Subscriber access provided by PEPPERDINE UNIV
Article
Untargeted LC-MS/MS profiling of cell culture media formulations for evaluation of High Temperature Short Time (HTST) treatment effects. Patrick Floris, Nicola McGillicuddy, Simone Albrecht, Brian Morrissey, Christian Kaisermayer, Anna Lindeberg, and Jonathan Bones Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02290 • Publication Date (Web): 19 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 56 57 58 59 60
Analytical Chemistry
Untargeted LC-MS/MS profiling of cell culture media formulations for evaluation of High Temperature Short Time (HTST) treatment effects. Patrick Floris†, Nicola McGillicuddy†, Simone Albrecht†, Brian Morrissey†, Christian Kaisermayer‡, Anna Lindeberg‡, Jonathan Bones†,§,* †
Characterisation and Comparability laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, Fosters avenue, Mount Merrion, Blackrock, Co. Dublin, A94 X099, Ireland. ‡Biomarin International Limited, Shanbally, Ringaskiddy, Co. Cork, P43 R298, Ireland. §School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, D04 V1 W8, Ireland. ABSTRACT: An untargeted LC-MS/MS platform was implemented for monitoring variations in CHO cell culture media upon exposure to High Temperature Short Time (HTST) treatment, a commonly used viral clearance upstream strategy. Chemically defined (CD) and hydrolysate-supplemented media formulations were not visibly altered by the treatment. The absence of solute precipitation effects during media treatment and very modest shifts in pH values observed indicated sufficient compatibility of the formulations evaluated with the HTST-processing conditions. Unsupervised chemometric analysis of LC-MS/MS data however, revealed clear separation of HTST-treated samples from untreated counterparts as observed from analysis of Principal Components and Hierarchical Clustering sample grouping. An increased presence of Maillard products in HTST-treated formulations contributed to the observed differences which included organic acids, observed particularly in chemically defined formulations, and furans, pyridines, pyrazines and pyrrolidines which were determined in hydrolysate-supplemented formulations. The presence of Maillard products in media did not affect cell culture performance with similar growth and viability profiles observed for CHO-K1 and CHO-DP12 cells when cultured using both HTST-treated and untreated media formulations.
reducing monosaccharides at elevated temperatures13. Therefore, amino acids and reducing monosaccharides (e.g. glucose) become unavailable for cellular nutrition and metabolism. Further complications due to the precipitation of inorganic salts within the fluidic pathway of HTST instrumentation have been also reported, which can potentially result in heat exchanger fouling and decreased media growth performance10,14. It is therefore crucial to monitor quality attributes at all stages of the bioprocess since often, the thermal stability of media formulations might not be fully known. Analytical techniques based on NMR15,16, Raman, SERS17, NIR18 and fluorescence spectroscopic measurements19,20 have been applied for screening chemically defined media formulations which, despite cost-saving benefits and the ability to provide rapid information on selected quality parameters, can suffer from low sensitivity and specificity. The applicability of these techniques for the analysis of complex formulations such as hydrolysate-supplemented culture media is limited due to the vast amount of chemically undefined trace-level components present. An attempt to build profiles of hydrolysates by NMR was reported21 however, Principal Component Analysis failed to demonstrate clear correlation between hydrolysates and antibody titers possibly due to the limited number of chemical entities originally identified. The ability to provide mass specific information on the chemical constituents present in complex biological systems
Adventitious contaminants represent a threat for biopharmaceutical companies involved in the production of therapeutic proteins from Chinese Hamster Ovary (CHO) cell lines1. Rodent retroviruses, such as Vesivirus 2117 and Mouse Minute Virus (MMV), can rapidly propagate in CHO cultures and, if not detected at early stages, can contaminate rapidly an entire manufacturing facility, therefore potentially jeopardizing proprietary cell lines and causing product supply disruptions2,3. Cell culture media represents a major pathway for the introduction of viral agents into bioprocesses4-6. Chemically defined (CD) media formulations, which contain only components of traceable origin, can minimize the risk of contamination but not completely remove it7,8. Furthermore, many established bioprocesses require supplementation with undefined components, such as plant-hydrolysates9, to obtain high protein titers which are sourced from unsterile environments, therefore increasing the risk of contamination. Upstream barriers for viral deactivation, such as High Temperature Short Time (HTST) treatment, have been widely reported10,11 whereby raw-materials are continuously processed and maintained at elevated temperatures of approximately 100°C for short time periods, generally 10 seconds12, resulting in the denaturation of viral capsid proteins. Depending on the nature and stoichiometric levels of components present, the quality of media can deteriorate upon treatment. The side chains of amino acids present, such as lysine and arginine, can react with
