Metabolomics study of CHO cell cultures

METABOLOMICS STUDY OF CHINESE HAMSTER OVARY CELL CULTURES CHONG POOI KAT WILLIAM B.Sc.(Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS This PhD thesis is a product that emerged from a long and sometimes arduous journey. It would not have been completed without the following people whom I would like to thank: To my supervisors, Dr Heng Chew Kiat, Professor Miranda Yap and Dr Niki Wong, I would like to extend my deepest gratitude for your guidance, patience and unwavering support. You also nudged me to put in the extra effort to make the results that more impactful and better conveyed. My thanks and gratefulness also to Dr Ho Ying Swan who performed like a supervisor by being much involved in discussions, and for providing numerous useful advices and leads. Many thanks go to Toh Poh Choo and Dr Lee Yih Yean from the Animal Cell Technology group for teaching me the fine art of setting up and operating a bioreactor culture. Janice Tan, Jessna Yeo and Wong Chun Loong were also essential for maintaining the smooth operation of the lab so that I could conduct my experiments successfully (some of the time). I would also like to thank Dr Lee Dong-Yup, Faraaz Yusufi and Satty Reddy for their technical assistance in implementing the bioinformatics software. My appreciation also to the people from Analytics group who helped me troubleshoot the HPLC systems when the machines didn’t behave, and who assisted me in amino acid analysis. They are Corrine Wan, Ong Boon Tee, Sim Lyn Chin, Gavin Teo, Eddie Tan and Hoi Kong Meng. In addition, the colleagues from Microarray and Proteomics groups were wonderful people to let me use their lab consumables without having to replenish them. Two attachment students under my supervision deserved special mentioning. Tan Liting from NUS rendered invaluable assistance to clone out the malate dehydrogenase gene. Chow Chung Ping from NTU helped to quantify some of the extracellular metabolites. Besides the students, Dr Goh Lin-Tang (formerly from Waters Asia Ltd) was instrumental to assist me in the operation of the SYNAPTTM HDMS. Last but not least, I would like to dedicate this thesis to my family. My lovely wife, Mandy, who was and will always be there by my side through the good and not so good times. My parents, Alan and Esther, who sacrificed a lot to provide me with a good education and a loving environment to grow up in. And to my five months old baby girl, Chloe, you are the icing on my cake at the end of this long and sometimes arduous journey! This work was supported by the Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. TABLE OF CONTENTS Page Acknowledgements Table of contents Summary List of tables 11 List of figures 12 CHAPTER Introduction 14 1.1 Background 14 1.2 Thesis objectives 15 1.3 Thesis organization 16 CHAPTER Literature Review 18 2.1 Metabolomics: the latest complement of functional genomics 18 2.1.1 Challenges of metabolomics analysis 20 2.1.2 Mass spectrometry-based metabolomics 21 2.1.3 Metabolite identification 23 2.1.4 Metabolomics to understand recombinant protein-producing cell cultures 25 2.2 Mammalian cell culture for the production of recombinant proteins 25 2.2.1 Accumulation of metabolites in mammalian cell culture medium 26 2.2.2 Induction of apoptosis by external stimuli 27 2.3 Metabolic engineering 28 2.3.1 Metabolic engineering in CHO 29 2.3.2 Malate dehydrogenase 30 CHAPTER 3.1 Materials and Methods CHO bioreactor cultures 32 32 Page 3.1.1 Bioreactor fed-batch culture operation 32 3.1.2 Bioreactor calculations 33 3.1.3 Enzyme-linked immunosorbent assay for IgG quantification 34 3.2 Metabolomics and targeted metabolite analysis 34 3.2.1 Liquid chromatography – mass spectrometry (LC-MS) analysis 34 3.2.2 MS data processing 36 3.2.3 MS data statistical analysis 37 3.2.4 MS2-based metabolite identification 39 3.2.5 MS-based targeted metabolite quantification 41 3.3 Cloning and overexpression of malate dehydrogenase II (MDH II) gene 43 3.3.1 Polymerase chain reaction (PCR) 43 3.3.2 Vector construction 44 3.3.3 Creation of stable cell line 45 3.3.4 Western blot analysis of MDH II protein level 46 3.4 Assessment of CHO mAb physiology 47 3.4.1 Assessment of enzymatic bottleneck at MDH II 47 3.4.2 Assessment of apoptosis induction 48 CHAPTER Development of strategy for the metabolomics analysis of CHO culture medium 50 INTRODUCTION 50 RESULTS 52 4.1 CHO mAb fed-batch bioreactor cultures 52 4.2 LC-MS analysis of CHO mAb culture medium 54 4.3 MS data pre-processing and statistical analysis 54 Page 4.4 Metabolite identification 56 DISCUSSION 60 4.5 LC-MS strategy produced mass accurate and reproducible data suitable for statistical analysis 60 4.6 Hierarchical cluster analysis 62 4.7 Metabolite identity confirmation was accomplished using two state-of-the-art MS instruments 64 66 CONCLUSION CHAPTER Overexpression of MDH II in CHO for improved cell growth 68 INTRODUCTION 68 RESULTS 69 5.1 Quantification of extracellular metabolites 69 5.2 Determination of the source of excess malate 71 5.3 Creation of CHO mAb clones stably overexpressing MDH II (CHO MDH II) 74 5.4 Characterisation of CHO mAb clones stably overexpressing MDH II 76 DISCUSSION 79 5.5 MRM quantification revealed malate was most abundant amongst the accumulated metabolites during cell culture 79 5.6 Malate efflux from CHO mAb cells was due to aspartate in the medium and enzyme bottleneck at MDH II 81 5.7 CHO mAb cells stably overexpressing MDH II demonstrated improved cell growth 83 CONCLUSION CHAPTER INTRODUCTION 84 Identification of apoptosis-inducing metabolites 85 in CHO culture medium 85 Page RESULTS 85 6.1 Profiling caspase activity of CHO mAb cells in triplicate fed-batch cultures 85 6.2 Correlation analysis of metabolites to the caspase activity profile 86 6.3 Assessment of apoptosis-induction by extracellular metabolites 89 DISCUSSION 91 6.4 Correlation analysis facilitated the short listing of metabolites of interest, whose trends required verification by absolute quantification 91 6.5 Nucleotides and nucleosides formed a major group of extracellular metabolites that correlated with caspase activities 92 6.6 Amino acid derivatives formed another major group of extracellular metabolites that correlated with caspase activities 95 6.7 Addition of GSSG to CHO mAb cells resulted in the greatest increase in caspase activity 96 98 CONCLUSION CHAPTER Conclusions and recommendations 99 7.1 Conclusions 99 7.2 Recommendations 101 7.2.1 Improve throughput of the metabolomics analysis 101 7.2.2 Account for mass peaks with no identities 103 7.2.3 Improvement in CHO MDH II culture performance 104 7.2.4 Intracellular metabolomics to verify mechanisms of apoptosis-inducing metabolites 105 7.2.5 Integration of “-omics” data 106 Abbreviations 107 Page 109 Bibliography Appendix A List of publications 124 Appendix B List of mass peaks in Cluster A4 (Figure 4.5) with putative metabolite identities. 