动物血浆中治疗性抗体检测方案(液相色谱仪)

thermoscientific APPLICATION NOTE 21692 R3 Rapid quantitation oftherapeutic antibodiesin animal plasma Author Xin Zhang1， John O'Grady2， KevinMeyer2 iThermo Fisher Scientific. Sunnyvale， CA， USA 2Perfinity Biosciences Inc.， West Lafayette， IN， USA Keywords Biopharmaceutical， bioanalysis，quantitation， peptide analysis，SRM， MRM， affinity proteomics，biotherapeutic，IgG， monoclonalantibody (mAb)， human lgG，immunoaffinity， immunocapture，Protein A， Protein G， Streptavidin，SMART DigestImmunoaffinity(IA) Kits， UHPLC， AccucoreC18， biotin， protein digestion，protein enzyme digestion，protein magnetic beads， samplepreparation， 96 well plates ·Rapid sample preparation workflow - 15 step process reduced to five steps·Simplified workflow - enrichment and digestion are carried out in one well · Significant time reduction， digestion normally complete in under one hour ·Immunoaffinity capture improves extract cleanliness and increasesLC/MS/MS method sensitivity more than ten-fold · Improved quantitative LC/MS/MS analysis of low-level protein applicable toa wide range of mAb Goal To demonstrate the rapid quantitation of a low-level human lgG in animalplasma by LC/MS/MS using the Thermo Scientific"SMART DigestImmunoaffinity (IA) kits (including magnetic and non-magnetic versions of theStreptavidin kit， Protein A kit， and Protein G kit)， which combine the IA captureand digestion process into a single well. The assay is required to be bothselective and accurate. Workflows for the quantitative analysis of proteins frombiological matrices are complex and time consuming.The most sensitive methods involve immunoaffinity(IA) enrichment and proteolytic digestion followedby liquid chromatography and mass spectrometricdetection. These IA-LC/MS/MS approaches allow forthe quantification of low abundance proteins in complexsample matrices. While sensitive， IA-LC/MS/MS methods are multistepworkflows that typically span across multiple days. Thedigestion of proteins has been the biggest bottleneckand one of the largest sources of variation in samplepreparation. Sources of variability arise from themultiple steps required to concentrate the compound ofinterest and remove matrix interferences， enzymaticallydigest proteins to surrogate peptides with better massspectrometric properties， and modify the sample matrixor buffering system to achieve compatibility with LCseparation and mass spectrometric detection systems. The bottlenecks and variability associated with theseworkflows can be removed by using the SMART Digest IAKit， which provides the following benefits： · Total time for protein enrichment and digestion isreduced by a factor of five compared to a conventionaltryptic digest? · Improves kinetics and drives the reaction to completion，improving digestion reproducibility2 · Simplifies sample pretreatment (three times fewer stepsthan traditional tryptic digestion) ·Compatible with automation3 The SMART Digest IA Kit contains immunocapturereagents and temperature-activated thermally stabletrypsin， which are co-immobilized onto beads (eithermagnetic or non-magnetic). During enrichment carried outat room temperature， the enzyme is inactive.Followingenrichment， the enzyme is activated by elevating thetemperature to 70℃， which also facilitates accelerateddigestion under protein denaturing conditions. Whendenaturation and digestion are performed simultaneouslyon the same stationary phase used for enrichment， theneed for elution， sample transfers， and pretreatment priorto digestion (e.g. denaturation， reduction， and alkylation) is eliminated. As such， the total time needed for thesample preparation workflow is decreased from around24 hours to 3-4 hours， depending on the target analyte.This represents a time savings of over 80%. This application demonstrates the use of each of thethree types of SMART Digest IA kits (SMART digest IAStreptavidin， Protein A， and Protein G). Streptavidin is abiotin-binding protein. Biotin is a small molecule that canbe conjugated to a protein without altering its biologicalactivity. The affinity of streptavidin for biotin results in thestrongest known non-covalent biological interaction.As such， capture reagents can be