The development and optimization of cell culture media for biotech applications is a fundamental step of process development. The composition of cell culture media requires an ideal blend of amino acids, vitamins, nucleosides, lipids, carbohydrates, trace elements and other components.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
Short Communication
Meire Ribeiro da Silva a , Izabela Zaborowska a , Sara Carillo a , Jonathan Bones a , b , ∗
a NIBRT – National Institute for Bioprocessing Research and Training, Dublin, Ireland
b School of Chemical and Bioprocess Engineering, University College Dublin, Dublin 4, Ireland
a r t i c l e i n f o
Article history:
Received 27 February 2021
Revised 1 June 2021
Accepted 4 June 2021
Available online 9 June 2021
Keywords:
Capillary electrophoresis mass spectrometry
Amino acid analysis
Spent media analysis
Cell culture
Monoclonal antibody
Upstream processing
a b s t r a c t
Thedevelopmentandoptimizationofcellculturemediaforbiotechapplicationsisafundamental step
ofprocessdevelopment.Thecompositionofcell culturemediarequiresanidealblendofamino acids, vitamins,nucleosides,lipids,carbohydrates,traceelements andothercomponents.Theabilityto mon-itortheseconstituents isrequiredto ensure thatcellsreceive sufficientnutrientsto facilitategrowth, viabilityandproductivity.Analysisofcellculturemediaischallengingduetotherangeanddiversityof compoundscontainedinthismatrixandnormallyrequirestimeconsumingmethods.Arapid,simpleand sensitivemicrofluidicchipCE-MSmethodisdescribedtomonitoraminoacidsinchemicallydefinedcell culturemediafromaChinesehamsterovarycell lineculturedoveraperiodof10days.Thedescribed platformenabledtheseparationof16aminoacidsinlessthan2minutesandwithouttherequirement forextensivesamplepreparation.Theanalyticalparametersevaluatedwereprecision,linearity,limitof detectionandlimitofquantification.Themajorityofessential aminoacidswerepresentincellculture growthinhighconcentrationscomparedtonon-essentialaminoacids.Overthecourseofthe10dayscell culturetheconcentrationofcertainaminoacidsdeclinedbyupto100%.MicrofluidicchipbasedCE-MS methodscan beusedeffectivelytoobtaintheconsumptionrates ofamino acidsincellculturemedia duringcellgrowthandtoperformat-linemonitoringandscreeningofcellculturestatus
© 2021TheAuthor(s).PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Cell culture media (CCM) is a key component of upstream
bio-processing and correct selection of an appropriate CCM is vital
to ensure optimal performance for both cell growth and
recombi-nant protein yield and quality CCM is composed of various amino
acids, vitamins, inorganic salts, nucleosides and anti-shear agents.
The quantity of each component present may affect cell growth,
specific productivity and other performance parameters Therefore,
the optimization and customisation of CCM have been extensively
studied to address nutrient demands of cell lines and to control
toxic metabolite production [1–5] Among these nutrients, amino
acids (AAs) are essential as they are the primary constituents of
proteins and intermediates in a variety of cellular metabolic
path-ways During cell growth, AAs can be both consumed from and
released into the CCM Monitoring AAs is crucial to understand
the dynamic conditions and to adjust the concentration of each
∗Corresponding author at: NIBRT – National Institute for Bioprocessing Research
and Training, Dublin, Ireland
E-mail address: Jonathan.bones@nibrt.ie (J Bones)
AA in the CCM accordingly to optimize recombinant protein yield and quality [6] The analysis of AA in CCM can be challenging due
to the potential presence of interfering compounds depending on CCM composition Additionally, AAs are typically amphoteric with significant differences in their chemical structures, different polar-ities from non-polar to highly polar and acidic to basic side chains, all these properties may affect detection and separation [7] Different approaches have been described to monitor AAs in spent media such as liquid chromatography with either optical (LC-UV or LC-fluorescence) or mass spectrometric detection (LC-MS) and gas chromatography with either flame ionisation (GC-FID)
or mass spectrometric detection (GC-MS) [8–13] The majority of these techniques include sample preparation steps such as derivati-zation, solid-phase microextraction (SPME), solid-phase extraction (SPE) and others, which increased the complexity of the method, the associated time required to perform the analysis and the risk of analyte loss during extraction and sample preparation [ 8 , 13 ] Rapid LC-MS methods have improved analysis time by avoiding derivati-zation steps, however, ion pairing reagents are often used that re-duce sensitivity and increase the risk of potential problems with quantitation due to ion suppression [ 8 , 9 ] As the ideal analytical
https://doi.org/10.1016/j.chroma.2021.462336
0021-9673/© 2021 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Trang 2approach to monitor AAs is to avoid complex sample preparation,
