Flash DigestTM Kit Next Generation Protein Digestion Plate Based, Temperature Stable Immobilized Enzyme Reactors Applications of Mass Spec In Protein Analysis Isoforms Conjugates Drug C- Terminal Sequence Function Humulin GFFYTPK Standard Humalog GFFYTKP Fast Acting Insulin Novolog GFFYTNK Fast Acting Insulin Levemir GFFYTPK-acyl Long Acting PTMs Wang et al. (2011), Mol. Immun. 48:860-866. Multiplexing Following recent advances in LCMS technology, sample preparation is the new bottleneck What is needed is something… 1) 2) 3) 4) Easy to use Reproducible Fast Standardized A Common Protein LC-MS Workflow Sample DTT, 60°C 1 hour IAA, RT 0.5 hour Reduced/alkylated analyte Trypsin, 37°C 18 hours Tryptic Digest Direct Inject LC-MS 96 samples, 12 hours Digestion has historically been biggest bottleneck, source of irreproducibility Complete digestion is kinetically unfavorable Digestion of Insulin Under Standard Conditions uV 5000000 4500000 4000000 GFFYPTK Undigested Protein 3500000 30 minutes 3000000 1 hour 3 hours 2500000 2000000 6 hours 1500000 12 hours 1000000 18 hours 500000 24 hours 0 4.0 4.5 5.0 Protein Product Sample: Column : Mobile Phase A: Mobile Phase B: Detection: 5.5 6.0 5ug insulin HALO 2.1x100mm RPC 2% ACN, 98% water, 0.1% Formic Acid 90% ACN, 10% water, 0.1% Formic Acid UV/VIS at 214nm 6.5 7.0 min Substrate -E -E -E EEE- - 100 nm [Enzyme] >> [Substrate] Immobilization Eliminates Autolysis Perfinity Immobilized Enzyme Technology Dramatically Reduces Digestion Time uV 2750000 Data1:Digest Data2:Digest Data3:Digest 2500000 Data5:Digest Insulin Insulin Insulin Insulin 50uL 50uL 50uL 50uL 4-185-03 4-185-03 4-185-03 4-185-03 400uLmin 60C May 17 Run 1.lcd Detector A:214nm 200uLmin 60C May 17 Run 3.lcd Detector A:214nm 100uLmin 60C May 17 Run 2.lcd Detector A:214nm 50uLmin 60C May 17 R un 3.lcd D etector A:214nm Undigested Protein 2000000 1750000 GFFYTPK 1500000 1250000 1000000 750000 500000 250000 0 19.0 1 minute 2 minutes 4 minutes 8 minutes 20.0 21.0 22.0 23.0 24.0 25.0 Conversion to Products (%) Perfinity IMER Digestion 2250000 26.0 min Protein Product Sample: Column 1: Column 2: Mobile Phase A: Mobile Phase B: Detection: 5ug insulin Perfinity Optimized Trypsin Column HALO 2.1x100mm RPC 2% ACN, 98% water, 0.1% Formic Acid 90% ACN, 10% water, 0.1% Formic Acid UV/VIS at 214nm 100 80 60 40 20 0 0 2 4 6 Digestion Time (min.) 8 A Common Protein LC-MS Workflow Sample DTT, 60°C 1 hour IAA, RT 0.5 hour Reduced/alkylated analyte Trypsin, 37°C 18 hours Tryptic Digest Direct Inject LC-MS 96 samples, 12 hours Denaturation, reduction and alkylation also a bottleneck, source of irreproducibility Digestion at High-Temperatures using a Thermally Stable IMER Simultaneous Denaturation/Digestion BSA - 17 Disulfide Bonds Digested in 5 Minutes uV 400000 Data1:50uL U ndig BSA R PC 5-70 in 30 min_R1.lcd Detector A Ch1:214nm Data2:FD_15uL_100ugmLtestP_BSA_70C 5 min_9_25_S1 R1.lcd Detector A Ch1:214nm Data3:FD_15uL_100ugmLtestP_BSA_70C 10 min_9_25_S1 R1.lcd Detector A Ch1:214nm Data4:FD_15uL_100ugmLtestP_BSA_70C 20 min_9_25_S1 R1.lcd Detector A Ch1:214nm 350000 Data5:FD_15uL_100ugmLtestP_BSA_70C 30 min_9_24_S1 R1.lcd Detector A Ch1:214nm Digest300000 Time 250000 0 min 200000 5 min 150000 10 min 100000 20 min 30 min 50000 0 15.0 17.5 20.0 22.5 Bovine Serum Albumin • 66 kD • 17 Disulfide Bonds • Hydrophobic 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 min Digestion Times for Various Samples Protein* Digest Time (min) IgG 45 IgG in 50uL plasma** 75 BSA <5 Carbonic Anhydrase <5 Lysozyme <5 Ribonuclease A 150 *All proteins digested directly (no denaturation, reduction or alkylation) **Beagle, mouse, monkey and human plasmas tested A Common Protein LC-MS Workflow Sample X Reduced/alkylated mAb ≤90C, 5-75 minutes Tryptic