Measure of HUMIRA®/ABP501 trough levels in spiked samples
Accuracy of the LISA-TRACKER was measured using 3 human serum matrix spiked with known levels of biosimilar, 3 levels spanning the dynamic range. For each level, the percentage of recovery was calculated (Percentage of recovery of Adalimumab assay = [Mean of the six obtained results/theoretical level of Adalimumab)] x 100). An imprecision intra-run has been realized. Imprecision was assessed by using 3 clinical levels (low, medium, high) for 3 human matrices and for each levels, 10 tests per run were performed. An imprecision inter-run has been realized. Imprecision was assessed by using 3 levels (low, medium, high) of clinical samples for 3 human matrices and for each levels, on 6 independent runs, 2 tests per run were performed with LISA-TRACKER Duo Adalimumab assay. Quantification of Adalimumab was performed according to the technical insert of LISA-TRACKER Adalimumab kits (product number LTA 005 or LTA 002). As described in the technical insert, a run performed with LTA kits was validated when the optic density (OD) of the highest standard was above 0.8 and when the result (µg/ml) of the positive control was within its target range. Then, the result of each sample could be interpreted.
Accuracy
Spiked samples made with batch1 gave percentages of recovery comprised between 90% and 120% and spiked samples made with batch2 gave percentages of recovery comprised between 93% and 105%. All results met the acceptance criteria (80–120%). “ABP501 spiked samples” were quantified with LISA-TRACKER Duo Adalimumab assay. (Table 1) Quantification of ABP501 was not affected by serum matrix. Similar results were obtained with Humira® during the development of LISA-TRACKER Duo Adalimumab assay.
Intra-run and inter-run imprecision
For the imprecision intra-run, the CV ranged from 4.1–11.1% for samples made with biosimilar batch1 and ranged from 3.8–16.5% for samples made with biosimilar batch2 (Table 2). For the imprecision inter-run, the CV ranged from 4.4–13.9% for samples made with biosimilar batch1 and 4.5–13.0% for samples made with biosimilar batch2 (Table 3). The acceptance criteria (CV < 20%) was met.
Low intra-run and inter-run imprecisions were reached with LISA-TRACKER Duo Adalimumab assay for the quantification of ABP501.
Measure of HUMIRA®/AB501 through levels in clinical samples
Clinical samples with detectable level of Adalimumab (Humira®) were spiked with Humira® or ABP501. Thus, Adalimumab (Humira® or ABP501) were added into the clinical samples in order to increase the level of Adalimumab of 4 µg/ml. All samples from the 3 preparations (3 × 30 “spiked clinical samples”) were quantified with LISA-TRACKER Adalimumab kit (LTI 002, batch: 1847). The results obtained with “ABP501 spiked clinical samples” were compared to the results obtained with “Humira® spiked clinical samples”. Furthermore, the same type of tests was launched with clinical samples with detectable level of adalimumab biosimilar. Thus, ABP501 clinical samples were spiked with Humira® in order to increase the level of 4 µg/ml; spiked samples were quantified and compared to the unspiked samples.
ABP501 levels in patients
There were 30 clinical samples with detectable level of Humira®. All results from clinical samples spiked with ABP501 were within +/-20% of the expected values (levels of Adalimumab from clinical samples spiked with Adalimumab (Humira®). (Tab. S1a). The coefficient of determination (R2) and the slope were calculated for the 3 different preparations. ABP501-batch1 vs Humira® (R2 = 0.95; slope: 0.94), ABP501-batch2 vs Humira® (R2 = 0.98; slope: 0.94), ABP501-batch1 vs ABP501-batch2 (R2 = 0.94; slope: 0.95). (Fig. 1a). Thus all the coefficients of determination and Slopes met the acceptance criteria (R² > 0.90 and Slope comprised between 0.9 and 1.1). Detection of ABP501 trough levels in the patients treated with ABP501 (34 clinical samples, ID: ABP501-1 to ABP501-34) with the LISA-TRACKER Adalimumab assay was compliant: all ABP501 positive samples were in the expected range after the addition of 4 µg/mL of R.A. (Tab. S1b). Therefore LISA-TRACKER Duo Adalimumab assay detect efficiently ABP501 trough levels as the R.A. in serum without any interference.
