High Throughput Bioreactor Mimetic in Early and

High throughput bioreactor mimetic in
early
l and
d late
l t stage
t
process development
d
l
t
American Chemical Society – Biochemical Technology (BIOT) Division
245th – ACS National Meeting, New Orleans, LA
Shahid Rameez, Ph.D.
Scientist I, Process Development
KBI Biopharma
p
Inc, Durham, NC
Overview
Bioreactor
Mimetic
Ambr System
KBI Evaluation
Experimental Design
in Earlyy and Late
Stage Process
Development
In pprocess
Activities
&
Design Space
Evaluation
C t l Strategies
Control
St t i
 A keyy bottleneck in biopharmaceutical
p
development
p
has been the
rapid development of robust and scalable manufacturing processes that
can permit accelerated progress of products into clinical trials.
 Innovations in this area can have a very significant impact on the
overall economics of biopharmaceutical drug development by
d
decreasing
i the
h time
i it
i takes
k to reach
h the
h clinic.
li i
 Mammalian cell culture p
processes typically
yp
y have the longest
g
experimental duration with 2-3 weeks being a typical duration for the
production bioreactor step with additional time spent on the seed
cultures.
cultures
 Shake flasks provide the capability to perform high throughput
experiments, but with an inability to control process parameters like
agitation rate, dissolved oxygen (DO) and pH.
 These
Th parameters
t play
l a huge
h
role
l when
h developing
d l i a robust,
b t efficient
ffi i t
cell culture process which dictates the product quality and yield.
 Moreover, reproducibility and scalability of process and culture
performance is a pre-requisite for successful and efficient process
development at a scale down level.
 At KBI we did case studies evaluating the ambr™ system (an
a tomated micro-scale
automated
micro scale bioreactor system)
s stem) to establish the role
this system could play in accelerating biotech drug development.
Automated Miniaturized Bioreactors
ambrTM Technology
1mL tips
1mL
or
4mL tips
Liquid
Handler
Used Tips
Discard
Culture stations; each holding 12 bioreactors
ambrTM Deck Layout
Culture stations
1ml tips
4ml tips
Plate lid
24 deep-well plate
Tip box lid
The Vessel
Vessel Cap:
In-line filter
on gas supply
DO sensor
Impeller
pH sensor
p
Culture Station Nomenclature
CS1-6
CS2-1
CS2-2
CS2-3
CS2-4
CS1-112
CS2-77
CS2-88
CS2-99
CS2-110
CS2-6
CS1-5
CS1-111
CS2-112
CS1-4
CS1-110
Things to consider:
– Feed strategy and Sampling strategy
– Low volumes – gas entrainment / vortexing
– High volumes – Limits kLa.
CS2-5
CS1-3
CS1-99
Id l working
Ideal
ki volume
l
iis 13mL; Working
W ki volume
l
range iis 11
CS2-111
CS1-2
Culture station 2 (CS2)
CS1-88
CS1-77
CS1-1
Culture station 1 (CS1)
– 15mL
PART 1:
Reproducibility for Results and Key Observations during Cell Culture Process Development
 The processes evaluated in ambrTM were previously developed from a rigorous
cell culture process development performed in classical bioreactors of various
scales. The process were successfully carried in bioreactors across various scales: 2L,
10L and
d 200L.
200L
 This study aimed at studying the reproducibility of the key observations of the
processes in ambrTM. Thus a reverse engineering approach was adopted, where we
were cognizant about the outcomes from most of the experiments as far as
inducing process variations was concerned.
 The reproducibility of key historical results in ambrTM would corroborate towards
its capability as a high throughput bioreactor mimetic in cell culture process
development.
Case Study 1: Reproducibility evaluation for the production of a monoclonal antibody in a
recombinant Chinese Hamster Ovary (CHO) cell line.
Key Observations
K
Ob
i
ffrom Hi
Historical
i lD
Data:
• Temperature Shift during the cell culture process was found to be the most important
process factor to regulate the productivity of the antibody titer.
•
The CHO cell line performed better at lower pH set point of 6.85 as compared to pH
set point of 7.00.
•
Feeding intermittently had shown to regulate growth and productivity in the process.
Intermittent feeding had showed better results than just Day 0 additions for feed.
•
A highly basic and a critical feed, referred here as FDX, had to be added without preneutralization with acids to avoid osmolality increase in cultures. Thus, a better control
had to be established in the bioreactors to control the pH drift with addition of FDX.
This
h was achieved
h
d in classical bioreactors
b
b tuning the
by
h PID controllers andd with
h
regulation in cascade feedback gassing of CO2 and Air.
Results: Both lower pH set-points and Temperature Shift showed higher cell growth, better
cell viabilities. DO as suspected at negligible effect on cell growth and viability.
Time courses for viable cell growth and viability for recombinant CHO cell line with changing
(A) Process pH (B) Temperature (C) Dissolved Oxygen (DO) levels and (D) Feeding Strategies. The
experimental data shows an average of 2-3 vessels in the ambr 24. The error bars show the standard
deviation.
deviation
Results: Both lower pH set-points and Temperature Shift showed higher cell titers. As
observed historically for this process, Temperature shift was found to be the most important
process factor to regulate the productivity of the antibody titer.
 The ambrTM system can be used as a high-throughput platform to make key
process decisions during the early process development phase of
bi h
biopharmaceutical
i l development.
d l
PART B:
Scalability Assessment in Cell Culture Process Development
Case Study 2: Comparison across scales for the production of a monoclonal antibody in a
recombinant CHO cell line.
