Cell Culture and Media
A total of 25 14-day fed-batch production cell culture conditions were
used to build a training dataset for the model. For all 25 batches, CHO
cells were cultured in AMBR® 15 (Sartorius Stedim, GE) mammalian
bioreactors seeded at 2E6 cells/mL with an initial working volume of 13
mL. Bioreactors were operated at dissolved oxygen of 30%, pH setpoint
of 6.95±0.15 controlled by either CO2 or base addition,
agitation at 900 RPM, and incubated at 36.5°C. Control cultures were fed
with a high nutrient feed based on a previously defined process. The
remaining cultures were fed varying amounts of the same high nutrient
feed following the same feeding schedule to obtain a varying
distribution of growth and productivity profiles for the training
dataset. Glucose was supplemented back to 6 g/L if extracellular glucose
levels fell below 3 g/L. Similarly, glutamic acid was supplemented as 3
mM bolus additions if the extracellular glutamic acid levels fell below
4 mM.
To validate the MVDA models for cell growth and mAb productivity, an
experiment containing 46-batches was performed. Three baseline controls
were designed in which cells were fed with three CD feed media
corresponding to high, moderate, and low nutrient levels. In addition to
the ranging nutrient feeds (high, moderate, and low), key amino acids
identified from either the growth or production model were formulated
into amino acid cocktail feeds and supplemented to the cultures at
model-informed time points.
Each cocktail feed contained a 100X concentration of the specific amino
acids with pH adjusted via 10N NaOH to maintain solubility. Previous
studies indicated that AMBR® 15 bioreactors incur a 1 – 1.5% of
evaporation rate (v/v per day), which can lead to inaccurate
quantification of cell growth and mAb productivity due to the small
working volume (Hsu, Aulakh, Traul, & Yuk, 2012). Therefore, 150 µL of
sterile water was supplemented daily to compensate for evaporation. To
compensate for osmotic and pH differences of amino acid cocktails,
control solutions with similar osmolality and pH to the most
concentrated amino acid cocktail feed were added at the same volume. In
addition, a control was included to confirm the impact of basal media
dilution.
Cell culture samples were taken on alternate days up to and including
day 14 for offline analysis of VCD, cell viability, cell diameter, pH,
pCO2, extracellular metabolites (lactate, ammonia,
glutamate, glutamine, and glucose), mAb titer, and amino acids. Viable
cell density, viability, and cell diameter were measured using the
trypan blue exclusion method on a CEDEX HiRes cell counter (Roche
Diagnostics GmbH, Mannheim, Germany). The ABL80 blood gas analyzer
(Radiometer, Denmark) was used to capture offline pH and pCO2 values.
The extracellular metabolites were measured using the RX IMOLA Clinical
Chemistry Tester (Randox, UK). Antibody titer was quantified using a
previously described protein-A reverse-phase high-performance liquid
chromatography (HPLC) method (Hoshan et al., 2019). Amino acids were
measured using Waters Acquity ultra‐performance liquid chromatography
(UPLC) H-Class Bio System with an AccQ‐Tag Ultra Derivatization Kit
(Waters, MA). The amino acid standard kit (A9781) was obtained from
Millipore Sigma (St. Louis, MO).