Chemical Identity and Mechanism of Action and Formation of a Cell Growth Inhibitory Compound from Polycarbonate Flasks

Article abstract of DOI:10.1021/acs.analchem.7b05102

This paper reports the chemical identity and mechanism of action and formation of a cell growth inhibitory compound leached from some single-use Erlenmeyer polycarbonate shaker flasks under routine cell culture conditions. Single-use cell culture vessels

Full text of DOI:10.1021/acs.analchem.7b05102

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Chemical identity and mechanism of action and formation of
a cell growth inhibitory compound from polycarbonate flasks
Jinlin Peng, Yaopeng Zhao, Yulong Hong, Robert S Burkhalter, Carrie L Hogue, Elizabeth Tran,
Lai Wei, Lori E. Romeo, Paula Dolley-Sonneville, Zara Melkoumian, Xinmiao Liang, and Ye Fang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05102 • Publication Date (Web): 08 Mar 2018
Downloaded from http://pubs.acs.org on March 8, 2018
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Analytical Chemistry
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Chemical identity and mechanism of action and formation of a
cell growth inhibitory compound from polycarbonate flasks
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Jinlin Peng,1 Yaopeng Zhao,2 Yulong Hong,1 Robert S. Burkhalter,3 Carrie L. Hogue,3 Elizabeth Tran,1
Lai Wei,2 Lori Romeo,4 Paula DolleyꢀSonneville,4 Zara Melkoumian,4 ,* Xinmiao Liang,2,* Ye Fang1,*
1Biochemical Technologies, Corning R&D Corporation, Corning Incorporated, Corning, NY 14831, USA
2Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian, Liaoning, China
3Characterization Sciences, Corning R&D Corporation, Corning Incorporated, Corning, NY 14831, USA
4Corning Life Sciences Development, Corning R&D Corporation, Corning Incorporated, Corning, NY 14831, USA
*
Corresponding authors: Z.M., Eꢀmail: MelkoumiZ@corning.com; Tel: +1ꢀ607ꢀ974ꢀ0455; X.L.: Email:
liangxm@dicp.ac.cn; Tel: +86ꢀ41ꢀ184379519; Y.F., Email: fangy2@corning.com; Tel: +1ꢀ607ꢀ974ꢀ7203.
1
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ABSTRACT: This paper reports the chemical identity and mechanism of action and formation of a cell growth inhibitory
compound leached from some singleꢀuse Erlenmeyer polycarbonate shaker flasks under routine cell culture conditions. Singleꢀuse
cell culture vessels have been increasingly used for the production of biopharmaceuticals; however, they often suffer from issues
associated with leachables that may interfere with cell growth and protein stability. Here, high performance liquid chromatography
preparations and cell proliferation assays led to identification of a compound from the water extracts of some polycarbonate flasks,
which exhibited sublineꢀ and seeding densityꢀdependent growth inhibition of CHO cells in suspension culture. Mass spectroscopy,
nuclear magnetic resonance spectroscopy, and chemical synthesis confirmed that this compound is 3,5ꢀdinitroꢀbisphenol A. Cell
cycle analysis suggests that 3,5ꢀdinitroꢀbisphenol A arrests CHOꢀS cells at the G1/Go phase. Dynamic mass redistribution assays
showed that 3,5ꢀdinitroꢀbisphenol A is a weak GPR35 agonist. Analysis of the flask manufacturing process suggests that 3,5ꢀ
dinitroꢀbisphenol A is formed via the combination of molding process with γꢀsterilization. This is the first report of a cell
culture/assay interfering leachable compound that is formed through γꢀirradiationꢀmediated nitric oxide free radical reaction.
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KEYWORDS: Leachables, polycarbonate, plastic labware,
3,5-dinitro-bisphenol A, cell cycle arrest
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Singleꢀuse plasticware is widely used in life sciences
butylphenyl)phosphate, a γꢀirradiationꢀinduced degradation
product of the antioxidant tris(2,4ꢀdiꢀtertꢀbutylphenyl)ꢀ
phosphite (branded as Irgafos 168®), was found to be a
leachable from some polyethyleneꢀfilm based singleꢀuse
bioreactors (SUBs) that can inhibit the growth of several
Chinese hamster ovary (CHO) cell lines in suspension
culture.14,15 A variety of CHO sublines have been widely used
as the host to produce recombinant proteins and antibodies for
therapeutic applications.19,20 Preꢀsterilized plastic SUBs have
been widely used for bioprocessing due to advantages
associated with reduced capital expenditure, increasing
manufacturing flexibility, and removal of costly cleanꢀ
sterilization steps between production runs.21,22
research, drug discovery, bioprocessing, and the storage of
consumer and medical products. However, when being
contacted with solutions, plastic labware can leach out trace
amount of contaminant chemicals, some of which may
interfere with biological testing results in an assay specific
manner,1ꢀ8 compromise the quality of consumer and medical
products,9ꢀ13 or cause the growth inhibition of cells during
bioprocessing14,15
.
