Gyrolab Protein A Kit Quantification of residual Protein A ligands in the presence of excess amounts of IgG
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1 Quantification of residual Protein A ligands in the presence of excess amounts of IgG Product number P P Product Name Gyrolab Protein A Kit for MabSelect SuRe Ligand Gyrolab Protein A Kit for Native Protein A 1. Intended use This document describes a protocol to quantify residual Protein A ligands in the presence of IgG on Gyrolab immunoassay platform, using Gyrolab Mixing CD 96 and ready-made reagents provided in a kit format. is for research only and not intended for diagnostic use. 2. Introduction Gyros Protein Technologies method for quantitative determination of residual Protein A ligands utilizes a sandwich immunoassay. The assay involves analyzing samples under acid conditions in the Gyrolab Mixing CD. The acid conditions used in the assay have been selected to make any Protein A ligand in the sample fully accessible in the assay while maintaining assay performance. This involves a completely automated acid treatment within Gyrolab Mixing CD 96 to dissociate Protein A from IgG, followed by quantification of Protein A in the presence of IgG. The assay described in this document can be used to detect native Protein A, recombinant Protein A with the native structure produced in E. coli, MabSelect, MabSelect SuRe and MabSelect SuRe LX - ligand. 3. Assay description The assay is intended for quantification of Protein A ligands in relation to a defined concentration of IgG. Results are typically reported as parts-per-million (ppm), i.e. the ratio of Protein A ligand in relation to the amount of IgG present in the sample. Standard Native Protein A or MabSelect Sure is provided as a standard (Reagent C) in the kit. Accurate quantification of protein A ligands in real samples may require using a standard that matches the Protein A from the affinity column material. Gyrolab method description To determine the concentration of Protein A ligand in the presence of IgG, the Gyrolab method involves acidification of samples in the mixing chamber (Figure 1). The sample is acidified at two ph levels in the mixing chamber: 1. A lower ph (Reagent D) is used to dissociate the Protein A from the IgG molecule, and 2. The ph of the acidified sample is adjusted using Reagent E to a ph compatible with the capture reagent. The dissociated sample is then passed through to the capture column. Page 1 of 11, Instruction For Use, D /H information@gyros.com 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 disclaims 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. Gyros, Gyrolab, Gyrolab xplore, Bioaffy, Rexxip and Gyros Protein Technologies 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 2017.
2 Figure 1: Gyrolab method to determine residual Protein A. 4. Limitations We recommend that you qualify or validate the assay for its intended use to ensure that the assay protocol yields acceptable performance, such as accuracy and precision, before using it to report residual Protein A or MabSelect SuRe, respectively. 5. Storage and Stability Reagents All reagents must be stored at +4 to +8 C for stability. Reagents should be used within 1 month after opening. Unopened CD package Refrigerate at +4 to +8 C, pouch unopened. Opened CD package A partially used CD saved for additional analysis at a later time, using unused microstructures, must be kept dark, dry and without exposure to heat. After the run is completed, place the CD in the CD-box, in the pouch. Re-seal the pouch with adhesive tape. Store at +20 to +28 C. Use within 1 week.
3 6. Reagents, Methods & Materials Kit Components Product # Reagents P or P Reagent A Capture Reagent, Biotinylated Anti-Protein A, Ready to use solution Reagent B Detection Reagent, Fluorophore-labeled Anti-Protein A, Ready to use solution Reagent C Standard Native Protein A or MabSelect SuRe, 1000 ng/ml Reagent D Acid Dissociation Buffer 1 Reagent E Acid Dissociation Buffer 2 Reagent F Acidic Wash Buffer Reagent G Wash Buffer Reagent H Sample Dilution Buffer Other components included in the kit Gyrolab CD Gyrolab Mixing CD 96 Wash solution for needles Gyrolab Wash Buffer ph 11 P P well plate 0.2 ml skirted PCR plate P Foil Microplate Foil P
4 Other Materials and Components required but not provided Gyrolab system with Gyrolab Control v.5.4 or later Gyrolab Evaluator v.3.3 or later PBS-T wash solution for Gyrolab wash stations and pump liquid see Gyrolab User guide Pipettes or pipetting equipment with disposable polypropylene tips Disposable polypropylene test tubes Lab centrifuge Vortex mixer CD design For instruments with Gyrolab Control software versions older than 7.1.3, a new CD design, Gyrolab Mixing CD 96, must be imported prior to the first analysis, please see Gyrolab User Zone at Gyrolab Protein A kit run v2 A standard assay protocol run can be downloaded from Gyrolab User Zone at or contact Gyros Protein Technologies. The run can be used as is or serve as a template for designing a new run. The installation procedure is described in Appendix 1 and in section F2 of the Gyrolab User Guide. Gyrolab Method The Gyrolab method: Protein A kit method v2 must be installed on the instrument being used for analysis. Note, if the Gyrolab Protein A kit run v2 is installed the method is automatically installed. If the method is not present in the database it must be installed. Please refer to Appendix 1 or Gyrolab User Zone, for instructions on how to install the method. 