PK SCIENCES Clinical Bioanalytics and Regulatory Science Qualification / validation of new lots of critical reagents for ADA assays: some practical examples Lydia Michaut EBF Training Day: Critical Reagents for LBAs 14 May 2018, Lisbon
Presentation outline 1. A really good example of a pretty bad case 2. Minimum practice for qualification / validation of new lots of critical reagents for ADA assays o o Minor change Major change 3. Qualification of a new monoclonal antibody positive control produced in a different facility but from the same hybridoma 4. Validation of a monoclonal antibody to replace a polyclonal antibody in a calibration curve-based titration assay 2
Assay principle: homogenous MSD assay Acid dissociation and neutralisation SENSITIVITY: 4 ng/ml of a polyclonal antibody DRUG TOLERANCE: 250 ng/ml of a polyclonal antibody can be detected in the presence of up to 56.8 μg/ml of drug Ru B POSITIVE CONTROL for sample analysis: Monoclonal anti-drug antibody (mab1) HPC: 1000 ng/ml LPC: 10 ng/ml (sensitivity of mab1: confirmed at 3 ng/ml) MRD: 1/2.5 in LowCross Buffer B Ru Biotin Ruthenium Streptavidin Streptavidin-MSD plate ADA (from sample or positive control) Drug 3
A very good example of a pretty bad case Assay validation Sept 2011 Mar- 2012 IA Apr-2013 Jul-2013 Sep-2013 1 st IA Mar-2014 2 nd IA Jun-2015 Critical reagent lot Core study analysis Initial lots of Bt- and Ru- drug Extension analysis New lots of Bt- and Ru-drug Cut-points (CP) used SSC: System Suitability Control IDC: Immunodepletion control TCP: Titer Control Positive AC: Acceptance Criteria PC: Positive Control mab: monoclonal Antibody pab: polyclonal Antibody SCPF: 1.19 CCP: 25.4% Retrospectively checked on 963 baseline samples from the core study: %age of baseline positive samples within expected The original Cut-points (CPs) were QUALIFICATION: applied in the extension since same SCPF: 1.19 patient population and SOP did not CCP: 25.4% ü Titration of new reagents specifically request to check the CP ü One run with two sets appropriateness of all SSCs (including for each study. IDCs and TCP) May-2014 with both old and new lots -> All SSCs analyzed with the new lot of Bt- and Rudrug analysis: met the 25% run acceptance of baseline samples criteria were (ACs) ADA positive 1 st IA result Investigation: the negative control (NC) signal was 2-3x lower with ÞThe newly labeled reagents were qualified and used the new lots of reagent for extension ð Determination sampleof analysis study-specific withcut-points the patientspecific cut-points SCPF: 1.59 CCP: 12.1% For the second IA, all the extension data were processed or reprocessed with the new cut-points 4
A very good example of a pretty bad case Root cause analysis All the controls met the sample analysis criteria with the new lot of Biotinylated and Ruthenylated drug. The negative control signal range was not a formal acceptance criteria of the assay, therefore it was not considered. Corrective action Specifically request external service providers to consider signal levels comparison in the qualification of new lots of critical reagents. Preventive action Creation of a mini-working group on critical reagents within Novartis Bioanalytics groups worldwide to define, strengthen and harmonize internal practice at ESPs. 5
General practice for qualification / validation of new lots of critical reagents in ADA assays 1. In case of a minor change ü If the previous lot is still available o o One run with two sets of all SSC *Question (includingto IDCs the Audience: and TCP) with both old and new lots -> Must meet the run acceptance criteria (AC) Signal intensities must beno considered* pre-defined acceptance criteria are currently set for signal comparison: acceptability is left at the scientific discretion of the BA monitor; Individual SCC values must in meet your opinion, the SA run is AC this and acceptable their inter-run or mean must meet the values established with the previous lot. not? Signal intensities must be compared with historical data obtained from previous runs*. ü If no more of old lot: three runs with three sets of all SSC (including IDCs and TCP) over two days o o ü Results are archived with the study raw data. No need to be reported separately, just mentioned in the special issues section of the bioanalytical data report. 6
General practice for qualification / validation of new lots of critical reagents in ADA assays 2. In case of a major change: ü Feasability runs to compare signals and adjust levels; reagent titration if necessary ü (Partial) validation and whenever possible, cross-validation with samples analyzed with the previous lot o If three tiered assay: the positive / negative status in screening / confirmatory must be identical (titer value is not cross-validated) o If titer-based assay: positive/negative status must be identical and the titer value must be in the same range (low, mid, high, very high) 7
Example 2: Qualification of a new monoclonal antibody positive control produced in a different facility but from the same hybridoma Julien Couturier PK 8 Sciences - Clinical Bioanalytics and Regulatory Science Business Use Only
Strategy Each assay «Identity Card» in terms of sensitivity and drug tolerance is established once for all using a polyclonal PC, but SSCs are prepared from a mab PC (approach accepted by Health Authrities) Partial validation in terms of SSC levels and acceptance: o One run with all SSCs to confirm that all AC are met and ensure signals and ratios are comparable (cf. previous question to the audience) o Appropriately redefine SSC levels: 12 independent titrations (4 runs by two analysts) to establish sentitivity and adequately define LSP concentration o «Two for the price of one» SSC: System Suitability Control IDC: Immunodepletion control TCP: Titer Control Positive AC: Acceptance Criteria PC: Positive Control mab: monoclonal Antibody pab: polyclonal Antibody 9
