Gyrolab ADA assay protocol

Transcription

1 Gyrolab ADA assay protocol Gyrolab ADA assay protocol D /G, June 2017

2 Table of Contents Abbreviations... 2 Introduction... 2 Prerequisites... 3 Procedure... 4 Gyrolab ADA protocol overview... 4 Without acid dissociation (overnight incubation, Gyrolab Bioaffy 200 CD)... 4 With acid dissociation (Gyrolab Mixing CD 96)... 5 Gyrolab ADA assay development... 5 ADA Experiment 1 Initial Screen... 5 ADA Experiment 2 Optimization... 6 ADA Experiment 3 - Screening cut point setting... 8 ADA Experiment 4 Drug Tolerance... 9 Further Characterizations Recipes Back-calculation to standard curve References Appendix Appendix Patents and disclaimer Gyrolab ADA assay protocol D /G 1

3 Abbreviations Abbreviation ADA CD FAS LOD MM NC PC PK SD S/B Explanation Anti-Drug Antibody Compact Disc Field Application Specialist Limit of Detection Master Mix Negative Control Positive Control Pharmacokinetic Standard Deviation Signal-to-Background ratio Introduction Anti-Drug Antibodies (ADAs) can be formed in a patient or experimental animal upon administration of a biotherapeutic. This immune response can be a safety concern, may neutralize the effect of the drug and/or may mask epitopes on the drugs used by the PK assay, resulting in lower than expected exposure values [1]. In all of these instances, it is important to monitor the ADA and an ADA assay needs to be developed [2, 3]. The assay format we recommend for ADA assays on a Gyrolab TM system is a homogeneous bridging format, which detects ADA of all isotypes and is not species dependent. This means that it can be used both for pre-clinical and clinical studies. In this assay format, the drug is used as both capture (biotinylated) and detection (fluorescently labeled) reagent and the sample and the reagents are mixed before addition to the streptavidin columns on the CD. A number of different complexes will be formed in this mixture. What will be detected in the assay are complexes where ADA or the positive control (PC) form a bridge between a biotinylated drug molecule and an Alexa Fluor 647 labeled drug molecule (see Figure 1). Figure 1. Detectable complexes in a homogenous bridging assay Gyrolab ADA assay protocol D /G 2

4 A general problem with ADA assays is that free drug forms complexes with the ADA and prevents it from being detected by the ADA assay. We present two approaches to solve this problem in a Gyrolab system: Over-night incubation of the ADA assay reagents with the sample before analysis to allow the unwanted complexes time to dissociate Automated acid dissociation protocol, using a dedicated CD with a mixing chamber The high binding capacity of the particles in the Gyrolab CD allows for high concentrations of labeled reagents to be used, that can compete with the free drug and give a stoichiometric advantage over interference from the free drug. Prerequisites Before the ADA assay development can begin, reagents must be prepared and suitable positive controls must be obtained. On the reagent side, biotinylated and Alexa Fluor 647 labeled drug must be prepared and their quality should be determined. Ideally, the binding characteristics of the labeled drug molecules should also be assessed to ensure that the capture and detection reagents have approximately the same affinity to maximize formation of the detectable complexes. The choice of PC is very important. The PC should be as similar to the actual ADA samples as possible and preferably polyclonal. A sample shown to be positive from previous studies would be a very good PC. A high affinity monoclonal antibody is not recommended as this would have low similarity to the actual ADA and might bind too strongly to the drug, possibly resulting in problems with free drug interference. Two different methods for ADA analysis are available to download on the Gyrolab User Zone if not already in your method database: Table 1. ADA methods Method Name Description 200-1W-004-A Homogenous assay on the Gyrolab Bioaffy 200 CD without acid treatment, designed for the ADA application Mixing96-1W-003-A Homogenous assay on the Gyrolab Mixing CD 96 with acid treatment inside the CD, designed for the ADA application Please note that Gyrolab Control version 7.2 or higher is required to run these methods. Gyrolab ADA assay protocol D /G 3

