HIGH IONIC STRENGTH DISSOCIATION ASSAY FOR HIGH DRUG TOLERANT TESTING
20230063268 · 2023-03-02
Assignee
Inventors
Cpc classification
International classification
Abstract
Herein is reported a method for the determination of the presence of anti-drug antibodies in a sample comprising the steps of incubating the sample with MgCl.sub.2 at a final concentration in the range of 1 N to 12 N; adding a tracer antibody and incubating the sample thereafter; incubating the isolated tracer antibody-anti-drug antibody-complexes with a detection antibody conjugated to a detectable label and determining the presence of anti-drug antibodies if a signal above a threshold level is obtained.
Claims
1. A method for the detection of a target antibody in a sample comprising the following steps: a) incubating the sample with a chaotropic salt at a final cation charge normality in the range of and including 1 N to 12 N; b) adding a tracer antibody to the sample obtained in step a) and incubating the sample thereafter to form a tracer antibody-target antibody-complex in the presence of the chaotropic salt; c) incubating the tracer antibody-target antibody-complex formed in b) with a detection antibody conjugated to a detectable label to form a tracer antibody-target antibody-detection antibody complex; whereby the target antibody is detected if a tracer antibody-target antibody-detection antibody-complex is detected in the sample obtained in step c).
2. The method according to claim 1, wherein the chaotropic salt is a medium strength chaotropic salt, preferably with a cation between potassium and calcium in the lyotrophic series according to Hofmeister and an anion between hydrogen phosphate and nitrate in the lyotrophic series according to Hofmeister.
3. The method according to any one of claims 1 to 2, wherein the chaotropic salt has a cation selected from the group of cations consisting of potassium, sodium, lithium, magnesium and calcium, and an anion selected from the group of anions consisting of (hydrogen)phosphate, acetate and chloride.
4. The method according to any one of claims 1 to 3, wherein the chaotropic salt is MgCl.sub.2 or LiCl.
5. The method according to any one of claims 1 to 4, wherein the final cation charge normality of the chaotropic salt is in the range of and including 6.5 N to 8.5 N.
6. The method according to any one of claims 4 to 5, wherein the final MgCl.sub.2 cation charge normality is 7.2 N +/−10% corresponding to a final MgCl.sub.2 concentration of 3.6 M +/−10% or the final LiCl cation charge normality is 8 N +/−10% corresponding to a concentration of 8 M +/−10%.
7. The method according to any one of claims 1 to 6, wherein the incubating in steps a) and b) is between 30 min. +/−10% to 60 min. +/−10%.
8. The method according to any one of claims 1 to 7, wherein in step b) further a capture antibody is added together with, before or after the tracer antibody but before incubating the sample.
9. The method according to any one of claims 1 to 8, wherein in step b) the tracer antibody is conjugated to a label.
10. The method according to any one of claims 8 to 9, wherein the capture antibody and the tracer antibody and the detection antibody are conjugated to different labels, whereby the label of the capture antibody does not interact with the label of the detection antibody and vice versa.
11. The method according to any one of claims 1 to 10, wherein the tracer antibody in step b) is added to a final concentration of from 0.9 μg/mL to 2.5 μg/mL.
12. The method according to any one of claims 8 to 11, wherein the capture antibody in step b) is added to a final concentration of from 0.9 μg/mL to 2.5 μg/mL.
13. The method according to any one of claims 1 to 12, wherein the detection antibody specifically binds to the label of the tracer antibody and is conjugated to horseradish peroxidase and step c) of the method is: c) incubating the isolated tracer antibody-target antibody-complex formed in b) with a detection antibody conjugated to horseradish peroxidase and ABTS or HPPA.
14. The method according to any one of claims 8 to 13, wherein step c) is c-1) transferring the sample obtained in step b) to a solid surface comprising a capture agent immobilized thereon that can specifically bind to the capture antibody; c-2) incubating the sample on the solid surface; c-3) removing substances not bound to the solid surface by washing; c-4) incubating the immobilized tracer antibody-target antibody-complex on the solid surface with a detection antibody conjugated to a detectable label; c-5) removing substances not bound to the solid surface immobilized tracer antibody-target antibody-complexes by washing; c-6) detecting the immobilized detectable label of the detection antibody.
15. The method according to any one of claims 1 to 14, wherein the target antibody is an anti-drug antibody or a therapeutic antibody.