Results: SSCs SSCs LSP (10 ng/ml) MSP HSP (1000 ng/ml) IDC Mean Signal (duplicate) or %inhibition # PC1 810 747 16215 14786 66994 65770 78.34 80.06 Intra-run mean 778 15500 66382 79.2 Mean Signal (duplicate) or %inhibition # PC2 550 538 10576 10433 41026 43659 74.68 69.89 Intra-run mean 544 10504 42342 72.3 % difference over PC1-32.06-27.93-34.78-29.44-38.76-33.62-4.67-12.71 % difference over PC1-30.08-32.23-36.21-8.73 SSCs LSP MSP HSP SP/NC ratios 4.81 4.44 96.44 87.94 398.47 391.20 Intra-run mean of ratios 4.63 92.2 395 SP/NC ratios 3.58 3.50 68.78 67.85 266.83 283.96 Intra-run mean of ratios 3.54 68.3 275 % difference over PC1-25.70-21.19-28.68-22.84-33.04-27.41 % difference over PC1-23.54-25.92-30.38 PC: Positive Control mab: monoclonal Antibody pab: polyclonal Antibody SSC: System Suitability Control IDC: Immunodepletion control LSP: Low-bevel Screening Positive control MSP: Mid-level Screening Positive control HSP: High-level screening Positive control 10
Results: Titration Typical titer curve comparison: mean of signals from 3 intra-run determinations 100000 Mean Titer mab1 run 15 SCP run 15 (176) Mean Titer mab2 (run 33) SCP run 33 (168) 10000 1000 signal (ECL) PC: Positive Control mab: monoclonal Antibody pab: polyclonal Antibody 1000 10 PC antibody concentration (ng/ml) 0,1 100 0,001 11
Results: Titration Run number Titer mab1 Titer mab2 14 / 33 3100 2100 3260 1840 9900 2130 15 / 34 11600 6080 1950 1930 2440 2070 19 / 35 2150 Ind. 14300 1690 5550 2950 38-3960 - 1840-1930 Titer Average 6028 2593 Precision (%) 78 51 Median Titer 3260 2070 Titer range lower limit # 1087 (690) Titer range upper limit ## 9780 (6210) # Median - one dilution step below: corresponds to 1/3 of the median titer ## Median + one dilution step below: corresponds to 3-fold the median titer SSC: System Suitability Control IDC: Immunodepletion control TCP: Titer Control Positive AC: Acceptance Criteria PC: Positive Control mab: monoclonal Antibody pab: polyclonal Antibody 12
Conclusion All SSC values met the acceptance criteria defined with the previous PC. Ratios and signal intensities were within 30% of each other. => The new PC lot produced from a different provider was qualified for use in sample analysis SSC: System Suitability Control IDC: Immunodepletion control TCP: Titer Control Positive AC: Acceptance Criteria PC: Positive Control mab: monoclonal Antibody pab: polyclonal Antibody 13
Qualification of new lots of critical reagents: «two for the price of one» Assay in use since 2011 -> provided the opportunity to «brush-up» the assay according to the 2016 FDA draft guidance and latest version of Novartis SOP o Inclusion of the Minimum Significant ratio (MSR) in the titer acceptance range calculation o LSP level to be precisely set up in order to have 1% failure rate during sample analysis o Influence of hemolysis and lipemia on assay sensitivity o Sensitivity < 100 ng/ml -> drug tolerance testing at 100 ng/ml with pab 14
Example 3: Validation of a monoclonal antibody to replace a polyclonal antibody in a calibration curve-based titer assay Nathalie Laurent and Igor Vostiar In the interest of time, can rather be used as interactive example for panel discussion PK 15 Sciences - Clinical Bioanalytics and Regulatory Science Business Use Only
Semi-quantitation of anti-target specific IgG in human serum in response to a therapeutic vaccine Goat anti-human IgG - HRP HRP AIM: Detect wanted immunogenicty, i.e patients who developped antibodies against the target and are therefore protected Target Anti-target specific IgG (serum) Typical standard curve METHOD Calibration curve-based ELISA assay using AA 1-40 of the target to coat the microtiter plate and a goat anti-human IgG coupled to Horseradish Peroxidase (HRP) for detection. Readout: colorimetric (OPD), 4PL-fit for calibration curve. QUANTIFICATION RANGE 15 (LLOQ) to 300 (ULOQ) arbitrary units in 0.1% serum. POSITIVE CONTROL Rhesus monkey polyclonal serum against target AA 1-6; purified IgG fraction. The undiluted PC was arbitrarily assigned a titer of 1 000 000 units, and then serially diluted to generate a calibration curve, against which the titers of the samples were assigned. 16
Strategy step 1: candidate selection Several monoclonal human IgG1 recognizing specifically the first 6 AA of the target were generated by phage display technology and assessed in preliminary runs. The match between signal level of the highest standard (C1) and consecutive dilutions with the pab response curve was evaluated with each candidate to select the closest candidate. The curve response of one higg1 monoclonal antibody (mab) starting with C1 at 30 ng/ml was the closest to the response obtained from the pab reference serum 17
Step 1: results 18
Strategy step 2: validation Intra-/inter-assay precision and accuracy Selectivity Precision evaluation with real samples Dilution linearity, parallelism Stability (coated plate, PC sub-stock solution (0.2 mg/ml) at -70 C, SSCs) 19
Strategy step 3: cross-validation Further correlation analysis with results generated from both assays will be conducted as part of the first part of the next clinical study by assessing an appropriate number of samples using both assays: 1 st assay using the previous pab reference serum pool which was used in all previous clinical studies, and the assay using the newly selected and validated mab. 20
21 Main contributors: Fabienne Deckert Julien Couturier Nathalie Laurent Thank you