CLIAMS:1. A method of detecting rituximab in a rheumatoid factor positive sample comprising;
a) preparing anti-rituximab antibodies in carbonic acid based buffer and coating of said antibodies onto a solid phase
b) washing the solid phase from step a) with a wash buffer containing 0.1 % polyoxyethylene sorbitan monolaurate
c) preparing samples containing rituximab in an assay diluent buffer comprising 0.1% polyoxyethylene sorbitan monolaurate
d) adding rituximab containing samples to the solid phase, followed by incubation, to allow binding of rituximab to anti-rituximab antibodies
e) washing the solid phase with 0.1% polyoxyethylene sorbitan monolaurate
f) adding a labelled anti-IgG antibody to the said solid phase to bind rituximab
g) detecting the said labelled antibody and in turn the bound rituximab, using enzyme linked immunosorbent assay, wherein, the said method significantly reduces non-specific interactions and/or RF interferences in the assay.
2. A method according to claim 1, the said sample is either processed or unprocessed sample.
3. A method according to claim 1, wherein concentration of said anti-rituximab antibody coated on to the said solid phase is 2 µg/ml.
4. A method of detecting rituximab in a rheumatoid factor positive rheumatoid arthritis patient’s sample using enzyme linked immune sorbent assay, wherein the method uses 0.1% poly-oxyethylene sorbitan monolaurate or its derivatives in the assay diluent and wash buffer to reduce non-specific interferences in the assay, and wherein the said method is sensitive in detecting rituximab present in unprocessed plasma sample.
,TagSPECI:FIELD OF THE INVENTION
The present invention relates to a method of detecting and quantifying rituximab in a rheumatoid factor positive sample.
BACKGROUND OF THE INVENTION
Pharmacokinetic study of a therapeutic drug measures the absorption, distribution, and elimination of the drug molecule in an individual over a course of time. Pharmacokinetic assessments are highly important as the results are directly applicable for a drug’s safe and effective therapeutic management. In particular, bioavailability (amount of unchanged drug present in systemic circulation) or toxic plasma concentrations of the drug are generally established from the results of pharmacokinetic studies. In addition, the pharmacokinetics of the administered drug helps in designing the dosage regimen of the therapeutic molecule. Hence regulatory bodies recommend the performance of pharmacokinetic studies during clinical trials, for better understanding of the fate of a therapeutic molecule, including its serum half-life.
The methodology of a pharmacokinetic assay involves several critical steps, such as timing and number of samplings, processing of the samples and the procedure employed for the assay that may specifically influence the performance and sensitivity of the assay.
Most importantly, it is necessary to collect sufficient number of samples over several time points for an accurate analysis of absorption, distribution and elimination rate of a therapeutic molecule. In other words, frequent sampling is essential to arrive at a more accurate value for comprehending the precise pharmacokinetics of a drug. Generally serum sample obtained by processing the blood plasma is preferred in any detection/analytical assay, since, a large number of plasma components that may interfere in the assay are removed and/or reduced during the processing step. However processing of plasma sample involves considerable time and resources, and pose difficulties, particularly in a pharmacokinetic assay that demands frequent sampling. Further, during this extended course of time, the analyte of interest may get disposed to proteolytic components present in the sample thereby resulting in degradation of the analyte. In addition, preparation of serum is complicated and is known for procedural variations caused by differences in clotting and processing of sample. These factors necessitated the use of plasma rather than the processed/serum samples.
Despite its advantageous factors, endogenous biological components present at relatively higher amounts in plasma (than in serum) may interact with the analyte or the detecting agent causing significant interferences in the assay. Hence, it is highly important to design an assay procedure that reduces such interferences, especially when the sample is a plasma pool.
Additionally, pharmacokinetic assays generally detect/measure the therapeutic molecule’s presence using the technique of an enzyme-linked immunosorbent assay (ELISA) which includes the steps of coating the solid phase with appropriate ligand, binding of ligand to analyte of interest and detection of ligand/analyte by complementary agents, with intermittent washing at the end of each step. These steps are essential in removing non-specific binding/interactions. Hence use of appropriate buffer or buffer component in these steps is highly critical, and is selected in a way that it stabilizes the interaction of detecting agent with the analyte, and concurrently minimizes the non-specific interactions/background noise.