Description
DESCRIPTION OF THE FIGURES
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Examples
Materials
Positive Controls
[0201] A polyclonal rabbit-derived antibody (pAb<mAb-1>Rb; Roche Diagnostics GmbH, Germany) directed against the therapeutic monoclonal mAb-1 was used as positive control (PC1) in the mAb-1-based ADA assay. It was dissolved at 2.0 mg/mL in 1× phosphate-buffered saline (PBS; Roche Diagnostics GmbH, Germany).
[0202] A monoclonal mouse-derived antibody (mAb<mAb-2>M; Roche Diagnostics GmbH, Germany) directed against the therapeutic monoclonal mAb-2 was used in the mAb-2-based ADA assay (PC2). It was dissolved at 5.4 mg/mL in an aqueous solution of 50 mM potassium phosphate (Merck Chemicals GmbH, Germany) and 150 mM potassium chloride (Merck Chemicals GmbH, Germany), pH 7.5.
Human Matrices
[0203] Human pooled sera and human pooled K3EDTA plasma, both from mixed gender, were obtained from TRINA Bioreactives AG, Switzerland.
Example 1
mAb-1-Based ADA Assay (With MgCl.SUB.2 .Treatment) According to the Invention
[0204] For the qualitative detection of antibodies directed against the therapeutic monoclonal mAb-1, a bridging enzyme-linked immunosorbent assay (ELISA) was used.
[0205] PC1 was used to generate quality control samples in human pooled sera. Quality control samples, negative control samples and test samples were diluted 1:10 (5 μL+45 μL) using a 4 M MgCl.sub.2*6H.sub.2O solution (VWR International bvba, Belgium) and incubated for 30 min. at room temperature with shaking at 450 rpm. Subsequently, all samples were diluted 1:10 (30 μL of the previous dilution+270 μL) using 1×PBS containing 1×Westem Blocking Reagent (Merck Chemicals GmbH, Germany), together with 900 ng/mL mAb-1-Biotin and 900 ng/mL mAb-1-Digoxygenin and incubated for 30 min. at room temperature with shaking at 450 rpm). Formed immune complexes (100 μL) were transferred to a streptavidin (SA)-coated microtiter plate (MTP) and incubated for one hour at room temperature with shaking at 450 rpm to immobilize immune complexes via the Biotin-labeled capture antibody.
[0206] After three washing steps each using 300 μL 1×PBS (phosphate buffered saline) containing 0.05% (v/v) Tween 20, 100 μL of a 25 mU/mL horseradish peroxidase (HRP)-labeled anti-Digoxygenin Fab fragments (<Digoxygenin>HRP; Roche
[0207] Diagnostics GmbH, Germany) diluted in 1×PBS containing 0.5% (w/v) BSA (Merck Chemicals GmbH, Germany) were added to the MTP and incubated for one hour at room temperature with shaking at 450 rpm.
[0208] After three washing steps, substrate reaction was carried out by adding 100 μL/well 20 mM 3-p-hydroxyphenyl propionic acid (HPPA; Merck Chemicals GmbH, Germany) supplemented with 0.02% (v/v) of a hydrogen peroxide solution 30% (w/w) (H.sub.2O.sub.2; Merck Chemicals GmbH, Germany), dissolved in 0.1 M tris(hydroxymethyl)aminomethane (TRIS; Merck Chemicals GmbH, Germany) solution, pH 8.5 and incubated for 10 min. at room temperature with shaking at 450 rpm [16]. Fluorescence intensity was determined using an excitation wavelength of 320 nm and an emission wavelength of 400 nm on a microplate reader (Infinite F200; Tecan, Switzerland) at optimal gain.
[0209] A typical calibration curve is shown in
TABLE-US-00001 serum 4000 2000 500 125 62.5 31 16 blank concentration [ng/mL] average 36804 24250 8021 2458 1471 982 721 476 emission [FU]
[0210] A sample was defined as “potentially ADA positive” if the associated signal was at or above the screening cut point value that was calculated to generate a false-positive rate of 5% based on the assumption of a coefficient of variation of 5% regarding the screening of individual donors. The assay had a sensitivity of at least 16 ng/mL PC1 in 100% human serum. Potentially ADA positive results were confirmed in a second, confirmatory assay which was identical to the screening assay with the difference that test samples were incubated with an excess of the therapeutic monoclonal mAb-1 (100 μg/mL final assay concentration) in the mAb-1-Bi and mAb-1-Dig containing buffer.
[0211] This method was developed and qualified according to recommendations [1,17] and was also successfully validated.
