Thermo Scientific Ligand Binding Mass Spectrometric Immunoassay (LB-MSIA)
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1 INSTRUCTIONS FOR USE OF PRODUCTS Thermo Scientific Ligand Binding Mass Spectrometric Immunoassay (LB-MSIA) Demonstration of the LB-MSIA Protocol for the Analysis of Intact Adalimumab from Mouse Plasma Utilizing Streptavidin MSIA D.A.R.T. S, MSIA TM, Versette Automated Liquid Handler, and Thermo Scientific Q Exactive LC/MS System 991STR11 991STR12 Product Number Description 991STR11 991STR MSIA Streptavidin MSIA D.A.R.T. S containing 1 rack of 96 units. Streptavidin MSIA D.A.R.T. S containing 1 pack of 24 units. MSIA Versette Automated Liquid Handler Table of Contents 1. Description 2 2. Important Product Information 2 3. LB-MSIA Workflow for Qualitative Analysis of Intact Adalimumab 3 A. Equipment and Materials Required 3 B. Versette LB-MSIA Program 5 1. Verify Essential Settings 5 2. Verify the Biotinylated Affinity Ligand Capture Settings 7 3. Verify the Analyte Capture Settings 8 4. Verify the Elution Settings 9 C. Derivatization of Streptavidin D.A.R.T. S with Biotinylated Anti-IgG Fc (Human) Preparation of Reagent Plates Preparation of the Versette 11 D. Analyte (Adalimumab) Capture Preparation of Reagent Plates Preparation of Adalimumab Dosing Curve and Controls Perform the LB-MSIA Analyte (Adalimumab) Capture 12 E. Elution of Immuno-Captured Adalimumab 15 F. LC-MS Detection of Intact Adalimumab Data Analysis Ordering Information E. Conference Dr., STE info.sandiego@thermofisher.com Tempe, AZ USA Page 1
2 1. Description Monoclonal antibodies (mab) and antibody-drug conjugates (ADC) are structurally complex and often exist as heterogeneous mixtures of molecules with varying degrees of modifications; examples include glycosylation variants or varying drug-to-antibody ratio (DAR) distributions. Furthermore, potential alterations in molecular structure may also occur in the biomatrix as a result of catabolism or other degradation mechanisms. These modifications and alterations may have a significant effect on the molecules toxicity, half-life and overall therapeutic efficacy. The classical ligand binding assays, ELISA, commonly applied for bio-analysis are not suited to assess molecular heterogeneity and potential structural changes. For bio-analysis of mabs and ADCs, hybrid assays that combine affinity capture with High Resolutions Accurate Mass Spectrometric (HRAM) detection are becoming more prominent in use. This concept of hybrid assays was first innovated as the Mass Spectrometric Immunoassay (MSIA ). Now available within Thermo Fisher Scientific, this workflow solution is accentuated through the use of Thermo Scientific MSIA D.A.R.T. S for fast, reproducible and accurate assaying capabilities. Combined with HRAM detection, unparalleled bio-analytical results are achieved at the intact level. Using software to deconvolve the complex full spectrum MS data, the molecular weights of the different species present can be calculated and the proportion of the molecules with different modifications determined. 2. Important Product Information Storage Conditions: Streptavidin MSIA D.A.R.T. S should be stored at 4 C for up to 1 year from the date of purchase. This product should never be frozen. The Streptavidin MSIA D.A.R.T. S are intended for single use only. Streptavidin MSIA D.A.R.T. S are not intended for the transference or measurement of liquids. This product is intended for micro-scale analyte purification prior to mass spectrometric detection. Technical literature is available at: msia.info@thermofisher.com Contact Sales: Contact Support: LB-MSIA Workflow for Qualitative Analysis of Intact Adalimumab Tempe, AZ USA Page 2