Monoclonal antibody drugs are in use for the treatment various oncological and rheumatological disorders. One such antibody is rituximab, which is a chimeric anti-CD20 monoclonal antibody, used in the treatment of Rheumatoid Arthritis (as well as Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia). As with any other therapeutic molecule, it is important to detect rituximab concentration in the patient’s sample at several time points, for accurately plotting the pharmacokinetics of rituximab. However in case of Rheumatoid Arthritis (RA), plasma or serum samples from patients often contain high concentrations of rheumatoid factors (RF), which are antibodies with high affinity to the Fc region of the administered antibody i.e., rituximab, which may interfere in a detection assay leading to improper estimation of the amount of rituximab present in a sample. This in turn additionally requires the detecting method to possess high RF tolerance, or mitigate RF interference to a level that it does not interfere in the performance/sensitivity of the assay.
Hence, the primary object of the invention to provide a method for detecting and measuring rituximab in RA patient’s sample, wherein the method is able to significantly remove non-specific interactions and/or the interferences from the assay. Another objective is to enable and qualify the method in detecting relatively lower levels of rituximab present in unprocessed plasma sample. A further objective of the invention is to optimize the method, such that the method substantially mitigates RF interference.
SUMMARY OF THE INVENTION
The present invention discloses an ELISA based method for detecting rituximab in rheumatoid factor positive rheumatoid arthritis patient’s sample. The method employs the use of polyoxyethylene sorbitan mono laurate and a carbonic acid based buffer separately in specific steps, wherein the method in entirety significantly reduces non-specific interactions and/or the interferences in the assay. Most importantly, the proposed inventive method does not require processing of the blood plasma and the sample could be used directly to detect rituximab, making it particularly advantageous in pharmacokinetics studies of the therapeutic antibody. Results show that the method is highly sensitive and is able to detect rituximab in relatively lower concentrations (~ 150 ng/ml) in unprocessed plasma samples.
Further, the method possess a dynamic, detection range of rituximab ( 156 ng/ml to 5 µg/ml ) with acceptable accuracy and precision and qualifies for its robustness and consistency as per the validation requirements of regulatory agencies .
In addition, the method mitigates RF interference substantially to a level, such that the results obtained for rheumatoid factor positive plasma sample and normal healthy individuals are comparable. In other words, the method is tolerant to RF levels as high as 1500 units /ml of the sample.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Illustrates a comparison between processed and unprocessed samples of NPHP and RAPHP samples (spiked with rituximab), wherein the samples were diluted in an assay diluent buffer containing 1% BSA in 0.1% PBST. Pro-Processed sample; Unp-Unprocessed sample
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a method for detecting rituximab in RF positive human plasma samples, wherein the samples were diluted in 1: 500 ratios in assay diluent buffer, and the components of diluent and coating buffer are selected to reduce non-specific interferences from the assay, leading to higher sensitivity.
In an embodiment, the invention discloses a method of detecting rituximab in a rheumatoid factor positive rheumatoid arthritis patient’s sample using enzyme linked immune sorbent assay, wherein the method uses 0.1% poly-oxyethylene sorbitan monolaurate or its derivatives in the assay diluent and wash buffer to reduce non-specific interferences in the assay, and wherein the said method is sensitive in detecting rituximab present in unprocessed plasma sample.