Ambr (n = 3).
2L (n = 1).
10 (n
10L
( = 4).
4)
200L (n = 1).
• Harvest Titers within 1.5
1 5 -1.7
1 7 g/L
across all scales.
Comparison of time courses for viable cell growth and viability for recombinant CHO cell line in ambrTM and other
scales bioreactors: 2, 10L Glass bioreactors and 200L disposable
p
bioreactor.
Case Study 3: Comparison across scales for the production of a protein molecule in a
recombinant Chinese Hamster Ovary (CHO) cell line.
Results: The cell growth, cell viability and cell
viabilities were comparable between ambrTM,10 and
200L Bioreactors.
Bioreactors
Case Study 3: This protein molecule had two Isodimers (A and B). The levels of Isodimers A
and B were a product quality attribute.
Results: Ratio of Isodimers A
and B were similar (± 5% of
mean values) across ambrTM , 10
and 200L Bioreactors.
 The process decisions and results from ambrTM were reproducible to the
results in other scales bioreactors.
Both the case studies (with antibody and a non antibody) demonstrate the
utility of the ambr™ system as a high throughput system for cell culture
process development.
p
p
PART C:
Control for process pH and DO during Cell Culture Process Development
 pH control in ambrTM is established using the
automated liquid handler based base additions
when pH drops below the pH set point.
point
 When the pH exceeds the pH set point, the
CO2 flow rate increases to establish control on
the pH drift.
CO2: 0 - 1.24 mL/min. Delivered on demand to control pH.
O2 : 0 - 1.24 mL/min. Delivered on demand to control dissolved oxygen.
N2 : 0 - 11.24
24 mL/min.
mL/min Flow rate is constant.
constant
 Online profiles for process pH (top
figure) and DO (bottom figure) levels
during the culture duration for CHO cell
line expressing a recombinant antibody
in ambrTM.
 The spikes in the DO profiles
corresponded to bioreactor sampling,
sampling
Liquid additions and Sampling.
 All these disturb the headspace and
alter
l the
h working
ki volume.
l
Th time
The
i
f
for
the DO traces to equilibrate to setpoint
after such manipulations would depend
on the controller setup.
p
Case Study 4: Artificial perturbations in pH and DO (by adding a basic feed and changing DO
set points respectively) during production of an antibody molecule in a recombinant CHO cell
process. Through adjustments to the PID control loop and gas flow rates the capability of
ambr™
b ™ system
t was evaluated.
l t d
Results: Tuning the gas flow limits and proportional gains in the PID loop of ambr™ system.
By changing the proportional gain by eight folds and CO2 gas limits by 1.25 and > 2folds as
opposed to default manufacturer values , the pH drifts were reduced by 23 and 47 % of initial
value, respectively.
Results: The DO set points were changed to 80% from 20 and 40%, respectively and
changed back to original values.
values The level for DO was maintained at 80% for duration of 6
hours and returned to original set points 20 and 40% in ≈ 90 and 120 mins, respectively.
 The
Th capability
p bilit off inducing
ind in deviations
d i ti n can
n help
h lp in designing
d i nin worst-case
r t
experiments. It enables to test operating limits with respect to particular
key operational parameters (DO, pH) in a process.
Conclusions
 The combination of pH and DO control and an automated liquid handling system in
ambrTM system overcomes major limitations of conventional small
small-scale
scale cultures vessels
especially shake flasks.
 The single-use, pre-calibrated, and instrumented vessels used in ambrTM system provides
a platform for high-throughput in cell culture process development while mimicking a
stirred-tank bioreactor environment.
 T
Thee reproducibility
ep od c b ty o
of key
ey obse
observations
vat o s obse
observed
ved in historical
sto ca p
process
ocess deve
development
op e t
demonstrated that ambr™ is capable of providing predictive results under bioreactor
relevant process conditions.
 Reproducibility,
R
d ibili scalability
l bili andd the
h ability
bili off the
h system to respondd to perturbations
b i
show
h
ambrTM to be adequate to consider this system for early and late stages of cell culture
process development.
 The studies at KBI aimed to demonstrate the utility of the ambr™ system as a
high- throughput bioreactors that can offer the realistic possibility of decreasing the
process development time for investigational biopharmaceuticals to reach the clinic.
Biopharmaceutical Development Process.
Discoveryy
Stage
Cell Line
Development
Process
Development
Manufacturing
Commercial Process
Development
Process
Characterization
and Validation
RAPID PRODUCT DEVELOPMENT AT KBI
ambrTM
Discovery Stage
Cell Line Development
Process Development
(Design Space & Optimization)
Manufacturing
• Platform Downstream Processes
• High-throughput Resin Screening
• Single - Use Technology
 The combination of methodologies such as ambrTM, Platform Downstream
Processes, High-throughput Resin Screening and use of Single-use technology
can significantly shorten the window for process development and
manufacturing.
f t i
Acknowledgements
• Joe McMahon
• Abhinav Shukla, Ph.D.
• Sigma Mostafa,
Mostafa Ph
Ph.D.
D
• Haiou Yang, Ph.D.
• Christopher Miller
• Anushya Mani
• Joe Jirka
President and CEO
VP, Process Development and Manufacturing
Director Process Development
Director,
Scientist II, Process Development
Scientist II, Process Development
Scientist I,
I Process Development
Product Specialist, TAP Biosystems
 Process Development Team at KBI
Thanks
Questions??