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Tools to Detect sub-1ppm Host Cell Proteins in Biological Products at Every Development Stage

In a Single-One Hour Run, SCIEX can:
PROFILE the HCP complement up to 1000s of proteins to sub-ppm level;
IDENTIFY HCPs without bias [without inclusion/ exclusion lists];
Provide a CATALOG of HCPs for a process;
Provide precursor and fragment information to allow easy MONITORING;
Easy transfer to (QQQ or QTRAP®) ABSOLUTE QUANTITATION of HCPs

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Tools to Detect sub-1ppm Host Cell Proteins in Biological Products at Every Development Stage

  1. 1. Tools to Detect sub-1ppm Host Cell Proteins in Biological Products at Every Development Stage Milla Neffling, Ph.D. SCIEX, Warrington, UK
  2. 2. 2 © 2015 AB Sciex SCIEX has a Strong Track Record with HCP Detection In a Single-One Hour Run, SCIEX can: PROFILE the HCP complement up to 1000s of proteins to sub-ppm level IDENTIFY HCPs without bias [without inclusion/ exclusion lists] Provide a CATALOG of HCPs for a process Provide precursor and fragment information to allow easy MONITORING Easy transfer to (QQQ or QTRAP®) ABSOLUTE QUANTITATION of HCPs
  3. 3. 3 © 2015 AB Sciex HCP SVA Modified peptide Nothing Hides from SWATH™ Host Cell Protein Studies Using Unbiased Methodology One data set can be used for multiple tasks
  4. 4. 4 © 2015 AB Sciex Time ROI for Single-Digit PPM Detection Post Purification Fraction 01 – 2Hrs Fraction 02– 2Hrs Fraction 03– 2Hrs Fraction 04– 2Hrs Fraction 05– 2Hrs Fraction 06– 2Hrs Fraction 07– 2Hrs Fraction 08– 2Hrs Fraction 09– 2Hrs Fraction 10– 2Hrs Post Purification One Run – 60 Minutes Total Time: ~ 1 Hour Total Time: ~ 20 Hours 2D Method
  5. 5. 5 © 2015 AB Sciex Return on Investment Based on Representative Example System Cost of MS per Day (Instrument, service, operator, overhead) # of cell lines (Assume ~1000 Proteins) # of Runs Time Taken Analysis cost 2D Method 1000 USD 10 10 Cell lines X 5 Samples X 10 fractions X 3 replicates = 1500 Runs 1500 Runs X 2Hr Ea =3000 Hr =125 Days 1000 USD/Day X125 Days = $125,000 SCIEX 1D Method 1000 USD 10 10 Cell lines X 5 Samples X 3 replicates = 150 Runs 150 Runs X 1 Hr Ea =150 Hr =6.25 Days. 1000 USD/Day X6.25 Days =$6,250
  6. 6. 6 © 2015 AB Sciex HCP analysis via SWATH™ Acquisition- Study Design • 48 model HCPs digested and analyzed by IDA MS and MS/MS to generate a peptide library via ProteinPilot™ software searches at upper concentration. • SWATH® acquisition used for all subsequent concentration levels for quantitative analysis • A range of concentrations of model proteins were spiked into 10 ug of IgG1 product digest. Digest LCMSMS Run (60’) Generate Library Analyze Dilution Series
  7. 7. 7 © 2015 AB Sciex UniProt Protein Name [Synonym] MW (Da) PPM At Lowest Dilution UniProt Protein Name [Synonym] MW (Da) PPM At Lowest Dilution Gelsolin 82,954 7.56 Ubiquitin-conjugating enzyme E2 C [UbcH10] 20,473 1.87 Lactotransferrin 78,289 7.14 Peptidyl-prolyl cis-trans isomerase A [Cyclophilin A] 17,947 1.64 Serotransferrin [Apotransferrin] 75,143 6.85 Tumor necrosis factor [TNF-alpha] 17,350 1.58 Serum Albumin 66,393 6.05 Myoglobin C 17,051 1.55 Catalase 59,583 5.43 Interferon gamma (IFN-gamma) 16,879 1.54 Histidyl-tRNA synthetase [Jo-1] 58,223 5.31 Leptin 16,024 1.46 Antithrombin-III 49,033 4.47 Cytochrome b5 16,021 1.46 Microtubule-associated protein tau [Tau protein] 46,810 4.27 Hemoglobin beta chain 15,867 1.45 Creatine kinase M-type [CK-MM] 43,070 3.93 Superoxide dismutase [Cu-Zn] 15,800 1.44 Small ubiquitin-related modifier 1 [SUMO-1] 37,420 3.41 Gamma-Synuclein 15,363 1.40 Annexin A 5 35,782 3.26 Hemoglobin alpha chain 15,127 1.38 NAD(P)H dehydrogenase [quinone] 1 [DT Diaphorase] C 30,984 2.82 Fatty acid-binding protein 14,716 1.34 Carbonic anhydrase 2 29,095 2.65 Lysozyme C 14,692 1.34 Carbonic anhydrase 1 28,738 2.62 Alpha-lactalbumin 14,070 1.28 Ribosyldihydronicotinamide dehydrogenase [quinone] [Quinone