Supplementary Material
Biotechnology Progress
Changes in Intracellular ATP-Content of CHO Cells as Response to Hyperosmolality
Jennifer Pfizenmaier, Jens-Christoph Matuszczyk and Ralf Takors*
Institute of Biochemical Engineering, University of Stuttgart, 70569 Stuttgart, Germany
*Corresponding author;
takors@ibvt.uni-stuttgart.de
Content
Figures:
-
metabolites
-
product titer
-
cell volume
-
intracellular ATP-concentration
-
intracellular ADP-content
-
oxygen uptake and carbon dioxide formation rate
Calculations:
-
Equations for carbon and oxygen balancing
-
Equations for the calculation of oxygen uptake and carbon dioxide formation rate
-
Equation for the calculation of ATP formation rate
-
Estimation of the required amount of ATP to compensate regulatory volume increase
Figure 1: Profiles of metabolites during batch cultivations with osmotic shift (arrow). Addition of sodium
chloride increased osmolality to ~ 390 mOsmol/kg ( ,
cultures (
,
replicates each.
,
,
) in comparison to ~ 290 mOsmol/kg for reference
). Error bars represent standard deviations for two biological replicates with three technical
Figure 2: Time evolution of antibody titer. Osmolality was increased by addition of sodium chloride (arrow)
during batch cultivation of CHO DP-12 cells. ELISA was used to analyze antibody titers of hyperosmolal
(~ 390 mOsmol/kg,
) and reference cultures (~ 290 mOsmol/kg,
). Error bars represent standard deviations
for two biological replicates with three technical replicates each.
Figure 3: Profile of cell volume during batch cultivation with osmotic shift. Addition of sodium chloride (arrow)
resulted in ~ 390 mOsmol/kg ( ) compared to ~ 290 mOsmol/kg ( ) in reference bioreactors. Cell diameters
were analyzed by Cedex XS cell counter (Roche) and were used to calculate cell volumes. Error bars represent
standard deviations for two biological replicates with three technical replicates each.
Figure 4: Effect of hyperosmolality on intracellular ATP-concentration (cATP). Osmolality was increased by
addition of sodium chloride (arrow) during batch cultivation of CHO. ~ 390 mOsmol/kg ( ).
~ 290 mOsmol/kg ( ). Fast filtration was used for cell sampling followed by methanol/chloroform extraction
and RP-HPLC-quantification of ATP. Cell volumes were calculated based on cell diameters which had been
analyzed by Cedex XS (Roche). Error bars represent standard deviations for two biological replicates
considering the technical error for the determination of ATP-content (n = 3) as well as of the cell volume (n = 3)
by Gaussian error propagation.
Figure 5: Profile of intracellular ADP-content during batch cultivation with osmotic shift (arrow). Addition of
sodium chloride increased osmolality to ~ 390 mOsmol/kg ( ) compared to ~ 290 mOsmol/kg for reference
culture ( ). Fast filtration was used for cell sampling followed by methanol/chloroform extraction and RPHPLC-quantification of ADP. Error bars represent standard deviations based on two biological replicates as well
as three technical replicates each.