miniaturization of analytical devices and the use of microfluidic
techniques provides an interesting analytical option that allows
fast analysis, high sensitivity and low consumption of reagents and
sample [14–18]
Microfluidic capillary electrophoresis (CE) is an attractive
an-alytical option as it offers low consumption of background
elec-trolyte and reagents, excellent performance, fast separation and is
suited to a wide range of applications ranging from small molecule
analysis to characterisation of intact proteins [19–24] As well, CE
has been widely used for AA analysis in a variety of complex
ma-trices [25] Microfluidic chip CE technology is versatile and can be
coupled to different detection options including optical,
electro-chemical and mass spectrometric detectors [25] Microfluidic chip
CE coupled to MS operates at low flow rates, similar to nanospray
ionisation, which enables maximization of MS sensitivity However,
CE and MS coupling is not without its challenges, due to the use of
low pressure, ensuring stability of the low operational flow rates
and compatibility of components of the background electrolyte
with MS detection [26] Many applications utilizing microfluidic
chip CE-MS using electrospray ionization have highlighted the
re-duction of analysis time and the increase in sensitivity [ 22 , 27 , 28 ].
The present study focuses on the analysis of AAs in spent
cell culture media using microfluidic chip CE-MS to generate a
rapid, simple and sensitive method to monitor AAs in spent media
from IgG1 monoclonal antibody expressing Chinese Hamster Ovary
(CHO) cells over a 10 day batch culture, employing stable
isotopi-cally labelled AAs as internal standards The optimised method
proved to be simple and rapid with minimal sample preparation
required, while the use of high resolution Orbitrap mass
spectrom-etry for the detection of the amino acids provided improved
se-lectivity and quantitative capability for those AAs that were
diffi-cult to separate during the rapid CE analysis The method is
ad-vantageous and sufficiently fast to provide an at-line analysis for
the identification and quantification of AAs consumed during cell
growth to ensure that cells are maintained in an optimal
environ-ment for proliferation and production.
2 Materials and methods
2.1 Chemicals and reagents
A ZipChipTM HS chip (cat# 810-0013) and ZipChip Metabolites
Kit (cat#850-0 0 033), including the ZipChip Metabolites BGE,
sam-ple diluent and acid were obtained from 908 Devices (Boston, MA,
USA) Stable isotope labelled AAs and Metabolomics Amino Acid
Mix Solution were obtained from Cambridge Isotope Laboratories,
Inc (MSK-A2-1.2 P/N 17K-628, UK) LC-MS Optima grade water was
purchased from Fisher Scientific (Dublin, Ireland).
2.2 Cell culture and sample preparation
CHO DP-12 cells [CHO DP12, clone#1934 aIL8.92 NB 28605/14]
(ATCC® CRL12445TM) were adapted to grow in suspension in
animal-component free, chemically defined medium BalanCD CHO
Growth A (Irvine Scientific, Wicklow, Ireland) with the addition of
4 mM L-glutamine (Sigma Aldrich, Wicklow, Ireland) Cells were
cultured using batch culture conditions in triplicate Cultures were
initiated by seeding 0.3 × 106 cells/mL in 250 mL polycarbonate
Erlenmeyer flasks (Corning, Amsterdam, The Netherlands)
contain-ing 100 mL of media supplemented with 4 mM of L-glutamine
on day 0 Cells were cultivated in a shaking incubator at 37 °C, 5%
CO2 for 10 days Cell density and viability were determined daily
by trypan blue exclusion assay using a Countess automated cell
counter (Invitrogen, Carlsbad, CA, USA) Cell culture was stopped
when the cell viability dropped below 70%, which generally oc-curred after day 9 Samples were collected on a daily basis by re-moving 2 mL aliquots from the cultures, which were centrifuged
to pellet the cells and the collected supernatant was stored at 4 °C prior to analysis Prior to analysis of analytical samples, the col-lected cell culture media samples were diluted 20 times with
LC-MS grade water and spiked with 1 μL of the IS stock solution de-scribed below to yield a final concentration of 1 μM of IS Data points for the concentration of each AA were calculated from trip-licate injection of the samples obtained from each biological repli-cate ( n = 9)
2.3 Preparation of calibration standards
Stable heavy isotope-labelled AA were employed as internal standards (IS) and were prepared as standard 500 μM solutions and subsequently diluted to obtain 50 μM, 5.0 μM, 0.5 μM and 0.05 μM calibration solutions, using fresh cell culture media di-luted 1:20 v/v with LC-MS water These stock solutions were di-luted 1:10 v/v with metabolite sample diluent provided in the ZipChip metabolite kit to obtain a standard calibration curve These standards were analysed in triplicate and the peak area obtained from the base peak chromatograms were used to estimate a cali-bration factor that was used in sample concentration calculations.
