Materials and
Methods
Cells, medium and shake-flask
cultures
Three clones derived from CHO Cell Line B (Wild-type Clone 47, BCAT1 KO
Clone 83, and BCAT1 KO Clone 90) utilizing a glutamine synthetase
expression system and expressing a recombinant antibody were used in the
present study (Mulukutla et al., 2019). Three types of proprietary
medium were used: “Medium A” is the production medium for traditional
and HiPDOG fed-batch production bioreactors. “Medium B” is an enriched
nutrient solution used as feed medium for both modes of production
bioreactor. Medium C is the passaging medium used for cultivation and
experimentation in shake-flask batch cultures. Medium A and B are
identical in composition to those used previously (Mulukutla et al.,
2019).
Medium A is a chemically defined, protein-free, amino acid fortified
version of DMEM:F12 medium with adjusted levels of vitamins, trace
elements, sodium bicarbonate and potassium chloride, also containing
polyvinyl alcohol. Medium A was modified with additional sodium
bicarbonate and glucose for non-HiPDOG fed-batch production bioreactors.
Medium B is a chemically defined, protein-free concentrated feed
composed of amino acids, vitamins, and trace elements (a subset of those
in Medium A). Medium B was supplemented with additional alanine,
cysteine, tyrosine, and zinc to prevent depletion of these compounds in
high-density cell culture processes.
The specific impact of lactate on cell growth of BCAT1 KO Clone 83 and
WT Clone 47 was assessed using shake-flask cultures. Medium C, a
chemically defined and protein-free lean passaging medium was modified
with 0 to 10 g/L lactate via sodium lactate (Sigma-Aldrich®, St. Louis,
MO) at concentrations similar to those achieved in fed-batch and HiPDOG
bioreactors. An osmolality control matching the 8 g/L lactate condition
was also made using Medium C modified with sodium chloride. WT Clone 47
sodium lactate concentrations used were a subset of those used for Clone
83, included for comparability. Cells were inoculated at 0.1 ×
106 cells/mL in triplicate 125mL shake-flasks for each
condition with a working volume of 30 mL, and cultivated for 6 days on
a shaking platform (Orbital Shaker, Bellco Glass, Inc.) in a humidified
incubator (ThermoFisher Scientific) maintained at 36.5 °C and 5%
carbon dioxide. Viable cell density, glucose, lactate, and ammonia
concentrations in the cell culture medium were measured on days 0, 3,
and 6 using a Nova Bioprofile FLEX Analyzer (Nova Biomedical, Waltham,
USA).
Bioreactor setup for fed-batch and
HiPDOG
cultures
Cell Line B clones were cultivated in pairs of bioreactors where the
first of each pair employed a typical titrant-based lower pH dead-band
and the second, the HiPDOG control strategy (Gagnon et al., 2011). For
HiPDOG bioreactors, pH dead-bands during and after HiPDOG were 7.125 +/-
0.025 and 7.10 +/- 0.20 respectively. Each fed-batch bioreactor had its
pH tightly controlled to match its HiPDOG partner during the HiPDOG
operational phase, while utilizing the same pH dead-band thereafter.
Medium A was used as the production medium, while Medium B was used as
feed medium for all vessels. Inoculation cell densities were 1.5 ×
106 cells/mL in an initial working volume of 1L, with
temperature and agitation set to 36.5 oC and 259 rpm
respectively. In HiPDOG reactors, feed medium was added based on upper
pH dead-band during HiPDOG control, operational from day 2 to day 6.
During this period each fed-batch reactor had constant semi-continuous
feed rates set daily to match the cell specific feed rate of its partner
HiPDOG reactor. Fed-batch reactors were additionally provided glucose to
target 2.5 g/L as-needed. Following the end of HiPDOG control, feed
rates for all vessels were set daily to target a final cumulative cell
specific feed rate of 1.75 pL/cell/day, resulting in a total feed medium
addition of 20 to 60% of the culture starting volume. Post-HiPDOG
glucose levels were targeted at 2.5 g/L by feeding additional glucose as
necessary for all vessels. Viable cell density, glucose, lactate, and
ammonia concentrations in the cell culture medium were measured daily
using a Nova Bioprofile FLEX Analyzer (Nova Biomedical, Waltham, USA).
Titer analysis was performed by Protein A HPLC (model 1100 HPLC, Agilent
Technologies, Inc., Santa Clara, CA, protein A column model 2-1001-00,
Applied Biosystems, Foster City, CA). WT clone 47 conditions were run in
duplicate, however data beyond day 11 for the duplicate HiPDOG condition
are unavailable due to a controlling probe failure.
Additional methods pertaining to amino acid concentration analysis via
UPLC and spent medium metabolite analysis via NMR are identical to those
utilized in the prior study (Mulukutla et al., 2019).