125 Appendix C Theoretical daughter ions of glutamylphenylalanine. 137 Appendix D Retention times and standard addition curve 139 equations of the extracellular metabolites quantified by multiple reactions monitoring scans. Appendix E List of putative extracellular metabolites that displayed strong correlation (R2>0.8) with intracellular caspase activities. 141 Metabolomics study of CHO cell cultures SUMMARY Chinese hamster ovary (CHO) cells are predominantly used for the production of recombinant proteins. Currently, with the exception of ammonia and lactate, there is little knowledge of other metabolites that are released by CHO cells during culture. This thesis describes the development of a liquid chromatography-mass spectrometry metabolomics strategy to identify extracellular metabolites in recombinant CHO fed-batch cultures. 11 out of 12 identified metabolites were reported for the first time in CHO culture medium. Amongst the metabolites, malate accumulation was the highest at 329 µM. Malate efflux was found to be due to the supply of aspartate and possibly an enzymatic bottleneck at malate dehydrogenase II (MDH II). Subsequent metabolic engineering to overexpress MDH II in CHO cells resulted in fold increases in intracellular ATP and NADH. In addition, integral cell number and product titer increased by up to 1.9 and 1.2 fold respectively. This is the first report of a metabolic engineering target that was successfully identified using a metabolomics approach. In addition, a panel of extracellular metabolites exhibited good correlation with intracellular caspase activity. Some of these metabolites, such as oxidized glutathione and AMP, were shown to induce apoptosis. This result could offer the first insight into why CHO cells in fed-batch cultures eventually senesce and die, even though nutrients were not limiting and ammonia and lactate were below inhibitory levels. These findings demonstrate how a metabolomics-based strategy enabled the identification of key cell growth and death-related metabolites in recombinant CHO cultures, thereby guiding metabolic engineering efforts to improve culture performance and product titer. 10 APPENDIX C Theoretical daughter ions of glutamylphenylalanine that matched with the observed MS2 product ions. Theoretical daughter ions were generated by MassFragment™ software. 137 138 APPENDIX D Retention times and standard addition curve equations of the extracellular metabolites quantified by multiple reactions monitoring scans at Day and of culture. The Limits of Detection were not determined as signal to noise ratios were more than 10 in all instances. Acetylphenylalanine Acetylspermidine Adenosine 5'monophosphate (AMP) Aspartylphenylalanine Dimethylarginine Formylmethionine Glutamylalanine Glycerophosphocholine (GPC) Guanosine 5'monophosphate (GMP) Malate Oxidized glutathione (GSSG) Retention time (min) 10.0 2.9 4.4 10.2 3.4 9.2 4.6 3.7 5.3 5.0 5.6 Day - Equations of standard addition curve Bioreactor Bioreactor Bioreactor Acetylphenylalanine Acetylspermidine AMP Aspartylphenylalanine Dimethylarginine Formylmethionine Glutamylalanine GPC GMP Malate GSSG y = 1.84E+17x + 1.18E+06 y = 2.82E+16x + 2.85E+05 y = 2.52E+16x + 7.40E+05 y = 6.81E+16x + 2.03E+05 y = 1.31E+16x + 3.45E+05 y = 3.09E+16x + 4.84E+05 y = 1.47E+16x + 5.70E+05 y = 8.06E+15x + 1.07E+05 y = 7.19E+14x + 1.36E+03 y = 4.17E+16x + 1.52E+06 y = 6.33E+15x + 6.84E+04 y = 2.33E+17x + 3.62E+05 y = 3.43E+16x + 2.41E+05 y = 3.64E+16x + 4.62E+05 y = 8.35E+16x + 2.01E+05 y = 2.29E+16x + 4.22E+05 y = 3.67E+16x + 4.28E+05 y = 2.01E+16x + 2.63E+05 y = 1.79E+16x + 4.47E+05 y = 1.39E+15x 1.25E+03 y = 4.04E+16x + 1.22E+06 y = 9.01E+15x + 1.61E+05 y = 2.53E+17x + 2.98E+05 y = 4.08E+16x + 1.60E+05 y = 3.74E+16x + 3.96E+05 y = 8.61E+16x + 1.98E+05 y = 2.70E+16x + 3.78E+05 y = 3.59E+16x + 1.12E+06 y = 5.84E+16x + 2.80E+05 y = 2.79E+16x + 3.17E+05 y = 1.62E+15x + 1.56E+02 y = 3.29E+16x + 7.58E+05 y = 1.05E+16x + 8.04E+04 139 Day - Equations of standard addition curve Bioreactor Bioreactor Bioreactor Acetylphenylalanine Acetylspermidine AMP Aspartylphenylalanine Dimethylarginine Formylmethionine Glutamylalanine GPC GMP Malate GSSG y = 2.32E+17x + 2.43E+06 y = 3.27E+16x + 3.02E+05 y = 4.30E+16x + 3.43E+06 y = 1.00E+17x + 5.01E+03 y = 1.81E+16x + 7.03E+05 y = 3.52E+16x + 1.03E+06 y = 2.45E+16x + 1.96E+06 y = 8.93E+15x + 9.91E+05 y = 6.05E+15x + 3.47E+05 y = 3.26E+16x + 1.18E+06 y = 1.25E+16x + 2.54E+05 y = 2.08E+17x + 1.61E+06 y = 3.06E+16x + 5.37E+05 y = 3.32E+16x + 1.47E+06 y = 8.45E+16x + 2.19E+05 y = 1.85E+16x + 9.29E+05 y = 3.01E+16x + 1.21E+06 y = 2.13E+16x + 2.78E+06 y = 1.25E+16x + 2.11E+06 y = 8.92E+15x + 3.32E+05 y = 3.72E+16x + 2.21E+06 y = 1.11E+16x + 5.32E+05 y = 2.32E+17x + 2.14E+06 y = 3.74E+16x + 5.59E+05 y = 3.39E+16x + 1.42E+06 y = 8.70E+16x + 4.06E+05 y = 2.69E+16x + 1.15E+06 y = 3.65E+16x + 1.19E+06 y = 2.21E+16x + 2.94E+06 y = 2.18E+16x + 2.42E+06 y = 9.38E+15x + 2.32E+05 y = 3.06E+16x + 1.72E+06 y = 1.13E+16x + 1.25E+05 140 APPENDIX E List of putative extracellular metabolites that displayed strong correlation (R2>0.8) with intracellular caspase activities. Reactor (Positive mode) S/N Mass (m/z) , Time (sec) 405.156303804333,475.094890117458 184.05995549446,1018.18140363111 247.139405491853,814.382773294245 10 11 12 13 14 15 16 17 18 19 20 21 296.065117208107,565.586204711917 130.085735091167,509.584401023825 375.063540646367,561.276469388601 355.288033894951,2189.54647584324 108.948183196035,534.507049509062 553.229291314455,1898.67608344188 811.450200355693,508.67987762175 360.242877690684,2189.53711265353 143.035107732922,419.589474590465 