3 This workflow has been optimized for the assaying of adalimumab from rodent plasma. While this protocol may be utilized as a model for other LB-MSIA monoclonal antibody assays it is recommended that the conditions of this protocol be individually optimized for each specific analyte and antibody. 3.A. Equipment and Materials Required Equipment and Software MSIA Versette Automated Liquid Handler (Thermo Scientific, Product No. 650-MSIA) Set of Finnpipette F1 Adjustable-Volume Pipettes (Thermo Scientific, Product No ) or equivalent ProSwift RP-4H LC Columns, 1 x 250mm (Thermo Scientific, Product No ) Liquid Chromatography System capable of flow rates of ml/min Column heater compatible to 250 mm length LC column and capable of 60 C Auto Sampler System with the ability to accommodate 100 µl sample volumes (100µL syringe and 100µL sample loop) and 96-well plates Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap LC-MS Thermo Scientific XCalibur Software, Version 2.2 Thermo Scientific Protein Deconvolution Software, Version 3.0 with the ReSpect algorithm Materials for Derivatization of Streptavidin MSIA D.A.R.T. S with Biotin Conjugate Affinity Ligand Streptavidin MSIA D.A.R.T. S (Thermo Scientific, Product No. 991STR12 CaptureSelect Biotin Anti-IgG-FC (Human) Conjugate (Life Technologies, Product No ) BupH Modified Dulbecco's Phosphate Buffered Saline Packs (PBS); 8mM sodium phosphate, 2mM potassium phosphate, 0.14M NaCl, 10mM KCl, ph 7.4. (Thermo Scientific, Product No ) Nunc 96-Well Polypropylene Plates, 500µL (Thermo Scientific, ) Tempe, AZ USA Page 3
4 Additional Materials for Sample Preparation and Capture Mouse Plasma (K2 EDTA) Abbvie Humira (adalimumab) A readily accessible mab alternative to adalimumab: SILu Lite SigmaMAb Universal Antibody Standard human (Sigma-Aldrich, Product No. MSQC4). This protocol is applicapable to SiluLite, however results may vary. Water, Optima LC/MS (Fisher Chemical, Product No. W6) Additional Materials for Adalimumab Elution MSIA Elution Buffer The MSIA Elution Buffer is a proprietary product recommended for all high throughput applications that reduces loss in signal due to absorption of the protein to the plastics. An alternate Elution Solvent may be used that requires that each sample be analyzed by LC-MS within 1 hour of elution. This manual will only outline the high-through put option. If utilizing the individual elution of each sample please refer to the Application Note: A Universal Pre-Clinical Solution for the Bio-analysis of Fully Human Therapeutic Monoclonal Antibodies in Plasma For any questions concerning appropriate elution solvents and the MSIA Elution Buffer please use the following contact information: Technical literature is available at: Contact Support: Outside North America Toll Free: msia.info@thermofisher.com Additional Materials for Adalimumab LC-MS Detection Acetonitrile, Optima LC/MS Grade (Fisher Chemical, Product No. A996) Formic Acid, Optima LC/MS Grade (Fisher Chemical, Product No. A117) Tempe, AZ USA Page 4
5 3.B. Versette LB-MSIA Programs The Complete_Streptavidin_MSIA_Workflow Versette Program may be downloaded at: 3.B.1. Verify Essential Settings for the LB-MSIA Versette Program The Verification Steps are for initial set up of a newly downloaded MSIA Versette program should not need to be repeated once the system is running the program properly. 1. Open the Complete_Streptavidin_MSIA_Workflow_GV Versette program with the ControlMate Software and uncollapse the Main Sequence. 2. Use Graphic 1 to verify that the Main Sequence settings and the Pipette Head & Tips Settings (Upper Right of Graphic 1) are properly set for the Complete_Streptavidin_MSIA_Workflow_GV Versette program. Refer to Steps 3 & 4 below to adjust the Pipette Head & Tips Settings otherwise Steps 3 & 4 may be skipped. Graphic 1: LB-MSIA Versette Program Main Sequence Settings and the Pipette Head & Tips Settings Tempe, AZ USA Page 5
6 3. To change the Pipette Head and Tips settings select the Change Pipette Head and Tips option from the Add-Ins drop down menu located at the Top-Mid-Left of the ControlMate window. 4. Adjust the Change To settings in the Addin: Change Pipette Head and Tips window to reflect the settings of Graphic 2 below. Then hit the Start button to initiate the changes. Graphic 2: Pipette Head & Tips Settings for the LB-MSIA Versette Protocol. Tempe, AZ USA Page 6