In another embodiment, the invention discloses a method of detecting rituximab in a rheumatoid factor positive sample comprising the steps of;
a) preparing anti-rituximab antibodies in carbonic acid based buffer and coating of the said antibodies onto a solid phase
b) washing the solid phase with wash buffer containing 0.1 % polyoxyethylene sorbitan monolaurate
c) preparing samples containing rituximab in an assay diluent buffer comprising 0.1% polyoxyethylene sorbitan monolaurate
d) adding rituximab containing samples to the solid phase, followed by incubation, to allow binding of rituximab to anti-rituximab antibodies
e) washing the solid phase with 0.1% polyoxyethylene sorbitan monolaurate
f) adding a labelled anti-IgG antibody to the said solid phase to bind rituximab
g) detecting the said labelled antibody and in turn the bound rituximab, using standard protocols of enzyme linked immunosorbent assay,
wherein, the said method significantly reduces non-specific interactions and/or RF interferences in the assay.
In another embodiment of the invention, the sample used in the assay can be either a processed or an unprocessed plasma sample.
In another embodiment of the invention, concentration of anti-rituximab antibody coated onto the solid phase is 2 µg/ml.
The method uses biotinylated anti-IgG antibody for detection using the principle of enzyme-substrate colorimetric reaction wherein, streptavidin coupled horseradish peroxidase is used as an enzyme and 3,3',5,5'-Tetramethylbenzidine (TMB) as a substrate. However, the anti-IgG antibody can be labeled with any commercially available suitable labelling agent (such as but not limited to ruthenium, iodine etc.,) and shall be detected using appropriate substrate using the principle of enzyme linked immunosorbent assay.

The method is able to detect 156 ng/ml of rituximab in unprocessed plasma samples. Sensitivity of the assay is validated with commercially available rituximabs such as Mabthera® and Rituxan® and is found to be 156 ng/ml. Further, the method mitigates RF interference to a significant level (RF tolerance as high as ~1500 RF units/ml) and the results obtained are comparable with that of the normal plasma sample.
As used herein, the unprocessed sample refers to a plasma sample which was obtained from blood using (The Royal Society of Chemistry, Lab Chip, 2006, 6, 776–781, DOI: 10.1039/b604145k) standard protocols. ‘Processed sample’ refers to a plasma sample that was obtained by centrifuging( at 3220g, for 30 mins at 4ºC) the unprocessed plasma sample and collecting the supernatant, followed by subjecting the collected supernatant to sequential filtration steps (using 0.45 µ filters and 0.22 µ filters) to obtain a clear liquid.

Definitions
The term ‘sample’ as used herein the invention refers to an RF positive plasma or serum sample obtained from patients who are not treated with rituximab and, in which case, the sample obtained is spiked with rituximab. However, the method as disclosed in the invention can be used for the detection of rituximab in RF positive plasma/serum sample obtained from patients treated with rituximab.
“Standard sample” as used here in the invention, is a sample (plasma from RF positive RA patients or from healthy volunteers) spiked with known amount of rituximab antibody to mimic the physiological condition of rituximab treated patient’s sample and/or to evaluate sensitivity of the assay.
“Blank Sample” is an unspiked sample (plasma from RF positive RA patients or from healthy volunteers) in which no rituximab has been added.
“RFPHP” is the rheumatoid factor positive pooled human plasma samples from rheumatoid arthritis patients obtained from authorized blood banks.
“NPHP” is normal pooled human plasma samples from healthy volunteers obtained from authorized blood banks.
“Sensitivity” of the assay is defined as the lowest concentration of standard sample antibody preparation which consistently provides signal in the assay.
“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.
“Upper level of quantification (ULOQ)” is the highest concentration of analyte that has been demonstrated to be measurable with stated levels of and precision.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.
EXAMPLES
During development of an ELISA based pharmacokinetic assay for detecting rituximab present in a rheumatoid factor positive rheumatoid arthritis patient’s sample various parameters were optimized to achieve good sensitivity and RF mitigation. One such critical parameter was choice of coating buffer and bicarbonate buffer was employed for diluting rat-anti rituximab antibody and the same was coated on 96 well micro titer plate.
Another critical parameter was choice of assay diluent buffer and 0.1% polyoxyethylene sorbitan monolaurate (Tween®-20) containing phosphate buffered saline solution (0.1% PBST) was selected. The assay diluent buffer was employed for the dilution of samples (RFPHP and NPHP).