199.074830377654,1925.15554295803 165.090529451394,2159.48864450567 364.064610128092,1075.22319300666 160.075214161572,1053.38104264894 451.204230870458,1740.84929989773 529.150087553702,1386.97970711382 219.097119254497,1013.03004936727 258.109401922703,571.800300397798 221.035817212321,590.326943050546 22 221.030784834692,590.920879625859 23 405.071025301995,1455.51557334676 24 25 26 27 28 29 30 31 32 33 34 35 36 37 190.107004456128,1679.08004311755 189.086583770707,892.70839182494 290.133981336291,1051.89302698412 361.234163843324,2002.29139800728 474.17172428244,1450.19070399129 266.083001264925,1386.97970711382 460.269470001165,2011.1251380665 144.101439857924,1679.37574655740 180.051195016390,1148.86450775063 260.054236422095,1114.40277576605 745.092498404786,480.401300618488 297.107517770278,1377.46905463763 298.096326390084,1506.06649549530 324.058182165802,708.942409823096 38 39 40 41 333.104650240044,1425.72528590694 435.100978557798,1132.66737721990 237.184464094544,1898.75505612708 227.117415764425,1758.36553362083 Putative Metabolite Mallotophenone 4-Pyridoxic acid L-N2-(2-Carboxyethyl)arginine 5-Aminoimidazole ribonucleotide Pipecolic acid Glucocochlearin Methyl acetyl ricinoleate Fe(CN)2 Clocapramine dihydrochloride Dianoside A Loxtidine trans-trans-Muconic acid 4-Hydroxybenzophenone 2-Phenylbutyric acid GMP Indoleacetaldehyde Geranyl-PP Mahuannin D Glutamylalanine Glycerophosphocholine 2-Chloro-4,4'-biphenyldiol 4-Carboxy-4-hydroxy-2oxoadipate 3-O-(3,6-Anhydro-alpha-Dgalactopyranosyl)-D-galactose 4-O-sulfate -(Butylamido)-4hydroxybutanoic acid N-Acetylglutamine Ophthalmic acid S 1033 N10-Formyl-THF Anhydrolycorinone Militarinone A Proline betaine Isoxanthopterin Glucosamine-1P NADP 4-Nitrophenyl-3-ketovalidamine 5'-Methylthioadenosine Cytidine monophosphate O-Ethyl-O-(5-methyl-4nitrophenyl)(1methylpropyl)phosphoramidothi oate Chlorohyssopifolin A Capsidiol Metyrapone R2 0.99 0.98 0.98 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.97 0.96 0.96 0.96 0.96 0.96 0.95 0.95 0.95 0.95 0.95 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.94 0.93 0.93 0.92 0.92 0.92 0.91 0.91 0.91 0.91 0.91 141 42 43 162.075546302208,1343.00072244339 329.109170993777,1405.37788253356 44 45 46 47 48 49 50 51 52 283.190016334417,1818.29738171306 178.070355498853,672.290235371551 348.069545233907,905.399900648569 267.195133785167,1968.6571871739 239.20007550096,2088.1311112666 187.132398631692,1744.06108555142 186.112043277127,1739.74529456842 225.184680646179,1830.38509038749 199.096220663182,1692.59986377793 53 54 55 56 57 58 59 60 61 62 63 64 65 249.053275065758,1330.67837022482 286.04924652933,1654.60274755205 158.117036887531,1739.74529456842 204.122601698452,1738.69291761407 192.101501058020,1767.42117984363 227.200152916627,2010.59741652288 589.193421503077,1178.56884303192 276.062092095312,1768.46058023918 267.158873664280,1781.09835259245 208.096178078208,1630.33773123778 213.148368381486,1759.61708523655 173.116781525388,1793.53191504162 330.058003931631,1078.23820488466 66 67 68 69 70 297.241971354835,2110.38125371833 197.153206703720,1897.40891348248 155.106155433968,1743.06677168382 274.091699425007,1081.94437037541 718.468820054624,1710.36829003806 71 72 73 74 75 76 271.190120994086,1828.42663798743 151.111240839242,1775.36957890567 225.14829112714,1812.13010180833 199.132474325752,1866.62785696438 222.078932701608,1652.82455254196 469.249003045701,1692.41997864857 77 78 79 80 81 82 405.260222409587,2092.10614817590 124.999214939745,570.272646205598 211.132539516274,1686.91700186648 279.231349850744,2110.39969698624 221.189614778959,2086.6891115331 213.138252160642,1764.10421007739 83 84 85 86 87 88 89 90 91 215.127470921126,1803.69034201014 145.132881147248,1761.94808189468 134.095900648707,1743.71771233964 153.090495323685,1655.18994777709 268.102995354649,1744.47232129231 202.143278537914,1794.14629596923 271.180152280103,1827.36493810472 219.133183968081,660.857641068698 185.153184407718,1822.47172732976 92 314.084075994883,1080.76095228948 Aminoadipic acid Branched chain fatty acid 12-trans-Hydroxy juvenile hormone III 4-Hydroxy-4-methylglutamate Adenosine monophosphate Juvenile hormone III Seiricardine A 2-Oxodecanoic acid pseudoecgonine 5,8-Tetradecadienoic acid Guaifenesin (Z)-2-(2-Furyl)-3-(5-nitro-2furyl)acrylamide Riccionidin A Lentiginosine L-Acetylcarnitine 5-Methoxytryptophol 5-Tetradecenoic acid Flavonoid Paraoxon Xanthoxic acid N-Acetyl-L-phenylalanine 12-Oxo-9(Z)-dodecenoic acid 9-Oxononanoic acid Cyclic AMP 13S-hydroxyoctadecadienoic acid Allyl cyclohexylpropionate Boschnialactone Brugine Erythromycin B (10S)-Juvenile hormone III acid diol Thymol (+)-7-Isomethyljasmonate Hercynine 6-Hydroxyindolelactate Gentamicin A 7a,12a-Dihydroxy-3-oxo-4cholenoic acid Phosphonoacetaldehyde (+)-7-Isojasmonic acid a-Linolenic acid Farnesal 3,3'-Dimethylbenzidine Methyl 2diazoacetamidohexanoate N-Acetylcadaverine Tranylcypromine 3-Isopropylcatechol Adenosine Capryloylglycine Doxylamine N2-(D-1-Carboxyethyl)-L-lysine Undecylenic acid Albendazole-gammahydroxysulphone 0.91 0.91 0.91 0.91 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 142 93 201.111790811483,1702.90581317465 94 95 96 97 98 99 100 101 102 103 104 174.075567950014,1654.22573064528 130.122078875456,1782.49313717882 139.07487002791,1846.69507970756 147.041940446579,1172.32075143759 195.137573978390,1763.12821610206 505.157476122195,562.750199179727 314.195636713511,1780.53468806642 153.126895660454,1934.53634745145 207.013663288633,563.290415695669 384.170136433079,1895.79913802191 338.105476500941,567.154233559837 105 106 107 108 109 110 111 112 113 235.129450141335,1760.69332081198 142.158668134477,1399.11194353164 587.180508968731,1365.09412334579 246.155177228521,1706.80479681423 366.114766778148,1181.55615757064 305.13532734446,1764.75672323541 243.122459270713,1774.98388805935 162.090824694975,1728.57228796342 201.086523810623,1018.18581605924 114 115 116 215.127530415382,1735.71635018540 259.091775127696,1125.09284652731 139.074825892822,1701.71104116258 117 118 119 120 121 122 123 124 125 433.101792952744,1121.54149752739 244.060902066882,1648.13458729031 277.140538142936,1872.79525263311 370.191021261845,1917.43310990145 129.090475768022,1711.48237787248 416.114377077968,924.678753505576 208.096535918104,1725.82483321109 