biotinylated， boundto streptavidin stationary phases， then readily applied toimmunoaffinity methods. Protein A and G specifically bind lgGs. As such， theyare frequently used for antibody capture. Protein A andG bind lgG subtypes with varying affinities. Protein Ais generally preferred for rabbit， pig， dog， and cat lgG.Protein G has better binding capacity for a broader rangeof mouse and human lgG subclasses (lgG1， lgG2， etc.).4 Depending on the type of immunocapture reagent (orbait) used， users can choose between these affinityreagents for customization of their immunoaffinityworkflow. This application demonstrates the use of eachof the three types of SMART digest IA kits (SMART digestIA Streptavidin， Protein A and Protein G). Human lgG wasused as a surrogate for a humanized biotherapeutic andits concentration was determined in mouse plasma. In the first example， anti-human lgG Fc was biotinylatedand adhered to the SMART Digest IA Streptavidin Kit.When combined with the plasma samples， human lgGis captured by the anti-human lgG Fc while all otherproteins are washed away. The enriched human lgG isthen digested prior to analysis (refer to Figure 1 for arepresentation of the process). While biotinylated anti-human lgG was used as the capture reagent in thisexample， streptavidin can be used to bind a wide varietyof capture reagents ranging from biotinylated proteins tosmall molecules. In the other two examples， the anti-human lgG Fc antibody was cross-linked to SMARTDigest IA Protein A and Protein G kits and then used toenrich human lgG. For LC/MS analysis， ‘universal’ surrogate peptidesfound in the Fc region of most human mAb candidates(which are absent from the proteomes of animals) Experimental Affinity and Digestion · SMART Digest IA kit， Streptavidin (Av) magnetic(P/N 60110-104)，non-magnetic (P/N 60110-101)* · SMART Digest IA kit， Protein A magnetic(P/N 60111-104)， non-magnetic (P/N 60111-101)* · SMART Digest IA kit， Protein G magnetic(P/N 60112-104)， non-magnetic (P/N 60112-101)* * Each SMART Digest IA kit includes beads，wash buffer，and digest buffer Chemicals ·Deionized water， 18.2 MQ·cm resistivity ·Fisher Scientific"Optima"acetonitrile (ACN) (A955-4) ·Fisher Scientific" formic acid (FA) (F/1900/PB08) · Thermo Scientific Pierce dimethylsulfoxide (DMSO)，LC/MS Grade (85190) ·Thermo ScientificEZ-LinkNHS-Biotin (20217) · Mouse plasma from a reputable supplier ·Anti-human IgG from a reputable supplier · Human lgG from a reputable supplier ( ·Glu t a r a l dehyde from a r e pu tabl e s up pl ier ) ( · pH 7. 4 phosph a te b u ffe r ed s al in e (PBS) (B P 2 4 38 4 ) ) · pH 7.4 tris buffered saline (TBS) (BP2471500) Sample handling · Thermo ScientificSepraseal (4463) ·Fisher ScientificDeepwell Plates 96 (E951032905) · Thermo ScientificNuncEZFlip Conical CentrifugeTubes 15 mL (362694)· Thermo Scientific"Graduated Safelock MicrocentrifugeTubes 1.5 mL (3457PK)·Thermomixer from a reputable supplier Preparation of calibration and quality control (QC)samples For Experiment 1， human IgG was spiked into mouseplasma at concentrations between 5 ng/mL and1.6 ug/mL. Quality control samples were prepared at5， 50， and 500 ng/mL in mouse plasma. For Experiments2 and 3， human lgG was spiked into mouse plasmaat concentrations ranging from 6 ng/mL to 6 pg/mL. Experiment 1 - Quantification of human IgG in animalplasma using SMART Digest IA Streptavidin beadsPreparing a biotinylated antibody/capture moietyTo prepare 0.5 mg of biotinylated anti-human mouselgG， 2.5 pL of 5 mg/mL NHS-Biotin (in DMSO) wasadded to 1 mL of 0.5 mg/mL anti-human mouse lgG.Then， 30 pL of SMART Digest IA Streptavidin beads and6 pL of biotinylated antibody were added to 36， 1.5 mLmicro centrifuge tubes. Capturing human lgG Four hundred microliters of the murine plasma samples，spiked with human lgG at varying concentrations， wasadded to each of the bead containing tubes. Sampleswere incubated at room temperature and shaking at1，400 rpm for 60 minutes. After incubation， the sampleswere washed 3 times with 600 uL of wash buffer. Trypsin digestion After the final wash， the sample volume was reducedto 50 pL by centrifuging the samples and decanting thesupernatant， then followed by the