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For singleꢀuse labware, leachables are substances that
migrate spontaneously into solution under routine conditions
of use and storage, while extractables are substances generated
under exaggerated temperature and time conditions in the
presence of an appropriate solvent.16 To date, two classes of
resultsꢀinterfering leachables have been identified. The first
type of bioactive leachables is plastic additives, chemicals that
are used to polymerize, process, or modify endꢀuse properties
of plastics. Common additives include antioxidants,
antiozonants, biocides, slip agents, and stabilizers. Several
additives have been found to leach out of specific disposable
plastic labware under routine conditions of usage and storage,
and contaminate assay results or products. The biocide di(2ꢀ
hydroxyethyl)methyldodecylammonium and the slip agent
oleamide both were identified as leachables from certain brand
polypropylene plastic tubes to be selective inhibitors of human
monoamine oxidaseꢀB (hMAOꢀB).1 Oleamide is also a known
endogenous signaling molecule that binds to several receptors
and ion channels.17,18 Erucamide, another slip agent, was found
as a leachable from specific lots of polypropylene pipette tips
during routine compound management liquid handling
processes to be an agonist of a G protein couple receptor
(GPCR) for fatty acids.2 An unknown leachable from specific
batches of plastic centrifuge tubes used to store compounds
was found to inhibit the tyrosine kinase Abl1.3 Several
Tergitol nonylphenol ethoxylate series surfactants were also
identified as leachables from some polypropylene 1ꢀmL
pipette tips to inhibit the mitochondrial respiratory chain
complex I, causing false patient diagnostic results.4 Chemical
PC Erlenmeyer flasks are one of the leading vessels widely
used for process/media optimization and seedꢀtrain scaleꢀup
culture in bioprocessing.23 Given that the sensitivity of cell
growth to leachables is strongly dependent on cell types and
culture conditions, we speculate that like some SUBs PC
flasks may also leach out cell growth inhibitory compound(s).
Here, we report a novel type of bioactive leachables from
some Erlenmeyer PC shaker flasks that are formed through a
γꢀirradiationꢀmediated free radical reaction. Specifically, 3,5ꢀ
dinitroꢀbisphenol A (3,5ꢀdinitroꢀBPA) was identified as a
leachable from these flasks, which arrests CHOꢀS cells at the
G1/Go phase and is also a weak agonist for the GPR35. We
also describe a strategy that combines multiple techniques
including bioassays and routine analytical tools to provide
orthogonal confirmation of the bioactive leachables identified.
EXPERIMENTAL SECTION
Materials. BPA, 3,3’ꢀdinitroꢀBPA, 3,3’,5,5’ꢀtetraꢀnitroꢀ
BPA, 4ꢀtertꢀbutylꢀ2,6ꢀdinitrophenol, 3,5ꢀdinitrophenol, 4ꢀ
chloroꢀ2,6ꢀdinitrophenol, 4ꢀtertꢀbutylꢀ2,6ꢀdinitroanisole, and
2,6ꢀdinitrohydroquinone were obtained from Sigma (St. Louis,
MO, USA). MLꢀ145 and zaprinast were obtained from Tocris
Chemical Co. (St. Louis, MO, USA). CHOꢀ5/9α cell line was
obtained from American Type Cell Culture (ATCC; Manassas,
VA, USA). FreeStyle™ CHOꢀS cell line, CD OptiCHO
medium, FxCycle™ PI/RNase staining solution, and USB®
Corp 4% para formaldehyde dolution were purchased from
Thermo Fisher (Boston, MA, USA). BD PharmigenTM staining
buffer was obtained from BD Biosciences (San Jose, CA,
USA).
leachables from disposable labware may represent
a
widespread problem for DNA and protein quantification
assays by interference with spectrophotometric5 or mass
spectroscopy (MS) based proteomics analysis6, or in some
cases directly inhibiting polymerase chain reaction7.
Antimony, a stabilizer and also a catalyst for the production of
polyethylene terephthalate (PET), can also leach into drinking
water from PET containers, causing potential environmental
contamination.9
Water extraction protocol. 2L Erlenmeyer PC shaker
flasks from Corning or ThermoFisher were incubated with 400
mL deionized water at 37 oC on an orbital shaker at 90
revolutions per minute (rpm) for 3 days. The water extraction
solution was then concentrated down to ~2 mL using a Rotaꢀ
The second type of bioactive leachables is certain
degradation products of plastic polymers and additives.
Leaching of bisphenol A (BPA) into foods and drinking water
from polycarbonate (PC) containers has generated great public
attention as BPA is an endocrine disrupter.10 Longꢀterm, lowꢀ
level exposure to endocrineꢀdisrupting chemicals such as BPA
and polybrominated diphenyl ether flame retardants was
estimated to cost the U.S. $340 billion in annual health care
spending and lost wages.11 The antioxidant dodecyl 3ꢀ(3ꢀ
dodecoxyꢀ3ꢀoxopropyl)ꢀsulfanylpropanoate and its two
degradation products, dodecanꢀ1ꢀol and dodecanoic acid, were
found as leachables from some plastic microplates to be
selective hMAOꢀB inhibitors.8 Recently, bis(2,4ꢀdiꢀtertꢀ
o
Vapor at 50 C. For PC resins, 20 g raw resin was placed
inside a glass vial and γꢀirradiated. Both raw and γꢀirradiated
resins were put in a 250 ml glass beaker, followed by
o
extraction with 100 mL water at 37 C on an orbital shaker at
90 rpm for 3 days. The collected water solution was then
concentrated down to 1 mL for further analysis. For pure BPA
monomer coated glass flasks, 5 mL of 0.5 mg/ml BPA in
methanol was used to coat a 250 mL glass flask by rotating it
for 10 min. After removal of excess solution, the coated bottle
was dried in air and γꢀirradiated. After γꢀirradiation, 5 mL
acetonitrile was added. The resultant acetonitrile solution was
then analyzed directly with high performance liquid
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first established. After compound addition, the cellular
chromatography (HPLC)/MS. All γꢀirradiation treatments
responses were recorded immediately. For antagonist
inhibition assays, cells were initially treated with ML145 for 1
hr, followed by stimulation with the agonist at a fixed dose.
The data represent mean ± std from two independent
measurements, each with four replicates (n = 8).
were performed at 18–36 KiloGray (kGy).
