Mitigating target interference challenges in bridging immunogenicity assay to detect anti-tocilizumab antibodies

Abstract

Aim: An assay to detect anti-tocilizumab antibodies in the presence of high levels of circulating target and drug is needed for immunogenicity assessment in comparative clinical studies.Methods: An assay was developed and validated using a combination of blocking agents and dilutions to overcome target interference challenges.Results: No false-positive signal was detected in serum samples spiked with 350-500 ng/ml of IL-6 receptor. As low as 50 ng/ml of positive control antibodies could be detected in the presence of either 500 ng/ml of IL-6 or 250 μg/ml of the drug product. Assay also demonstrated high sensitivity, selectivity and precision.Conclusion: A robust, easy to perform immunogenicity assay was developed and validated for detecting anti-tocilizumab antibodies.

Keywords: anti-drug antibodies; electrochemiluminescence; serum IL-6; serum IL-6 receptor; tocilizumab; validation.

Figure 1.

Schematic representation of anti-drug antibody assay to detect anti-tocilizumab antibodies in human serum. Stepwise representation of principle of the assay is shown (1) Samples containing drug (tocilizumab), anti-drug antibodies and drug target (IL-6R). (2) 300 mM acetic acid is added to dissociate anti-drug antibody and drug complexes along with tocilizumab and IL-6R complexes at MRD of 1/80. (3) Acidified samples containing free drug, target and anti-drug antibodies are neutralized in master mix solution containing biotin-tocilizumab, sulfo-tag labelled-tocilizumab along with fixed concentration of IL-6 to quench IL-6R. (4) Neutralized samples are incubated to allow formation of complexes, followed by (5) washing of plate and addition of read buffer (6) data acquisition on MSD reader and (7) data analysis. IL-6: Interleukin-6; IL-6R: Interluekin-6 receptor; MRD: Minimum required dilution.

Figure 2.

Evaluation of IL-6R target tolerance at 1/20 minimum required dilution in screening and confirmatory assays. (A) Mean RLUs of the analysis of unspiked (0 ng/ml) and spiked with 50–300 ng/ml of IL-6R, NC pooled (purple), individual-1 (blue), Individual-2 (orange) and PCH (500 ng/ml) (green) obtained in screening assay. (B) % inhibition values of the analysis of unspiked (0 ng/ml) and spiked with 50–300 ng/ml of IL-6R; NC pooled (purple), individual-1 (blue), Individual-2 (orange) and PCH (500 ng/ml) (green) obtained in confirmatory assay. Plate Specific Screening cut point and Confirmatory cut points are represented in black dotted lines. IL-6R: Interleukin-6 receptor; MRD: Minimum required dilution; NC: negative control; PCH: Positive control high; PCL: Positive control low; RLU: Relative light unit.

Figure 3.

Strategies employed to mitigate target IL-6R interference in the assay. (A) RLUs of NC pooled unspiked 0 ng/ml (orange), spiked with 750 ng/ml of IL-6R (green), 500 ng/ml of IL-6R (yellow) and with 100 ng/ml of IL-6R (blue) with varying concentrations of anti-IL-6R antibody (0–4000 ng/ml). Plate Specific Screening cut point (black dotted line). (B) Analysis of PCH 500 ng/ml (green), PCL 50 ng/ml (yellow), NC pooled spiked with 350 ng/ml of IL-6R and 50 μg/ml of anti-IL-6R antibody (blue), unspiked NC (purple) and plate specific screening cut point (black dotted line) with varying conditions. All samples were acidified 20-fold with 300 mM acetic acid. Condition-1 acidified samples were incubated to allow acid dissociation followed by neutralized in master mix containing 1M tris and 50 μg/ml of anti-IL-6R antibody. Condition-2 acidified samples were incubated to allow acid dissociation followed by neutralized in master mix containing 60 mM tris and 50 μg/ml of anti-IL-6R antibody. Condition-3 acidified samples were incubated post acid dissociation followed by addition of 50 μg/ml of anti-IL-6R diluted in 60 mM tris, followed by incubation and addition of samples to master mix containing 60 mM tris. Condition-4 post acid dissociation of samples, 50 μg/ml of anti-IL-6R antibody was added followed by incubation and addition of master mix containing 1M tris. (C) Mean RLUs of the analysis of PCH (500 ng/ml), PCL (50 ng/ml), NC-unspiked, NC spiked with 50, 100 and 350 ng/ml of IL-6R with acid dissociation (blue) and without acid dissociation (orange). Plate Specific Screening cut point without acid dissociation is represented in red dotted lines and with acid dissociation in black dotted lines (D) Mean RLUs of analysis of PCH 500 ng/ml (green), PCL 50 ng/ml(yellow)), NC (purple), NC spiked with 100 ng/ml of IL-6R (orange), NC spiked with 350 ng/ml of IL-6R (blue), NC spiked with 750 ng/ml of IL-6R (pink) at three different MRD's 1/20, 1/40 and 1/80 in presence of 5 μg/ml of IL-6 in master mix. Plate Specific Screening cut point is represented in black dotted lines. (E) % inhibition values of analysis of PCH 500 ng/ml (green), PCL 50 ng/ml (yellow), NC (purple), NC spiked with 100 ng/ml of IL-6R (orange), NC spiked with 350 ng/ml of IL-6R (blue), NC spiked with 750 ng/ml of IL-6R (pink) at three different MRD's 1/20, 1/40 and 1/80 in presence of 5 μg/ml of IL-6 in master mix. Confirmatory cut point is represented in black dotted lines. IL-6R: Interleukin-6 receptor; MRD: Minimum required dilution; NC: negative control; PCH: Positive control high; PCL Positive control low; RLU: Relative light unit.

