Page 13 - Pharmaceutical M5 Biopharma


Sponsored Feature  13

Culture Media Comprehensive analysis made fast and easy

Primer eBook: Poster Presentation (WCBP Application News:
LC-MS/MS Makes Cell Culture 2018): Simultaneous Analysis of Culture
Media Analysis Fast, Easy and A Novel Cell Culture Media Supernatant of Mammalian Cells
Effective Analysis Platform for Culture Using Triple Quadrupole LC/
Process Development MS/MS

LAAN-A-LM-E077$
A novel cell culture media analysis platform for culture process development
Takashi Suzuki 1 , Kohei Yamamoto 1 , Tomonori Nozawa 1 , Tatsuya Nishio 1 , Kenichi Toyoda 1 , Tairo Ogura 2 , Yasuhiro Mito 1 , Hajime Bungo 1 , Masatoshi Takahashi 1
1 Shimadzu Corporation, Kyoto, Japan and 2 Shimadzu Scientific Instruments, Columbia, MD Application Liquid Chromatography Mass Spectrometry
Introduction Cell Culture Media Analysis Platform, C2MAP Results and Discussion News Simultaneous Analysis of Culture Supernatant of
Mammalian Cells Using Triple Quadrupole LC/MS/MS
Optimization and control of cell culture processes are essential to Cell Culture Media Analysis Platform, C2MAP, is configured from A dedicated software, C2MAP software, can control both Pluripotent stem cells (PSCs) have a feature maintaining
increase production efficiency of biopharmaceuticals. In the field of pretreatment module (C2MAP-2000), UHPLC system (Nexea-X2), pretreatment module and LC/MS/MS system, making it possible to undifferentiated state. In this experiment, C2MAP system was used to No.C106$
and triple quadrupole mass spectrometer LCMS-8060/-8050 (Fig.1).
cell therapy including regenerative medicines, enhanced control of the
the
culture
in
changes
supernatant
carry out seamless analysis and to associate the treated sample and
compare
temporal
the
culture process is also becoming important to reduce cell variability the measurement results easily because pretreatment and analysis components in undifferentiated human iPS cells and its differentiated
and improve consistency of mass production of the cells. UHPLC Nexera X2 are carried out with the common sample ID. The progress of counterparts. As a result, significant difference could be found in the Industrial fermentation for the production of biofuels or bioprocess if monitored altogether.
Comprehensive monitoring of culture supernatant components gives C2MAP-2000 pretreatment and analysis is easily confirmed (Fig. 3). time course of some compounds (Fig.5). We think these compounds biopharmaceutics requires routine monitoring of To meet the demand for comprehensive analysis of
researchers useful information for these purposes. However, current LCMS-8060 can be marker candidates for culture process management. medium conditions such as pH, dissolved gas, carbon medium component, we optimized the analytical
technologies for process monitoring are limited to measurement of pH, source (glucose) and nitrogen source (glutamine) for conditions and developed this “Method Package for
dissolving gases, and some small compounds such as glucose,
glutamine, lactate, and ammonia in culture supernatant. optimization and control of the fermentation process. Cell Culture Profiling” that can monitor relative
However, culture media also consist of various other abundance of 95 compounds listed herein. Using this
nucleic acids and other primary metabolites, which
Cell Bank biologically important compounds such as vitamins, Method Package, we demonstrated the change in
abundance of culture medium components associated
would lead to more detailed understanding of the with hybridoma growth over a period of 5 days.