2.4 Microfluidic chip CE-MS settings
All analyses were performed using a ZipChipTM device and au-tosampler from 908 Devices (Boston, MA), that was interfaced with
a Q ExactiveTM Plus hybrid quadrupole Orbitrap mass spectrome-ter (Thermo, Bremen, Germany) The mass spectrometer settings included: mass resolution 17,500 at m/z 200, 5 micro scans, an ac-quisition gain control (AGC) target 1 × 106, maximum inject time
100 ms, spray voltage 0 kV, sheath gas flow rate 2 arbitrary units (au), auxiliary and sweep gas flow rate 0 au, capillary tempera-ture at 200 °C, S-lens radio frequency (RF) level 50, with data ac-quired over a mass range of 70–500 m/z Data acquisition was accomplished through the XcaliburTM tune page, which was trig-gered by the ZipChip software The microfluidic chip CE settings were: injection load time 30 at 2 Pa (4 nL), analysis run time
3 minutes, pressure assist start time 0.5 minutes and field strength
10 0 0 V/cm.
3 Results and discussion
3.1 Rapid separation of amino acids using microfluidic chip CE-MS
Fig 1 a depicts the base peak electropherogram following injec-tion of the 50 μM of AAs standard mix solution In this instance, since the BGE is acidic, all AAs are positive charged and the re-sulting separation of the 16 AAs is based on the difference of elec-trophoretic mobility that is related to the charge and size of the analytes As expected, the amino acid migration order observed was highly charged AAs migrating first, followed by neutral AAs and then the acidic residues The 16 AAs could be separated using
a three minute method, with a separation window spanning less than two minutes and with sample requirements of 4 nL per injec-tion The method resulted in symmetrical and narrow peaks with asymmetry values ranging between 0.96 and 1.32, average asym-metry 1.07, peak width at half height ranges between 0.006 and 0.036 minutes, average peak width 0.0133 minutes and good res-olution Some of the AAs were not fully resolved within the sepa-ration window with comigrating pairs observed at migration times from 1.4 to 2.0 minutes correspond to methionine and threonine, phenylalanine and proline as showed in Fig 1 A Comigration of certain amino acids such as methionine and threonine, asparagine
Trang 30.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Time (min) 0
10
20
30
40
50
60
70
80
Lys
90
Arg
Gly
Ala
Val
Iso Leu
Ser Met/Thr Phe/Pro
Asp
(A
(B
Lys His Arg Gly Ala Val Iso/Leu Ser Met Thr Phe Pro Glu Tyr Asp
Fig 1 (a) Electropherogram of the mixture of 16 heavy labelled AAs (50 μM) used as internal standard (b) Extracted ion chromatogram of the individual heavy labelled
amino acids The figure shows the excellent resolution of the analytes in less than 2 minutes of analysis time
and proline, tyrosine and glutamic acid, glutamic acid and cysteine
are commonly described in the literature with both LC and CE
sep-aration; this behaviour can be attributed to respective pKa
val-ues and similarities in electrophoretic mobility behaviour of these
compounds [ 7 , 24 ] Despite observed co-migration, coupling of the
chip based separation with MS detection facilitated identification
and quantitation of these comigrating pairs as shown in the
ex-tracted ion electropherogram in Fig 1 b.
A concern when using microfluidic chip CE can relate to low
performance with regard to reproducibility and robustness,
how-ever, the migration time reproducibility for the AAs analysed in
this study was excellent, with determined %RSD values lower than
1.10% over 6 injections.