5 Both methods in Table 1 use Gyrolab Wash Buffer ph 11 as a second wash solution for the needles. This procedure washes the needles with a high ph solution, which helps to prevent carry-over problems and is extra important when labeled reagents are loaded with sample needles as is the case for homogeneous bridging assays. Procedure Optimizing an ADA assay differs from optimizing other assays. Instead of looking primarily at the Signal-to-Background ratio (S/B), it is more relevant to optimize the most robust assay in the cut point area and to include free drug at a concentration close to what can be expected in study samples. Our recommendation is that this is done by back-calculating a mini-cut point based on seven treatment naïve individuals against a standard curve obtained with your positive control in a relevant drug concentration. This way, both the blank precision, sensitivity and individual variability is taken into consideration in the optimization. The parameters to optimize are: (1) Master Mix (MM) concentration and (2) sometimes sample dilution. In general, a higher MM concentration gives better drug tolerance but lower sensitivity (without drug). A low sample dilution gives good sensitivity without drug but poor drug tolerance. As in most cases, presence of free drug can t be avoided so optimizing the assay without addition of free drug might be miss-leading and will generate an assay with sub-optimal drug tolerance. We recommend that the ADA assay is simultaneously optimized on a Gyrolab Bioaffy 200 CD with overnight incubation and on a Mixing CD 96 with acid dissociation (first two experiments) as this saves time and allows for a direct comparison between the two assay designs. Ready-made runs for the ADA experiments in this protocol can be obtained from your local Field Application Specialist (FAS) or can be downloaded from Gyrolab UserZone. Gyrolab ADA protocol overview Without acid dissociation (overnight incubation, Gyrolab Bioaffy 200 CD) 1. Dilute your samples and positive and negative controls in Rexxip ADA 2. Prepare a MM solution in Rexxip ADA 3. Mix 10 µl of each sample/pc/nc with 10 µl of MM 4. Incubate overnight in a non-binding plate, without shaking, at 4 C 5. Analyze on Gyrolab Bioaffy 200 CD in duplicate Gyrolab ADA assay protocol D /G 4

6 With acid dissociation (Gyrolab Mixing CD 96) 1. Dilute your samples, PC s and NC s in Rexxip ADA 2. Prepare a MM solution in Neutralization Buffer (see Recipes) 3. Load samples, PC s, NC s on PCR plate along with Acidic Buffer and analyze on Gyrolab Mixing CD 96 in duplicate Gyrolab ADA assay development ADA Experiment 1 Initial Screen Aim To assess assay sensitivity, drug tolerance and effect of sample dilution at three MM concentrations. Method A schematic of this experiment can be found in Appendix 1. Prepare a stock solution of your PC at 80 g/ml and prepare a dilution series of PC at 2x final concentration in 100% matrix according to Table 2. Table 2 Preparation of standard curve for Initial Screen Experiment Prepare 100 µl of unlabeled drug at 2x the final concentration in 100% matrix. Mix 20 µl 2x standard curve and blank with 20 µl 2x free drug or matrix resulting in two curves with/without drug. Incubate for one hour at room temperature to allow complexes to form. Dilute the standards (with and without drug) according to Table 3. Table 3 Dilution of standard curve in Rexxip ADA Prepared PC (ng/ml) Dilution Vol. Standard (µl) Vol. Rexxip ADA (µl) 1: : Vol. of previous standard ( L) Added vol. matrix ( L) Stock x Standard_ Intermediate dilution (not analyzed) x Standard_ x Standard_ x Blank Gyrolab ADA assay protocol D /G 5