Seeding pH, dO 2 , Glc/Gln/Lac/NH 3 monitoring Q List of Compounds
No. $PNQPVOE /BNF $MBTT No. $PNQPVOE /BNF $MBTT No. $PNQPVOE /BNF $MBTT
Expansion Analysis platform that can Fig.1 Overview of C2MAP system Fig.5 Biomarker screening for potential critical 1 2 2-Isopropylmalic acid *4 $BSCPIZESBUF 33 34 N-Acetylaspartic acid "NJOP BDJE "NJOP BDJE 65 66 Cytidine Cytidine monophosphate /VDMFJD BDJE /VDMFJD BDJE
N-Acetylcysteine
Gluconic acid
perform multi components Fig.3 C2MAP software 3 Glucosamine $BSCPIZESBUF 35 Ornithine "NJOP BDJE "NJOP BDJE 67 Deoxycytidine /VDMFJD BDJE /VDMFJD BDJE
Phenylalanine
Sucrose
analysis of culture sup. is process parameters 4 5 Hexose (Glucose) $BSCPIZESBUF $BSCPIZESBUF 37 36 Oxidized glutathione "NJOP BDJE 69 68 Guanosine Guanine /VDMFJD BDJE
Hypoxanthine
culture supernatant (400 to 500 mL) are set into the sample rack of
Product necessary. After removal of the cells from culture fluid, vials containing cell Fetal bovine serum (FBS) often affect cell growth. In this experiment, 6 7 Threonic acid 2-Aminoadipic acid $BSCPIZESBUF "NJOP BDJE 38 39 Pipecolic acid Proline "NJOP BDJE "NJOP BDJE 70 71 Guanosine monophosphate /VDMFJD BDJE /VDMFJD BDJE
C2MAP-2000 (Max. 65 samples). Pretreatment and measurement detection of component amount variation among the product lots was 8 4-Aminobutyric acid "NJOP BDJE 40 Serine "NJOP BDJE 72 Inosine /VDMFJD BDJE
We have developed a “Cell Culture Media Analysis Platform, C2MAP flow of C2MAP system are shown in Figure 2. Temporal changes in each component can be graphed with the tested. Three different lots of FBS were analyzed by C2MAP system. 9 10 4-Hydroxyproline "NJOP BDJE "NJOP BDJE 41 42 Threonine Tryptophan "NJOP BDJE "NJOP BDJE 73 74 Thymidine Thymine /VDMFJD BDJE /VDMFJD BDJE
We could detect 56 compounds from FBS sample. Overall pattern of
5-Glutamylcysteine
system” that combines automated pretreatment module for culture dedicated viewer software, C2MAP TRENDS, using LC/MS/MS data mass chromatogram from each lot was similar, whereas significant 11 5-Oxoproline "NJOP BDJE 43 Tyrosine "NJOP BDJE 75 Uracil /VDMFJD BDJE
supernatant samples with LC/MS/MS. This system can perform C2MAP-2000 Filter aging secreted metabolites during cultivation, as well as display graphs of differences were detected in some compounds (Fig.6). 12 Alanine "NJOP BDJE 44 Valine "NJOP BDJE 76 Uric acid /VDMFJD BDJE
set. Analysts can monitor variations in basal media components and
automated sample pretreatment and simultaneous analysis of up to component comparisons with samples from different culture series. 13 Alanyl-glutamine "NJOP BDJE 45 4-Aminobenzoic acid 7JUBNJO 77 Uridine /VDMFJD BDJE
95 compounds including basal medium components and secreted Addition of internal std. (Reagent probe) These observations can provide useful insights into considerations 14 Arginine "NJOP BDJE 46 Ascorbic acid 7JUBNJO 78 Xanthine /VDMFJD BDJE
metabolites (The list of target compounds are shown below). This Addition of culture sup. (Sample probe) of the optimal culture conditions and the culture process. Lot 1 15 Asparagine "NJOP BDJE 47 Ascorbic acid 2-phosphate 7JUBNJO 79 Xanthosine /VDMFJD BDJE
system contains a software that can visualize temporal change in Lot 2 16 Aspartic acid "NJOP BDJE 48 Biotin 7JUBNJO 7JUBNJO 80 Penicillin G 0UIFS "OUJCJPUJDT
each culture supernatant components through the cell culture. Addition of organic solvent (Reagent probe) Lot 3 17 18 19 Cystathionine Citrulline Cysteine "NJOP BDJE "NJOP BDJE "NJOP BDJE 50 49 51 Cyanocobalamin 7JUBNJO 7JUBNJO 82 81 83 2-Ketoisovaleric acid 0UIFS
Choline
2-Aminoethanol
3-Methyl-2-oxovaleric acid 0UIFS