1
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 56 57 58 59 60
resulted in mass spectrometry becoming a method of choice for profiling culture media formulations22-25. Untargeted approaches by LC-MS and GC-MS have been successfully implemented for screening plant hydrolysates used for CHO culture supplementation, facilitating the identification of distinctive features, specifically nucleosides, amino acids and organic acids, which correlated to IgG titer levels26,27. Targeted approaches were similarly applied for monitoring the formation of photo-induced degradation products of tryptophan and riboflavin in CD media28. In the study described herein, chemically defined and hydrolysate-supplemented CHO culture media formulations were subjected to HTST-treatment and analyzed via an untargeted LC-MS/MS approach to determine variations caused by heatinduced effects. Multivariate data analysis techniques were applied to identify relationships between HTST-treatment and media quality alterations. Furthermore, CHO culture experiments were performed using both treated and untreated formulations to evaluate the effects of the treatment on cell growth performance.
Page 2 of 9
defined formulations in liquid format were supplemented with 4 mM L-glutamine only. HTST treatment of media samples. Treatment involved pre-heating media from room-temperature (~20°C) to 100°C over a period of 10s. The media was maintained at 100°C for 10s, followed by a 10s cooling step whereby the temperature of the media was reduced to ~20°C. Treatments were performed in triplicate for CD media and four times for hydrolysate-supplemented media. Samples were filtered using 0.45/0.1µm membranes and centrifuged using Sartorius Vivaspin 2 cartridges with a MWCO of 3,000 Da prior to vialing for analysis. Media samples were analyzed immediately after HTST-treatment to minimize the risks of visible light-induced degradation Chromatographic conditions for media analysis. Reversed-phase analysis of media samples was performed on a Cortecs C18 column (2.1 × 150 mm, 1.6 µm particle size) at 0.2 mL/min with gradient conditions starting at 98% water with 0.1% FA (line A) and 2% acetonitrile with 0.1% FA (line B). The gradient was switched to 95% A-5% B from 5 min to 24 min and to 5% A-95% B from 24 min until 34 min, followed by a final equilibration period under starting conditions. Triplicate injections of media samples were performed in randomized order. MS/MS detection settings. Media samples were analyzed in positive resolution mode with a standard ESI source using the following settings. Acquisition time, 18min. Source and lockspray capillary voltage, 3kV; sampling cone, 40V; extraction cone, 4V. Temperature settings: source, 120°C; desolvation, 450°C. Desolvation gas flow-rate, 800 L/hr; cone gas flowrate, 20L/hr. 0.5 s MS survey scans were performed followed by data dependent MS/MS fragmentation of the top 10 most abundant precursor ions, scan time 0.1 s, with collision energy voltage ramps of 10-30 V for LM and 30-75 V for HM. Detector calibration was performed using a NaI solution (2 µg/µL in 2-propanol-water) to cover a 100-1200 m/z mass range resulting in typical resolution values of 16,000 (FWHM) at 200 m/z. Leucine enkepthalin solution (2ng/µL in 50:50 acetonitrile:water + 0.1% formic acid) was used as an accurate lockmass calibrant. Mass accuracy was determined at 15%) were removed prior to chemometric evaluation. Normalized compound abundance values were averaged from triplicate injections and two-tailed paired t-tests were performed to determine features which were considerably altered by HTST-treatment. Compounds with a p value < 0.05 and outside an arbitrary fold-change criteria of 0.8 and 1.2 were considered as significantly impacted. False positives were minimized by selecting for identification only compounds which were found to be repetitively impacted upon multiple HTST-treatment experiments. The MetaScope plugin in Progenesis QI was linked to the HMBD and Metlin libraries for compound identification which was performed at 95%) even after 6 days from the start of the culture study and CHO-DP12 cells revealing signs of decline after day 4 but still remaining at acceptable levels (85-90%). The culture profiles obtained indicated that the Maillard products formed in media upon HTST-treatment were either not cytotoxic towards CHO cells or their levels were not sufficiently high to disrupt standard cell metabolism activities.