Example 2
mAb-2-Based ADA Assay (With MgCl.SUB.2 .Treatment) According to the Invention
[0212] For the qualitative detection of antibodies directed against the therapeutic monoclonal mAb-2, a bridging ELISA was used.
[0213] PC2 was used to generate quality control samples in human pooled K3EDTA plasma. 5 μL of quality control samples, negative control samples and test samples were diluted with 45 μL 4 M MgCl.sub.2*6H20 solution resulting in a dilution of 1:10 and incubated for 30 min at room temperature with shaking 450 rpm. Subsequently, 30 μL of all samples were diluted 1:10 using 270 μL Roche Universal Buffer (Roche Diagnostics GmbH, Germany), together with 2000 ng/mL mAb-2-Biotin and 2000 ng/mL mAb-2-Digoxygenin and incubated for 30 min. at room temperature with shaking at 450 rpm. Formed immune complexes were transferred to a SA-coated MTP (100 μL/well) and incubated for one hour at room temperature with shaking at 450 rpm.
[0214] After three washing steps each using 300 μL 1×PBS containing 0.05% (v/v) Tween 20, 25 mU/mL anti-Digoxygenin antibody-HRP conjugate diluted in Roche Universal Buffer (100 μL/well) were added to the MTP and incubated for one hour at room temperature with shaking at 450 rpm.
[0215] After three washing steps, substrate reaction was carried out by adding 100 μL/well 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid solution (ABTS; Roche Diagnostics GmbH, Germany) and optical density was measured at a wavelength of 405 nm with a reference wavelength at 490 nm on a microplate reader (Sunrise; Tecan, Switzerland) until the quality control sample containing 1600 ng/mL PC2 reached 2.0±0.1 absorbance units. The final absorbance was calculated as follows: absorbance (405 nm)−absorbance (490 nm).
[0216] A sample was defined as “potentially ADA positive” if the associated signal was at or above the screening cut point value that was calculated to generate a false-positive rate of 5% based on the assumption of a coefficient of variation of 5% regarding the screening of individual donors. The assay had a sensitivity of at least 25 ng/mL PC2 in 100% human plasma. Potentially ADA positive results were confirmed in a second, confirmatory assay which was identical to the screening assay with the difference that test samples were incubated with an excess of the therapeutic monoclonal mAb-2 (100 μg/mL final assay concentration) in the mAb-2-Bi and mAb-2-Dig containing buffer.
[0217] This method was developed and qualified according to recommendations [1,17].
Example 3—Comparative Example
Acid Treatment
[0218] Acid dissociation was performed using the mAb-1-based and mAb-2-based ADA assay according to corresponding assay protocols with the following difference to treatment with MgCl.sub.2 of Examples 1 and 2: 3 μl quality control samples, negative control samples and test samples were diluted with 17 μL corresponding assay buffer followed by addition of 100 μL 0.1 M Glycine-HCl pH 2.0 (Merck Chemicals GmbH, Germany) resulting in a final dilution of 1:40 and incubated for 30 min. at room temperature with shaking at 450 rpm. Subsequently, all samples were adjusted to neutral pH by 2.5-fold dilution with 0.5 M Tris-HCl pH 8.5 (Merck Chemicals GmbH, Germany) together with corresponding concentrations of Biotin- and Digoxygenin-labeled assay reagents and incubated for 30 min. (120 μL acidified sample; 30 μL labeled reagent, 150 μL 0.5 M Tris buffer) at room temperature with shaking at 450 rpm. Subsequently, formed immune complexes (100 μL/well) were transferred to a SA-coated MTP as described in the corresponding MgCl.sub.2 assay protocol of Examples 1 and 2.
Example 4—Comparative Example
Over-Night Incubation
[0219] The assay variant “over-night incubation” was performed using the mAb-1-based and mAb-2-based ADA assay according to corresponding assay protocols with the following difference to treatment with MgCl.sub.2 of Examples 1 and 2: 3 μL quality control samples, negative control samples and test samples were diluted 1:50 using 147 μL of the corresponding assay buffer. Subsequently, all 50-fold diluted samples were further diluted 1:2 by addition of 150 μL assay buffer containing corresponding concentrations of Biotin- and Digoxygenin-labeled assay reagents and incubated over-night at room temperature with shaking at 450 rpm. The next day, formed immune complexes were transferred to a SA-coated MTP (100 μL/well) as described in the corresponding MgCl.sub.2 assay protocol of Examples 1 and 2.