addition of 150 pL ofSMART Digest Buffer. The samples were capped thenincubated for 60 minutes at 70℃ and 1，400 rpm (seeResults and discussion). The samples were acidified with200 uL of 1% trifluoroacetic acid， decanted， and placedinto a new 96-well plate for analysis. Experiments 2 and 3-Quantification of human lgGin animal plasma using SMART Digest IA Protein Abeads (Experiment 2) and SMART Digest IA Protein Gbeads cross-linking anti human lgG (Experiment 3)Preparation of the SMART Digest ImmunoaffinityProtein A and G beads -cross-linking anti human lgGThe following components were added to a 15 mLcentrifuge tube， which were 750 pL SMART Digest IAProtein A or G beads， 250 pL of 0.5 mg/mL mouse anti-human lgG antibody， and 500 pL of PBS. The beads weremixed at 1，400 rpm for 30 minutes at room temperature，centrifuged， decanted (1，300 pL of solution) and washedthree times with PBS (add 1，500 uL of PBS， decant1，500 pL PBS). After the final wash， 450 pL of PBS and2，500 pL of 0.01% glutaraldehyde in PBS were added tothe beads， then mixed at 1，400 rpm for 2.5 hours at roomtemperature. The cross-linking reaction was quenchedby the addition of 250 uL of 0.5 M TBS and the beadsmixed at 1，400 rpm for 10 minutes at room temperature.The beads were centrifuged and 2，750 pL of solutiondecanted. 30 pL of modified SMART Digest IA Protein Aor G beads were added to 24， 1.5 mL micro centrifugetubes. Capturing human lgG Five hundred microliters of the murine plasma samples，spiked with human lgG at varying concentrations， wasadded to each of the bead containing tubes. Sampleswere incubated at room temperature and shaking at1，400 RPM for 60 minutes. After incubation， the sampleswere washed three times with 650 uL of wash buffer. Trypsin digestion As in Experiment 1， after the final wash， the samplevolume was reduced to 50 pL by centrifuging thesamples and decanting the supernatant， then followedby the addition of 150 pL of SMART Digest Buffer. Thesamples were capped， then incubated for 60 minutes at70°℃ and 1，400 RPM (see Results and discussion). Thesamples were acidified with 200 pL of 1% trifluoroaceticacid， decanted， and placed into a new 96-well plate foranalysis. Separation conditions Instrumentation Thermo ScientificUltiMate3000 Rapid Separation DualSystem equipped with： ·SRD-3600 Solvent Racks with Degasser(P/N 5035.9230) ·DGP-3600RS Rapid Separation Pump (P/N 5040.0066) ·WPS-3000TRS Rapid Separation Thermostatted WellPlate Autosampler (P/N 5841.0020) ·TCC-3000RS Rapid Separation Thermostatted ColumnCompartment (P/N 5730.0000) Column Thermo ScientificAccucoreC18 column 2.1 mm x 50 mm， 2.6 um (P/N 17126-052130) LC settings Table 1. LC gradient conditions. Time (min) %A %B 0 90 10 1 90 10 5 30 70 5.1 10 90 6.5 10 90 6.6 90 10 8 90 10 MS conditions Data processing Thermo ScientificVelos Promion trap massspectrometer. The LC/MS instrument was controlled by ThermoScientificXcalibursoftware. Instrumentation MS SettingsHESIModePositiveHeater Temp350°CSheath GasAux GasSpray Voltage4 kVCapillary Temp375°CS-Lens RF Level55% MS Fragment (See Table 2) m/z 603.4， 937.7 Note： Flow is diverted to waste using the divert valve until1.5 minutes into the gradient. Flow is sent to the sourcefrom 1.5 minutes to 3 minutes into the gradient， and thensent to waste again at 3 minutes into the gradient Table 2. MS fragment information. Q1 Mass Q3 mass Act Q Act time (ms) CE Peptide sequence 603.4 805.4 0.25 10 35 VVSVLTVLHQDWLNGK 937.7 836.5 0.25 10 35 TTPPVLDSDGSFFLYSK Results and discussion To quickly and efficiently quantify human lgG，optimization for immunocapture and digestion time wereinvestigated with the SMART Digest IA Streptavidin kit. Determination of digestion time The optimal digestion time for human lgG wasdetermined by dispensing 200 pL of 1 ug/mL humanlgG， and 30 uL of SMART Digest IA Streptavidin beads(biotinylated anti-human mouse lgG)， into each of eightwells. The samples were vortexed then placed into theheated block set to 70℃. Samples were removed at15 minute intervals， centrifuged， and decanted， andthe resulting peptides collected and analyzed byLC/MS. It was determined that the digestion of lgGreaches completion after 60 minutes (Figure 2). Figure 