587.180161721378,1332.96928590865 293.139780932200,1820.50419579301 126 127 128 129 251.118692947053,1698.10518793347 226.044327076837,1184.37543186182 613.157477675421,1112.92925862756 243.122368054840,1652.61215328981 130 131 132 133 134 135 136 352.123512683529,565.033171448663 587.328107076613,549.678427189694 167.055934974609,1172.32075143759 123.043501498484,1027.64849450970 307.151040403820,1784.52476293507 294.148414003534,1821.20658214065 388.202230069163,1784.41210009547 137 441.208456698984,1908.69285739775 138 139 303.119641326411,1682.84127613657 309.106955381376,784.748703469636 140 374.107354503791,1164.02419615749 Decenedioic acid N-Acetyl-L-glutamate 5semialdehyde N-Methylhexanamide Tyrosol 3-Oxo-3-ureidopropanoate 4-Hexyloxyphenol Clindamycin phosphate Heliotrine (-)-trans-Carveol 3-Oxalomalate Prazosin S-(Hydroxymethyl)glutathione 2,6-Diamino-7-hydroxy-azelaic acid (+)-N-Methylconiine Phyllanthostatin A beta-Alanyl-L-arginine CGP 28-392 2'-Deoxymugineic acid Diphenylcarbazide Tryptophanol Dihydroclavaminic acid Methyl 2diazoacetamidohexanoate Ribothymidine Tyrosol 7a-Hydroxy-o-carbamoyldeacetylcephalosporin C Maculine Saccharopine Trimetrexate beta-EMGBL Chelating agent N-Acetyl-L-phenylalanine Phyllanthostatin A SB 206553 3-(m-Aminophenyl)-2-(pmethoxyphenyl)acrylonitrile Furazolidone Oxidized glutathione Diphenylcarbazide N-Acetyl-7-O-acetylneuraminic acid Presqualene diphosphate Arabinonic acid Benzoic acid Lupinate Melosatin B Terazosin 15-hydroxynorandrostene-3,17dione glucuronide 3''-Deamino-3''oxonicotianamine Indicaxanthin (2R)-2-beta-DGlucopyranosyloxy-4-hydroxy7-methoxy-(2H)-1,4- 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.85 0.85 143 141 273.05866498855,1435.35671931498 142 143 144 282.097468120616,1122.54825294019 283.114593734317,1647.74136083916 350.21663624863,1809.75543234741 145 146 147 148 149 150 151 152 153 586.176431695291,1420.04124359136 137.070350682007,551.488674124936 302.080229788715,1768.10402407452 256.153950113589,1754.32732203658 294.100048740798,1045.98489372482 359.094146457194,562.917075893992 160.059879166155,599.737408565759 403.12917926895,953.524343750824 229.143566308215,1707.84932014793 154 155 156 157 158 159 131.052201198176,1125.09284652731 277.144808934016,1917.50707784183 127.074809727183,1697.43087724616 169.085428709876,1650.67419693595 258.107865583508,1117.99480242089 457.203327596485,1905.30605789482 160 161 162 220.050306792569,1792.64014516566 160.107586567623,1039.23521511989 232.153923308267,1730.88183782532 163 164 165 166 167 168 169 187.096066868242,1701.35389765061 403.027496614591,564.822073873009 312.110614332024,1045.98489372482 325.123874702677,1688.79040165571 233.114277669198,1784.59541225881 192.070836545343,1642.62847106634 207.050250121157,500.215149602426 170 171 219.047019442496,1792.43903156127 253.104173513436,1794.47418656093 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 200.102377666030,1123.57208543475 244.153922726093,1735.00612759212 174.112006701863,1711.20353936126 332.167769028274,1416.59000483478 384.170315452649,1970.65168849791 388.202346757684,1719.15239689452 189.122830291274,1208.16383694896 311.145950149145,1840.39528502891 364.09984311117,1194.08365478389 311.145903014763,1773.88103670964 324.149239725833,1730.48556148620 128.106466190325,1763.51824260978 156.101425428010,1711.29887352778 309.107214887663,1252.77925167902 218.138293870239,1099.34454201724 279.119751373554,1672.98398958456 325.161539135554,1755.05835339335 182.080601525564,1169.93531582442 benzoxazin-3(4H)-one 1-Phenyl-3-(phenylsulfonyl)-2propen-1-one 4Hydroxyphenylacetylglutamine 2,3-Diphenyl-1-indanone Cetiedil SN38 glucuronide carboxylate form N-Methylnicotinamide Peonidin Meteloidine (+)-2,7-Dideoxypancratistatin Miraxanthin-I N-Methyl-2-oxoglutaramate Saphenamycin Traumatic acid 1-Fluorocyclohexadiene-cis,cis1,2-diol Scorpioidin Cyclohex-1-enecarboxylic acid MTIC 5-Methylcytidine Delavirdine 4-Amino-2-methyl-5phosphomethylpyrimidine delta-Guanidinovaleric acid Isobutyryl-L-carnitine cis-2-Carboxycyclohexyl-acetic acid dTDP Zierin Chromanol 293B N2-Succinyl-L-ornithine N-Acetylmethionine 2-Methylcitric acid cis-1,2-Dihydroxy-1,2dihydrodibenzothiophene Benzo[e]pyrene gamma-Glutamyl-betaaminopropiononitrile Tiglylcarnitine Hexanoylglycine Melochinone Prazosin Terazosin N6-Acetyl-L-lysine Mannopine Cefadroxil Mannopine Naftifine hydrochloride N-Cyclohexylformamide Scopoline Indicaxanthin Propionylcarnitine Dibenz[a,h]anthracene Galactosylhydroxylysine L-Tyrosine 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.85 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 144 190 191 237.109298624230,1736.01798024898 654.288694838438,547.105553767843 192 327.140765405064,1803.52789267673 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 184.084090245208,1169.93531582442 259.091669153157,655.413455945196 277.104089633448,1663.69839536024 136.075092244863,1169.93531582442 264.128110556775,1715.15321086012 206.138280935091,1057.07773045738 323.06208394853,1122.55786804322 123.04350438116,1169.93531582442 169.085455153284,1556.19774567957 139.097077238122,658.77558259828 165.054136602844,1169.93531582442 267.08790795507,1743.45513226123 422.237422534538,2010.36838311499 238.070291489298,1130.15221166132 422.237447061496,1969.66476793263 330.190572277441,1830.26580740300 224.091461365408,1596.11722932703 216.087481078844,468.722353553428 211 212 213 214 215 216 217 251.116096916520,1702.25813878662 269.135373227675,1807.45934576538 170.095947990875,2054.42879999766 266.123988334061,1690.30692811327 307.082570634243,1111.91777106554 217.084798470466,468.998373395135 281.073648148650,1121.54252678999 218 219 220 221 222 237.109300888043,1803.99920544884 320.095826146344,1355.25674358550 215.087239385345,468.762956195253 162.075547805176,1141.28218296619 249.109309278237,1832.36636140245 223 296.063635419675,1116.47778705187 224 225 226 227 228 188.1277375892,1057.07773045738 187.132372634283,1862.50594750455 185.031862940243,562.714699054012 197.153284178869,1960.09718600052 422.237358163069,1897.18792251583 229 230 231 232 233 234 235 236 187.096181211051,1555.68856163223 262.128017703303,866.54180053589 350.104807423102,583.483239235635 249.109271315391,1675.20283360866 653.261072232217,549.184913278494 