7 3.B.2. Verify the Biotinylated Affinity Ligand Capture Settings 1. Uncollapse the group named Affinity Ligand Capture and the subsequent groups that make up the Affinity Ligand Capture Steps. Individually compare the program values with Graphic 4. Specifically double check that the correct Vessel Type is selected under each Move to Stage function. i. All stages should utilize the 96 MicroWell Plates U Bottom PP as displayed in Graphic 4. Tempe, AZ USA Page 7
8 Graphic 4: Biotinylated Affinity Ligand Capture Settings for the LB-MSIA Versette Protocol. 3.B.3. Verify the Analyte Capture Settings 1. Uncollapse the group named Analyte Capture and the subsequent groups that make up the Analyte Capture Steps. Individually compare the program values with Graphic 5. Specifically double check that the correct Vessel Type is selected under each Move to Stage function. i. All stages should utilize the 96 MicroWell Plates U Bottom PP as displayed in Graphic 5. Tempe, AZ USA Page 8
9 ii. Note: Graphic 5 shows two rinses in their collapsed state. These two rinses share identical settings to the rinse performed directly before them with the exception of the Stage. Graphic 5: Analyte Capture Settings for the LB-MSIA Versette Protocol. 3.B.4. Verify the Elution Settings i. Uncollapse the group named Elution and the subsequent groups that make up the Elution Steps. Individually compare the program values with Graphic 6. Specifically double check that the correct Vessel Type is selected under each Move to Stage function. Tempe, AZ USA Page 9
10 Graphic 6: Elution Settings for the LB-MSIA Versette Protocol. Tempe, AZ USA Page 10
11 3.C. Derivatization of Streptavidin D.A.R.T. S with Biotinylated Anti-IgG Fc (Human) The procedure below outlines the preparation of ten of the Streptavidin MSIA D.A.R.T. S to prepare a 7-point dosing curve and 3 controls. If initial testing of the system is necessary prior to running a curve then prepare only 1-3 D.A.R.T. S and reduce the reagents appropriately for each step. The recommended sample concentration for testing the MSIA Workflow is 4µg/mL of adalimumab in 200µL of mouse plasma + 200µL of 10mM PBS. Further sample preparation is described in detail below within the protocol. 3.C.1. Preparation of Reagent Plates Use the color coded plate maps represented below to add the affinity ligand capture reagents to the corresponding wells of six 96-well microplates. Reference Table 1 for the color key: Table 1: Key Code for the 96-Well Plate Maps for the LB-MSIA Biotinylated Affinity Ligand (Human Anti-IgG Fc) Capture Representative Versette Reagent Reagent Plate Texture Stage Rinse Buffer Squares 200µL of PBS 1, 3 1, 3 Biotinylated Affinity Ligand Solution Solid Black 125 µl of 4 µg/ml Biotinylated Anti-IgG-Fc (Human) in 10 mm PBS 2 2 Tempe, AZ USA Page 11
12 3.C.2. Preparation of the Versette for the Derivatization of Streptavidin D.A.R.T. S with Biotinylated Anti-IgG Fc (Human) Conjugate 1. Refer to Graphic 7 for the 6 positions that make up the Versette stage. Graphic 7 Numbering of Versette Stages 2. Secure plates 1-3 onto their corresponding positions on the Versette stage as specified in Table 1 and Plate Map (Section 3.D.1). Plates should be positioned with well A1 located at the upper left of the Stage. 3. Load ten Streptavidin MSIA D.A.R.T. S into the 96-D.A.R.T. S Magazine using positions H1-H10 as depicted in Graphic 8. Then fill the remaining empty magazine positions with blank or cut D.A.R.T. S in order to balance the pressure the Versette will be applying to them. Tempe, AZ USA Page 12