2(A). SMART Digest IA Streptavidin kit sample processingmethod optimization. Fragment peak area at different digest times. Determination of immunocapture time Optimization of capture time was determined bydispensing 400 pL of mouse plasma containing 1 pg/mLhuman lgG，and 30 pL of SMART Digest IA Streptavidinbeads (biotinylated anti-human mouse lgG)， into each ofeight tubes. The samples were placed into a shaker atroom temperature. Samples were removed at 15 minuteintervals and rinsed four times. Following the additionof digest buffer， samples were digested at 70°℃ for60 minutes. Samples were then centrifuged anddecanted， and the resulting peptides collected andanalyzed by LC/MS. It was determined that the captureof lgG reaches completion after 60 minutes. Figure 2(B). SMART Digest IA Streptavidin kit sample processingmethod optimization.Fragment peak area at different incubation(immunocapture) times. Calibration and quantification for human IgG withSMART Digest IA Streptavidin kit The SMART Digest IA Streptavidin kit provided excellentreproducibility and linearity across a wide dynamic range.Calibration and quality control samples were preparedas previously described in the materials and methodssection. Calibration ranges were from 5 ng/mL to1，580 ng/mL. Quality control samples were preparedat low， medium， and high ranges. Six-point calibrationcurves of lgG in plasma yielded linear fits for R? greaterthan 0.995 for both front and back calibration curves(Figure 3 shows both， front in blue and back in red).Quality control samples prepared at 5，50， and500 ng/mL were accurate and precise (Table 3). Variabilitywas determined and CV values were obtained for lgG inmouse plasma ranging from 6% at the LLOQ to 2.3% atthe ULOQ (n=6) even without internal standard. Figure 3. Extraction of human lgG from mouse plasma usingSMART Digest IA Streptavidin kit， front (blue) and back (red)calibration curve. Calibration of human IgG with SMART Digest IAProtein A/G kits The SMART Digest IA Protein A and G kits also providedexcellent reproducibility and linearity across a widedynamic range from 6 ng/mL to 6000 ng/mL. For bothkits， 6 ng/mL was readily and reproducibly detected.Linear fits of all six calibration curves with three lines ineach kit were 0.99 or better (Figure 4). Figure 4. Calibration curve for extraction human IgG from mouseplasma (A： SMART Digest IA Protein A kit； B： SMART Digest IAProtein G kit). Without cleanup， the targeted analysis of mAbs incomplex biological matrices is generally limited toconcentrations above 100 ng/mL.5 Historical attemptsto add IA enrichment have resulted in time-consuming，multi-step workflows and highly variable results. TheSMART Digest IA kits streamline the process， reducingthe sample preparation time to less than three hourswith the detection limit reduced to <10 ng/mL. Thisworkflow also combined the use of a general anti-Fcantibody and the monitoring of ‘universal' surrogatepeptides. This combination results in a single methodthat can be used for the measurement of a wide rangeof monoclonal antibodies， keeping method developmenttime to a minimum. Additionally， three stationary phases(Streptavidin， Protein A， and Protein G) allow for the useof a variety of capture moieties as required. The SMART Digest IA workflow has been repeated usingmagnetic beads (similar protocol， linearity ranged from10 ng/mL to 10 pg/mL with R2>0.99)， 1 mL deep wellplates， and SepraSeal caps. No significant changes insensitivity or reproducibility were observed while themagnetic bead format allowed for further simplificationof the wash steps. Furthermore， these results suggestthat the process is readily amenable to automation usinga number of existing instruments such as the ThermoScientificKingFishersystem as long as the heating unitis capable of heating the plate evenly. Table 3. Extraction of human IgG from mouse plasma using SMARTDigest IA Streptavidin kit， quality control results (N=6). Replicates 5 (QCL) 50 (QCM) 500 (QCH) R1 4416 30074 284410 R2 4087 29392 272466 R3 4301 30199 268620 R4 4101 28610 270032 R5 4004 30550 267826 R6 3714 29413 270273 Average 4104 29706 272271 Std Dev 244 704 6156 CV (%) 6.0 2.4 2.3 Conclusions · The