316.118823991014,559.162758572317 299.161267697691,2121.69452183347 305.023186326542,853.57699597242 237 291.119691546134,1660.08664280801 Saminomethyldihydrolipoamide Rifamycin W-hemiacetal 2,2,4-Trimethyl-3-(4fluorophenyl)-2H-1benzopyran-7-ol acetate 2,6-Diamino-4-hydroxy-5-(Nmethyl)formamidopyrimidine Ribothymidine Triphenylsilanol N-Methylformamide 7-Mercaptoheptanoylthreonine Pantothenol 5-Thymidylic acid Benzoic acid MTIC Pentylenetetrazole Phenylpyruvic acid Ungeremine Ancistrotectorine Cartap Ancistrotectorine Europine N-Acetyl-L-tyrosine Succinyl proline 3-(m-Aminophenyl)-2-(pmethoxyphenyl)acrylonitrile 3-Methylcholanthrene Diphenylamine N6-Methyl-2'-deoxyadenosine (-)-Epigallocatechin Allenolic acid Sulfamethopyrazine Saminomethyldihydrolipoamide Methylene blue Methyl bisnorbiotinyl ketone Aminoadipic acid 5,6-Dihydrouridine 5-Aminoimidazole ribonucleotide (E)-2-Butenyl-4-methylthreonine 2-Oxodecanoic acid 5-Hydroxyisourate Allyl cyclohexylpropionate Ancistrotectorine cis-2-Carboxycyclohexyl-acetic acid Mycosporine S-(Formylmethyl)glutathione 5,6-Dihydrouridine Dehydroisocoproporphyrinogen Hippeastrine Trimeprazine 7-Chloroemodin N-Succinyl-LL-2,6diaminoheptanedioate 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 145 238 239 240 183.101314581000,2159.90026554180 163.026345369298,413.423753342909 227.107965245682,1740.78456140949 241 242 198.103869274832,542.865112105971 173.091592649560,617.192683758578 243 244 792.593657916177,1716.97541139551 290.159216222725,1641.40663850589 245 246 247 248 249 373.043714354181,479.658441911456 263.124846022558,1716.5241714783 145.048977079388,1036.86838683224 297.106385792523,1097.3225083513 249.109247216813,1743.51820985732 Dihydroconiferyl alcohol Allicin Favan-3-ol 3-Amino-1-methyl-5Hpyrido[4,3-b]indole Glycylproline PC(20:4(5Z,8Z,11Z,14Z)/dm18: 1(11Z)/0:0) 3-Methylglutarylcarnitine 4-(NMaleimido)benzyltrimethylamm onium iodide D-Ornaline 3-Hexenedioic acid 4-Nitrophenyl-3-ketovalidamine 5,6-Dihydrouridine 0.81 0.81 0.81 0.81 0.81 0.81 0.80 0.80 0.80 0.80 0.80 0.80 Reactor (Negative mode) S/N Mass (m/z) , Time (sec) 245.125315438635,797.921527935529 606.073132849658,955.050123790132 383.121641837342,1143.65110495099 226.083244683312,709.702725313609 158.045996329012,696.332107421682 385.12760774032,1143.69922913366 175.061224138245,1160.43029333231 140.108429707808,1745.71984440929 10 11 12 155.046000632417,797.809604556845 505.182829305546,1601.75786626710 144.103223285620,1680.99429555012 119.050607919163,1143.06034221537 13 14 15 188.092843104672,1681.37264520067 472.158035613718,1439.19450607794 217.082934917715,976.517488620585 16 17 236.145503425594,1772.97851900352 160.061536489796,1330.67103901179 18 19 20 21 22 23 24 25 26 27 28 29 30 31 189.090994602343,1682.88558907056 158.045986174511,785.091881297664 164.071752975945,1407.03792082323 192.103151894201,1773.42359338084 348.106014101789,1773.12167599259 181.069854424922,1143.06034221537 130.087779518811,1748.00620236382 180.066618763896,1143.241529697 200.129410272072,1801.87638275258 362.049836299529,1033.58876625693 158.118809400989,1745.62376861905 581.268484117549,1739.97363699971 302.085228877085,1257.30042190065 247.093350169020,761.061206031691 Putative Metabolite L-N2-(2-Carboxyethyl)arginine Uridine diphosphate-Nacetylgalactosamine Acetyl-maltose Deoxycytidine N-Methyl-2-oxoglutaramate 2-(4-Ethoxyphenyl)-5,6,7,8tetramethoxy-4H-1benzopyran-4-one 2-Isopropylmalic acid Trachelanthamidine 5-Hydroxymethyl-4methyluracil Piperaduncin B 3-Dehydroxycarnitine 4-Hydroxystyrene -(Butylamido)-4hydroxybutanoic acid N10-Formyl-THF Glutamylalanine N-Methyl-(R,S)tetrahydrobenzylisoquinoline Aminoadipic acid 3-Dimethylallyl-4hydroxybenzaldehyde N-Methyl-2-oxoglutaramate L-Phenylalanine (R)-N-Methylsalsolinol Voriconazole Galactitol L-Isoleucine L-Tyrosine Capryloylglycine GMP DL-2-Aminooctanoic acid 2'-Norberbamunine Gynocardin 5,6-Dihydrouridine R2 0.97 0.97 0.97 0.97 0.96 0.96 0.96 0.96 0.96 0.95 0.95 0.95 0.94 0.94 0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.92 0.92 0.92 0.92 0.92 0.91 0.91 0.91 0.91 0.90 146 32 33 34 35 36 37 38 39 40 41 465.138032827698,1632.16444460293 154.062342564082,542.292051618472 328.044816196359,1151.14070638161 465.142598111859,1633.19734886264 202.108483630515,1745.46509474965 289.05546919682,1655.91322529006 322.043950947443,723.74896623235 253.090753288457,1633.45299860492 356.168489329232,1738.56679257927 114.056398245515,783.433944581958 42 228.048828814209,1330.59981707238 43 44 45 46 47 48 759.42513436602,550.90180656736 399.095461171925,1144.47746618156 221.068139397619,1655.65877709822 206.082241013972,1733.00759960855 222.077026697116,1600.32264455042 110.036401527426,1198.45420386468 49 50 51 52 53 54 55 56 57 58 59 60 325.043429189554,548.771786513289 555.129782184167,562.776987032933 130.087696723802,1714.13624560936 368.115729844096,553.259626531935 209.081176578692,568.707169121934 288.05196527669,1605.52515426899 157.077148478030,1288.35437442055 172.097972723861,1715.22111361307 304.079859060831,1773.61064957725 346.054904371792,863.591916371592 134.061348348267,1157.30560151460 288.051883740223,1655.82405806810 61 62 63 64 65 66 333.058583721148,702.946558472944 171.006509873782,615.137991936559 290.064136152865,1600.17492294538 479.116482612061,734.49847406915 360.142590377082,1732.14011025755 205.035330905851,1160.75828632561 67 388.061798808094,1633.50541865703 68 69 70 267.095887793245,1391.76661401943 244.037202235167,1143.25174715183 447.092105749820,1143.66122980031 Agnuside L-Histidine Cyclic AMP Agnuside L-Acetylcarnitine Isoprothiolane Cytidine monophosphate Galactosylglycerol Uplandicine L-Proline 5-(L-Alanin-3-yl)-2-hydroxycis,cis-muconate 6semialdehyde Avermectin A2a monosaccharide S-Acetylphosphopantetheine Ethyl glucuronide (Ethanol) N-Acetyl-L-phenylalanine N-Acetyl-L-tyrosine Cytosine 2,4-Dioxotetrahydropyrimidine D-ribonucleotide Aurasperone D L-Isoleucine Corpaine Multifidol PMEG Nicotyrine Hexanoylglycine Convicine Adenosine monophosphate N-Methylformamide PMEG 2-(beta-D-Glucosyl)-sn-glycerol 3-phosphate Glyceryl phosphate Lycoricidine Cefepime 3-Acetylnerbowdine 2-Methylcitric acid N-Acetylneuraminic acid 9phosphate 2-p-Tolyl-5,6,7,8tetrahydrobenzo[d]imidazo[2,1b]thiazole Chlorphenesin carbamate Aureusidin 