Cell culture. Suspension culture was routinely maintained
using an Infors Multitron Pro 25 mm orbital shaker at 37 °C, 8
% CO2, and 85 % humidity in 1X CD OptiCHO™ medium
supplemented with 8 mM Corning glutagro™ (Corning) and
Pyrolysis sample preparation. Commercial 2L PC
flasks were cut into several pieces, which were then placed
into a 250 mL glass flask. 100 mL dichloromethane was added
to the glass flask to completely dissolve the PC pieces. The
resulted solution was poured into approximately 400 mL
methanol (used as an antiꢀsolvent) to precipitate out the
polymers. After filtration, the remaining solution was
concentrated to dryness and then reꢀdissolved in methanol.
The final pyrolysis sample solution was used for subsequent
HPLC/MS analysis.
1X hypoxanthine and thymidine using
a
protocol
recommended by the medium manufacturer. The shaking
speed was set to be 130 rpm for flasks of various sizes from
different vendors, or 225 rpm for 24 well deepwell plates (24
DW, Corning). The orbital diameter of the shaking platform
was 15 mm and 50 mm for the flasks and the 24 DW plates,
respectively. The culture volume was 2.5 mL for 24 DW
plates. To study the effects of the water extracts of the flasks
on cell growth, aliquots of the 200x concentrated water
extracts from the flasks were added to the culture media in
24DW cell culture plates. To determine the IC50 values of
different chemicals, aliquots of serial dilutions of testing
chemicals were added to CHO 24DW cell culture. The seeding
density was 0.15 x 106 cells/mL for culture in the deepwell
plate, but was variable for flasks of different size. The relative
percentage of cell growth under a specific condition was
calculated by normalizing to the dimethyl sulfoxide (DMSO)
control. The IC50 values were analyzed with a nonꢀlinear
regression method using GraphPad Prim 5 software.
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HPLC/MS analysis. HPLC was performed on an Acquity
UPLC H class system (Waters Corp.) with PDA and QDA
detectors using a Waters BEH C8, 1.7 µm, 2.1 X 100 mm
column and eluted with an acetonitrile/water reverse phase
system. Mobile phase A was 30ꢀ80% (v) acetonitrile/0.1% (v)
TFA; while B was 80% ꢀ100% (v) acetonitrile/0.1% (v) TFA,
C was 100% (v) acetonitrile/0.1% (v) TFA. The gradient of
separations was generally A (0ꢀ4 min), B (4ꢀ5.5 min), and C
(5.5ꢀ9 min), unless specifically mentioned. Flow rate was 0.5
mL/min. UV detector at 254, 280, or 360 nm was used to
separate leachable molecules in the extract samples. Individual
components were given structural assignments based on both
retention time and molecular weight data obtained from the
negative ion mass spectrum of the peak, in comparison to
standard compounds when available.
Cell viability analysis. The number and viability of cells
were monitored daily using trypan blue exclusion assay and a
ViꢀCell™ cell viability analyzer (Beckman Coulter).
Cell cycle analysis. CHOꢀS cells were cultured in 125
mL shaker flasks with an initial seeding density of 0.15 x 106
cells/mL in the presence or absence of 3 µM 3,5ꢀdinitroꢀBPA.
The number and viability of cells were monitored daily. At
different time points, 106 cells were collected and then pelleted
by centrifugation at 300x g for 5 min. The cells were washed
once with Dulbecco’s phosphate buffered solution, and then
fixed by incubating with 2% para formaldehyde solution for
10 min at room temperature. The cells were then pelleted
again and the fixative solution was removed. The cells were
reꢀsuspended in the staining buffer from FxCycle™ PI/RNase
staining kit according to the manufacturer instruction. The
stained cells were then analyzed using a BD FACS Calibur
Flow Cytometer (BD Biosciences). 30,000 events were
collected for each sample at FL2 channel for propidium iodide
fluorescence (linear, 532 nm excitation and 584 nm emission).
The percentages of cells at G1/G0, S, and G2/M phases were
analyzed using the BD Cell Quest Pro software.
High-resolution MS analysis. Highꢀresolution MS was
carried out at the Chemical Core facility of Cornell University.
In addition, direct infusion electrospray ionization mass
spectrometry on a Thermo LCQ Deca XP mass spectrometer
operating in both positive and negative ion modes was also
used. Relative concentrations were determined by the ion
current value in nA of a given species and comparing to
another reference peak from the same sample. For
quantification, a calibration curve for 3,5ꢀdinitroꢀBPA was
established in the negative mode of ionization on the
synthesized compound of known concentration over a range of
3 orders of magnitude. Furthermore, the tandem MS was
performed using an Orbitrap Elite mass spectrometer under
high duty cycle mode at 180 volts (ThermoFisher).
Nuclear magnetic resonance (NMR) analysis.