Further, to check the role of Tween®-20 in assay diluent buffer in reducing non specific interactions which in turn improves percentage recovery of rituximab present in a sample, samples were diluted in an assay buffer with and without Tween®-20 and, results from ELISA based pharmacokinetic assay were compared.

Example 1
Sample Preparation:
Plasma samples (unprocessed) obtained from various rheumatoid factor positive individual RA patients were pooled and spiked with various concentrations of rituximab antibodies (Standard samples–rheumatoid factor positive individual human plasma samples from RA patients (RFPHP)) to mimic physiological conditions of rituximab treated patients. To compare the study with normal samples, plasma samples (unprocessed) were collected from normal healthy individuals, pooled, and spiked with various concentrations of rituximab antibodies (Standard samples – Normal Pooled Human Plasma (NPHP)). Further, to compare the results of unprocessed plasma samples with processed plasma samples, some of the plasma samples of NPHP and RFPHP were processed by the procedure defined in the description.
RFPHP used in all experiments were estimated to contain not less than 1500 units of rheumatoid factors/ml of sample.
Various concentrations of rituximab were spiked in both processed and unprocessed plasma samples of NPHP and RFPHP for the preparation of standard samples. Some of the NPHP and RFPHP samples were not spiked with rituximab as to maintain the blank sample. Further, the spiked and unspiked samples were diluted in the selected assay diluent buffer containing 1% bovine serum albumin in 0.1% phosphate buffer saline Tween®-20 (PBST) solution in 1: 500 ratio.
Example 2:
Detection of rituximab:
Individual wells of 96 micro titer plate were coated with 100 µl of 2 µg/ml rat-anti-rituximab antibody in bicarbonate buffer (pH 9.4) and the plate was sealed and incubated for overnight at 2-8 ºC. After incubation, the plate was washed with 0.1 % PBST for three times to remove uncoated rat-anti-rituximab antibody. Followed by, addition of 250 µl of 1% BSA in PBS to block additional sites and incubated for 2 h at 37 ºC at 300 rpm. After the incubation period, the blocking buffer was discarded and 100 µl of spiked, unspiked samples of processed and unprocessed samples prepared from example 1 were added to each well and incubated under shaking conditions at 37 ºC for 1h. After incubation, the plate was washed with 0.1 % PBST to remove unbound material from the wells, then 100 µl of biotinylated goat-anti-human IgG Fc specific antibody (which was diluted in assay diluent buffer in 1: 175000) was added to each well and the mixture was incubated at 37 ºC for 1 h at 300 rpm. Followed by, washing of the plate with 0.1 % PBST for three times. 100 µl of streptavidin-horse radish peroxidase antibody (which was diluted in assay diluent buffer in 1: 400) was added to each well and incubated at 37 º C for 20 mins under shaking conditions. The plate was washed with 0.1% PBST for three times and then, 100 µl/well of 3,3’,5,5’-Tetramethylbenzidine (TMB) was added to the plate and the mixture was incubated for 20 mins at 37 º C at 300 rpm. 100 µl of stop solution was added to stop the reaction and absorbance was measured at 450 nm using a micro plate reader.
The results of the assay for processed and unprocessed samples of NPHP and RFPHP were represented in terms of blank subtracted mean absorbance at 450 nm and % difference in below table 1 and also represented in figure 1.
% difference of the samples were calculated using the following formula:
% Difference = Mean concentration of test (T) - Mean concentration of reference (R) / Mean concentration of reference (R) X 100

Table 1: Mean subtracted blank values and % difference of processed and unprocessed plasma samples.