6-O-glucoside 0.90 0.90 0.90 0.89 0.89 0.89 0.89 0.89 0.88 0.88 0.88 0.88 0.87 0.87 0.87 0.86 0.86 0.86 0.85 0.85 0.85 0.84 0.84 0.84 0.84 0.84 0.84 0.83 0.83 0.82 0.82 0.82 0.82 0.81 0.81 0.81 0.81 0.80 0.80 Reactor (Positive mode) S/N Mass (m/z) , Time (sec) 405.208744677028,949.864815964166 349.054080846104,1316.80653872206 299.084623161657,921.293561050504 201.065723066094,1481.13624451603 278.106014191219,921.683836389939 Putative Metabolite 15beta-Hydroxy-7alpha-mercaptopregn-4-ene-3,20-dione 7-acetate Inosinic acid Mefenacet Tegafur Metazachlor 147 R2 0.97 0.96 0.96 0.96 0.96 249.107916656645,856.101449686559 291.129584897821,825.882325312638 10 11 12 13 14 15 16 17 18 19 20 21 22 23 333.105264656125,1549.91665206029 247.13983548559,920.984992324388 364.065003556992,1255.47149425414 219.097360842242,942.627470789645 277.102843652784,922.019115484223 491.133343554277,1833.01229420378 253.179707925295,2107.04743911242 160.060089070835,876.573207270106 245.174502270376,1925.23022411896 330.059550726791,1320.63184483530 258.109819837163,635.808139618513 557.141135706719,1210.22588860319 228.097703919631,776.336214027739 151.061120955036,1452.63353318222 153.127046783366,1927.07881199448 325.042941562930,1215.45164493394 24 25 26 27 28 29 30 31 32 33 34 35 36 142.049513337326,876.671940220274 126.066416603011,863.807906073225 469.151678179699,1832.32485195111 178.070730039504,764.814693693839 137.070554690683,596.222821643837 205.081689679798,688.419790383946 152.056275140188,918.945004771705 261.144361638650,1675.33153700299 429.239719579977,2206.73425725054 312.13006108831,1524.33563319280 144.101562714442,1770.88357019653 260.026754672129,625.585728486487 129.065641996769,1378.21976816020 37 38 39 40 41 42 43 335.073485722063,694.839025983308 346.124377785056,1174.95148297785 247.056364404506,1210.62423969364 189.136363291040,597.317679522397 113.035017168873,1478.95888465468 291.069746441788,1376.57027168546 296.065684417339,637.004880865748 44 45 46 47 218.06570610225,672.663318894887 247.057568144223,855.548884816825 327.206593099507,1677.89993230488 204.122885252391,1831.37891664341 48 49 50 51 52 53 54 55 56 57 142.049512411968,672.562664852878 296.127276352536,1672.73054099601 173.020687445994,692.819107222576 295.12860837022,1677.53124643878 184.096560887538,1522.14560421619 180.087728736979,1525.07797132334 298.114502465376,1439.10652659218 520.106807310613,623.549557190417 324.058811858578,1536.21364771214 348.070036203428,1137.34747016568 5,6-Dihydrouridine Argininosuccinic acid O-Ethyl-O-(5-methyl-4nitrophenyl)(1methylpropyl)phosphoramidothioate L-N2-(2-Carboxyethyl)arginine GMP Glutamylalanine Triphenylsilanol L-2-Aminoadipate adenylate Juvenile hormone III acid N-Methyl-2-oxoglutaramate 1,11-Undecanedicarboxylic acid Cyclic AMP Glycerophosphocholine Aurasperone D Deoxycytidine D-Xylose (-)-trans-Carveol Uridine 5'-monophosphate 2-Aminomuconic acid semialdehyde 5-Methylcytosine 104-2 4-Hydroxy-4-methylglutamate N-Methylnicotinamide Levamisole Guanine Methaphenilene Aspidoalbine 1,7-Dimethylguanosine Proline betaine M2 Dihydrothymine 2-(beta-D-Glucosyl)-sn-glycerol 3phosphate Omeprazole Glycerophosphoglycerol Homo-L-arginine Uracil Isoprothiolane 5-Aminoimidazole ribonucleotide 2-(Hydroxymethyl)-3(acetamidomethylene)succinate Glycerophosphoglycerol (-)-Tortuosamine L-Acetylcarnitine 2-Aminomuconic acid semialdehyde Japonine Glyceryl phosphate Glutamylphenylalanine Epinephrine Glucosamine 1-Methylguanosine Ceforanide Cytidine monophosphate Adenosine monophosphate 148 0.96 0.95 0.95 0.95 0.95 0.93 0.93 0.93 0.92 0.92 0.92 0.91 0.91 0.91 0.90 0.90 0.90 0.90 0.90 0.90 0.89 0.89 0.89 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.87 0.86 0.86 0.86 0.86 0.86 0.85 0.85 0.85 0.85 0.85 0.85 0.84 0.84 0.84 0.84 0.84 0.84 58 59 60 61 62 63 64 65 66 67 68 69 234.097044264610,651.631392909725 224.105446418166,783.647599728556 166.072097612075,1140.39295964769 206.102414040153,782.106293053761 158.117258794902,1831.90709550955 384.089245695107,975.137988996092 216.062928784678,627.449503089929 324.058917277167,813.973484411815 208.096647152544,1710.59699957291 258.108297163766,1216.46716846458 298.114500614027,1476.55967990729 160.060121656137,667.710585200794 70 71 72 73 74 75 76 269.111524854384,1523.6123180812 192.101775789838,1851.98151321540 172.06017139819,672.663318894887 613.158661213141,1227.61477101320 244.061271859842,1746.21695819302 103.075118945779,2084.63101966833 136.061387022849,1135.48856707772 77 78 79 615.152346453233,1227.91825249556 589.187441651162,1490.63810185726 126.054550471398,723.722779183426 80 81 82 83 84 85 86 87 88 89 90 174.075849957835,1752.11335025437 138.054567542654,726.867492794891 276.118800725055,720.942429658164 155.044763645736,1050.44514982405 225.148404279091,2033.57085141505 264.053316363717,1362.69288666452 204.086428775281,728.328382688048 319.066357350550,1956.82792181659 171.005029192357,1006.82137153939 474.172963949319,1578.02064116714 587.181610041133,1491.48669883872 Dihydroxycoprostanoic acid 2-Acetamidofluorene 7-Methylguanine N-Acetyl-D-quinovosamine Lentiginosine Tetraphenylarsonium Glycerylphosphorylethanolamine Cytidine monophosphate N-Acetyl-L-phenylalanine Glycerophosphocholine 1-Methylguanosine N-Methyl-2-oxoglutaramate 2-p-Tolyl-5,6,7,8tetrahydrobenzo[d]imidazo[2,1b]thiazole 5-Methoxytryptophol AminoDHS Oxidized glutathione Maculine Isovaleric acid Adenine Cytidine monophosphate Nacetylneuraminic acid Flavonoid 3,6-Dihydronicotinic acid N-Acetyl-L-glutamate 5semialdehyde 2-Aminobenzoic acid Norophthalmic acid 3-Imidazole-2-oxopropanoate (+)-7-Isomethyljasmonate Epinephrine sulfate N2-Acetyl-L-aminoadipate DMPP D-Glyceraldehyde 3-phosphate N10-Formyl-THF Phyllanthostatin A 0.84 0.84 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 Reactor (Negative mode) S/N Mass (m/z) , Time (sec) 362.050497629887,1262.52187156089 306.076330855691,793.092980441906 R2 0.82 0.81 Putative Metabolite GMP Glutathione Reactor (Positive mode) S/N Mass (m/z) , Time (sec) 482.126193640135,646.853084924961 296.065953391978,632.405106780217 146.116999788116,611.660996777122 129.065714041388,1353.26382316772 218.065937277071,668.221473357155 608.088703047946,1100.80675445852 258.109891677245,636.859877700314 124.999460945329,631.954605503475 R2 0.94 