times followed by 100 µl of anti digoxigenin horse radish peroxidase (which was diluted in 1% BSA in the ratio of 1: 2500) was added to all the wells and was incubated for 1 hour at room temperature with shaking at 300rpm. After incubation with secondary detection reagent, wells were washed with wash buffer for three times and 100 µl of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate was added to each well and was incubated for 15 minutes at room temperature. 100 µl of stop solution (1N H2SO4) was added to stop the reaction and absorbance at 450 nm was measured using a micro plate reader.
The assay sensitivity was determined from six independent assay runs, by two analysts on four separate days. The sensitivity for each run was determined from the dilution curve as the S/N ratio of positive control antibody equivalent to or above the cut point or screening cut point.
The screening cut point for this experiment is set to be 1.1. “Screening Cut point” of the assay is the level of response at or above which a sample is identified to be positive and below to be negative and is determined using a formula, Mean+1.645*standard deviation, wherein the mean value is a ratio between negative control (NC) and negative quality control (NQC). Negative control is either plasma/serum collected from a healthy individual, not spiked with any positive control antibody. Results of the assay are mentioned in table 3. S/N ratio of a sample above the cut point is considered to be contain anti drug antibodies in ADA assays.
Table 3: S/N ratios of samples containing various concentrations of anti PEG GCSF antibody
PC1 (anti PEG GCSF antibody) Concentration (ng/mL) Mean absorbance at 450 nm S/N ratio; (P=Positive signal, N=NQC) Cut point
1000 2.319 44.22 1.1
500 1.318 25.11
250 0.711 13.54
125 0.386 7.35
62.5 0.221 4.20
31.3 0.135 2.57
15.6 0.093 1.78
7.81 0.072 1.38
3.91 0.063 1.20
1.95 0.058 1.10
0.977 0.056 1.06
0.488 0.055 1.04
0.244 0.052 1.00
0.122 0.051 0.98
0.0610 0.053 1.01
NQC-1 0.053
NQC-2 0.052