Specificity Assessment: Adalimumab Assay
“ABP501 spiked samples” inhibition assays were performed with LISA-TRACKER Adalimumab kit (product number: LTA 002, batch: 1847). In order to confirm the positivity of “ABP501 spiked samples” and the positivity of clinical samples, polyclonal antibodies directed against Humira® were added to the kit’s dilution buffer: “poly-anti-ada-buffer”. On one hand, “ABP501 spiked samples” (“ABP501 spiked samples” and “ABP501 + Humira® spiked samples”) and clinical samples were diluted with “poly-anti-ada-buffer”, and on the other hand they were diluted with the kit’s dilution buffer. Quantification of the samples was performed after 60 minutes of incubation at room temperature. For each sample, the percentage of inhibition was calculated. (Percentage of inhibition of Adalimumab assay = [1-(level of Adalimumab from sample diluted with poly-anti-ada-buffer/level of Adalimumab from sample diluted with sample’s dilution buffer)] x 100). Spiked samples (“ABP501 spiked samples” and “ABP501 + Adalimumab (Humira®) spiked samples”) and clinical samples with a percentage of inhibition greater than 50% were considered “positive” (detectable level) for ABP501.
Specificity
For “ABP501 spiked samples”, the levels of Adalimumab were below the limit of quantification when the kit’s dilution buffer spiked with polyclonal was used. All percentages of inhibition were at least above 77%. For “ABP501 + Adalimumab spiked samples” made with Humira® in addition with ABP501, the levels of Adalimumab were below the limit of quantification when the kit’s dilution buffer spiked with the polyclonal antibodies was used. (Tab. S2a). For “ABP501 clinical samples” (ID: ABP501-35 to ABP501-68), the levels of Adalimumab were below the limit of quantification when the kit’s dilution buffer spiked with the polyclonal antibodies was used. All percentages of inhibition were at least above 92%. (Tab. S2b). Acceptance criteria (percentage of inhibition > 50%) were met. “ABP501 spiked samples” and “ABP501 + Adalimumab spiked samples” and clinical samples gave similar results. Anti-Adalimumab antibodies generated with Humira® have the capacity to block Humira® and ABP501. LISA-TRACKER Duo Adalimumab assay should be able to detect ABP501 as it does for Humira®.
a. |
ADALIMUMAB Samples | SAMPLES spiked with ABP501-Batch1 |
No-inhibited* | Inhibited** | % of inhibition*** |
µg/ml | mean | µg/ml | mean |
Low | 1,3 | 1,3 | < 0,3 | < 0,3 | > 77% |
1,3 | < 0,3 |
Medium | 4,8 | 5,1 | < 0,3 | < 0,3 | > 94% |
5,4 | < 0,3 |
High | 18,6 | 18,5 | < 0,3 | < 0,3 | > 98% |
18,4 | < 0,3 |
ADALIMUMAB Samples | SAMPLES spiked with ABP501-Batch2 |