Ergocalciferol
In this poster, we present features of C2MAP system and its Stirring 20 Cystine "NJOP BDJE 52 Folic acid 7JUBNJO 84 4-Hydroxyphenyllactic acid 0UIFS
applications. 21 Glutamic acid "NJOP BDJE 53 Folinic acid 7JUBNJO 85 Citric acid 0UIFS
Suction filtration 22 Glutamine "NJOP BDJE 54 Lipoic acid 7JUBNJO 86 Ethylenediamine 0UIFS
Delivery of filtrate to the HPLC autosampler 23 24 Glutathione Glycine "NJOP BDJE "NJOP BDJE 55 56 Niacinamide Nicotinic acid 7JUBNJO 7JUBNJO 87 88 Fumaric acid Glyceric acid 0UIFS 0UIFS
1
1
3
3
2
2
1
2
1
25 Glycyl-glutamine "NJOP BDJE 57 Pantothenic acid 7JUBNJO 89 Histamine 0UIFS
3
2
3
Autosampler 26 27 Histidine Isoleucine "NJOP BDJE "NJOP BDJE 58 59 Pyridoxal Pyridoxine 7JUBNJO 7JUBNJO 90 91 Isocitric acid Lactic acid 0UIFS 0UIFS
SIL-30AC 28 Kynurenine "NJOP BDJE 60 Riboflavin 7JUBNJO 92 Malic acid 0UIFS
Dispensing into 96 well MTP 29 31 30 Leucine Methionine Lysine "NJOP BDJE "NJOP BDJE "NJOP BDJE 61 62 63 Tocopherol acetate /VDMFJD BDJE 7JUBNJO /VDMFJD BDJE 93 94 95 O-Phosphoethanolamine 0UIFS 0UIFS 0UIFS
Pyruvic acid
Putrescine
Adenine
Adenosine
Dilution by pure water 1 Fig.6 Evaluation of lot to lot variation of FBS 3 1 2 3 32 Methionine sulfoxide "NJOP BDJE 64 Adenosine monophosphate /VDMFJD BDJE 96 Succinic acid 0UIFS
2
1
3
2
1
3
2
Sample injection HPLC Conditions MS Conditions (LCMS-8050)
Conclusion
Ionization
: 53 &ROXPQ
: 0.1 % Formic Acid in Acetonitrile
Mobile Phase B
LCMS-8060 Through multicomponent monitoring of the culture supernatant using Column Mobile Phase A : 0.1 % Formic Acid aq. Nebulizer Gas Flow : 3.0 L/min. : ESI (Positive / Negative)
: 10.0 L/min.
Drying Gas Flow
Heating Gas Flow
: *UDGLHQW HOXWLRQ
: 10.0 L/min.
0RGH
Measurement of relative abundance of C2MAP system, various useful information can be obtained. This Flowrate : 0.35 mL/min. DL Temp. Block Heater Temp. : 400 °C : 250 °C
95 compounds in 17 min information provides useful insights into optimization of the culture Interface Temp. : 300 °C
media composition and the culture process.
Fig.4 C2MAP TRENDS
Fig.2 Pretreatment and measurement flow Disclaimer: C2MAP, other products, and applications in this presentation are intended for
research Use Only (RUO). Not for use in diagnostic procedures.
Poster Presentation (ASMS 2017): Poster (ASMS 2018): Application News:
Using LC-MS/MS to Non-invasive LC-MS/MS analysis A Compilation on the
Simultaneously Determine 95 for evaluation of undifferentiated Application of Cell Culture
Compounds in Mammalian Cell state of human iPS cells Supernatant and Medium
Culture Supernatants Component Analysis
WCBP2018 P-219-W
Bioanalytical Platform for Monoclonal Antibodies at ng/mL Concentrations Using Microflow LC-MS/MS
in Combination with nSMOL Proteolysis
Masateru Oguri 1 , Wataru Fukui 1 , Shinya Imamura 1 , Noriko Iwamoto 2 , Takashi Shimada 2 ,Toshiya Matsubara 1 , Atsuhiko Toyama 3 , Kyoko Watanabe 1 , Masahide Gunji 1 , Youske Iwata 1 , Kazuo Mukaibatake 1 , and Ichiro Hirano 1
1 Analytical & Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan. 2 Technology Research Laboratory, Shimadzu Corporation, Tokyo, Japan. 3 Marketing Innovation Center, Shimadzu Corporation
1. Introduction 2-2. System Configuration 3. Results
The newly developed micro-LC system by Shimadzu Corporation, Nexera Mikros (Fig. 2), consists of
increasingly used for pharmacokinetic studies in the preclinical, clinical, and therapeutic phases. One major (1) LC-Mikros, the ‘micro to semi-micro flow’ pump with 1-500 μL/min range and 800 bar pressure tolerance, Calibration curve in plasma matrix showed good linear response in the range 7.6 ng/mL to 62.5 μg/mL (Fig 4).