Conc. of rituximab (µg/ml) Blank subtracted mean absorbance values at 450 nm % Difference between T and R
Plate 1 (pro-
samples) Plate 1 (Unp-samples) Proc Unp NPHP RFPHP
NPHP RFPHP NPHP RFPHP NPHP (R) –RFPHP (T) NPHP (R) –RFPHP (T) Pro (R) –Unp (T) Pro (R) –Unp (T)
10 2.261 2.241 2.346 2.371 -1 1 4 6
5 1.598 1.523 1.609 1.599 -5 -1 1 5
2.5 1.059 1.004 1.062 1.065 -5 0 0 6
1.25 0.635 0.592 0.656 0.620 -7 -5 3 5
0.625 0.372 0.341 0.360 0.356 -8 -1 -3 4
0.313 0.197 0.172 0.195 0.187 -13 -4 -1 9
0.156 0.104 0.1 0.112 0.1 -4 -11 8 0
0.078 0.063 0.051 0.059 0.053 -19 -10 -6 4
Blank 0.153 0.170 0.167 0.183 11 10 9 8
Pro-processed samples; Unp-unprocessed samples;
From the above results, it is evident that the mean absorbance values obtained for processed and unprocessed samples of NPHP and RFPHP are similar. The assay is sensitive such that it is able to detect 156 ng/ml of rituximab present in the samples. The result was checked for accuracy and precision, and found to be consistent across samples (validated). The results also indicate that the method is sensitive in detecting rituximab, as low as 78 ng/ml, however the consistency of this lower detection limit has not been validated. Further, the dynamic range of the assay is found to be 156 ng/ml (LLOQ) to 5 µg/ml (ULOQ).
In addition, % difference between NPHP and RFPHP were in acceptable ranges. Amount of RF present in NPHP samples were =25 units/ml whereas, amount of RF present in RFPHP samples were ~ 1500 units/ml. However, the obtained results for both processed and unprocessed samples of NPHP and RFPHP are comparable, which is clearly indicating that, the method mitigates RF interference significantly.
Example 3: Assessment of role of Tween®-20 in % recovery of rituximab
156 ng/ml (LLOQ) of rituximab was prepared in unprocessed RFPHP samples and the dilutions of the samples were performed in two different assay diluent (AD) buffers, 1% BSA in 0.1% PBST and 1% BSA in PBS by diluting the samples in the respective assay diluent buffers in 1: 500 ratio. Further, 0.1% PBST and PBS were used as wash buffers (WB) respectively during ELISA based pharmacokinetic assay for detection of rituximab present in the sample. Further, all samples were prepared in triplicates to assess accuracy of the assay. All prepared samples were subjected for ELISA assay as described in example 2 for the detection of rituximab present in the sample and the results were mentioned in terms of blank subtracted mean absorbance value (measured at 450 nm) and as percentage recovery in table 2. The amount of rituximab present in the samples was estimated by extrapolating their absorbance values to 4 logistic curve fit prepared from10 µg/ml to 78 ng/ml of rituximab. Percentage of recovery was calculated using the following formula:
% recovery= (estimated concentration ÷ nominal concentration) X 100
Table 2: % recovery of rituximab in various assay diluent buffers and wash buffers
Sample details Conc.
of rituximab (nominal. conc) 1% BSA in PBS (AD)
and PBS (WB) 1% BSA in 0.1% PBST (AD) and 0.1 % PBST (WB)
Mean Value at 450 nm Mean
Conc (est) (µg/ml) %
CV %
recovery Mean Value Mean
Conc
(est)
(µg/ml) %CV %
recovery
LLOQ1 0.156 µg/ml 0.059 0.107 2 69 0.085 0.136 8 87
LLOQ2 0.068 0.118 7 76 0.091 0.145 12 93
LLOQ3 0.066 0.116 8 74 0.097 0.153 2 98

From the above results, it is evident that the percentage recovery of rituximab in RFPHP unprocessed samples are in acceptable limit (80-120%) when diluted in assay diluent buffer containing 0.1% Tween®-20. Whereas % recovery of rituximab is less (<80-unacceptable limit) in samples diluted in assay diluent buffer without Tween®-20.
In an assay, blank value directly correlates to the interferences in the assay occurring due to non-specific interactions. However ‘higher percentage recovery’(i.e., in acceptable limit) measured in terms of mean absorbance value which is a blank subtracted value, mentioned in table 2, indicates significant reduction of non-specific interactions/interferences in the assay when Tween®-20 is used as a buffer component.