No-inhibited* | Inhibited** | % of inhibition*** |
µg/ml | mean | µg/ml | mean |
Low | 1,3 | 1,3 | < 0,3 | < 0,3 | > 77% |
1,2 | < 0,3 |
Medium | 5,1 | 5,0 | < 0,3 | < 0,3 | > 94% |
4,9 | < 0,3 |
High | 15,8 | 16,1 | < 0,3 | < 0,3 | > 98% |
16,3 | < 0,3 |
ADALIMUMAB Samples | SAMPLES spiked with ABP501-Batch1 + ADALIMUMAB (Humira) |
No-inhibited* | Inhibited** | % of inhibition |
µg/ml | mean | µg/ml | mean |
Low | 3,8 | 3,7 | < 0,3 | < 0,3 | > 92% |
3,5 | < 0,3 |
Medium | 12,4 | 12,7 | < 0,3 | < 0,3 | > 98% |
13,0 | < 0,3 |
High | > 20 | > 20 | < 0,3 | < 0,3 | > 98% |
> 20 | < 0,3 |
ADALIMUMAB Samples | SAMPLES spiked with ABP501-Batch2 + ADALIMUMAB (Humira) |
No-inhibited* | Inhibited** | % of inhibition*** |
µg/ml | mean | µg/ml | mean |
Low | 3,6 | 3,8 | < 0,3 | < 0,3 | > 92% |
4,0 | < 0,3 |
Medium | 13,6 | 13,9 | < 0,3 | < 0,3 | > 98% |
14,1 | < 0,3 |
High | > 20 | > 20 | < 0,3 | < 0,3 | > 98% |
> 20 | < 0,3 |
| | ANTI-ADALIMUMAB SAMPLES DILUTED WITH BUFFER SPIKED WITH : |
ANTI-ADALIMUMAB CLINICAL SAMPLES | ADALIMUMAB (Humira) | ABP501-Batch1 | ABP501-Batch2 |
ID | Anti-ADALIMUMAB (ng/ml) | Anti-ADALIMUMAB (ng/ml) | % of inhibition* | Anti-ADALIMUMAB (ng/ml) | % of inhibition* | Anti-ADALIMUMAB (ng/ml) | % of inhibition* |
AADA1 | 48 | < 10 | 79% | < 10 | 79% | < 10 | 79% |
AADA2 | 39 | < 10 | 74% | < 10 | 74% | < 10 | 74% |
AADA3 | 43 | < 10 | 77% | < 10 | 77% | < 10 | 77% |
AADA4 | 31 | < 10 | 68% | < 10 | 68% | < 10 | 68% |
AADA5 | 52 | < 10 | 81% | < 10 | 81% | < 10 | 81% |
AADA6 | 42 | < 10 | 76% | < 10 | 76% | < 10 | 76% |
AADA7 | 49 | < 10 | 80% | < 10 | 80% | < 10 | 80% |
AADA8 | 26 | < 10 | 62% | < 10 | 62% | < 10 | 62% |
AADA9 | 46 | < 10 | 78% | < 10 | 78% | < 10 | 78% |
AADA10 | 41 | < 10 | 76% | < 10 | 76% | < 10 | 76% |
AADA11 | 58 | < 10 | 83% | < 10 | 83% | < 10 | 83% |
AADA12 | 44 | < 10 | 77% | < 10 | 77% | < 10 | 77% |
AADA13 | 38 | < 10 | 74% | < 10 | 74% | < 10 | 74% |
AADA14 | 33 | < 10 | 70% | < 10 | 70% | < 10 | 70% |
AADA15 | 49 | < 10 | 80% | < 10 | 80% | < 10 | 80% |
AADA16 | 44 | < 10 | 77% | < 10 | 77% | < 10 | 77% |
AADA17 | 48 | < 10 | 79% | < 10 | 79% | < 10 | 79% |
AADA18 | 67 | < 10 | 85% | < 10 | 85% | < 10 | 85% |
AADA19 | 53 | < 10 | 81% | < 10 | 81% | < 10 | 81% |
AADA20 | 54 | < 10 | 81% | < 10 | 81% | < 10 | 81% |
AADA21 | 41 | < 10 | 76% | < 10 | 76% | < 10 | 76% |
AADA22 | 63 | < 10 | 84% | < 10 | 84% | < 10 | 84% |
AADA23 | 31 | < 10 | 68% | < 10 | 68% | < 10 | 68% |
AADA24 | 57 | < 10 | 82% | < 10 | 82% | < 10 | 82% |
AADA25 | 39 | < 10 | 74% | < 10 | 74% | < 10 | 74% |
AADA26 | 64 | < 10 | 84% | < 10 | 84% | < 10 | 84% |
AADA27 | 54 | < 10 | 81% | < 10 | 81% | < 10 | 81% |