CONCLUSIONS The impact of HTST-treatment on CHO culture media was evaluated. Untargeted profiling of defined and undefined for-
7
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 56 57 58 59 60
Page 8 of 9
(13) Fan, Y.; Jimenez Del Val, I.; Muller, C.; Wagtberg Sen, J.; Rasmussen, S. K.; Kontoravdi, C.; Weilguny, D.; Andersen, M. R. Biotechnol. Bioeng. 2015, 112, 521-535. (14) Cao, X.; Stimpfl, G.; Wen, Z. Q.; Frank, G.; Hunter, G. PDA J. Pharm. Sci. Technol. 2013, 67, 63-73. (15) Aranibar, N.; Reily, M. D. Methods Mol. Biol. (N. Y., NY, U. S.) 2014, 1104, 223-236. (16) Yen, S.; Sokolenko, S.; Manocha, B.; Blondeel, E. J.; Aucoin, M. G.; Patras, A.; Daynouri-Pancino, F.; Sasges, M. Biotechnol. Prog. 2014, 30, 1190-1195. (17) Calvet, A.; Ryder, A. G. Anal. Chim. Acta 2014, 840, 58-67. (18) Kirdar, A. O.; Chen, G.; Weidner, J.; Rathore, A. S. Biotechnol. Prog. 2010, 26, 527-531. (19) Li, B.; Shanahan, M.; Calvet, A.; Leister, K. J.; Ryder, A. G. The Analyst 2014, 139, 1661-1671. (20) Calvet, A.; Li, B.; Ryder, A. G. Anal. Chim. Acta 2014, 807, 111-119. (21) Luo, Y.; Pierce, K. M. Biotechnol. Prog. 2012, 28, 1061-1068. (22) Aretz, I.; Meierhofer, D. Int. J. Mol. Sci. 2016, 17. (23) Kovarikova, P.; Pasakova-Vrbatova, I.; Vavrova, A.; Stariat, J.; Klimes, J.; Simunek, T. J. Pharm. Biomed. Anal. 2013, 76, 243-251. (24) McElearney, K.; Ali, A.; Gilbert, A.; Kshirsagar, R.; Zang, L. Biotechnol. Prog. 2016, 32, 74-82. (25) Want, E. J.; Wilson, I. D.; Gika, H.; Theodoridis, G.; Plumb, R. S.; Shockcor, J.; Holmes, E.; Nicholson, J. K. Nat. Protoc. 2010, 5, 1005-1018. (26) Gupta, A. J.; Hageman, J. A.; Wierenga, P. A.; Boots, J.-W.; Gruppen, H. Process Biochem. 2014, 49, 309-317. (27) Richardson, J.; Shah, B.; Bondarenko, P. V.; Bhebe, P.; Zhang, Z.; Nicklaus, M.; Kombe, M. C. Biotechnol. Prog. 2015, 31, 522-531. (28) Zang, L.; Frenkel, R.; Simeone, J.; Lanan, M.; Byers, M.; Lyubarskaya, Y. Anal. Chem. 2011, 83, 5422-5430. (29) Mathew, A. G.; Parpia, H. A. B. In Adv. Food Res., C.O. Chichester, E. M. M.; Stewart, G. F., Eds.; Academic Press, 1971, pp 75-145. (30) Kim, K. W.; Lee, S. B. Appl. Environ. Microbiol. 2003, 69, 4325-4328. (31) Vinaixa, M.; Schymanski, E. L.; Neumann, S.; Navarro, M.; Salek, R. M.; Yanes, O. TrAC, Trends Anal. Chem. 2016, 78, 23-35. (32) Katayama, H.; Tatemichi, Y.; Nakajima, A. Food Chem. 2017, 228, 279-286. (33) Röper, H.; Röper, S.; Meyer, B. IARC Sci. Publ. 1984, 101-111. (34) Hoogenkamp, H.; Kumagai, H.; Wanasundara, J. P. D. In Sustainable Protein Sources; Academic Press: San Diego, 2017, pp 47-65. (35) Pokorný, J.; Pánek, J. In Handbook of Herbs and Spices (Second edition); Woodhead Publishing, 2012, pp 51-71. (36) Cid, M. C.; de Peña, M. P. In Encyclopedia of Food and Health; Academic Press: Oxford, 2016, pp 225-231. (37) Antony, S. M.; Han, I. Y.; Rieck, J. R.; Dawson, P. L. J. Agric. Food Chem. 2000, 48, 3985-3989. (38) Nursten, H. In The Maillard Reaction: Chemistry, Biochemistry and Implications; The Royal Society of Chemistry, 2005, pp 62-89. (39) Ho, C.-T.; Riha Iii, W. E. In The Maillard Reaction in Foods and Medicine, Nursten, H. E.; Crabbe, M. J. C.; Ames, J. M., Eds.; Woodhead Publishing, 2005, pp 187-192. (40) Tamanna, N.; Mahmood, N. Int. J. Food Sci. 2015, 2015, 6.
