Optimizing Xuri W25 bioreactor parameters
To further shorten the end-to-end process time, we evaluated whether
several bioreactor parameters, including seeding density, dissolved
oxygen (DO) level and rocking agitation, could accelerate cell growth.
The same donor apheresis was split into five tested bioreactor
conditions listed in Table 1 .
Xuri#1, the standard condition where cells were seeded at lower density
(3.5e6 cells/ml) with lower agitation (3 rpm x 3°), yielded <
4e9 engineered T cells at day9 while Xuri#2, Xuri#3, and Xuri#5 all
significantly promoted cell growth to around 10e9 cells through
increasing rocking agitation or gassing oxygen directly (DO feedback
loop control) to promote a greater level of oxygen transfer(Fig. 3A) . Moreover, Xuri#2, Xuri#3, and Xuri#5 with
increased rocking speed and angle demonstrated that enhancing agitation
also improved in-process cell viability from day6 to day9 compared to
Xuri#1 (Fig. 3B) . In addition, shortening the bioprocess from
11 days to nine days did not adversely affect T cell purity, as
comparable % CD3 T cells were found for all conditions upon harvest(Fig. 3C) .
While enhanced oxygen mixing through increasing agitation or direct
oxygen input promoted cell growth, those two approaches contrasted in
their impact on transduction efficiency. Having direct and steady oxygen
delivery through DO feedback loop control from the start (Xuri#3)
compromised transduction efficiency and resulted in the lowest TCR
surface expression (Fig. 3D) . In contrast, DO feedback loop
control at a later stage when the bioreactor was fully scaled up
(Xuri#5) or enhancing oxygen mixing through rocking agitation at 500 mL
volume (Xuri#2) led to TCR levels similar to standard conditions
(Xuri#1) (Fig. 3D) . In
addition, a higher inoculation density at 7e6 cells/ml (Xuri#4) led to
a ~15% increase in transduction efficiency compared
with conditions at 3.4e6 cells/ml (Xuri#2) (Fig. 3D) .
Correlating with higher TCR expression in cells from Xuri#4 and % Tcm
for cells from Xuri#2, INF-ɣ release was also modestly enhanced
compared with Xuri#1 when co-cultured with MAGE-B2 peptide loaded T2
cells for 24 hours at E:T ratio at 1 (Fig. 3E) . No distinct
differences were found for % T2 cells lysis at 48 hours due to
saturation of T2 cell lysis above 99% (Fig. 3F) .
T cell activation and expansion usually comes at the cost of
differentiation into effector phenotype (CD45RO+, CCR7-, CD95+) from
naïve (CD45RA+, CCR7-, CD95-), stem (CD45RA+, CCR7+, CD95+) and central
memory (CD45RO+, CCR7+, CD95+) pool, a phenomenon associated with a
shortened life span, T cell exhaustion and compromised therapeutic
persistence in the patient(Henning, Roychoudhuri, & Restifo, 2018;
Wherry, 2011). Cells from Xuri#2, Xuri#3, and Xuri#5, which had
greater oxygen/mass mixing compared to Xuri#1, contained significantly
higher % Tcm cells than cells from Xuri#1 and Xuri#4 when compared at
yield of 10e9 cells (Fig. 3G) . It is also notable that while
cells from #Xuri4 had the highest TCR surface expression (Fig.
3D) among all the conditions tested, cell growth and %Tcm was lower
than its counterpart conditions tested. On the other hand, while cells
from Xuri#3 showed higher % Tcm and enhanced cell growth, TCR surface
expression of those cells was 3-fold less than cells from Xuri#2(Fig. 3D) . This suggests that while oxygen promotes T cell
growth, it may negatively impact transduction when supplied in excess
during T cell activation. Overall, higher inoculation density and
greater rocking agitation adopted at 0.5 L culture volume enables a
shorter end-to-end bioprocess time with optimal T cell phenotype and
potency.