The microfluidic chip CE-MS approach was next applied to eval-uate samples of conditioned cell culture media, however, prior to proceeding with the analysis of a real samples, calibration curves for each amino acid were prepared L-cysteine was observed to
be unstable in the cell culture media and was easily oxidized to form cystine in aqueous solution [ 29 , 30 ] Accordingly, it was not included for further study The range of concentrations analysed in this study was overall well above the limit of detection and sen-sitivity of the instrument; however, internal standard (0.1 μM for all AAs) measurements were used to evaluate the precision of the method, which showed %RSD values < 10%, except for methionine (%RSD = 13.6), bringing the LOQ and LOD well below this concen-tration level This value is in the range of what reported before
Trang 4Fig 2 Relative abundance trends of the amino acids included in this study (a-q), when compared to viable cell density growth curve (r)
for CE analysis of AAs, using either optical or MS based detection
methods [25] The presented microfluidic chip CE-MS method
of-fers excellent sensitivity with low sample and reagent
consump-tion and enables rapid and simple method development, requiring
only dilution when compared to previously reported methods [ 8–
13 , 23 , 24 , 31 ].
3.2 Application of the developed microfluidic chip CE-MS method for monitoring AA in CCM
To demonstrate the applicability of the developed microfluidic chip CE-MS method for monitoring AAs in conditioned cell cul-ture media, IgG1 mAb producing CHO cells were cultured in shake flasks and samples were collected daily over the 10 days of batch culture Samples were diluted with the IS containing diluent and analysed by microfluidic chip CE-MS as outlined above.
Trang 5Table 1
Summary of the analysis parameters on the analysed AAs and their calibration curves
Amino Acid [M + H] + Monoisotopic mass [M + H] + Internal Standard Migration time (min) RSD (%)
Fig 3 Base peak chromatogram (BPC) of the electropherograms of spent media analysed at day 1 (a) and day 10 (b) of cellular growth
Across the 10 days of cell culture the majority of the 16 AAs
present in the chemically defined CCM used in the present study
show similar range of concentration The consumption rates of AA
in cell culture medium are influenced by the genetic composition
of cells, the cell cycle, environment and other characteristics The
consumption of AA was clearly observed from day 1 to 10 where
the concentration declined up to 87% for the AAs quantified in
this study as shown in Fig 2 In the study, for three amino acids
(asparagine, glutamine, tryptophan) it was not possible to obtain
a heavy labelled standard; as a consequence, their concentration
trends were obtained as relative abundance compared to day 1,
rather than as absolute amount in the media, and showed total
consuption of the AA in two cases ( Table 2 ).
Fast consumption rate of phenylalanine, methionine, serine,
leucine, isoleucine, valine, histidine and lysine was observed from
day 5 to day 8 suggesting that supplementation of these amino
acids is necessary using associated feeding strategies.
Limiting supply of phenylalanine, methionine, leucine, serine
and others have been noted to result in inhibition of cell growth
as they interact with key metabolic processes such as the TCA cy-cle [4] As observed in the current study from day 7 to day 9, the number of viable cells reduced associated with the observed re-duction in the levels of phenylalanine, methionine, leucine and ser-ine, leading to potential nutrient stress, as shown in Fig 2 Other non-essential AA showed a different trajectory with alanine and glutamic acid observed to increase over the 10 days of cell culture, which can be explained by reversible transamination reactions that occur as part of the TCA cycle based on interaction with other intermediates such as alpha-ketoglutarate, pyruvate and oxaloac-etate As a result the net levels of glutamic acid can be related to aspartate and if the concentration of aspartic acid in the media is
in abundance, the equilibria of the metabolic reactions are altered
to release glutamate which in turn results in interconversion and release of alanine [ 3 , 32 , 33 ].
Among the AAs quantified, arginine levels were noted to be sta-ble during the 10 days of cell culture suggesting a balance between the cellular production and consumption of this amino acid.
