Materials and Methods
Cell Culture and Bioreactor Operation
The cells used for protein production were Chinese hamster ovary (CHO) cells, and all data presented are from one protein,
product 1. All cell culture was performed in custom media and feed developed by Irvine Scientific. Atypical seed train was
used for manufacturing runs. The original seed train consisted of WAVE reactors up to the 100-L and 500-L working volume scale
for manufacturing. The current seed train in the new facility uses WAVE bioreactors for scale-up and SUBs for production vessels
at the 50-L, 250-L, and 1,000-L scale. Development runs were performed in four 2-L Applikon glass vessel reactors with ADI
1030 controllers and two 5-L Sartorius Stedim bioreactors with A+ and B+ controllers. Scaled-up runs were performed in 50-,
250-, and 1,000-L SUBs. The 50-L SUB was integrated with an Applikon I controller, and the 250- and 1,000-L SUBs were integrated
with a Finesse TruLogic RDPD controller. The parameters that were logged and optimized included feeding strategy (i.e., schedule,
volumes), pH, temperature, DO, and agitation set points, and flow rates. Partial pressure of carbon dioxide (pCO2) levels also were monitored and controlled. Viable cell density and viability were measured using a Cedex automatic cell
counter (Innovatis) or a BioProfile Flex (Nova Biomedical). Protein production was determined using a Protein A assay on an
Agilent 1100 series high performance liquid chromatography (HPLC) instrument. Some nutrients, metabolites, and electrolytes
(L-glutamine, glucose, glutamate, lactate, ammonium, and sodium, calcium, and potassium ions) were monitored offline using
the BioProfile Flex.
Multiple scale-up parameters were analyzed to transfer the optimized process from the glass vessels to the SUBs. Initial values
for gas flow rates were calculated using vessel volumes per minute. For agitation speed, tip speed, power per volume, and
torque, calculations were performed and evaluated. Power per volume was chosen to scale up the agitation speed. After the
flow rates and agitation speed had been approximated, oxygen mass transfer coefficient (kLa) studies were performed in the 50-LSUB to determine the efficiency of the calculated values compared with small-scale studies.
Fiber Optic Autoclavable and Disposable Probes
Different configurations of fiber optic technology were evaluated. Autoclavable oxygen probes for in-line measurement from
PreSens and Finesse (TruDO Optical) were studied in the 5-L and 2-L glass vessels, respectively. Measurements were taken every
minute and compared directly to the Mettler-Toledo (InPro6800) polarographic probes integrated with the controllers. Preliminary
studies also were performed in shaker flasks using the PreSens non-invasive oxygen sensors based on the same fiber optic technology.
The values from these disposable probes were compared to offline measurements of the BioProfile Flex. Lastly, the disposable
option available from Finesse (TruFluor DO) was evaluated in the 50-L SUB and compared directly with autoclavable polarographic
DO probes. The TruFluor DO is available in a sleeve that is manufactured with HyClone's bioprocess container (bag) for the
SUBs. The sleeve contains the disposable sensor, and inserted into the sleeve is a non-invasive optical reader connected to
a transmitter.
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