oxidoreductase 2] [NQO2] 25,817 2.35 Thioredoxin 12,424 1.13 Glutathione S-transferase A1 [GST A1-1] 25,482 2.32 Platelet-derived growth factor B chain 12,286 1.12 Glutathione S-transferase P [GST] 23,220 2.12 Beta-2-microglobulin 11,729 1.07 C-reactive protein 23,030 2.10 Cytochrome c[Apocytochrome c] 11,608 1.06 Ubiquitin-conjugating enzyme E2 I [UbcH9] 22,907 2.09 Ubiquitin 9,387 0.86 Ubiquitin-conjugating enzyme E2 E1 [UbcH6] 22,222 2.03 Neddylin [Nedd8] 9,071 0.83 Peroxiredoxin 1 22,106 2.01 Interleukin-8 8,381 0.76 BH3 Interacting domain death agonist [BID] 21,978 2.00 Complement C5 [Complement C5a] 8,266 0.75 GTPase HRas [Ras protein] 21,292 1.94 Insulin-like growth factor IA 7,643 0.70 Retinol-binding protein 21,065 1.92 Insulin-like growth factor II 7,464 0.68 Ubiquitin-conjugating enzyme E2 C [UbcH10] 20,473 1.87 Epidermal Growth Factor 6,211 0.57 48 Proteins Ranging From 7.56 ppm Down to 0.57 PPM at the Lowest Dilution Level The Number of Proteins Analyzed is Not Limited
  8. 8. 8 © 2015 AB Sciex SWATH™ HCP Analysis - Optimized LC Method LCMSMS Run (60’) Divert Valve 50 Micron Peeksil 25 Micron Peeksil To Waste Pump Column Oven@ 55℃ Autosampler Time %B 0 5 4 5 49 35 50 90 55 90 56 5 60 5 Flow Rate 7 ul/min Easily Transferable Method! 0.3x 150mm ChromXP™ C18 Column 10 ug Product on Column
  9. 9. 9 © 2015 AB Sciex SWATH Acquisition: 45 Minute Run @ 7 uL/min on 0.3mm x 15cm column visualized in PeakView® software HCP Analysis via SWATH™ - Visualization Informatics Ion Library Listing - Proteins Replicates of Chromatographic Runs Overlaid Fragment Ion Display Linked to Peptide Display Spectrum Display Linked to Peptide Display Ion Library Listing - Peptides
  10. 10. 10 © 2015 AB Sciex Statistical Analysis for Tracking and Trending MarkerView™ software: trending HCPs across different runs
  11. 11. 11 © 2015 AB Sciex 8.55 ppm4.28 ppm2.14 ppm1.07 ppm Beta-2 Microglobulin PEPTIDE: VNHVTLSQPK 137 ppm68.4 ppm34.2 ppm17.1 ppm Example Data: 1 ppm Detection
  12. 12. 12 © 2015 AB Sciex Complement C5 [Complement C5a] 90 ppm45 ppm22.5 ppm11.25 ppm 5.63 ppm2.81 ppm1.40 ppm0.70 ppm PEPTIDE: AFTEC[MSH]C[MSH]VVASQLR Example Data: sub-1 ppm Detection
  13. 13. Methods Developed for all Departments
  14. 14. 14 © 2015 AB Sciex HCP Workflows to Support Different Groups Sample-Limited Environments Routine High-Throughput
  15. 15. 15 © 2015 AB Sciex MicroFlow HCP Analysis: CASSS Mass Spec Conference
  16. 16. 16 © 2015 AB Sciex HCP Workflows to Support Different Groups: High Flow • Typically load 30-40 ug product per run • 2mm x 250mm C18 Column or UPLC column • Gradients between 60 and 90 min long
  17. 17. 17 © 2015 AB Sciex High Flow HCP Analysis: WCBP Conference Poster presented at WCBP 2015, Washington DC
  18. 18. 18 © 2015 AB Sciex HCP Workflows to Support Different Groups: Ultra low Flow • Early development sample- constrained (~1 ug) • Very low flow rate = Lower ion suppression.
  19. 19. 19 © 2015 AB Sciex Analyzed using CESI-MS on a TripleTOF®5600 System Transitions 536.3 (MS) and 814.5 (MS/MS) m/z Concentration (ppm) 316 ppb Linear scale Myoglobin Peptide VEADAGHGQEVLIR (+3) CESI-MS works on all TripleTOF® Systems
  20. 20. 20 © 2015 AB Sciex MSMS Quantitation of All Product Peptides and Modifications
  21. 21. 21 © 2015 AB Sciex MSMS Quantitation of All Product Peptides and Modifications
  22. 22. 22 © 2015 AB Sciex Conclusions Unbiased and Comprehensive Analysis Sub-Single Digit PPM HCP Detection Simple, 1D Generic Methodology Substantial Return on Investment

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    Apr. 29, 2016

In a Single-One Hour Run, SCIEX can: PROFILE the HCP complement up to 1000s of proteins to sub-ppm level; IDENTIFY HCPs without bias [without inclusion/ exclusion lists]; Provide a CATALOG of HCPs for a process; Provide precursor and fragment information to allow easy MONITORING; Easy transfer to (QQQ or QTRAP®) ABSOLUTE QUANTITATION of HCPs

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