Multiple NMR instruments were used. For the synthesized
Dynamic mass redistribution (DMR) assays. Human
colorectal adenocarcinoma HTꢀ29 cells, obtained from ATCC,
were cultured in the complete medium (McCoy’s 5a medium
modified supplemented with 10% fetal bovine serum, 4.5 g/L
1
3,5ꢀdinitroꢀBPA, HꢀNMR data were collected on a Bruker
AVANCE III 400MHz NMR spectrometer. For the peak #18
sample collected from 60 2L PC flasks, 1HꢀNMR spectra were
obtained using an Agilent DD2 700MHz NMR in conjunction
glucose,
2 mM glutamine, and penicillinꢀstreptomycin
1
antibiotics) at 37oC under 5% CO2. For DMR assays HTꢀ29
cells were seeded in a 384well biosensor microplate at 30,000
cells per well suspended in 50 ꢁL of complete medium and
with a 16.4 T superconducting magnet. The H spectra were
acquired with a delay time of 1 s, a 45 degree pulse width and
1
as a composite of 32 to 252 free induction decays. H,1Hꢀ
o
homonuclear correlation spectroscopy (COSY) data were
acquired by collecting 16 transients over 128 increments of t1
with a d1 of 1 s and a d2 of 20 ms. Data in both direct and
indirect dimensions were apodized with squared sinebell
weighting functions. Nuclear Overhauser effect spectroscopy
(1D NOESY) data were acquired with a delay time of 1 s, a 90
cultured at 37 C under 5% CO2 overnight. The plates were
washed twice using a plate washer (BioꢀTek Microplate
Washers ELx405), then maintained in 30 µL of the 1x HBSS
(Hank’s balanced salt solution containing 20 mM HEPES)
assay buffer, and further incubated inside an Epic BT system
(Corning) for 1 hr. For agonist profiling, a 2 min baseline was
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degree pulse width, a mixing time of 500 ms and as a
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composite of 800 free induction decays. Samples were
dissolved in CDCl3 (Cambridge Isotope Laboratories,
Andover, MA, USA). A 5 mm Shigemi NMR tube (Sigma),
which allowed the solvent volume to be reduced to ~1/3 of
that required by conventional 5 mm NMR tubes, was used in
order to achieve high sensitivity. For commercial compounds,
solution 1HꢀNMR spectra were obtained using a Varian
UnityInova 300MHz NMR in conjunction with a 7 T
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2L PETG,
500K cells/mL
2L PC,
500K cells/mL
6
2L PETG,
100K cells/mL
3
2L PC,
100K cells/mL
0
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superconducting magnet. The H spectra were acquired with a
0 1 2 3 4 5
delay time of 1 s, a 45 degree pulse width and as a composite
of 32 free induction decays. Theoretical spectra were obtained
using Advanced Chemistry Development ACD/Labs software.
Chemical shifts (δ) were reported in parts per million (ppm),
and determined relative to the internal standard
tetramethylsilane at δ 0.0 ppm. Spin multiplicities were
assigned as s (singlet), d (doublet), and br (broad).
Culture duration (day)
Figure 1. The growth pattern of CHO-S cells in 2L PC vs.
PETG flasks. Cells from the midꢀlogarithmic growth stage were
seeded at 1 x 105 cells/mL or 5 x 105 cells/mL seeding density
into Corning plain bottom 2L PETG (Cat. No. 431280) or 2L PC
flasks with 400 mL working volume. The flasks were incubated in
an orbital shaker at an agitation rate of 130 rpm until the
maximum viable cell density was achieved (5 days). The cells
were enumerated and assessed for viability using the trypan blue
exclusion assay. Data represents mean ± std (n=3).
Synthesis of 3,5-dinitro-bisphenol A. All reagents
were received from commercial sources and used without any
further purification or drying. All solvents were purified and
dried by standard methods. The reactions were monitored by
thin layer chromatography. The target products were
determined by NMR, and ESIꢀmass spectra obtained on an
LCꢀMS instrument (Waters Acquity UPLC, Waters
Micromass ZQ 2000). The purity of the final compounds was
≥ 95%. Detailed synthesis procedure with characterization
data was provided in Supplementary Materials.
Eighteen peaks in total were collected and tested
individually for their effects on the proliferation of CHOꢀS
cells in 24ꢀwell deepwell plates for 3 days. Results showed
that only peak #18 greatly suppressed cell growth, while all
peaks had little impact on cell viability (Fig.2b). Peak #18
alone was found to contribute to almost 100% of the overall
cell growth inhibition produced by the total extract obtained
(Fig.2c). These results indicate that the peak #18 leachable is a
primary contributor to the inhibition of CHOꢀS cell growth.
Statistical analysis. The statistical significance reported
was obtained using Oneꢀway ANOVA with the Prism 6.0.
RESULTS AND DISCUSSION
Identification of peak #18 as 3,5-dinitro-BPA.
To ascertain the chemical identity of peak #18, we purified
and obtained about 100 µg in total of peak #18 from the water
extracts of 60 2L flasks. Although the peak #18 obtained was
not highly pure (see below), high resolution orbitrap negative
ion mode MS showed that peak #18 has a main m/z of
317.07688 (Fig.S1). This result, together with the isotopic
distribution and the abundance of the A+1 peak, indicates that
C15H14N2O6 represents the most probable formula. Chemical
mining through several public databases including ChemBL,
PubChem, and ChemSpider led us to speculate one of seven
possible dinitroꢀBPA isoforms as a candidate for peak #18,
given the identical formula, the possible presence of BPA
derivatives in the extract, as well as documented toxicity of
certain dinitrophenol compounds24.
Identification of
a cell growth inhibitory
compound from the water extracts of PC flasks.
Cell culture conditions, such as seeding density, cell passage
number and media type can affect the sensitivity of cells to
growth inhibitory leachables. We first assessed the growth of
suspension CHOꢀS cells in 2L PC and polyethylene
terephthalate (PETG) Erlenmeyer flasks using 2 different
seeding densities. Results showed a cell density and flask
dependent growth pattern (Fig. 1). Compared to PETG flasks
under the same culture conditions, for the high seeding density
(500K cells/ml) viable cell density obtained was 15% less at
Day 3 but similar at Day 4 and 5 in PC flasks; however, for
the low seeding density (100k cells/ml) viable cell density
obtained was 39% less at Day 3, 23% less at Day 4, but
similar at Day 5 in PC flasks. On the other hand, the cell
viability was greater than 98% throughout the culture under all
conditions. This result indicates the presence of cell growth
inhibitory, but not cell toxic, leachable(s) in the PC flasks.