7 Prepare three twofold concentrated MM solutions with equal concentrations of biotinylated and Alexa Fluor 647 labeled drug as a serial dilution in Rexxip ADA according to Table 4. Table 4 Preparation of 2x MM solutions Conc. (µg/ml) Vol. previous dilution (µl) Vol Rexxip ADA (µl) 2x MM32 64 Prepare 180 µl 2x MM x MM Prepare 1xMM solutions (at 32, 8 and 2 µg/ml) in Neutralization Buffer by mixing 25 µl of each 2xMM with 25 µl 2 M Tris-HCl, ph 8.0 (See Recipes). Load the diluted standards on a PCR plate according to the loading list for experiment ADA Exp 1 - Initial Screen Mix96 along with MM (prepared above) and Acidic Buffer (see Recipes) and run the experiment on a Gyrolab Mixing CD 96. Mix 90 µl of each 2xMM with 90 µl Rexxip ADA to use in the experiment on the Bioaffy 200 CD. Mix 10 L of the diluted standards with 10 L 1xMM of each concentration on a nonbinding plate according to the Gyrolab loading list for the experiment ADA Exp 1 - Initial Screen BA200. Seal the plate and protect it from light. Incubate the plate without shaking overnight at 4 C to allow drug-ada complexes to dissociate so that labelled drug from the MM can bind. Analyze on a Gyrolab Bioaffy 200 CD. Evaluation Estimate the sensitivity of the assay in the presence of drug. Compare with the sensitivity without the drug to distinguish between poor sensitivity and poor drug tolerance Determine if the lower or higher Master Mix range works best in presence of the drug Determine if matrix dilution has a major effect on sensitivity in presence of the drug ADA Experiment 2 Optimization Aim To find the most suitable Master Mix concentration and, if needed, matrix dilution and to set a mini-cut point based on seven individuals. Experiment design Perform one run for each CD type, matrix dilution and drug concentration to be tested. A single 1:10 dilution is usually enough (a lower dilution will generally improve sensitivity without drug but makes the assay less drug tolerant). Optimization only in the presence of Gyrolab ADA assay protocol D /G 6

8 free drug is recommended and one drug concentration is often sufficient. If higher Master Mix concentrations were more promising in the Initial Screen experiment, examine at e.g. 3, 9, and 27 µg/ml in the optimization. If lower Master Mix concentrations were more promising, examine e.g. 1, 3 and 9 µg/ml. Method A schematic of this experiment can be found in Appendix 2. Prepare a standard curve of your PC in 100% matrix spiked with a relevant concentration of drug, according to Table 5, with 100% matrix with drug as diluent. Allow the PC and free drug to form complexes by incubating the mixture for about 1 h at room temperature. Dilute the PC:drug mixture along with seven samples from treatment naïve subjects 1:10 in Rexxip ADA (5 L mix + 45 L Rexxip ADA). Load the diluted standards according to the loading list for experiment ADA Exp 2 - Optimization along with MM (prepared below) and Acidic Buffer on a PCR plate and run the experiment on a Mixing CD 96. Table 5. Preparation of standard curve for Optimization Experiment Prepared PC (ng/ml) Vol. of previous standard ( L) Added vol. matrix with free drug ( L) Stock Stock Standard_ Standard_ Standard_ Standard_ Standard_ Blank Prepare three 2xMM with equal concentrations of biotinylated and Alexa Fluor 647 labeled drug as a serial dilution in Rexxip ADA. Start with preparing 110 L of a mixture of 2x the highest MM concentration (e.g. 2x9 or 2x27µg/mL). Dilute down 1:3 according to Table 6. Prepare 1xMM solutions (at e.g. 1, 3 and 9 µg/ml) in Neutralization Buffer by mixing 25 µl of each 2xMM with 25 µl of 2 M Tris-HCl, ph 8.0 (See Recipes) and run the experiment ADA Exp 2 Optimization Mix96. Table 6 Preparation of 2x MM solutions Vol. previous dilution (µl) Vol Rexxip ADA (µl) 2x MM1 - High Prepare 110 µl 2x MM2 - Mid x MM2 - Low Gyrolab ADA assay protocol D /G 7