7. Assay protocol The assay protocol has been tested with several approved therapeutic IgG and Fc-fusion proteins. To characterize and optimize the assay run conditions for a new IgG molecule, we recommend that you run a serial dilution of samples unspiked and spiked with a known concentration of Protein A or MabSelect SuRe. See Section 9 and 10 for an example on how to prepare the samples and analyze the results. When optimal assay conditions have been established, follow the protocol below. Sample preparation: Dilute the sample to an IgG concentration determined in the serial dilution run using Reagent H (Sample dilution Buffer). Note that to ensure optimal analytical performance, minimum dilution with Reagent H is 1:3 (1 part sample + 2 parts Reagent H). Prepare reagents: Reagents and standard curve should be prepared according to Tables 1 2 below. We recommend that at least one QC sample has a concentration close to the Lower Limit Of Quantitation (LLOQ) and is prepared in sample matrix with your product antibody. See the example in Table 2. Design a run: Use Gyrolab Protein A kit method v2 when designing a run. For optimal use of the CD it is recommended that the samples, standard curve and QC samples are run in duplicate. Create a sample list for the experiment. For more information, please visit Gyrolab User Zone at Appendix 1 (Gyrolab Protein A kit run v2) includes an example of a Protein A run design.
5 Prepare a run: Prime the Gyrolab with PBS-T as Wash Solution 1, freshly prepared Gyrolab Wash Buffer ph 11 (included in the kit) as Wash Solution 2 and PBS-T as pump liquid. Prepare the micro titer plates according to the Sample list and Gyrolab Control loading list. Start the run. Note that Reagent F is placed in two microplate wells and named F and F2 see appendix 1. Analyze run: When the run is finished, analyze the result using the Gyrolab Evaluator software. A concentration of Protein A ligands is usually expressed in relation to the IgG concentration (parts per million or ppm). 8. Preparation of reagents Note! Briefly spin all vials in a micro-centrifuge before opening Capture Reagent The Capture Reagent (Reagent A) is ready-to-use and is transferred directly to the microplate according to the Gyrolab Control Loading List Detection Reagent The Detection Reagent (Reagent B) is ready-to-use and is transferred directly to the microplate according to the Gyrolab Control Loading List Standard curve Table 1 shows an example of how to prepare a standard curve assuming the stock solution of Native Protein A or MabSelect SuRe (Reagent C) is 1000 ng/ml. We recommend that the Protein A standard curve is in the range of 0.03 to 120 ng/ml. Dilute the standard curve in Sample Dilution Buffer (Reagent H). Prepare the dilutions in polypropylene tubes and vortex the tubes throughout the dilution series before transferring to the PCR plate. Table 1 Example of a standard curve for Protein A Conc. Protein A Volume Protein A stock [µl] Volume higher Std conc. [µl] Volume Reagent H [µl] Std Std Std Std Std Std Std Blank
6 QC samples Table 2 shows an example of how to prepare QC samples assuming the stock solution of Native Protein A or MabSelect SuRe (Reagent C) is 1000 ng/ml. Prepare the dilutions in polypropylene tubes. Vortex before transferring to the PCR plate. Table 2 Example of dilutions of QC samples Conc. Protein A Volume Protein A stock [µl] Volume higher QC conc. [µl] Volume Reagent H [µl] QC QC QC Serial dilution to establish optimal assay performance conditions Start by diluting the sample down to an IgG concentration of 10 mg/ml with sample dilution buffer (Reagent H). Mix the sample 1:1 with a Protein A spiked sample dilution buffer containing 10 ng/ml Protein A (Reagent C) to obtain a sample with 5 mg/ml IgG and 5 ng/ml Protein A to be used as stock. It is recommended that the Protein A spike concentration is similar to the expected Protein A concentration, typically 5-10 ng/ml will be sufficient to obtain accurate recovery results. Prepare serial dilution series according to Table 3 and Table 4, below, with spiked and unspiked sample. Prepare the dilutions in polypropylene tubes. Vortex before transferring to the PCR plate. Table 3 Example of serial dilution of spiked samples Serial Dilution Conc. IgG [mg/ml] Spiked Protein A conc Volume higher sample conc. [µl] (stock) Volume Reagent H [µl] 1: : : Table 4 Example of serial dilution of unspiked samples Serial Dilution Conc. IgG [mg/ml] Volume higher sample conc. [µl] Volume Reagent H [µl] (10 mg/ml) 20 1: : : Determine the highest IgG concentration that passes your recovery specification for the spiked + unspiked samples. Examples of spike recovery and dilution linearity experiments are explained above there may be other approaches that work well.