Mass spectrometric (LC-MS/MS) determination of therapeutic monoclonal antibodies in serum or plasma is
LCMS-8060), switching to the Nexera Mikros system contributed to sensitivity improvement by nearly one order of
advantage of this approach over conventional ligand binding assay (LBA) is high specificity for the target (2) CTO-Mikros, the new-design column oven that couples any analytical column directly to the ion source by Compared to the LLOQ of 0.06 μg/mL as previously reported for Trastuzumab (also using nSMOL proteolysis and
the UF-Link™ technology (Fig. 3) to minimize post-column void volume,
antibodies that can be achieved by selecting tryptic peptides derived from the complementarity-determining (3) Micro ESI-8060, the camera-equipped and X-Y adjustable ESI ion source for maximum ionization magnitude. Notably, the chromatographic peak shape and elution band was equivalent to UHPLC system with
region (CDR) as the antibody signature peptide and subjecting it to LC-MS/MS quantitation. Moreover, LC- efficiency and usability. average W 0.5h of 3.7 seconds, most likely due to near-zero post-column dead volume achieved by the UF-Link.
MS/MS approach requires much less assay developmental work than LBA, which completes within days rather 1 0 0
than several months (Table 1). Our recent advancement of sample preparation strategy, namely nano-surface
and molecular-orientation limited (nSMOL) proteolysis (Fig 1), have further simplified the method [LC] Nexera Mikros Analytical Column : Shim-Pack MC C18 (0.175 mm I.D. x 50 mm L.) Peptide MRM transition Objectives
development process. nSMOL proteolysis yields extremely clean CDR peptide mixture thereby alleviating the
need to address interference from biological matrix. Trap column : CERI L-column2 Micro (0.3 mm I.D. x 50 mm L.) 1 0 IYPTNGYTR 542.8>808.4 (y7+) 542.8>404.7 (y7++) Qualifier Quantifier
: (Analytical) 50 deg.C, (Trap) 40 deg.C
Solvent A Oven Temp. : 0.1% Formic Acid in water R 2 =0.9998 542.8>610.3 (y5+) Qualifier
Solvent B : 0.1% Formic Acid in Acetonitrile 1 Peptide MRM transition Objectives
: 0.00-0.50 min 5%B  4.50 min 22%B
Gradient
 4.51 min 95%B  5.50 min 95%B C a lc u la te d C o n c . [u g/ m l] 512.1>292.3 (b3+) Quantifier
Inj. Volume Analytical flow Rate  5.60 min 5%B  11.00 min STOP 0 .1 0 .0 1 P 14 R (IS) 512.1>660.4 (b6+) 512.1>389.3 (b4+) Qualifier Qualifier
: 10 μL
: 4 μL/min
Ionization
[MS] LCMS-8060 with Micro ESI-8060 : ESI Positive 0 .0 1 S e t c o n c . [u g/ m l] 1 1 0 1 0 0 宔宸室宱宷宬宷室宷宬宲宱季宵室宱宪宨季宬宱季宫宸宰室宱季害宯室家宰室季孽 孳孱孳孳孺孹孶 宷宲季孹孵孱學季廘宪孲宰宏
宄容宨宵室宪宨宧季室宦宦宸宵室宦宼季孽季孴孳孴季孨
0 .1
DL Temp. : 250 deg.C
ESI Temp. Heat Block Temp. : 400 deg.C : 100 deg.C 宅宯室宱宮 孳孱孳孳孺孹孶季廘宪孲宰宏 孳孱孳孹孴孳季廘宪孲宰宏 孶孱孼孴季廘宪孲宰宏
Nebulizer Gas Drying Gas : 2 L/min. : OFF 800 700 800 700 3000 2500 150000 125000
Heating Gas : 3 L/min. 500 600 500 600 2000 75000 100000