0.92 0.92 0.92 Putative Metabolite Yersiniabactin 5-Aminoimidazole ribonucleotide 3-Dehydroxycarnitine Dihydrothymine 2-(Hydroxymethyl)-3(acetamidomethylene)succinate Uridine diphosphate-Nacetylgalactosamine Glycerophosphocholine Phosphonoacetaldehyde 0.91 0.91 0.91 0.91 149 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 333.105602008385,1524.64439057979 249.1078177976,834.715371391954 247.139943672678,904.389341611605 244.092495335922,779.490764871678 587.181690387151,1462.12136572055 180.087850282312,1500.3667191939 151.061238408187,1425.90678103492 278.106339290235,888.153472027563 348.145526745079,1216.57941925976 277.10282639783,886.87536484527 346.124452062069,1137.01073866382 166.072214672495,1415.97310531828 427.094768107023,769.648138122121 295.128725699998,1689.63568076734 137.070689400238,604.445685051152 219.097349626211,911.615151630284 364.065173307106,1197.18768156939 234.097176106224,654.037805114137 296.115953998463,1658.45982989428 489.114256067285,679.467865537584 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 335.07362120587,674.78466727482 312.130230422668,1499.98559989127 282.119562869258,1457.88555110347 230.042612612782,1380.02380286800 304.150331864797,1371.50155819568 325.04305637683,1126.09691783652 321.060102343258,1422.54149686969 228.097615219327,766.322141472743 295.095509530922,779.729445826843 201.032692415396,1348.10552477721 261.144351216299,1680.27314621506 166.072252551601,1102.54285176035 324.059156080882,1504.36851913028 162.075935025847,1449.93078405849 557.140932076059,1182.65173186647 198.052486483926,632.59173057616 135.080079786541,1516.24653213718 189.086835695851,998.974277500032 224.107945609726,769.654181708985 327.119696152675,1657.69505989344 348.070173509619,1046.35311227843 152.056473032004,1448.80824865638 298.114603456576,1414.38950212972 150.077102029205,889.338385695188 260.05297692996,668.539362536275 245.076718461663,980.186473973895 189.039214836879,1251.38190366632 O-Ethyl-O-(5-methyl-4nitrophenyl)(1methylpropyl)phosphoramidothioate 5,6-Dihydrouridine L-N2-(2-Carboxyethyl)arginine Cytidine Phyllanthostatin A Glucosamine D-Xylose Metazachlor 3-Epimacronine Triphenylsilanol Omeprazole 7-Methylguanine Cysteineglutathione disulfide Glutamylphenylalanine N-Methylnicotinamide Glutamylalanine GMP Dihydroxycoprostanoic acid Triadimenol CDP-choline 2-(beta-D-Glucosyl)-sn-glycerol 3phosphate 1,7-Dimethylguanosine 1-Methyladenosine 5-Phosphoribosylamine Nicotianamine Uridine 5'-monophosphate 2,3-Dihydrogossypetin Deoxycytidine Dehydrocycloguanandin CPP Methaphenilene 7-Methylguanine Cytidine monophosphate Aminoadipic acid Aurasperone D 2-Amino-4,6-dinitrotoluene Phenylacetone N-Acetylglutamine 2-Acetamidofluorene Humilixanthin Adenosine monophosphate Guanine 1-Methylguanosine 6-Methyladenine Glucosamine-1P Uridine cis-2-Methylaconitate 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.81 0.81 0.81 Putative Metabolite Cefixime Myxochelin A Quassimarin R2 0.93 0.93 0.92 0.90 0.90 0.90 0.89 0.89 0.89 0.89 0.88 0.88 0.88 0.88 0.87 0.87 0.86 0.86 0.86 0.86 0.86 0.85 0.85 Reactor (Negative mode) S/N Mass (m/z) , Time (sec) 452.029267275568,636.284396483681 403.148191694835,639.504130179963 521.20105526775,611.715482349183 150 187.072642694630,1003.19934127627 497.101159857302,820.71393579727 606.074434199389,1086.73717871138 247.093287834271,834.166085085148 10 11 12 13 14 15 16 17 18 19 234.081090226424,668.129371681934 332.09435299014,1525.95979134239 276.091284025098,894.117728583731 346.126544574254,1218.62508527499 402.164152174844,630.114304174015 565.046883603266,1039.72324465759 275.087946779782,888.767320846037 362.050010724345,1362.88141320574 233.139435207348,1793.52518761543 436.176363503418,911.75623281297 362.04979093512,1196.87465592065 556.182783896364,1495.25268280017 20 21 22 23 24 25 26 27 28 29 331.090659112269,1526.03496999399 346.054910705902,1051.72669561031 301.044055204339,685.173331617939 344.039651843854,1421.91686872057 345.060478082626,840.973823505815 284.088792171563,1506.29545423431 577.161096372049,1495.19054252024 217.082930924837,915.186921804888 239.065001920810,910.635979858994 425.079999161214,766.432162499062 30 31 32 33 34 35 36 37 38 39 333.058286207599,666.296478561693 293.113782060655,1688.39428017905 293.080825492955,778.460498042604 274.103949719173,710.889375493859 226.083342034261,768.578094982161 556.116311175187,1183.14944877378 307.082195000155,780.471600077454 259.022216529608,669.633352184295 327.094089328842,1426.85262127866 315.049851740226,914.658298570536 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 333.058450674350,734.897726553698 256.093863472674,1207.96426869577 258.038351337047,663.554965197322 382.073883333488,944.629708548273 555.126159733476,1183.56284890871 458.158389773921,1688.81082519220 160.061575331618,1449.40717635508 310.114876453115,1501.83500310857 243.061976338331,990.480596891427 282.114943017067,1681.14131337609 302.134788544769,1369.95106418694 153.030703595644,987.889815032554 409.085120950062,1368.63044841297 227.067241209839,1330.76054628490 188.056717576861,1280.78730281658 N-Acetylglutamine Bis-gamma-glutamylcystine Uridine diphosphate-Nacetylgalactosamine 5,6-Dihydrouridine p-N,N(Dimethylamino)phenyldiazonium fluoroborate Norchelerythrine Metazachlor 3-Epimacronine Myxochelin B Uridine diphosphate glucose Triphenylsilanol GMP Benzoylagmatine Azocyclotin GMP Rhodomycin D O-Ethyl-O-(5-methyl-4nitrophenyl)(1methylpropyl)phosphoramidothioate Adenosine monophosphate Olsalazine Cyclic GMP Syringetin N4-Acetylcytidine Scolymoside Glutamylalanine Dinoseb Cysteineglutathione disulfide 2-(beta-D-Glucosyl)-sn-glycerol 3phosphate Glutamylphenylalanine Dehydrocycloguanandin Norophthalmic acid Deoxycytidine Cefpodoxime proxetil Allamandin Fructose 6-phosphate Branched chain fatty acid Isorhamnetin 2-(beta-D-Glucosyl)-sn-glycerol 3phosphate Glycerophosphocholine Glucosamine-1P Tetraphenylarsonium Aurasperone D Doronine Aminoadipic acid 1,7-Dimethylguanosine Uridine N-Phenylacetylphenylalanine Nicotianamine Diethylphosphoric acid Bensulfuron methyl Deoxyuridine Glutarylglycine 151 0.91 0.91 0.91 0.91 0.90 0.90 0.90 0.90 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.89 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.88 