Example 5
Drug Tolerance
[0220] The ability to detect ADA in the presence of therapeutic drug (assay drug tolerance) was determined using the respective PC and drug in each ADA assay. Tested combinations were PC1/mAb-1 and PC2/mAb-2. PC concentrations were chosen to comply with the FDA's ADA assay sensitivity recommendation of at least 100 ng/mL [1] and drug concentrations were chosen based on the expected levels of circulating drug in study samples.
[0221] Different quantities of respective PC were added into ADA-negative human serum or plasma samples in the presence or absence of different concentrations of drug and incubated for 3 hours at room temperature with shaking at 450 rpm to allow immune complex formation. Subsequently, samples were frozen and stored over-night at −80° C. and analyzed the next day according to corresponding assay protocols. The highest concentration of drug resulting in a mean signal at or above the screening cut point was considered the assay drug tolerance for a given PC concentration.
Example 6
Determination of Screening Cut Points
[0222] According to the description provided for mAb1 (Example 1) the sensitivity of the assay was assessed by screening of 64 healthy volunteer sera samples. Signals were normalized with the corresponding sera pool value. The normalized values are presented in the Table below.
TABLE-US-00002 ID normalized signal 1 1.105 2 1.041 3 1.053 4 1.058 5 1.103 6 1.070 7 1.055 8 1.060 9 1.091 10 1.055 11 1.008 12 1.062 13 1.103 14 1.120 15 1.081 16 1.054 17 1.150 18 1.018 19 1.001 20 1.025 21 1.001 22 1.034 23 1.540 24 1.077 25 1.232 26 1.006 27 0.994 28 1.008 29 0.985 30 0.995 31 1.030 32 1.011 33 1.118 34 1.019 35 1.033 36 1.047 37 1.054 38 1.057 39 1.057 40 1.012 41 1.081 42 1.007 43 0.994 44 1.015 45 1.013 46 1.010 47 0.994 48 1.050 49 1.170 50 1.007 51 0.978 52 0.985 53 0.977 54 0.965 55 0.977 56 1.019 57 1.115 58 1.015 59 0.995 60 0.982 61 0.966 62 0.982 63 0.999 64 0.980
[0223] Normalized values were checked for normal distribution using R (version 3.5.1 (2018-07-02) “Shapiro-Wilk normality test” and a non-normal distribution was determined. The 64 values were then analyzed using an outlier-test based on 1.5 IQR and three values were excluded (ID: 23, 25 and 49) and retested for normal distribution with p value=0.01, still non-normal distributed. Due to the non-normal distribution, the sensitivity calculation was based on the 95 quantil of the outlier removed values and was calculated to 1.115 (Excel, quantil function, Microsoft Office Standard 2016) and a back calculation (extrapolated) resulted in a sensitivity of 4.96 ng/mL PC1 in 100% serum. Calibration data is shown in following Table, based on normalized signal versus PC1 concentration.
TABLE-US-00003 concentration 4000 2000 500 125 63 31 16 0 PC1 in serum S/B 82.94 49.12 17.16 5.24 3.15 2.05 1.53 1.00
Example 7
Binding Disruption by the Use of LiCl and Maintaining Activity of the Analyte
[0224] An antibody of 145.8 kDa molecular weight was used as analyte. The antibody is capable to bind recombinant human (rh) mesotheline either in a “one to one” or “one to two” complex. To evaluate the complex disruption properties of LiCl the antibody was incubated at different concertation with different ratios of rh-mesotheline forming in sum 50% of total free and partial free antibody based on the total used concentration of the antibody in the sample. Horse serum was used as sample matrix. These samples were analyzed by a homogenous ligand binding assay using biotinylated rh-mesotheline as capture reagent and human IgG specific detection antibody conjugated to ruthenium for signal generation. The QC samples were analyzed in two ways. Once with incubation with 8 M LiCl to disrupt the complexes and analyzing total antibody concentration and once without LiCl to analyze free antibody concentration by confirming the stability of the complexes during assay procedure.
TABLE-US-00004 preparation of the QC samples free (50% to total; total (with LiCl incubation) without LiCl incubation) target target QC concentration analyzed total concentration analyzed free sample [ng/mL] [ng/mL] recovery [ng/mL] [ng/mL] recovery #1 200000 160772 80% 100000 85081 85% #2 40000 43508 109% 20000 20211 101% #3 10000 10413 104% 5000 4637 93% #4 1000 1074 107% 500 559 112% #5 280 293 105% 140 129 92% #6 100 110 110% 50 53 105%
[0225] The results indicate a sufficient complex disruption by maintaining the binding properties of the antibody in the investigated range of 100 to 200,000 ng/mL in serum.