Figure 2. Nexera Mikros system, equipped with additional modular pumps for Trap & Elute 300 400 300 400 1000 1500 50000
Figure 1. The working principle of Fab-selective reaction of IgG by nSMOL proteolysis. 100 200 0 2.75 3.00 3.25 3.50 100 200 0 2.75 3.00 3.25 3.50 500 0 2.75 3.00 3.25 3.50 25000 0 2.75 3.00 3.25 3.50
Figure 4. Calibration curve for Trastuzumab bioanalysis and representative MRM chromatograms
Despite various advantages, one drawback of LC-MS/MS assays is that the level of sensitivity depends on the
mass spectrometric response (efficiency of ionization and fragmentation) of the signature peptide, which is
essentially unpredictable and uncontrollable. For example, recently reported bioanalyses of therapeutic mAb As part of assay validation, intra-day repeatability (%RSD) was evaluated using two sets of QC samples. The
[ref.1-8] showed varying LLOQ levels ranging from 0.06-0.58 μg/mL in plasma. Currently there is risk that a newly- results are shown in Table 2. Good repeatability was observed (<20% for LLOQ and otherwise well under 15%)
developed assay might not fulfil the sensitivity requirement for pre-clinical trials. and accuracies fell under 85-115% range, which are the commonly accepted criteria for quantitative adequacy
Here we aim to overcome this issue by implementation of a robust microflow LC-MS/MS system to measure from FDA Bioanalytical Method Validation.
signature peptides at increased sensitivity than conventional semi-microflow systems, while maintaining the UF-Link TM Easy, Dependable Column Installation Table 2. Results of assay repeatability evaluation using QC samples.
same level of robustness, analysis turnaround time and ease of system configuration.
宖宨宷季宦宲宱宦孱季 宔宆季家宨宷季孴季孫宑宀學季宩宲宵季宨室宦宫季宯宨容宨宯孬 宔宆季家宨宷季孵季孫宑宀學季宩宲宵季宨室宦宫季宯宨容宨宯孬
Table 1. Comparison of nSMOL+LCMS and LBA Standard Ferrule Seal 孫廘宪孲宰宏孬 孳孱孳孳孺孹孶 宇宨宷宨宵宰宬宱宨宧季 孳孱孳孳孺孷孴 宄宦宦宸宵室宦宼 孼孺孱孴孨 宕宨害宨室宷室宥宬宯宬宷宼 學孱孹孼孨 宇宨宷宨宵宰宬宱宨宧 孳孱孳孳孺孹孵 宄宦宦宸宵室宦宼 孴孳孳孨 宕宨害宨室宷室宥宬宯宬宷宼 孴孴孱孶孨
宱宖宐宒宏孮宏宆宐宖 宏宅宄 Development of LCMS bioanalysis in Zero Dead Volume Seal (UF-Link TM ) 孳孱孳孵孵孼 孳孱孳孵孶孷 孴孳孵孨 孹孱孹孻孨 孳孱孳孵孶孵 孴孳孴孨 孵孱孻孷孨
宄宥 宩宲宵季 宑宲宷季宱宨宨宧宨宧 孹孮孮季宰宲宱宷宫家季 combination with nSMOL proteolysis Ferrule Male nut Adapter 學孱孻孹 孹孱孴孼 孴孳孹孨 孵孱孹孺孨 學孱孻孶 孼孼孱孷孨 孶孱孴孵孨
is much faster, and can dramatically
宆宲宯宯宨宦宷宬宲宱孲宇宨宷宨宦宷宬宲宱 宆宵宲家家孰宵宨室宦宷宬容宬宷宼季宷宨家宷 宑宲宷季宱宨宨宧宨宧 宐室宱宧室宷宲宵宼季室宱宧季宷宵宬宦宮宼 宷宲季宧宨容宨宯宲害 accelerate the total R&D workflow 學孳孱孳 孷孹孱孼 孼孷孨 孹孱孶孹孨 孷學孱孻 孼孴孱孺孨 孺孱孵孶孨
宓宵宨孰容室宯宬宧室宷宬宲宱 孴 孰 孶季宧室宼家 孵 孰 孶季宺宨宨宮家 period of biologics by alleviating the
安宸宯宯 容室宯宬宧室宷宬宲宱 宖室宰害宯宨 害宵宨害孱 孶 孰 孷季宺 孶季孰 學季宫 孶季孰 孷季宺 孵季孰 孷季宫 bottleneck that typically occur when 4. Conclusion
 Combination of Nexera Mikros TM and nSMOL TM Antibody BA Kit achieved single digit ng/mL
entering the preclinical and clinical
宋宬宪宫宯宼 家宨宯宨宦宷宬容宨季室宱宧季宵宨宯宬室宥宯宨孯 宋宬宪宫宯宼季宧宨害宨宱宧宨宱宷季宲宱季宴宸室宯宬宷宼季 phase. Dead volume Sealing part Sealing part LLOQ in the bioassay of Trastuzumab in 11 minutes of analysis runtime.