9 Mix 40 µl of each 2xMM with 40 µl Rexxip ADA to use on the experiment on a Gyrolab Bioaffy 200 CD. Mix 10 L of the diluted standards and samples with 10 L 1xMM of each concentration on a non-binding plate according to the Gyrolab loading list for the experiment ADA Exp 2 - Optimization BA200. Seal the plate and protect it from light. Incubate the plate, without shaking, overnight at 4 C to allow drug-ada complexes to dissociate, so labelled drug from the Master Mix can bind. Analyze on a Gyrolab Bioaffy 200 CD. Drug (µg/ml) Equimolar Master Mix conc. (µg/ml) Serum dilution 25% 25% 25% 10% 10% 10% Av response 7 negative individuals %CV response 7 negative individuals 12% 32% 27% 6% 9% 12% Av response Negative control (pool serum) %CV response (triplicate) Negative control (pool serum) 12% 12% 12% 2% 16% 8% Mini cut point concentration (ng/ml) ng/ml Figure 2. Example of results from Experiment 2. Two serum dilutions were tested. In this example, 9 µg/ml Master Mix and a 1:10 serum dilution was found to be the optimal condition (lowest estimated cut point concentration) Evaluation Compare the responses from the individuals to the standard curve blank (diluted matrix pool) to make sure the individuals and pool correlate Set a mini-cut point using the response values from the seven individuals (e.g. 2 SD above average individual response as a normal distribution of responses from as few as seven individuals is difficult to obtain). Use the standard curve to back-calculate the cut point responses to concentrations, see Back-calculation to standard curve. A low cut point concentration (estimated sensitivity in presence of drug) is desired At this point a decision can be made on which MM concentration (and matrix dilution if that was assessed in this experiment) to use and if acid dissociation is required or not ADA Experiment 3 - Screening cut point setting Aim To set a cut point using a larger number of treatment naïve individuals. Gyrolab ADA assay protocol D /G 8

10 Method Use the conditions selected in the previous experiments (MM concentration, matrix dilution, with or without acid dissociation) to do a number of runs with many individuals to calculate a cut point. We recommend using three PCs (e.g. 100 ng/ml PC diluted in Rexxip ADA) and three negative controls (matrix pool diluted in Rexxip ADA) for each CD and run the analysis in duplicate. A Gyrolab Bioaffy 200 CD will then fit 50 individuals and the Mixing CD 96 will accommodate 42. Use of a floating cut point is recommended. Evaluation Example of a cut point run is shown in Figure 3 Set a screening cut point according to industry guidelines [4] An estimate of the assay sensitivity in the presence of drug can now be calculated using the standard curve from ADA Experiment 2 Optimization and the correction factor set in this experiment Figure 3. Example of a cut point run on the Mixing CD 96. Sample 26 was found to be an outlier and was excluded from the cut point calculation ADA Experiment 4 Drug Tolerance Aim To calculate the maximum concentration of free drug a certain concentration of PC (e.g. 100 ng/ml) can tolerate before being classified as negative in the screening ADA assay. Method Prepare PC in matrix at 2x concentration. Prepare a 10-fold serial dilution of drug in matrix to cover the range of e.g. 10 ng/ml to 1 mg/ml (prepare 2x concentrated drug dilutions and mix with an equal amount of PC or NC). Incubate at room temperature for one hour to allow for complexes to form. In the ADA Exp 4 Drug Tolerance experiments two PC levels (100 and 800 ng/ml) and one NC is tested and the run requires ½ Gyrolab Mixing CD 96 or 6 segments of a Gyrolab Gyrolab ADA assay protocol D /G 9