7 10. Performance characteristics Precision and standard curve The data below show both standard curves and precision data from Native Protein A and MabSelect SuRe runs. Each standard curve has been repeated three times and the curves are shown together in the same diagram. The precision data for one curve is shown in the table for each respective ligand. Native Protein A Sample Concentration Response Signal/ (ng/ml) (n=2) Background CV% Blank
8 MabSelect SuRe Sample Concentration Response Signal/ (ng/ml) (n=2) Background CV% Blank
9 Intra and inter precision for Native Protein A and MabSelect Sure Intra and inter precision data are presented below for both native Protein A and MabSelect SuRe. The intra CV has been determined for duplicates of each control sample and the inter CV precision has been determined with duplicates on 3 CDs. Intra- and inter-cd CV% for Gyrolab Protein A assays in presence of human recombinant IgG at 5 g/l. MabSelect SuRe ligand Native Protein A ligand Concentration (ng/ml) Intra CV % (n=2) Inter CV % (3 CDs) Concentration (ng/ml) Intra CV % (n=2) Inter CV % (3 CDs) n=2. Kit buffer containing 5 mg/ml IgG (commercial MAb) Sensitivity The LLOQ for MabSelect Sure and Native Protein A are presented in the table below. The LLOQ is defined as the lowest concentration at which Relative Error (%RE) + Precision (%CV) = Total Error (TE) is below 30%. MabSelect SuRe Native Protein A Assay Characteristics (ng/ml) (ppm) (ng/ml) (ppm) LLOQ (5 mg/ml IgG*) *Commercially available IgG molecule (MAb)
10 Recovery data Spike recovery experiments where MabSelect SuRe at 5 ng/ml were spiked into different clinical grade therapeutics at mg/ml concentrations followed by further dilution and analysis for protein A using Gyrolab Protein A kits for MabSelect SuRe to determine spike recovery. Sample Dilution Calc conc SPIKED CV Conc Calc conc UNSPIKED CV Conc Spiked conc MabSSR conc Spike recovery ppm AVG MabSSR conc ppm IgG 1 20 mg/ml IgG 2 20 mg/ml IgG 3 60 mg/ml % % % % % % % % % % % %
11 Appendix 1 Gyrolab Protein A kit run v2 Preparation before Run 1. Check if the Gyrolab Protein A kit Run v2 is present in the database. If not open the Administrator tool and import the Run: Gyrolab Protein A kit Run v2 the method will be imported automatically together with the Run. Close the admin tool. For more detailed instruction see User Guide section F2. 2. Prime Gyrolab. Use PBS-T as wash solution 1 and freshly prepared Gyrolab Wash Buffer (GWB) ph 11 as wash solution Prepare reagents for the Run. a. Prepare a standard curve according to Table 1 in Section 8. b. The appropriate number of QC samples should be prepared in sample dilution buffer, Reagent H. Use Native Protein A or MabSelect SuRe, Reagent C, to spike to the correct concentrations: 0.5, 5 and 50 ng/ml. c. Load Reagent A (capture). Reagent B (detect). Reagent D and E (acid dissociation buffers) and wash buffers onto the microplate according to the Gyrolab Control loading list (shown below). Note that Reagent F (acid wash) is placed in two microplate wells and named F and F2. Run: Gyrolab Protein A kit run v2 1. Open Gyrolab Control and select Execute Run. 2. Select the Run: Gyrolab Protein A kit run v2. 3. Prepare a microplate according to the Gyrolab control loading table below. Note that Reagent A is the Capture reagent and Regent B is the detect reagent. 4. Load the CD and microplate and start the run. 5. After the run is completed, open Gyrolab Evaluator and analyze the result.
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