From the above results, it is evident that sensitivity of the assay in detecting anti PEG GCSF antibody is approximately 4 ng/mL.
In addition, the same assay was repeated with the samples containing anti PEG antibodies to assess the detection of Ig M ADA using the optimized double antigen bridging assay. Various concentrations of anti PEG antibodies obtained from ANP technologies were prepared by diluting it from 2000 ng/mL to 62.5 ng/mL in pooled human serum and all samples were prepared in duplicates. Further, the samples were diluted in 1: 10 ratio in assay buffer (1% BSA in PBS) prior to the subjecting of samples to ADA assay. Assay procedure was described above (step iii of example 1) was employed to detect anti PEG antibodies. Results of the assay are given in table 4 and 5.
The same assay was performed on three different days to generate the date and validate the results.
Table 4: Mean absorbance values of samples containing anti PEG antibodies
PC2 (anti PEG antibody) Concentration
(ng/mL) Mean OD Mean OD Inter Day variation
Day 1 Day 2 Day 3
2000 2.534 2.015 2.975 2.508 19
1000 0.822 0.768 0.961 0.850 12
500 0.293 0.309 0.334 0.312 7
250 0.152 0.172 0.149 0.158 8
125 0.11 0.105 0.104 0.106 3
62.5 0.09 0.094 0.083 0.089 6
NQC 0.079 0.1 0.081 0.0870 13
NQC-Negative quality control
Table 5: S/N ratio of anti PEG antibodies
PC2 (anti PEG ab) Conc. (ng/mL) S/N ratio
Day 1 Day 2 Day 3
2000 31.1 20.6 34.8
1000 10.1 7.8 11.2
500 3.6 3.2 3.9
250 1.9 1.8 1.7
125 1.3 1.1 1.2
62.5 1.1 1.0 1.0
It is evident from the above results that, the optimized assay is capable of detecting 250 ng/mL of anti PEG antibodies in assay serum.
The optimized method is capable of detecting both Ig M and Ig G antibodies.
Example 2: Assay method for detection of anti darbepoetin alfa antibodies and evaluating the sensitivity of the assay
Prior to the performance of ADA, antibodies against darbepoetin alfa were raised in a rabbit and purified using darbepoetin conjugated sepharose column. All the positive and negative control samples were prepared as described in example 1 instead of anti PEG GCSF antibody anti darbepoetin alfa antibody was added to pooled human serum obtained from healthy individuals.
Step 1
Various concentrations of biotinylated darbepoetin alfa ranging from 5000 ng/mL to 3.91 ng/mL was diluted in assay buffer containing 1% BSA in PBS.
96 well micro titer plate was coated with was coated with 100 µl/well of 5 µg/well of streptavidin and plate was washed with 0.05% PBST (wash buffer) and wells were blocked by adding 250 µl/well of 1% BSA (blocking buffer) and were incubated at room temperature for 1 hour. Various concentrations of biotinylated darbepoetin alfa (250 ng/mL to 0 ng/mL) was added to each well and incubated at room temperature for 1 hour with shaking at 300 rpm. All the wells were washed with wash buffer. 100 µl of sample containing various concentrations of anti darbepoetin alfa antibody were added after diluted to 1: 20 in assay buffer and incubated for 1 hour at room temperature with shaking at 300 rpm. After incubation, wells were washed with wash buffer for three times. 100 µl of 200 ng/mL of digoxigenylated darbepoetin alfa was added to each well and was incubated for 1 hour at room temperature with shaking at 300 rpm. After incubation, all the wells were washed with wash buffer for three times followed by 100 µl of anti digoxigenin horseradish peroxidase (which was diluted in 1% BSA in the ratio of 1: 2500) was added to all the wells and was incubated for 1 hour at room temperature with shaking at 300rpm. After incubation with secondary detection reagent, wells were washed with wash buffer for three times and 100 µl of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate was added to each well and was incubated for 15 minutes at room temperature. 100 µl of stop solution (1N H2SO4) was added to stop the reaction and absorbance at 450 nm was measured using a micro plate reader.
Results of the assay are mentioned in terms of mean absorbance and S/N ratio in table 6.
Table 6: Mean absorbance and S/N ratio of samples containing varying concentration of anti darbepoetin alfa antibody in presence of varying concentration of Biotinylated darbepoetin alfa antibody.

From the above results it is evident that, 31.25 ng/mL concentration of biotinylated darbepoetin alfa shows higher assay response and S/N ratio. Hence, this concentration is selected as an optimized coating concentration for further experiment.
Step II
To assess the sensitivity of the assay in detecting anti darbepoetin alfa antibody using the above disclosed assay format, various concentrations of anti darbepoetin alfa antibody ranging from 100 ng/mL to 6.25 ng/mL was prepared by diluting it in assay medium. Detection of anti darbepoetin alfa antibody was performed as described in above section iii of example 2. Results of the assay are measured in terms of mean absorbance and S/N ratio and given in below table 7.
The screening cut point for this experiment is set to be 0.996. The assay was performed in two different days.
Table 7: Mean absorbance and S/N ratios of samples containing varying concentrations of anti darbepoetin alfa antibody.
Anti darbepoetin alfa Conc. (ng/mL) Mean absorbance at 450 nm S/N ratio
Day 1 Day 2 Mean % CV
100 1.047 0.936 0.991 8 14.573
50 0.590 0.537 0.563 7 8.279
25 0.316 0.304 0.310 3 4.558
12.5 0.195 0.186 0.190 3 2.794
6.25 0.138 0.131 0.134 3 1.970
NQC 0.073 0.063 0.068 11 1