Carbon and oxygen balancing
Equation 1: C-molar carbon balances for metabolites
∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑪−𝒎𝒐𝒍 = 𝑵𝑪,𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏) × (𝒄𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝒕=(𝒊+𝟏) × 𝑽𝒕=𝒊+𝟏 − 𝒄𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝒕=𝒊 × 𝑽𝒕=𝒊 )
Δnmetabolite (n), C-mol : change in amount of metabolite n in C-mol
NC,metabolite(n)
: number of C-atoms of metabolite n
cmetabolite(n),t = i
: molar concentration of metabolite n at time i
Vt=i
: working volume in bioreactor at time i
Equation 2: Calculation of total carbon or oxygen influx
𝒄𝒂𝒓𝒃𝒐𝒏𝒊𝒏𝒇𝒍𝒖𝒙 = ∑ ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑪−𝒎𝒐𝒍
𝒘𝒊𝒕𝒉 ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑪−𝒎𝒐𝒍 < 𝟎
𝒐𝒙𝒚𝒈𝒆𝒏𝒊𝒏𝒇𝒍𝒖𝒙 = ∑ ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑶−𝒎𝒐𝒍
∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑶−𝒎𝒐𝒍 < 𝟎
Equation 3: Calculation of carbon or oxygen efflux
𝒄𝒂𝒓𝒃𝒐𝒏𝒆𝒇𝒇𝒍𝒖𝒙 = ∑ ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑪−𝒎𝒐𝒍
𝒘𝒊𝒕𝒉 ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑪−𝒎𝒐𝒍 > 𝟎
𝒐𝒙𝒚𝒈𝒆𝒏𝒆𝒇𝒇𝒍𝒖𝒙 = ∑ ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑶−𝒎𝒐𝒍
∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑶−𝒎𝒐𝒍 > 𝟎
Equation 4: Determination of the amount of carbon dioxide or oxygen
𝒏𝑪𝑶𝟐 = (−𝟏) × 𝒄𝒂𝒓𝒃𝒐𝒏𝒊𝒏𝒇𝒍𝒖𝒙 − 𝒄𝒂𝒓𝒃𝒐𝒏𝒆𝒇𝒇𝒍𝒖𝒙
𝒏𝑶𝟐 = (−𝟏) × 𝒐𝒙𝒚𝒈𝒆𝒏𝒊𝒏𝒇𝒍𝒖𝒙 − 𝒐𝒙𝒚𝒈𝒆𝒏𝒆𝒇𝒇𝒍𝒖𝒙
Equation 5: Determination of oxygen influx or efflux of metabolites
∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑶−𝒎𝒐𝒍 = 𝒙𝑶⁄𝑪,𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏) × ∆𝒏𝒎𝒆𝒕𝒂𝒃𝒐𝒍𝒊𝒕𝒆(𝒏),𝑪−𝒎𝒐𝒍
Δnmetabolite(n), O-mol : change in the amount of metabolite n in O-mol
xO/C,metabolite(n)
: ratio of oxygen and carbon in metabolite n
xO/C,biomass = 0.487
Δnmetabolite (n), C-mol : change in the amount of metabolite n in C-mol
Table 1: Data of carbon balance. Values were calculated according to equations 1 – 4. Negative values represent
carbon influx while positive values represent carbon efflux (positive values).
Table 2: Data of oxygen balance. Values were calculated according to equations 1 – 5. Negative values
represent carbon influx while positive values represent carbon efflux (positive values).
Calculation of specific oxygen uptake (qO2) and carbon dioxide formation (qCO2) rates
Equation 6: Calculation of differential specific oxygen uptake (qO2) and carbon dioxide formation (qCO2) rate
𝒒𝑶𝟐 =
1
∆𝑡
×
𝒏𝑶𝟐
̅
𝑿
or 𝒒𝑪𝑶𝟐 =
𝟏
∆𝒕
×
𝒏𝑪𝑶𝟐
̅
𝑿
nO2 , nCO2
: amount of oxygen or carbon dioxide
X
: geometric mean of viable cell density
∆t
: difference in time
Figure 6: Effect of hyperosmolality on qO2 and qCO2. During batch cultiviations of CHO DP-12 osmolality
was increased by the addition of sodium chloride about 70 h after inoculation (arrow) resulting in an osmolality
of ~ 390 mOsmol/kg ( ,
) compared to ~ 290 mOsmol/kg for reference cultures ( ,
). Oxygen uptake and
carbon dioxide formation rates were calculated according to equation 6.
Equation 7: Calculation of specific ATP formation rate (qATP) based on respiration activity
𝒒𝐴𝑇𝑃 = 𝑃⁄𝑂 × 𝒒𝑂
2
P/O
: number of ATPs produced per oxidized NADH
q O2
: specific oxygen uptake rate
Estimation of the required amount of ATP to compensate regulatory volume increase
time interval
: day 3 to day 5
increase in cell volume
: ∆Vcell = 0.075 pL/cell/d
intracellular ATP-concentration
: cATP = 3.4 mM
required qATP to maintain cATP
: qATP,+ = ∆Vcell × cATP = 0.26 fmol/cell/d
difference in qATP observed in Figure 3 (manuscript) : qATP = 4910 fmol/cell/d
qATP ≫ qATP,+