The possibility that peak #18 is a dinitroꢀBPA was further
supported by evidence from tandem MS. Negative ion tandem
MS under high duty cycle mode at 180 volts showed that peak
#18 gave rise to m/z values of 317.32 (ꢀH), 302.35 (ꢀCH3),
300.22 (ꢀOH), 287.20 (ꢀNO), 285.25 (ꢀOH, ꢀCH3), and 272.28
(ꢀNO, ꢀCH3). This fragment pattern is similar to, although its
ion abundance pattern is different from, commercial 3,3’ꢀ
dinitroꢀBPA (Fig.S2). Furthermore, derivatization of peak #18
with N,Oꢀbis(trimethylꢀsilyl)trifluoracetamide to form
trimethylsilyl derivatives revealed that the peak #18 obtained
is a mixture of dinitroꢀBPA (~80%) and a possible chloroꢀ
dinitroꢀBPA (~20%) (Fig.S3). This result was further
confirmed by reꢀanalyzing the collected peak #18 sample
using UPLC/MS (Fig.S3).
To identify cell growth inhibitory leachable(s), water
extracts were obtained using the water extraction protocol
described above. This water extraction protocol was chosen to
mimic the routine condition of use for suspension cell culture,
and at the same time to facilitate followꢀup analysis. HPLC
profiling of the 200x concentrated water extracts revealed a
complicated leachable profile (Fig.2a), indicating that many
different compounds can be released into the media during cell
culture.
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a
b
6
0.010
0.008
0.006
0.004
0.002
0.000
ꢀ0.002
3,5ꢀdinitroꢀBPA
m/z 317.13 (ꢀ)
Viability
VCD
3,3'ꢀdinitro
ꢀBPA
m/z 317.13 (ꢀ)
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m/z 229.12 (+H)
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C: control
T: total extract
**
0
1
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4
5
6
Retention time (min)
Figure 2. 2L Erlenmeyer PC shaker flask leachable profile and impact on CHO-S cell growth. (a) HPLC/UV chromatogram at 254
nm of the 200x concentrated water extract from a flask. (b) The effect of different leachable peaks on CHOꢀS cells. (c) Viable cell density
(VCD) in the presence of total peak #18 only versus total water extracts from a single flask. Control: water extract from glass bottle. (b,c)
Cells were cultured in 24ꢀwell deepwell plates for 3 days at 225 rpm and 37 °C. The seeding density was 0.15 x 106 cells/mL. The final
concentration of water extracts added was 8x. Data represents mean ± std (n=3). ***, pꢀvalue <0.001; **, pꢀvalue <0.05.
Scheme 1. Synthesis of 3,5-dinitrobisphenol A (6). Reagents and Conditions: (a) 1, anhydrous K2CO3, acetone; CH3I, acetone, room
temperature (rt); yield, 95%; (b) 2, THF; CH3MgBr, diethyl ester, 0oC; rt, 84%; (c) 3, anisole, CH2Cl2; AlCl3, diethyl ester, 0oC; rt, 60%;
(d) 4, Cu(SO3CF3)2, KNO2, DMSO; 130oC, 54%; (e) 5, CHCl3; trimethylsilyl iodide (TMSI); 50oC, 10%.
Given the small quantity and impurity of the peak #18
8.16 (s, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 8.0 Hz, 2H),
4.96 (br, 1H), 1.71 (s, 6H)) of the synthesized 3,5ꢀdinitroꢀBPA
almost completely matched peak #18 (Fig.S6). Together, these
analyses confirmed that peak #18 is 3,5ꢀdinitroꢀBPA.
1
obtained, we performed HꢀNMR with 2000 scans using the
PreꢀSAT technique, an effective approach to suppress residual
water and solvents in NMR.25 Results showed that there are
clearly three major aromatic proton environments (8.15, 7.16
and 6.88 ppm), and peaks at 7.16 and 6.88 ppm are doublets
with J coupling of 8 MHz, indicating protons in the ortho
position (Fig.S4). 1D NOESY data did not indicate any
interactions between protons, while COSY data did not
indicate any correlation of the protons at 8.15 ppm with the
protons at 7.06 or 6.80 ppm (Fig.S5). These results suggest
that the prominent component of peak #18 is highly likely 3,5ꢀ
dinitroꢀBPA (Fig.3a).