AADA28 | 48 | < 10 | 79% | < 10 | 79% | < 10 | 79% |
AADA29 | 56 | < 10 | 82% | < 10 | 82% | < 10 | 82% |
AADA30 | 39 | < 10 | 74% | < 10 | 74% | < 10 | 74% |
AADA31 | 43 | < 10 | 77% | < 10 | 77% | < 10 | 77% |
| | DETECTION REAGENT SPIKED WITH : |
ANTI-ADALIMUMAB CLINICAL SAMPLES | ABP501-Batch1 | ABP501-Batch2 |
ID | Anti-ADALIMUMAB (ng/ml) | Anti-ADALIMUMAB (ng/ml) | % of inhibition* | Anti-ADALIMUMAB (ng/ml) | % of inhibition* |
AADA32 | 48 | < 10 | 79% | < 10 | 79% |
AADA33 | 39 | 14 | 64% | < 10 | 74% |
AADA34 | 32 | < 10 | 69% | < 10 | 69% |
AADA35 | 27 | < 10 | 64% | < 10 | 64% |
AADA36 | 27 | < 10 | 64% | < 10 | 64% |
AADA37 | 24 | < 10 | 58% | < 10 | 58% |
AADA38 | 37 | < 10 | 73% | < 10 | 73% |
AADA39 | 62 | < 10 | 84% | < 10 | 84% |
AADA40 | 31 | < 10 | 68% | < 10 | 68% |
AADA41 | 23 | < 10 | 56% | < 10 | 56% |
AADA42 | 36 | < 10 | 72% | < 10 | 72% |
AADA43 | 94 | < 10 | 89% | < 10 | 89% |
AADA44 | 28 | < 10 | 65% | < 10 | 65% |
AADA45 | 74 | < 10 | 86% | < 10 | 86% |
AADA46 | 35 | < 10 | 71% | < 10 | 71% |
AADA47 | 18 | < 10 | 44% | < 10 | 44% |
AADA48 | 55 | < 10 | 82% | < 10 | 82% |
AADA49 | 30 | < 10 | 67% | < 10 | 67% |
AADA50 | 14 | < 10 | 30% | < 10 | 30% |
AADA51 | 74 | < 10 | 86% | < 10 | 86% |
AADA52 | 53 | < 10 | 81% | < 10 | 81% |
AADA53 | 29 | < 10 | 65% | < 10 | 65% |
AADA54 | 64 | < 10 | 84% | < 10 | 84% |
AADA55 | 52 | < 10 | 81% | < 10 | 81% |
AADA56 | 39 | < 10 | 74% | < 10 | 74% |
AADA57 | 30 | < 10 | 67% | < 10 | 67% |
AADA58 | 64 | < 10 | 84% | < 10 | 84% |
AADA59 | 27 | < 10 | 63% | < 10 | 63% |
AADA60 | 51 | < 10 | 80% | < 10 | 80% |
AADA61 | 70 | < 10 | 86% | < 10 | 86% |
AADA62 | 25 | < 10 | 60% | < 10 | 60% |
AADA63 | 29 | < 10 | 65% | < 10 | 65% |
AADA64 | 21 | < 10 | 51% | < 10 | 51% |
AADA65 | 34 | < 10 | 71% | < 10 | 71% |
AADA66 | 59 | < 10 | 83% | < 10 | 83% |
AADA67 | 29 | < 10 | 65% | < 10 | 65% |
AADA68 | 20 | < 10 | 51% | < 10 | 51% |
AADA69 | 40 | < 10 | 75% | < 10 | 75% |
| ADALIMUMAB (µg/ml) |
a. | Kit’s stability |
Levels | Unexposed | Acc. Criteria (+/- 20%) | 7d + 37°c |
target Low | target High |
PC (c+) | 4,1 | 1,9 | 5,6 | 5,1 |
ADALIMUMAB ABP501-batch1 | Low | 1,8 | 1,4 | 2,2 | 1,6 |
Medium | 5,3 | 4,2 | 6,4 | 6,2 |
High | 19,6 | 15,7 | 23,5 | 20,0 |
ADALIMUMAB ABP501-batch2 | Low | 1,1 | 0,9 | 1,3 | 1,3 |
Medium | 4,3 | 3,4 | 5,2 | 4,8 |
High | 18,0 | 14,4 | 21,6 | 17,5 |
b. | Specimen’s stability |
Levels | Unexposed | Acc. Criteria (+/- 20%) | 7d + 4°c | 3d RT | 5 x f/t cycles |
target Low | target High |
ADALIMUMAB - ABP501-Batch1 | Low | 1,2 | 1,0 | 1,4 | 1,2 | 1,0 | 1,1 |
Medium | 4,0 | 3,2 | 4,8 | 4,2 | 4,2 | 4,6 |