cells as no detrimental effects on cell culture performance were observed, suggesting that key nutrients required for cellular growth were not impacted by the heat-treatment.
ASSOCIATED CONTENT Supporting Information Additional information as described in the text which includes summary of media pH values, figures showing overlaid TIC chromatograms, chart bars with abundance values of compounds affected by HTST-treatment and volcano plots showing distribution of compounds based on HTST-treatment effects. A separate file containing relevant MS/MS annotations is included. The Supporting Information is available free of charge on the ACS Publications website. AC_HTST_untargeted_media_analysis_SI (.doc) AC_HTST_MSMS_annotations (.ppt)
AUTHOR INFORMATION Corresponding Author Email:
[email protected] Tel: +353 12158105
ACKNOWLEDGMENT The authors would like to thank Enterprise Ireland for research funding under grand number: IP/2014/0309, co-funded by the European Union through the European Regional Development Fund (ERDF) 2014-2020 programme. Special thanks to Dr. Colin Clarke for the assistance with the statistical evaluation of analytical data.
REFERENCES (1) Merten, O. W. Cytotechnology 2002, 39, 91-116. (2) Qiu, Y.; Jones, N.; Busch, M.; Pan, P.; Keegan, J.; Zhou, W.; Plavsic, M.; Hayes, M.; McPherson, J. M.; Edmunds, T.; Zhang, K.; Mattaliano, R. J. Biotechnol. Bioeng. 2013, 110, 1342-1353. (3) Moody, M.; Alves, W.; Varghese, J.; Khan, F. PDA J. Pharm. Sci. Technol. 2011, 65, 580-588. (4) Garnick, R. L. Dev. Biol. Stand. 1998, 93, 21-29. (5) Garnick, R. L. Dev. Biol. Stand. 1996, 88, 49-56. (6) Nims, R. W. Dev. Biol. (Basel, Switz.) 2006, 123, 153-164; discussion 183-197. (7) Hodgson, J. Nat. Biotechnol. 1995, 13, 333-343. (8) Gronemeyer, P.; Ditz, R.; Strube, J. Bioengineering 2014, 1, 188. (9) Lobo-Alfonso, J.; Price, P.; Jayme, D. In Protein Hydrolysates in Biotechnology, Pasupuleti, V. K.; Demain, A. L., Eds.; Springer Netherlands: Dordrecht, 2010, pp 55-78. (10) Pohlscheidt, M.; Charaniya, S.; Kulenovic, F.; Corrales, M.; Shiratori, M.; Bourret, J.; Meier, S.; Fallon, E.; Kiss, R. Appl. Microbiol. Biotechnol. 2014, 98, 2965-2971. (11) Murphy, M.; Quesada, G. M.; Chen, D. Biologicals 2011, 39, 438-443. (12) Schleh, M.; Romanowski, P.; Bhebe, P.; Zhang, L.; Chinniah, S.; Lawrence, B.; Bashiri, H.; Gaduh, A.; Rajurs, V.; Rasmussen, B.; Chuck, A.; Dehghani, H. Biotechnol. Prog. 2009, 25, 854-860.
Insert Table of Contents artwork here
8
ACS Paragon Plus Environment
Page 9 of 9
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 56 57 58 59 60
9 Environment ACS Paragon Plus