Digest Direct Inject LC-MS 96 samples, 12 hours Denaturation and digestion can occur simultaneously Flash Digest Thermally Stable Immobilized Enzyme in PCR Tubes/Plates Up to 96 Samples, ≤ 75 Minutes No Pretreatment Necessary 1 Undigested IMER Protein Resin 2 ≤ 90°C, ≤75 Minutes 3 LC/MS Filter or Centrifuge RT: 0.00 - 30.02 8.21 575.80 100 9.08 547.83 95 90 85 80 75 4.95 395.65 7.15 464.70 70 Relative Abundance 65 9.17 501.28 3.49 441.13 60 55 14.92 683.01 50 45 10.53 490.56 1.96 538.26 40 12.17 657.02 13.62 672.57 35 30 17.23 802.27 25 20 0.63 515.64 10 19.36 813.31 15.38 812.42 15 21.32 1398.41 20.05 823.40 17.78 821.23 5 22.60 23.33 974.78 1353.92 0 0 2 4 6 8 10 12 14 16 Time (min) 18 20 22 24 •Complete Digest of Native Proteins in ≤ 75 Minutes • Pretreatment (Reduction and Alkylation) – Optional • Equipment Required – ThermoMixer C w/ PCR block • Multiple Patents Pending Confidential 26.92 2 316.75 3 26 Case Study: Quantification of IgG in Plasma “Universal” surrogate peptides to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody (mAb) in preclinical studies Peptide "Source" Anal. Chem. 2012, 84, 1267−1273 Anal. Chem. 2012, 84, 1267−1273 Anal. Chem. 2012, 84, 1267−1273 DVDMDG -Jemal, BMS DVDMDG -Jenkins, PPD Amino Acid Sequence Location SGTASVVC(-CM)LLNNFYPR Light Chain TVAAPSVFIFPPSDEQLK Light Chain NQVSLTC(-CM)LVK Heavy Chain VVSVLTVLHQDWLNGK Heavy Chain TTPPVLDSDGSFFLYSK Heavy Chain Optimization of Digest Time T= 15 min T= 30 min T= 45 min T= 60 min T= 75 min T= 90 min Use in Quantification • Performed in Multi-well PCR Plate Format • No reduction or alkylation necessary 50 Front Curve 144 Back Curve 151 158 441 512 IgG1 Concentration in ng/mL 500 1580 5000 15800 1498 4733 18472 61747 1619 5545 18769 66195 50000 195555 218685 • Great precision even without an internal standard (IS) Confidential Quantification in Plasma Peak Areas Front Curve Peak Areas Back Curve (ng/mL) 100 3475 3458 IgG in Plasma (ng/mL) (ng/mL) (ng/mL) 316 1000 3160 12657 40477 131633 9190 41114 117060 (ng/mL) 10000 407025 375038 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Average StDev CV(%) IgG in Plasma (ng/mL) (ng/mL) (ng/mL) 100 1000 10000 3938 35256 391028 3843 30713 400077 3725 31881 368354 4173 34346 362317 3449 27114 381921 3286 29304 424200 4033 30125 400099 4112 34953 388384 3820 31712 389548 317 2940 19550 8.3 9.3 5.0 Evaluation of digestion time for IgG in monkey, mouse and beagle plasma was determined to be 75 minutes. Without internal standard or immunocapture, the CV values for IgG in beagle plasma ranged from 8.3% at the LLOQ to 5% at the ULOQ. A five point calibration curve of IgG in plasma yielded a linear fit (R2 = 0.999 and 0.998). Use in Peptide Mapping uV 900000 875000 850000 30 Minutes – Flash Digest 825000 800000 775000 750000 725000 700000 675000 650000 625000 600000 24 Hours – 10:1 Protein:Trypsin 575000 550000 525000 500000 475000 450000 425000 400000 375000 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min Confidential Testing Within Batch Variation QC 1 QC 2 QC 3 QC 4 Average StDev CV (%) Peak Areas (mAu*s) GFFYTPK Protein Product Undigested 767938 1402420 1303565 778171 1450228 1263443 800800 1509982 1231390 792000 1498508 1263783 784727 14561 1.9 1465285 49262 3.4 1265545 29550 2.3 Resin per Well (μg) Plate 1 14.89 Plate 2 14.9 Plate 3 14.95 Plate 4 14.98 Plate 5 15 Plate 6 15.01 Average 14.95 StDev 0.05 CV(%) 0.35 Lot-to-Lot Variation GFFYTPK Batch 1 Batch 2 Batch 3 Average StDev CV (%) 722052 736262 739016 732443 9104 1.2 Digested Undigested Protein Protein 1338093 1395263 1414963 1382773 39928 2.9 1508363 1507108 1481433 1498968 15199 1.0 Current State of Technology 1) 2) 3) 4) Reproducible Easy to use Fast Standardized
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