Cell line and cell culture
A suspension-adapted recombinant CHO-DG44 cell line expressing mAb1 in
the dihydrofolate (dhfr) selection system was cultivated in FB, SS
perfusion and NSS perfusion in Boehringer Ingelheim’s proprietary
in-house media. Fed-batch material was derived from a 12-kL stainless
steel bioreactor inoculated at a seeding density of 3.0 x
105 cells/ml. Dissolved oxygen (DO) was maintained at
60% and the temperature and agitation were 36.8oC and
29 rpm, respectively. Nutrient feed media was continuously fed at 10%
cell culture volume starting from day 3 and repeated every 48 hours with
a targeted final glucose concentration at 4 g/L. Culture supernatant
collected from the bioreactor was centrifuged at 5000 rpm for 55 L/min
and stored at -80oC pending further analysis.
Perfusion cultures (SS, NSS) were established with a 2 L working volume
and subsequently seeded at 12.4 x 106 cells/ml and
12.1 x 106 cells/ml, respectively. In-house perfusion
growth medium (1x concentration) started perfusing immediately following
inoculation on day 0 at the rate of 1 vessel volume per day (1 vvd) and
increased gradually to 2 vvd on day 2. The SS reactor maintained a
constant 2 vvd perfusion rate from day 3-14 using a combination of three
concentrated feeds and diluent, the respective rates thereof adjusted
accordingly to maintain a residual culture osmolality of 310 ± 15 mOsm.
A culture bleed controlled by an Incyte permittivity probe (Hamilton,
Reno, NV) was used to maintain the viable cell density (VCD) target of
40 x 106 cells/ml. The NSS reactor did not utilize a
cell bleed; cell culture proliferated to the maximum VCD attainable by
the system (feed, DO control, etc.). The addition rate of the three
concentrated feeds remained constant at a total of 0.5 vvd; the addition
rate of the diluent varied accordingly to maintain a residual culture
osmolality target of 330 ± 50 mOsm. Permeate was harvest by 0.2 µM
hollow fiber filtration throughout culture.