High | 12,8 | 10,2 | 15,4 | 12,1 | 13,0 | 11,2 |
ADALIMUMAB - ABP501-Batch2 | Low | 1,2 | 1,0 | 1,4 | 1,1 | 1,1 | 1,2 |
Medium | 4,3 | 3,4 | 5,2 | 4,1 | 4,5 | 4,3 |
High | 12,8 | 10,2 | 15,4 | 13,7 | 11,8 | 12,3 |
Measure Of Anti-adalimumab In Clinical Samples: Inhibition Assay
Clinical samples with detectable level of Anti-Adalimumab antibodies were diluted with the kit’s dilution buffer previously spiked with Adalimumab (Humira® or ABP501 were added to kit’s dilution buffer in order to prepare 2 types of “ada-buffer”). Also, the clinical samples were diluted with the kit’s dilution buffer. The 3 preparations (2 preparations made with Adalimumab and 1 preparation made without Adalimumab) were incubated 60 minutes at room temperature and quantified with LISA-TRACKER Anti-Adalimumab kit (product number LTA 003, batch: 1844). For each sample, the percentage of inhibition was calculated (percentage of inhibition of Anti-Adalimumab assay = [1-(level of Anti-Adalimumab antibodies from sample diluted with “ada-buffer”/level of Anti-Adalimumab antibodies from sample diluted with kit’s dilution buffer)] x 100). The capacity of ABP501 to block the detection of ATA was measured. Clinical samples with a percentage of inhibition greater than 50% were considered to be inhibited by Adalimumab.
Adalimumab and ATA inhibition assay
There were 31 clinical samples (ID: AADA1 to AADA31) with detectable level of Anti-Adalimumab antibodies. Samples with high levels of Anti-Adalimumab antibodies gave high percentages of inhibition because they were well above the LLOQ. In any case, all percentages were above 50% and both batches of ABP501 and Adalimumab gave similar results. Acceptance criteria were met. (Tab. S3). ABP501 is able to block Anti-Adalimumab antibodies from patients treated with Humira®.
Measure Of Specificity Detection Step In Spiked Clinical Samples
Clinical samples with detectable level of Anti-Adalimumab antibodies were quantified with LISA-TRACKER Anti-Adalimumab kit (product number: LTA 003, batch: 1849). In order to confirm the capacity of ABP501 to block antibodies directed against Humira®, detection step was performed with or without the addition of ABP501 into the detection reagent (biotinylated Humira®) used for the detection step of Anti-Adalimumab antibodies during the assay). For each sample, the percentage of inhibition was calculated (percentage of inhibition of Anti-Adalimumab assay = [1-(level of Anti-Adalimumab antibodies in the presence of ABP501 into the detection reagent / level of Anti-Adalimumab antibodies)] x 100). Clinical samples with a percentage of inhibition greater than 50% were considered to be inhibited by ABP501.