3,5-dinitro-BPA is a cell cycle arrest agent. To
elucidate the mechanism of action of 3,5ꢀdinitroꢀBPA in
CHOꢀS cell growth inhibition, we first performed a structureꢀ
activity relationship analysis of a small set of nitrophenol
compounds, except for 3ꢀnitroꢀBPA which was not available
(Fig.3a). Results obtained using 24ꢀwell deepwell suspension
culture showed that among the nine compounds tested, 4ꢀ
chloroꢀ2,6ꢀdinitrophenol,
2,6ꢀdinitroꢀhydroquinone, and 4ꢀ
tertꢀbutylꢀ2,6ꢀdinitroꢀanisole all had little impact on the
proliferation of CHOꢀS cells, while the other six compounds
gave rise to doseꢀdependent inhibition of CHOꢀS cells after 3
days culture (Fig.3b,c). Interestingly, the six active
compounds appear to fall into two categories. The first group,
comprised of 4ꢀtertꢀbutylꢀ2,6ꢀdinitrophenol, 3,3’ꢀdinitroꢀBPA,
and 3,5ꢀdinitroꢀphenol, almost completely inhibited cell
To further confirm this, we attempted to synthesize 3,5ꢀ
dinitroꢀBPA. Although it has never been reported in the
literature, we successfully synthesized 3,5ꢀdinitroꢀBPA based
on nitrite replacement of the bromo groups of 3,5ꢀdibromoꢀ
BPA (scheme 1). The UPLC retention time, MS (m/z of
1
317.15), and HꢀNMR (400 MHz, CDCl3: δ 11.32 (s, 1H),
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b
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1
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BPA
50
3,5ꢀdinitroꢀBPA
3,3'ꢀdinitroꢀBPA
3, 3', 5, 5'ꢀ tetranitroꢀBPA
25
0
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ꢀ9
ꢀ8
ꢀ7
ꢀ6
ꢀ5
ꢀ9
ꢀ8
ꢀ7
ꢀ6
ꢀ5
Compound, log M
Compound, log M
d
e
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60
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3,5ꢀdinitroꢀBPA:
G1/G0
125
100
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0
Control: G1/G0
Control: S
Control: G2/M
CHOꢀS
3,5ꢀdinitroꢀBPA:
G2/M
CHOꢀ5/9α
3,5ꢀdinitroꢀBPA: S
ꢀ9
ꢀ8
ꢀ7
ꢀ6
ꢀ5
0
12
24
Compound, log M
Time (hr)
Figure 3. Compounds and their effects on cell growth inhibition and cell cycle. (a) Chemical structures of bisphenol A (BPA) and diꢀ
nitrophenol (DNP) compounds tested. (b,c) Doseꢀdependent inhibition of CHOꢀS cells by different compounds, including BPA compounds
(b), DNP compounds including 1: 4ꢀtertꢀbutylꢀ2,6ꢀDNP; 2: 3,5ꢀDNP; 3: 2,6ꢀdinitroꢀhydroquinone; 4: 4ꢀchloroꢀ2,6ꢀDNP; 5: 4ꢀtertꢀbutylꢀ
2,6ꢀdinitroanisole (c). (d) The sensitivity of two CHO sublines to 3,5ꢀdinitroꢀBPA. For (bꢀd) Cells were cultured in a 24ꢀwell deepwell
plate for 3 days. Cell seeding density: 0.15 x 106 cells/mL. Data represents mean ± std (n=3). (e) CHOꢀS cell cycle analysis as a function of
treatment time with 3 µM 3,5ꢀdinitroꢀBPA. Control: equal amount of dimethyl sulfoxide (0.3%).
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growth, indicating cytotoxicity. In contrast, the second group,
comprised of 3,5ꢀdinitroꢀBPA and 3,3’,5,5’ꢀtetranitroꢀBPA,
only suppressed cell growth, indicating that both compounds
may act as cell cycle arrest agents with little cytotoxicity. BPA
at high doses (~10 µM) only partially suppressed cell growth.
The potency rank order was found to be 4ꢀtertꢀbutylꢀ2,6ꢀ
dinitrophenol (logIC50: ꢀ7.37±0.04) > 3,5ꢀdinitroꢀBPA (ꢀ
6.82±0.04) > 3,3’,5,5’ꢀtetraꢀnitroꢀBPA (ꢀ6.46±0.03) > 3,3’ꢀ
dinitroꢀBPA (ꢀ6.20±0.02) > 3,5ꢀdinitrophenol (ꢀ5.42±0.02) >
BPA (~10 µM) (n=3 for all). The lack of effect by 4ꢀtertꢀ2,6ꢀ
dinitroanisole suggests that the OH group of 2,6ꢀdinitrophenol
compounds is essential for the inhibition of cell growth.
These data also suggest the importance of the 4ꢀposition
modification of 2,6ꢀdinitrophenol compounds for cell growth
inhibition.
of 3,5ꢀdinitroꢀBPA, we performed a chemical similarity search
within the ChemBL database.31 Chemical similarity analysis is
a
useful approach to predict novel compoundꢀtarget
interactions, based on the principle that similar compounds
will likely have similar properties and bind to same proteins.32ꢀ
36 Our results showed that in the ChemBL database only 3,3’ꢀ
dinitroꢀBPA and 3,3’,5,5’ꢀtetraꢀnitroꢀBPA are two known
compounds similar to 3,5ꢀdinitroꢀBPA. Examining the
annotated biological activity data in the database showed that
3,3’ꢀdinitroꢀBPA is known to inhibit lysineꢀspecific
demethylase 4A (KDM4A), geminin, and chromobox protein
homolog 1 (CBX1), all with low micromolar potency. Gene
knockdown or chemical inhibition of KDM4A37, or geminin38,
or CBX139,40 has been shown to cause cell cycle arrest and
even cell death. 3,3’ꢀdinitroꢀBPA was found to display no
estrogenic activity, but high genotoxicity.41 These results led
us to hypothesize that the three compounds may cause cell
growth inhibition at least partially through these molecular
targets. Further study is warranted to test this hypothesis.
Next, we compared the sensitivity of two different CHO
sublines, CHOꢀS and CHOꢀ5/9α, to 3,5ꢀdinitroꢀBPA. Several
sublines derived from CHOꢀK1 cells have been adapted to
suspension culture and used as the host for the bioproduction
of recombinant proteins and antibodies; because each subline
has its unique genome and metabolic properties,26ꢀ28 each
would be expected to have different sensitivity to cell growth
inhibitory compounds.14,15 Our results showed that CHOꢀ5/9α
cells were less sensitive to 3,5ꢀdinitroꢀBPA than CHOꢀS cells
(Fig.3d).
Together, these results suggest that 3,5ꢀdinitroꢀBPA is a
potent cell cycle arrest agent, thus inhibiting the growth of
CHOꢀS cells; and different CHO sublines have distinct
sensitivity to this leachable.
Agonistic activity of 3,5ꢀdinitroꢀBPA at the GPR35.