11 Bioaffy 200 CD. Additional concentration levels for both drug and PC can be added if required. Note that the standard curve concentrations in this experiment is concentration of free drug and not PC! a) b) Figure 4. Example of drug tolerance for a) Bioaffy 200 CD with overnight incubation and b) Mixing CD 96 with acid dissociation Evaluation Investigate the effect of high drug concentrations on the NC. There can be a slight decrease in the background signals at high drug concentrations which is not considered a problem. Calculate the cut point by using the correction factor established in ADA Experiment 3 - Screening cut point setting using the NC data in the run and estimate the upper limit of drug tolerance concentration for a given PC (e.g. 100 ng/ml) by extrapolating the 100 ng/ml PC curve to the cut point using Back-calculation to standard curve. This level is an estimate of the maximum drug tolerance for the PC, as seen in Figure 4. Gyrolab ADA assay protocol D /G 10

12 Further Characterizations When an assay has passed through the four experiments described in this protocol you have established that you have a working ADA assay, but you probably need to do some further experiments to fully characterize your assay. Here are some examples: Full drug tolerance evaluation. Run the drug tolerance experiment, but with smaller steps between the different drug concentrations (e.g. 1:2 dilutions of drug) to determine the maximum concentration of free drug to keep the PC above the cut point Hook effect (prozone effect). A homogeneous bridging assay will give lower signals at very high analyte concentrations. This is not a problem as long as the assay doesn t give false negative responses at high ADA concentrations. Higher Master Mix concentrations will shift the hook to even higher analyte concentrations. Run a standard curve of PC starting with as high concentrations as possible to investigate the hook effect Recipes Name Preparation 1 M Glycine 7.51 g Glycine ml H2O 1 M HCl 12.1 ml 25% HCl (8.2 M) ml H2O Acidic Buffer (0.5 M Glycine-HCl, ph 2.6) 12.5 ml 1 M Glycine ml 1 M HCl ml H2O 2 M Tris-HCl, ph g Tris + 30 ml H2O. Adjust ph to 8.0 with 25% HCl. Fill up to 50 ml with H2O Neutralization Buffer Equal parts 2 M Tris-HCl, ph 8.0 and Rexxip ADA Back-calculation to standard curve Back-calculations of response values to the standard curve can be done in Gyrolab Evaluator using Quantification in the ADA analysis module. This is done by entering a response value in the curve intersection dropdown below the standard curve graph, see Figure 5. Note: this functionality is only available in the Quantification module of the ADA software. Gyrolab ADA assay protocol D /G 11

13 Figure 5. Back-calculation to standard curve References 1. Schellekens H, Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clinical Therapy 24(11), EMEA Guideline on Immunogenicity Assessment of Biotechnology-derived Therapeutic proteins. Draft 3. FDA Guidance for Industry. Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products. DRAFT GUIDANCE 4. Shankar G, Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal 48(5): Gyrolab ADA assay protocol D /G 12

14 Appendix 1 ADA Experiment 1 Initial Screen Gyrolab ADA assay protocol D /G 13

15 Appendix 2 ADA Experiment 2 Optimization Gyrolab ADA assay protocol D /G 14

16 Patents and disclaimer Gyros, Gyrolab, Gyrolab xplore, Bioaffy, Rexxip and Gyros logo are trademarks of Gyros Protein Technologies Group. All other trademarks are the property of their respective owners. Products and technologies from Gyros Protein Technologies are covered by one or more patents and/or proprietary intellectual property rights. All infringements are prohibited and will be prosecuted. Please contact Gyros Protein Technologies AB for further details. Products are for research use only. Not for use in diagnostic procedures. Gyros Protein Technologies AB Gyros Protein Technologies will use reasonable efforts to include accurate and up-to-date information in this document, but Gyros Protein Technologies makes no warranties or representations of any kind as to its accuracy, currency or completeness. Gyros Protein Technologies disclaim all warranties, expressed or implied, including warranties of satisfactory quality or fitness for a particular purpose. Neither Gyros Protein Technologies nor any party involved in creating, producing or delivering this document shall be liable for any damages arising out of use of this document, or any errors or omissions in the content thereof. Gyrolab ADA assay protocol D /G 15