Trang 6Table 2
Daily values for the concentrations of the AAs included in the study during the 10 days of cell culture growth exper- iment Values correspond to the average from 9 measurements (3 biological replicates and 3 technical replicates on each samples)
Concentration (mg/L) Amino Acid Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Lysine 12.86 15.63 14.72 14.03 14.17 10.60 10.36 8.42 8.50 8.98 Arginine 32.79 36.25 34.98 34.89 33.36 31.81 31.56 28.53 28.19 33.00 Histidine 7.42 9.96 8.71 8.45 9.08 6.80 6.83 5.14 5.51 5.47 Glycine 15.21 10.21 9.12 9.01 12.60 10.20 8.45 8.47 12.13 7.85 Alanine 1.05 1.60 2.26 3.46 5.38 6.67 8.46 7.90 6.88 7.25 Valine 5.10 5.93 5.00 4.97 4.67 3.27 2.44 1.00 1.06 1.39 Isoleucine 8.01 9.50 8.34 8.30 8.04 6.30 5.08 2.95 2.96 2.84 Leucine 10.06 12.41 10.46 10.07 9.41 6.90 5.24 2.00 1.88 2.07 Serine 7.83 9.08 8.50 8.34 8.11 6.13 4.80 1.71 0.94 0.96 Threonine 9.76 12.20 10.96 10.89 11.20 8.79 8.83 6.85 6.95 7.79 Methionine 1.93 2.27 2.00 1.82 1.66 1.07 0.70 0.26 0.34 0.48 Phenylalanine 3.29 3.85 3.49 3.25 3.08 1.99 1.41 0.48 0.56 0.79 Proline 5.44 6.36 5.96 5.85 5.88 4.98 5.38 5.11 5.68 6.17 Glutamic Acid 2.80 4.02 4.42 5.31 6.65 6.67 7.76 7.70 9.43 10.88 Tyrosine 3.61 4.35 3.85 3.60 3.60 2.37 1.95 1.33 1.24 1.49 Aspartic Acid 22.14 21.26 16.70 16.77 15.76 13.97 13.94 7.11 4.82 3.56 Asparagine ∗ 100 123.4 103.5 79.4 50.0 8.8 0.2 0.0 0.0 0.0 Glutamine ∗ 100 151.8 131.0 103.8 60.5 11.2 1.6 37.2 1.8 0.0 Tryptophan ∗ 100 128.1 116.6 114.2 119.1 80.0 65.4 32.7 20.6 28.0
∗Values for these AAs were calculated as relative amount in media compared to day 1
The ability to monitor CHO cell growth when cultured in batch
mode without AAs supplementation facilitates identification of
amino acids levels throughout the duration of culture to identify
which amino acids remain at stable levels within the media and
which potentially become limiting and thereby require
supplemen-tation through feeding These experiments could contribute to
op-timization of supplementation feeds for recombinant protein
pro-ducing cell lines and suggest that some of the AA may require fine
tuning to ensure sufficient nutrient availability to promote cell
pro-liferation and protein expression.
4 Conclusions
A microfluidic chip CE-MS method was developed for the
quan-titative determination of AAs in chemical defined CCM that
en-abled quantification and excellent reproducibility The use of stable
isotope labelled amino acids as internal standards for quantitative
determination enhanced the accuracy and precision of the method
especially when applied to conditioned media samples This fast
analytical approach with low sample consumption may be
im-portant during early-stage upstream process development or
me-dia optimisation using multiplexed microbioreactors where sample
availability can be challenging The method described in this study
could be used as a rapid screening method to identify the AAs
contained in the cell culture media, identify or monitor the
con-sumption of AAs during the cell growth to support and enhance
the productivity of recombinant proteins.
Author contribution
Meire Ribeiro da Silvia: Methodology, Investigation, Formal
analysis, Writing – Original Draft; Izabela Zaborowska:
Investiga-tion; Sara Carillo: Supervision, visualization, Writing – Review &
editing; Jonathan Bones: Conceptualization, Funding acquisition,
Supervision, Writing – Review & editing
Declaration of Competing Interest
This research was part-funded by Thermo Fisher Scientific as
part of a funded collaborative agreement between NIBRT and
Thermo Fisher Scientific.
Acknowledgements
The authors gratefully acknowledge funding from Thermo Fisher Scientific as part of a funded collaborative agreement with NIBRT Ashley Bell, Erin Redman and Trent Basarsky from 908 De-vices are gratefully acknowledged for instrument access and tech-nical assistance with the ZipChip instrument used to generate the experimental data Authors acknowledge collaborative funding sup-port from Thermo Fisher Scientific and access to the Q Exactive MS instrumentation used during the current study.
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