13 Graphic 8 Preparation of MSIA D.A.R.T. S Magazine 4. Load and run the Complete_Streptavidin_MSIA_Workflow Versette program. When prompted, insert the loaded D.A.R.T. S Magazine into the 96-Channel Versette Head as depicted in Graphic 9. The magazine will click into place when inserted correctly. Click the Continue button and the program should take ~40 minutes to complete the affinity ligand capture. Then the program will pause allowing for stages to be setup for the analyte capture steps. Graphic 9 Inserting D.A.R.T. S Magazine into Versette 96-Channel Head Tempe, AZ USA Page 13
14 3.D. Analyte (Adalimumab) Capture 3.D.1. Preparation of Reagent Plates Use the color coded plate maps represented below to add the analyte capture reagents to the corresponding wells of six 96-well microplates. Reference Table 2 for the color key: Table 2: Key Code for the 96-Well Plate Maps for the LB-MSIA Analyte (Adalimumab) Capture Representative Reagent Reagent Texture Plate Versette Stage Rinse Buffer Squares 200µL of PBS 4, 6, 7 1, 3, 4 Sample* Solid Gray *Sample preparation specified in Step 3.D Water Dots 200µL of Optima LC/MS Grade Water 8, 9 5, 6 Tempe, AZ USA Page 14
15 3.D.2. Preparation of Adalimumab Dosing Curve and Controls 1. Add 200µL of 10mM PBS to wells H1-H10 (Reference Table 2 and corresponding Plate Maps in Section 3.D.1) of the Sample Plate. 2. Prepare the sample dilutions for the adalimumab dosing curve and controls by following the dilution table outlined in Table 3 Table 3 - Quick Reference Dilutions for Adalimumab Dosing Curve and Controls Final Volume and Concentration of Adalimumab Combine Adalimumab Dosing Curve Initial Adalimumab Volume and Concentration Mouse Plasma (µl) 500µL of 0.5mg/mL 5µL of 50mg/mL µL of 50µg/mL 20µL of 0.5mg/mL µL of 8000ng/mL 160µL of 50µg/mL µL of 4000ng/mL 500µL of 8000ng/mL µL of 2000ng/mL 500µL of 4000ng/mL µL of 1000ng/mL 300µL of 2000ng/mL µL of 500ng/mL 300µL of 1000ng/mL µL of 250ng/mL 300µL of 500ng/mL µL of 125ng/mL 300µL of 250ng/mL Final Volume and Concentration of adalimumab Combine Adalimumab Controls Initial Volume and Concentration of Adalimumab Mouse Plasma (µl) 800µL of 750ng/mL 300µL of 2000ng/mL Follow Table 4 to add 200µL of each of the adalimumab curve and control samples to their corresponding wells in column 2 of the Sample Plate. Plate Map Adalimumab Dosing Table 4 Curve and Control Samples Adalimumab Sample Well 125 ng/ml H1 250 ng/ml H2 500 ng/ml H ng/ml H ng/ml H ng/ml H ng/ml H7 750 ng/ml Controls 1-3 H8-H10 Tempe, AZ USA Page 15
16 3.D.3. Perform the LB-MSIA Analyte Capture of Adalimumab 1. Secure plates 4-9 onto their corresponding positions on the Versette stage as specified in Table 2 and corresponding Plate Maps (Section 3.D.1). Plates should be positioned with well A1 located at the upper left of the Stage. 2. Once the plates are properly staged, press the Ok button on the pause prompt. This will continue the program performing the analyte capture step of the LB-MISA workflow. The program should take ~40 minutes to complete the analyte capture and will pause allowing for stages to be setup for the elution step. 3.E. Elution of Immuno-Captured Adalimumab The MSIA Elution Buffer is a proprietary product recommended for all high throughput applications that reduces loss in signal due to absorption of the protein to the plastics. An alternate Elution Solvent may be used that requires that each sample be analyzed by LC-MS within 1 hour of elution. This manual will only outline the high-through put option. If utilizing the individual elution of each sample please refer to the Application Note: Ligand Binding Mass Spectrometric Immunoassay (LB-MSIA) Workflow for Therapeutic Antibodies: A Universal Pre-Clinical Solution for the Bio-analysis of Fully Human Therapeutic Monoclonal Antibodies in Plasma For any questions concerning appropriate elution solvents and the MSIA Elution Buffer please use the following contact information: Technical literature is available at: Contact Support: Outside North America Toll Free: msia.info@thermofisher.com 1. The elution will be performed in a 96-Well Polypropylene Plate, 500µL (Elution Plate). Add 50 µl of the MSIA Elution Buffer to wells H1- H10 of the Elution Plate as depicted in the plate map below: Tempe, AZ USA Page 16