From the above results, it is evident that the assay is sensitive enough to detect 6.25 ng/ml of anti darbepoetin alfa antibody present in a biological sample.
Example 3:
Assessment of serum tolerance
To assess the biological matrix interferences, anti darbepoetin alfa samples were prepared in both assay buffer (1% BSA) and pooled human serum (PHS). Various concentrations ranging from 1000 to 1.5625 ng/mL of anti darbepoetin alfa antibodies were diluted in assay buffer as well as PHS and diluted to 1: 10 ratio in the respective buffer. In addition to that, different dilutions of PHS including neat sample was assessed in the assay.
Further, the assay was performed as described in section 2 of example 2.
Results of the assay are given in below table 8 and table 9.
Table 8: Mean absorbance of samples containing varying concentration of anti darbepoetin alfa antibody prepared in both assay buffer (1% BSA) and pooled human serum (PHS).
Anti darbepoetin alfa antibody Concentration (ng/mL) Anti darbepoetin alfa
1% BSA in PBS (1:10) Pooled Human Serum
(PHS-1:10)
Mean OD %CV S/N Mean OD %CV S/N
2000 3.879 0 38.1 3.838 0 42.0
1000 3.572 1 35.1 3.390 2 37.1
500 2.604 3 25.6 2.268 4 24.8
250 1.639 2 16.1 1.391 2 15.2
125 0.925 4 9.1 0.807 2 8.8
62.5 0.565 5 5.6 0.506 2 5.5
31.25 0.367 4 3.6 0.312 2 3.4
Blank 0.102 11 1.0 0.091 17 1.0

Table 9: Mean absorbance of negative quality control samples prepared in various dilutions of PHS.
NQC sample PHS neat Mean OD
PHS Dilution (1:2) Mean OD

PHS
Dilution (1:5) Mean OD

PHS
Dilution (1:10) Mean OD

PHS
Dilution (1:20) Mean OD

PHS
Dilution (1:40) Mean %CV
NQC1 0.060 0.061 0.063 0.071 0.069 0.071 0.066 8
NQC2 0.061 0.060 0.064 0.068 0.069 0.077 0.066 9
NQC3 0.066 0.064 0.073 0.084 0.068 0.072 0.071 10

From the above results (table 8), it is evident that the samples prepared in 1% BSA and pooled human serum are showing similar results. Hence, the method is tolerant to interferences present in a serum sample.
From table 9, it is evident that, pooled human serum neat is not interfering the assay, as percentage variation between neat, 1:2, 1:5, 1:10, 1:20 and 1:40 diluted pooled human serum (un spiked with positive control) found to be = 10.
,CLAIMS:1). A method for detecting anti-drug antibodies raised against a drug comprising:
a). coating optimized concentration of the biotinylated drug on a micro titer plate
b). addition of a biological sample comprising anti-drug antibodies and incubating for a first period of time
c). addition of the digoxigenylated drug and incubating for a second period of time and,
d). addition of anti digoxigenin horseraddish peroxidase conjugate to detect the said digoxigenylated drug and bound anti-drug antibody.
2). A method according to claim 1, wherein the drug is a therapeutic protein molecule which includes GCSF, PEG GCSF, erythropoietin, darbepoetin or the like.
3). A method according to claim 1, wherein the biological sample is plasma or serum.
4). A method according to claim 1, the concentration of the biotinylated drug is from about 20 ng/mL to 40 ng/mL.
5). A method according to claim 1, the concentration of digoxigenylated drug is selected from the range of about 25 ng/mL to 200 ng/mL.
6). A method according to claim 1, the first period of time is a sufficient time which allows binding of anti drug antibody present in a biological sample to biotinylated drug.
7). A method according to claim 1, the second period of time, is a sufficient time which allows binding of digoxigenylated drug to the ADA captured by biotinylated drug.
8). A method according to claim 1, the anti drug antibody is IgG or IgM antibody.
9). A method according to claim 1, the biological sample is diluted from 1:10 to 1:20 prior to the subjecting of sample to said assay.