Figure 4.

Evaluation of IL-6R target tolerance in individual serum samples at 1/80 minimum required dilution. % inhibition values of analysis of PCH (500 ng/ml) (green), PCH (500 ng/ml spiked with 500 ng/ml of IL-6R) (red), NC/Individuals + 250 ng/ml of IL-6R (blue), NC/individual + 350 ng/ml of IL-6R (yellow) or NC/individual + 500 ng/ml of IL-6R (orange). Confirmatory cut point is represented in red-dotted lines. IL-6R: Interleukin-6 receptor; NC: Negative control; PCH: Positive control high.

Figure 5.

Evaluation of drug tolerance of the assay at 1/80 minimum required dilution. Mean RLUs of the analysis of NC (purple), PC 50 ng/ml +250 μg/ml of drug (green), PC 50 ng/ml +100 μg/ml of drug (yellow), PC 50 ng/ml +50 μg/ml of drug (grey), PC 50 ng/ml +10 μg/ml of drug (blue), PC 50 ng/ml +5 μg/ml of drug (pink), PC 50 ng/ml + 0 μg/ml of drug (orange). Plate-specific screening cut point is represented in (red dotted line). Drug here denotes data obtained with all the three drug products DRL_TC, Actemra^® and RoActemra^®. DRL_TC: Dr. Reddy's tocilizumab; NC: Negative control; PC: Positive control; RLU: Relative light unit.

Figure 6.

Evaluation of specificity of the assay in presence of higher concentrations of IL-6 at 1/80 minimum required dilution. % inhibition values of the analysis of PCH (500 ng/ml) (green), PCH spiked with 500 ng/ml of IL-6 (yellow), NC spiked with 500 ng/ml of IL-6 (blue), individuals (1–5) spiked with 500 ng/ml of IL-6 (orange). NC spiked with 500 ng/ml was below the confirmatory cut point. Confirmatory cut point is represented in red dotted line. IL-6: Interleukin-6; NC: Negative control; PCH: Positive control high.

Figure 7.

Evaluation of antigenic equivalence of the three drugs in precision, sensitivity and drug tolerance assay. (A) % inhibition values of analysis of PCH (500 ng/ml), PCM (50 ng/ml), PCL (20 ng/ml) and DPC (250 ng/ml + 50 μg/ml of drug). Three drugs evaluated in confirmatory assay were Actemra^® (blue), RoActemra^®(red) and DRL_TC (green). Confirmatory cut point (purple dotted line). (B) % inhibition values of analysis of sensitivity of the assay. Three drugs evaluated to assess sensitivity in confirmatory assay were Actemra^® (blue), RoActemra^®(red) and DRL_TC (green). Confirmatory cut point (purple dotted line). (C) Mean RLUs values of analysis of drug tolerance samples, samples were generated by adding varying concentrations (0–1000 μg/ml) of three drugs; Actemra^® (blue), RoActemra^®(Red) and DRL_TC (green) along with 100 ng/ml of positive control. Plate-specific screening cut point; RoActemra^® (red dotted line), DRL_TC (purple dotted line), Actemra^® (solid black line). DRL_TC: Dr. Reddy's tocilizumab; PCH: Positive control high; PCL: Positive control low; PCM: Positive control mid; RLU: Relative light units.