17 2. Remove the Analyte Capture Plates from the Versette Stages. Place the Elution Plate onto the Versette Stage Click the Ok button to continue the Elution steps of the LB-MSIA Versette program, which should take ~1 minute. 4. Seal the plate. Then spin the samples down. The eluates are ready for LC-MS analysis of intact adalimumab and should be directly loaded onto the LC-MS from the elution plate. 3.F. LC-MS Detection of Intact Adalimumab LC Parameters: Column: ProSwift RP-4H LC Column, 1 x 250mm (Thermo Scientific, Product No ) Column Temperature: 60 C Mobile Phase A: 0.2% Formic Acid in Water Mobile Phase B: 0.2% Formic Acid in Acetonitrile Gradient Table: Time (mins) % Mobile Phase B Flow Rate (µl/min) % % % % % % % % % % % % % 200 Tempe, AZ USA Page 17
18 Mass Spectrometric Detection: The optimization of the LB-MSIA analysis of adalimumab was performed on a Thermo Scientific Q Exactive Mass Spectrometer. Equivalent instruments to the Q Exactive may require further optimization of the MS Conditions. For all samples, full-scan MS data were acquired over the range of m/z ,500 in positiveion mode on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with a resolving power of 17,500 (FWHM) at m/z 200 and the AGC (Automatic Gain Control) set to a target value of 3.00E6. 4. Data Analysis All LC-MS raw data was collected using Thermo Scientific s XCalibur Software, Version 2.2. From the raw MS data an extracted ion chromatogram was generated for the five most abundant charge states of adalimumab, which were then integrated to obtain the AUC (Area Under the Curve) value for each sample analyzed. Further characterization of adalimumab, specifically in reference to the presence of glycosylation, was obtained from processing the MS raw data using Thermo Scientific s Protein Deconvolution Software Version 3.0 with the ReSpect algorithm. Tempe, AZ USA Page 18
19 5. Ordering Information MSIA D.A.R.T. S for Immunoaffinity Capture Compatible with the Thermo Scientific Versette Automated Liquid Handler and Thermo Scientific Finnpipette Novus i Multichannel Electronic Pipette Cat. No. Description Packaging 991CUS02 300µl MSIA D.A.R.T. S, Custom Pack of 96 units 991PRT11 300µl MSIA D.A.R.T. S, Protein A Pack of 96 units 991PRT12 300µl MSIA D.A.R.T. S, Protein A Pack of 24 units 991PRT13 300µl MSIA D.A.R.T. S, Protein G Pack of 96 units 991PRT14 300µl MSIA D.A.R.T. S, Protein G Pack of 24 units 991PRT15 300µl MSIA D.A.R.T. S, Protein A/G Pack of 96 units 991PRT16 300µl MSIA D.A.R.T. S, Protein A/G Pack of 24 units 991STR11 300µl MSIA D.A.R.T. S, Streptavidin Pack of 96 units 991STR12 300µl MSIA D.A.R.T. S, Streptavidin Pack of 24 units µl MSIA D.A.R.T. S, Insulin Pack of 96 units µl MSIA D.A.R.T. S, Insulin Pack of 24 units 991R 300 µl MSIA D.A.R.T. S, Reloadable Rack 1 reloadable rack, D.A.R.T. S are not included Automated Liquid Handling Platform Cat. No. Description 650-MSIA MSIA Versette Automated Liquid Handler Multichannel Pipettes and Pipette Stand Cat. No. Description Packaging 991S Finnpipette Novus i Adjustable Pipette Stand 1 pipette stand 991SP12 Finnpipette Novus i Electronic 12-Channel Pipette, µl and Pipette Stand 1 pipette and 1 pipette stand Liquid Chromatography Cat. No. Description Thermo Scientific Dionex UltiMate 3000 UHPLC System ProSwift RP-4H Monolith Column, 1.0 x 250 mm Mass Spectrometry and Software Description Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer Thermo Scientific Pinpoint Software Thermo Scientific XCalibur Software Thermo Scientific Protein Deconvolution Software, Version 3.0 with the ReSpect algorithm thermoscientific.com/msia Products are intended for research use only Thermo Fisher Scientific Inc. All rights reserved. North America: info.sandiego@thermofisher.com Outside North America: info.sandiego@thermofisher.com Tempe, AZ USA Page 19
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