宺宬宧宨季宧宼宱室宰宬宦季宵室宱宪宨孯
宨室家宼季宷宲季宰宸宯宷宬害宯宨宻孯
宇室宷室季宩宨室宷宸宵宨家 宬宱宧宨害宨宱宧宨宱宷季宲宩季室宱宷宬宥宲宧宬宨家 宲宩季宧宨宷宨宦宷宬宲宱季宄宥孱季
rate, while peak shape was maintained by the UF-Link column connection at ion source. The
2. Methods Connection Procedure  Enhancement in sensitivity may be attributed to increased ionization efficiency at lower flow
system is also suitable for routine analysis without the use of specialized tubings that
2-1 Sample and Pretreatment typically suffer from clogging.
Pooled human plasma sample was purchased from Kohjin Bio (Saitama, Japan). Trastuzumab was spiked at  Assuming same level of sensitivity enhancement for other signature peptides of therapeutic
various concentrations (0, 0.00763, 0.0153, 0.0305, 0.0610, 0.122, 0.0244, 0.488, 0.977, 1.95, 3.91, 7.81, 15.6, mAbs, it now became highly probably that a developed LC-MS/MS assay will satisfy the
31.3, 62.5 μg/mL) for calibration curve and independently at four concentration set for QC samples. QC set 1 1. Attach the adapter to column. 2. Place the column in the UF- 3. Swing the lever to the sensitivity required for both preclinical and clinical studies.
and 2 were prepared and ran on two separate days. Standard threads on the adapter link slot inside the oven. right to connect & lock.
Spiked and blank plasma samples were pretreated after keeping at -80°C for 24 h or longer using the variety of columns. make it compatible with a wide Acknowledgement References
nSMOL™ Antibody BA Kit (Shimadzu Corporation, Japan) in accordance with the instruction manual.
Figure 3. The sealing mechanism of UF-Link and its facile attachment The applications of nSMOL™ Antibody BA Kit in this I. II. Iwamoto N et al, Analyst. 2014 139(3):576-80
Iwamoto N et al., Anal. Methods. 2015; 21:9177-9183.
presentation are the results of cooperative research between
Shimadzu and National Cancer Center Japan. We are deeply III. Iwamoto N et al., Drug Metab. Pharmacokinet. 2016 31(1): 46-50
Iwamoto N et al., Biol. Pharm. Bull. 2016 39(7): 1187-94
VII. Iwamoto N et al., Clin. Pharmacol. Biopharm. 2016, 5-4
Disclaimer : nSMOL™ Antibody BA Kit, Nexera Mikros TM and LCMS-8060 is intended for Research Use Only (RUO). Not for use in diagnostic procedures. grateful for providing us with the data and kind supports. V. IV. Iwamoto N et al., J Chromatogr. B 2016 1023-24:9-16
VI. Iwamoto N et al., Bioanalysis, 2016 8(10):1009-20
VIII. Iwamoto N et al., J Pharm. Biomed. Anal. 2017, 145: 33-39
Video: Video:
C2MAP Cell Culture Media Advancing Cell Culture Media
Analysis Platform Analysis and Monitoring by LC-
MS/MS
www.shimadzu.com