Given that certain nitrophenol compounds are GPR35
agonists,42 we examined the activity of 3,5ꢀdinitroꢀBPA at the
GPR35, which is endogenously expressed in HT2943. Profiling
using DMR assays showed that 3,5ꢀdinitroꢀBPA triggered a
dose dependent DMR signal (Fig.4), but with a potency well
below 3,3’,5,5’ꢀtetraꢀnitroꢀBPA (EC50 of 2.35 µM)42. Of note,
3,5ꢀdinitroꢀBPA at 100 µM did not reach the maximal
Lastly, we examined the cell cycle distribution of CHOꢀS
cells in the presence of 3 µM 3,5ꢀdinitroꢀBPA using flow
cytometery. Our results suggest that this compound arrests the
cells predominately at the G1/G0 phase of the cell cycle with
no effect on cell viability (Fig.3e).
Cell cycle arrest can be induced through multiple
molecular mechanisms.29,30 To elucidate possible mechanisms
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a
100ꢁM
50ꢁM
0.008
200
150
100
50
3,5ꢀdinitroꢀBPA
m/z 317.13 (ꢀ)
BPA
25ꢁM
12.5ꢁM
6.25ꢁM
0.006
0.004
0.002
0.000
PC resin + γ
PC resin
PC flask + γ
PC flask
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4.5
5.0
Time (min)
Retention time (min)
Figure 4. The dose dependent dynamic mass redistribution signal
(wavelength shift in picometer, pm) of 3,5ꢀdinitroꢀBPA in HT29
cells. Data represents mean + std (n = 8).
b
BPA
m/z 229.12 (+H)
3ꢀnitroꢀBPA
m/z 272.10 (ꢀ)
0.27
0.21
0.15
0.09
0.03
ꢀ0.03
response (~250 pm in amplitude) arising from the activation of
GPR35 as determined by the known GPR35 agonist
zaprinast.42,43 However, MLꢀ145 at 1 µM was found to
completely block the DMR of 100 µM 3,5ꢀdinitroꢀBPA (data
not shown). MLꢀ145 is a known GPR35 antagonist.44
Together, these results suggest that 3,5ꢀdinitroꢀBPA is a weak
GPR35 agonist.
3,3'ꢀdinitroꢀBPA
m/z 317.07 (ꢀ)
?
3,5ꢀdinitroꢀBPA
m/z 317.05 (ꢀ)
m/z 274.06 (ꢀ)
m/z 249.02 (ꢀ)
?
m/z 447.27 (ꢀ)
Quantification of 3,5-dinitro-BPA per flask. Next,
we quantified the amount of 3,5ꢀdinitroꢀBPA per flask using
UPLC. Our results showed that the water extraction protocol
extracted 1.52±0.14 µg (n=3) 3,5ꢀdinitroꢀBPA from each
flask, suggesting that its effective concentration could reach as
high as ~5 µM when the 2L flasks are used in 1L culture,
which is sufficient to cause the maximal inhibition of CHOꢀS
growth. However, compared to the 2L PETG flasks after 3
days suspension culture, we only observed 15% or 39% less
cells in the PC flask when the seeding density was 5x or 1x
105 cells/mL, respectively (Fig.1), suggesting that less 3,5ꢀ
dinitroꢀBPA is released into cell culture media during 3 days
culture compared to the water extraction.
2
3
4
5
6
7
8
Retention time (min)
Figure 5. (a) HPLC/UV chromatogram at 254 nm of the water
extracts from molded shaker flasks and PC resin before and after
gammaꢀirradiation. (b) HPLC/UV chromatogram at 254 nm of a
water extract from a γꢀirradiated thin film of pure BPA monomer
coated on a glass flask.
Fourth, we performed γꢀirradiation treatment of a thin
film of pure BPA monomer coated on a glass flask. Results
showed that γꢀirradiation induced a high amount of 3,3’ꢀ
dinitroꢀBPA and 3ꢀnitroꢀBPA, but a much smaller amount of
3,5ꢀdinitroꢀBPA (Fig.5b), also suggesting that nitroꢀBPA
compounds may form upon γꢀirradiation after polymer
degradation.
Mechanism of formation of 3,5-dinitro-BPA. We
next examined how 3,5ꢀdinitroꢀBPA is formed in 2L PC
flasks. First, we performed MS analysis of the extracts
using a negative ion scan. Results showed that besides 3,5ꢀ
dinitroꢀBPA the water extracts also contain 3,3’ꢀdinitroꢀ
BPA (m/z of 317.13; retention time of 5.88), which was at
trace amounts (Fig.2) as confirmed with the commercial
compound. This result suggests a nitric oxide free radical
reaction is a common mechanism to form nitroꢀBPAs.
Fifth, we examined the possibility that the formation of
3,5ꢀdinitroꢀBPA is a flask surface event, as indicated by the
low amount of 3,5ꢀdinitroꢀBPA formed in the bulk BPA
monomer film. Assuming that the 3,5ꢀdinitroꢀBPA formed in a
flask can reach as high as a monolayer surface coverage and
has a molecular size similar to 3,3’ꢀdinitroꢀBPA, we would
expect to obtain ~20 µg 3,5ꢀdinitroꢀBPA per flask. This is
much higher than what was actually extracted (1.25 µg per
flask, see above), suggesting less than monolayer coverage.
3,3’ꢀdinitroꢀBPA has a maximal planar area of 1.95 nm2, and a
molecular volume of 1.44 nm3.45 The surface area of a flask
exposed to water extraction is about 800 cm2. Furthermore,
our HPLC/MS analysis showed that a second round of
repeated water extraction of the same flask only led to <5% of
the amount of 3,5ꢀdinitroꢀBPA obtained in the first round
water extraction. This analysis suggests that the formation of
Second, we examined the effect of γꢀirradiation.