rapid processing of antibodies using the SMARTDigest IA kits allows development of faster methodscompared to conventional protein enrichment anddigestion by simplifying sample preparation andenabling a reproducible， sensitive， and fully integratedLC/MS/MS workflow， ·This application shows a rapid， simple， and sensitivemethod for the quantification of human IgG in mouseplasma using the SMART Digest IA kits with a total timeof 3-4 hours that compares to a process that can takeup to 24 hours. ·The method described provides a significantsimplification for the affinity proteomics process withina single well providing increased sensitivity andreduced sample preparation time. · This Application Note provides a general approach forquantifying low abundance mAbs with reproducibleresults across a broad dynamic range for all three typesof SMART Digest (IA) kits (SMART digest IAStreptavidin， Protein A， and Protein G). References 1. Ewles， M.；Mannu， R.； Fox，C.； Stanta， J.； Evans， G.； Goodwin， L.； Duffy，J.； Bell， L.；Estdale， S.； Firth， D. LC-MS/MS strategies for therapeutic antibodies and investigationinto the quantitative impact of antidrug-antibodies. Bioanalysis， 2016， 8(24)，2565-2579. 2. Bardsley， J.； Jones， J.； Humphryes， P. Thermo Scientific Application Note AN21198：Improvement in Speed and Reproducibility of Protein Digestion and PeptideQuantitation， Utilizing Novel Sample Preparation Technology in a Full Solution Workflowhttps：//tools.thermofisher.com/content/sfs/brochures/AN-21198-SP-SMART-Digest-Peptide-AN21198-EN.pdf 3. SMART Digest IA USERS MANUAL： https：//tools.thermofisher.com/content/sfs/manuals/Man-21549-SMART-Digest-IA-User-Man21549-EN.pdf 4. Comparison of Antibody lgG Binding Proteins https：//www.thermofisher.com/us/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/comparison-antibody-igg-binding-proteins.html 5. Berna， M.； Ackermann， B. Increased Throughput for Low-Abundance ProteinBiomarker Verification by Liquid Chromatography/Tandem Mass Spectrometry，Anal. Chem. 2009， 81，3950-3956. Find out more at thermofisher.com/SMARTDigest ( For R esearch Use Only.Not for use in diagnostic pro c edu r es. 2017 T hermo Fish e r Scien t ific In c . All r i gh t s r e serve d . Al l t r ad e ma rk s ar e t he p rope rt y o f Th ermo Fi s h er Sc ie nt i fic a nd i ts s u bsidia r ies. T his inf o rmation is pr e sentedas an e xampl e o f th e capabi l iti e s of The rmo Fi sher Scie n t if i c pr odu c t s . I t i s not in ten d e d t o en courage u se of t hese p r odu c ts in a n y m an ne rs tha t m i ght inf ri nge t he i n tel l ectu a l p r o p er ty right s of ot h ers. Spe ci ficati on s， t er ms a n d p ric i ng a r e s ub j ect to ch a n ge . No t all products a r e av a il a bl e i n all c ou nt r i e s. Please c o n sult y o ur l o ca l sa le s re pre sentat iv e s for de t ails. AN21692-EN 0817S ) To demonstrate the rapid quantitation of a low-level human IgG in animal plasma by LC/MS/MS using the Thermo Scientific™ SMART Digest™ Immunoaffinity (IA) kits (including magnetic and non-magnetic versions of the Streptavidin kit, Protein A kit, and Protein G kit), which combine the IA capture and digestion process into a single well. The assay is required to be both selective and accurate.  • The rapid processing of antibodies using the SMART  Digest IA kits allows development of faster methods  compared to conventional protein enrichment and  digestion by simplifying sample preparation and  enabling a reproducible, sensitive, and fully integrated  LC/MS/MS workflow.• This application shows a rapid, simple, and sensitive  method for the quantification of human IgG in mouse  plasma using the SMART Digest IA kits with a total time  of 3–4 hours that compares to a process that can take  up to 24 hours.• The method described provides a significant  simplification for the affinity proteomics process within  a single well providing increased sensitivity and  reduced sample preparation time.• This Application Note provides a general approach for  quantifying low abundance mAbs with reproducible  results across a broad dynamic range for all three types  of SMART Digest (IA) kits (SMART digest IA  Streptavidin, Protein A, and Protein G).