Specificity detection step
There were 38 clinical samples (ID: AADA32 to AADA69) with detectable levels of Anti-Adalimumab antibodies. Samples with low levels (around 2 x limit of quantification (LLOQ)) of Anti-Adalimumab antibodies gave percentages of inhibition near 50% because they were near the limit of quantification (10 ng/ml). Samples with high levels of Anti-Adalimumab antibodies gave high percentages of inhibition because they were well above the LLOQ. In any case but two, all percentages were above 50%. Both batches of ABP501 gave similar results: acceptance criteria were met. (Tab. S4). ABP501 is able to compete with biotinylated Humira®. Anti-Adalimumab antibodies induced by Humira® were able to detect ABP501 during the detection step. LISA-TRACKER Duo Adalimumab assay should be able to detect Anti-Adalimumab antibodies induced by ABP501.
Measure Of Anti-adalimumab/anti-abp501 In Spiked Samples
Clinical samples with detectable level of Anti-Adalimumab antibodies were quantified with the 3 pairs of reagents made with the 3 types of raw materials (pair1: Humira® coated microplate and biotinylated Humira®; pair2: ABP501-batch1 coated microplate and biotinylated ABP501-batch1; pair3: ABP501-batch2 coated microplate and biotinylated ABP501-batch2). Results from the 3 pairs were compared.
Measurement of levels of Anti-adalimumab in patient and spiked samples according to the Anti-adalimumab standard curve
There were 20 clinical samples (ID: AADA70 to AADA89) with detectable level of Anti-Adalimumab antibodies. The coefficient of determination (R2) and the slope were calculated for the 3 different combinations of reagents. ABP501-batch1 vs Humira® (R2 = 0.98; slope: 1.05), ABP501-batch2 vs Humira® (R2 = 0.93; slope: 1.04), ABP501-batch1 vs ABP501-batch2 (R2 = 0.97; slope: 0.97) (Fig. 1b). Thus all the coefficients of determination and Slopes met the acceptance criteria (R² > 0.90 and Slope comprised between 0.9 and 1.1). Reagents made with ABP501 give the same performances as reagents made with Humira® for the detection of Anti-Adalimumab antibodies (from patients treated with Humira®). This demonstrates the similarity of ABP501 and Humira® towards Anti-Adalimumab antibodies. LISA-TRACKER Anti-Adalimumab assay should be able to detect Anti-Adalimumab antibodies induced by ABP501 as it does for Anti-Adalimumab antibodies induced by Humira®.
Measure Of Kit And Sample Stability
In order to evaluate the kit’s stability, LISA-TRACKER Duo Adalimumab kit was stored under stress thermic condition (7 days at + 37 °C). Then, “ABP501 spiked samples” were tested with this “stressed” kit. Results were compared to the results obtained with the unexposed kit (stored between + 2 °C and + 8 °C). For specimen’s stability, spiked samples from patients treated with adalimumab biosimilar were stored in different conditions until quantification: at -20 °C (unexposed samples), 7 days between + 2 °C and + 8 °C (+ 4 °C storage condition), 3 days between + 18 °C and + 24 °C (room temperature (RT) storage condition), and 5 freeze/thaw cycles undergone.
Kit’s stability
The percentages of variation (results from the stressed kit compared to the unexposed kit) were comprised between − 11% and 17% for spiked samples made with ABP501-batch1, and between − 3% and 18% for spiked samples made with ABP501-batch2. All results were within +/-20% from the unexposed kit: acceptance criteria were met. LISA-TRACKER Duo Adalimumab assay is robust. Quantification of ABP501 was not disrupted even if the kit was stored after a long period of elevated temperature (delivery, storage, etc.). (Tab. S5a)
Specimen’s stability
For all storage conditions (+ 4°c, RT or freeze/thaw cycles), all samples gave levels of Adalimumab comprised within +/- 20% compared to the unexposed samples. ABP501 serum samples can be stored, 7 days at + 4°c, or 3 days at RT, or can undergo up to 5 freeze/thaw cycles, before being quantified with LISA-TRACKER Duo Adalimumab assay. (Tab. S5b)