UPLC/MS didn’t detect any nitroꢀBPA compounds in the
200x concentrated water extracts from flasks that had not been
treated with γꢀirradiation (Fig.5a), suggesting that this
treatment is necessary to form 3,5ꢀdinitroꢀBPA.
Third, we examined the effect of molding process on 3,5ꢀ
dinitroꢀBPA formation. UPLC/MS analysis of the water
extracts of γꢀirradiated PC resin showed no 3,5ꢀdinitroꢀBPA
(Fig.5a). This result suggests that the molding process also
contributes to the formation of 3,5ꢀdinitroꢀBPA, presumably
via degradation of PC during the manufacturing process.
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to develop a supply chain/quality process to secure high
3,5ꢀdintroꢀBPA is highly likely a surface event upon γꢀ
quality products for academic and industrial applications. In
this regard, we would like to point out that as PC flasks are
available in multiple formats and made using different grad
resins or manufacturing processes from different
manufacturers, it is highly likely that these commercially
available PC flasks may have different leachable profiles
including 3,5ꢀdintroꢀBPA. With the present discovery,
together with the approaches to identify, quantify and assay, of
3,5ꢀdinitroꢀBPA as a cell growth inhibitory leachable from
some PC flasks, vendors shall collaborate with polymer resin
suppliers to develop high quality products and with end users
to develop solution to minimize or eliminate any resultsꢀ
interference effects of specific leachables including 3,5ꢀ
dinitroꢀBPA.
irradiation of PC flasks.
Finally, we prepared and analyzed the bulk pyrolysis
sample of γꢀirradiated 2L PC flasks by first dissolving several
flask pieces and then precipitating out the polymers. Results
showed that the pyrolysis sample contained little 3,5ꢀdinitroꢀ
BPA (Fig.S7). Of note, our analysis of the pyrolysis sample
(Fig.S7) also confirmed the Fries type free radical reactions,
leading to the destruction of the carbonate linkages and the
formation of a number of phenol coupling products and
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salicylate esters, as reported previously46,47
.
Together, these results suggest that 3,5ꢀdinitroꢀBPA is
only formed on the surface of PC flasks and via a combination
of high temperature molding processꢀinduced polymer
degradation with a γꢀirradiationꢀmediated nitric oxide free
radical reaction.
ASSOCIATED CONTENT
Supporting Information
Conclusion
The study described here exemplifies our strategy to
identify resultsꢀinterfering leachables from disposable plastic
labware/vessels, and our commitment as a labware tool
provider to address the increasing demand from academic and
industrial labs for better quality singleꢀuse products.
The Supporting Information is available free of charge on the
ACS Publications website.
Supporting UPLC/MS/NMR data, and synthesis procedure and
characterization of 3,5ꢀdinitroꢀBPA (PDF).
Here, we applied multiple analytical tools (such as HPLC,
MS, NMR), together with chemical mining and synthesis, to
discover 3,5ꢀdinitroꢀBPA as a bioactive leachable from certain
Erlenmeyer PC shake flasks.
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: MelkoumiZ@corning.com (Z.M); liangxm@dicp.ac.cn
(X.L.); fangy2@corning.com (Y.F.).
We also employed multiple bioassays (e.g., cell
proliferation, viability, and functional assays), together with
chemical similarity analysis, to examine the mechanism of
action of the bioactive leachables identified. The results
showed that 3,5ꢀdinitroꢀBPA acts as a potent cell cycle arrest
agent to inhibit the proliferation of CHOꢀS cells in suspension
culture, as well as a weak agonist to activate the GPR35 in
HT29 cells. We also found that the sensitivity of cells to 3,5ꢀ
dinitroꢀBPA varied between different CHO sublines as well as
different cell seeding density, which explains why the majority
of end users do not observe growth inhibition with
commercially available PC vessels.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding Sources
This work was partly supported by the State Key Program of
National Natural Science of China (Grant No.U1508221 ) and the
External Cooperation Program of BIC, Chinese Academy of
Science (121421KYSB20130013) (X. Liang).
ACKNOWLEDGMENT
Finally, we applied materials science knowꢀhow and
precision manufacturing process controls to identify the
mechanism of formation of the bioactive leachables. As a
result, 3,5ꢀdinitroꢀBPA was identified as a novel resultsꢀ
interfering leachable that is formed through γꢀirradiationꢀ
mediated nitric oxide free radical reaction. Our results also
point to possible solutions, including increasing initial seeding
density, preꢀwashing the flasks, and switching to PETG flasks,
when a specific cell line under culture is highly sensitive to
this leachable compound.
The authors acknowledge that the high resolution mass
spectroscopy, NOE and ROESY NMR experiments were carried
out at the Chemistry Core facility of Cornell University. The
authors also acknowledge the assistance of Natalya Zaytseva,
Cindy Wilson, Chris Dover, Chris Schweiger, Seth Goodreau, Ed
Fewkes, and Anthony G. Frutos in discussions and followꢀup
studies.
Conflict of Interest Disclosure
Corning is a manufacturer of plastic labware including single use
polycarbonate and PETG flasks.
The presence of bioactive substances that can leach into
bioassay solutions may present an underestimated issue for the
quality of biological testing results reported in the literature,
given the wideꢀspread use of disposable plasticware in life
sciences research and drug discovery. The possibility of
certain leachables to diminish the productivity of
bioprocessing by inhibiting cell growth,14,15 or to decrease the
quality of biologics by increasing aggregation or triggering in
vivo responses16,48 has called for collaborations among
polymer resin suppliers, labware manufacturers, and end users
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