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.85 0.85 0.85 0.85 0.85 0.84 0.84 0.84 0.84 0.84 55 56 57 58 497.101443578986,1168.20625443795 114.056387062517,852.306956616933 329.022959772051,1641.66162803352 291.083019840859,939.658048490753 59 60 61 62 63 64 267.095780502766,1502.81944992843 323.027662553987,1122.19003148340 258.074696384023,768.252757539958 472.157686028774,1549.88446914941 177.034098052187,777.326513512832 328.044840795153,1278.34819098511 65 66 67 68 69 188.092826185598,1766.93548528908 296.099418823138,1450.39396179614 162.040179187740,795.471541803994 322.043755435066,791.040880383243 176.038697514207,1694.46479329315 70 71 72 73 74 75 76 77 78 79 80 335.085333776208,1503.09135842423 206.082129780537,1816.59255269229 349.098672201548,1680.12912452603 351.103779827380,917.720493957431 241.011565025273,664.303461289325 172.097846891626,1796.13486439193 281.088567007266,1462.75525925907 338.987805630169,672.435274052116 158.118701653453,1831.03288113448 256.059134455645,714.621329900247 295.093252191920,1199.62019633290 Bis-gamma-glutamylcystine L-Proline Fenarimol EDTA 2-p-Tolyl-5,6,7,8tetrahydrobenzo[d]imidazo[2,1b]thiazole Uridine 5'-monophosphate Skimmianine N10-Formyl-THF Cysteinylglycine Cyclic AMP -(Butylamido)-4-hydroxybutanoic acid 1-Methylguanosine Pterin Cytidine monophosphate N-Formyl-L-methionine 4,4'-Stilbenedicarboxamidine dihydrochloride N-Acetyl-L-phenylalanine Biapenem Benzylpenicilloic acid Inositol cyclic phosphate Hexanoylglycine 1-Methylinosine 1D-Myo-inositol 1,4-bisphosphate DL-2-Aminooctanoic acid N-Benzoyl-4-hydroxyanthranilate 4-Nitrophenyl-3-ketovalidamine 152 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.81 0.81 0.81 0.81 0.81 0.81 [...]... between parental CHO mAb ( ) and CHO MDH II (Clone A: Δ; Clone B: O) cells in fed-batch shake flask cultures (B) Cell viability comparison of the same cultures 77 Figure 5.10 Growth comparison between CHO mAb ( ) and CHO MDH II-A (Δ) cells in fed-batch bioreactor cultures (n=2) 78 Figure 6.1 Growth curves (A) and intracellular caspase profiles (B) of the triplicate CHO mAb fed-batch bioreactor cultures 87... Extracellular concentration of malate in CHO mAb fed-batch bioreactor cultures (n=3) 71 12 Page Figure 5.3 Proposed relationship between aspartate and malate in CHO mAb cell culture 72 Figure 5.4 Relationship between amount of aspartate in the medium and the amount of malate released by CHO mAb cells one hour postinoculation in shake flask cultures (n=2) 72 Figure 5.5 Intracellular concentrations of. .. Chapter 4, the development of a metabolomics strategy to analyze metabolites in culture medium from fed-batch cultures of CHO expressing monoclonal antibodies (CHO mAb) In Chapter 5, the results of the overexpression of malate dehyrdogenase II in CHO mAb are reported Chapter 6 describes the 16 identification of extracellular metabolites that induces apoptosis in the CHO mAb cells Chapter 7 summarizes... fed-batch cultures (n=3) as isolated by metabolomics analysis, and their average concentrations 70 Table 5.2 Comparison of metabolism and intracellular metabolite levels between parental CHO mAb and CHO MDH II-A cells in fed-batch bioreactor cultures (n=2) 79 Table 6.1 List of extracellular metabolites that displayed strong correlation (R2 > 0.8) with intracellular caspase activities in at least two of the... Figure 4.1 Metabolomics strategy that was adopted for the analysis of CHO mAb fed-batch culture medium 52 Figure 4.2 (A) Average growth data of three CHO mAb fed-batch bioreactor cultures (B) The transition points of the various phases of growth (exponential, stationary and death) were determined by taking the natural log of the viable cell density 53 Figure 4.3 Typical MS total ion count of culture... producing to lactate consuming state in cultures of NSO and CHO cells expressing monoclonal antibodies (Ma et al., 2009) Most recently, metabolic profiling with GC-MS enabled the differentiation of baby hamster kidney cell cultures based on cell source, cell age and bioreactor scale (Chrysanthopoulos et al., 2010) However, specific applications which enhanced cell culture performance did not emerge... metabolites of interest 42 Table 3.3 Components used in the reaction mixture of the PCR 44 Table 3.4 List of metabolite standards used in the apoptosis induction assay, and their effective concentrations in the CHO mAb 6 well-plate cultures 49 Table 4.1 Extracellular metabolites with their identities confirmed via MS2 experiments 57 Table 5.1 List of extracellular metabolites accumulating in the media of CHO. .. for the analysis of metabolites in culture medium from recombinant CHO fed-batch cultures (2) Identifying an extracellular metabolite that was associated with growth limitation and performing metabolic engineering in CHO cells for improved cell growth and recombinant protein yield (3) Establishing a methodology for the evaluation of extracellular metabolites that were associated with cell death, thereby... NS0 cells (Khoo and Al-Rubeai, 2009) However, these studies did not result in any applications to improve cell growth or protein production 1.2 THESIS OBJECTIVES The main aim of the thesis is to determine new strategies for improving the growth of CHO cells and consequently improve the recombinant protein yield, through a metabolomics approach The scope of the thesis involved: 15 (1) Developing a metabolomics. .. C13-fumarate (Δ) after exposure of CHO mAb cells to C13-aspartate 73 Figure 5.6 PCR amplification of MDH II A single PCR product (arrowed) with expected size of 1131bp was generated 74 Figure 5.7 Digestion of (A) TOPO-MDH II and (B) pcDNA3.1(-)/Hygro using Apa I and Hind III restriction enzymes 75 Figure 5.8 Western blot comparison of MDH II protein level between CHO mAb parental and two CHO MDH II clones 77 . with intracellular caspase activities. 8 Metabolomics study of CHO cell cultures SUMMARY Chinese hamster ovary (CHO) cells are. Development of strategy for the metabolomics 50 analysis of CHO culture medium INTRODUCTION 50 RESULTS 52 4.1 CHO mAb fed-batch bioreactor cultures 52 4.2 LC-MS analysis of CHO mAb. activity of CHO mAb cells in triplicate 85 fed-batch cultures 6.2 Correlation analysis of metabolites to the caspase 86 activity profile 6.3 Assessment of apoptosis-induction by extracellular