There is a strong impetus towards rapidly advancing an increasing number of novel biotherapeutics to clinical trials. However, development of cell culture processes is labor intensive and time consuming. KBI focuses on a high throughput process development (HTPD) approach using high-throughput miniaturized bioreactors and high throughput analytics that generate growth, productivity and product quality data that match those seen with classical systems. This approach enables a significant reduction in the cell culture process development timeline and costs for investigational biopharmaceuticals to reach the clinic.

1. 1
High-throughput Cell Culture Process Development
Integrated utilization of high-throughput bioreactors and high-throughput
analytics for rapid and robust cell culture process development.
Shahid Rameez, Srivatsan Gopalakrishnan, Carl Zhang, Jaspreet S. Notey, Christopher Miller,
Derek Ryan, Nathan Oien, James G. Smedley, Sigma S. Mostafa and Abhinav A. Shukla
KBI Biopharma Inc., 2 Triangle Drive, Research Triangle Park, NC 27709.
Executive Summary
There is a strong impetus towards rapidly advancing an increasing number of novel
biotherapeutics to clinical trials. However, development of cell culture processes is labor
intensive and time consuming. KBI focuses on a high throughput process development (HTPD)
approach using high-throughput miniaturized bioreactors and high throughput analytics that
generate growth, productivity and product quality data that match those seen with classical
systems. This approach enables a significant reduction in the cell culture process development
timeline and costs for investigational biopharmaceuticals to reach the clinic.
We integrated three technologies; (1) ambrTM miniaturized disposable bioreactors controlled by
an automated workstation for cell culture experiments. (2) ForteBio's Octet® for rapid and
accurate analysis of antibody concentrations that utilizes biolayer interferometry based
biosensors for antibody quantification. (3) The LabChip® separation system that utilizes
reusable micro-fluidic chips for rapidly screening N-glycan, protein charge and molecular
weight profiles.
This HTPD approach has demonstrated the ability to match results from classical systems. The
integrated utilization of high-throughput bioreactors and high-throughput analytics can be
implemented during various stages of cell culture process development for a range of biologic
therapeutics. This includes non mAb proteins that require more detailed process development
as opposed to implementation of a platform approach and biosimilars that need to match a pre-
determined product quality profile. In addition, this approach significantly increases knowledge
of the process and the influence of upstream process parameters on product quality and process
performance and facilitates more robust scale-up into manufacturing scales for any product
class.
With the increase in prominence of
biopharmaceuticals in the clinic (> 900)
and a steady increase in approvals (> $ 100
billion in annual sales), there is a strong
impetus is put on strategies to accelerate
clinical entry.1 In the current regulatory
landscape it often takes ten years and
billions of dollars to bring a drug candidate
from development to the shelves.2 While it
is typically desired to keep CMC (chemistry,
manufacturing and controls) activities off
the critical path for drug development, this
situation cannot be avoided prior to clinical
entry. Hence, there is increased interest in
pursuing methodologies that can shorten
the window for process development and
manufacturing. Some of these have arisen in
the form of platform processes, high
throughput methods and single-use
manufacturing technologies.3-6 At KBI, we

2. 2
High-throughput Cell Culture Process Development
have pursued all of these methodologies.
This white paper focuses on increasing
experimental throughput in process
development utilizing high throughput
methodologies.
Platform approaches have been successfully
adapted for the rapid development of
certain classes of therapeutics such as
monoclonal antibodies (mAbs). However,
even for this well-established product class,
what is gained in terms of speed is often
lacking in terms of process knowledge and
the influence of various process parameters
on process and product quality outcomes.
Biosimilar processes present an even
greater challenge. In this situation, a
comparable bioanalytical profile is critical to
achieve and is significantly influenced by
cell culture process parameters. Thus the
challenge in process development is finding
the right process conditions to produce a
molecule with matching product quality
attributes to the innovator.
With conventional laboratory scale
bioreactors and shake flasks being the
dominant forms of experimentation, the cell
culture development stage becomes a
resource and time intensive step.
Mammalian cell culture processes typically
have the longest experimental duration with
inoculum seed train and production culture
stretching between 4-6 weeks. In order to
test critical process parameters such as pH,
dissolved oxygen and agitation, bioreactors
must be used since shake flasks lack the
necessary control capabilities. During
optimization of a typical cell culture process,
at least 3-4 rounds of 10-12 bioreactor runs
need to be performed. This combination of
experimental duration and the extensive
resources required to run multiple reactors
in parallel makes the cell culture process
development stage a key bottleneck step
during process development. More
importantly, to develop a robust cell culture
process that ensures batch to batch product
quality consistency, Design of Experiment
(DOE) based studies have to be
implemented during cell culture process
development to reveal the effect of cell
culture changes on homogeneity, purity and
post translational modifications. These
studies provide for a comprehensive process
understanding which in turn enables the
production of more consistent batches.
However employing this approach produces
a large number of bioreactor runs and a
large number of samples. This in turn can
exceed the resources and capacity of cell
culture and analytical laboratories which
primarily depend on conventional small
scale glass bioreactors (1-15L in size) and
HPLC and CE based separations to monitor
protein quantification and product quality.
As a result there is a compelling demand for
a HTPD platform which enables key process

3. 3
High-throughput Cell Culture Process Development
decisions during the early process
development phase.
In the paper above, we have demonstrated7
the ability to employ the ambr™ system to
make key process decisions during the
development of a biopharmaceutical
manufacturing process. The capability to
fine-tune process controls with 24-48
single-use miniature bioreactor vessels
provides for a platform to employ fractional
factorial and minimum-run designs to
enable identification of key process
parameters and interactions of those
process parameters. Moreover, the
reproducibility and scalability of the system
enable its use for high throughput
experiments for cell culture process
development during the first in human
(FIH) phase of biopharmaceutical drug
development, offering a significant
possibility of decreasing the development
timeframe prior to clinical entry (Figure 1).

4. 4
High-throughput Cell Culture Process Development
Figure 1: Clinical and process development/manufacturing activities during biopharmaceutical
development and role of ambr™ in accelerating product development during the FIH phase of the
biopharmaceutical development lifecycle.
In addition to the ambr™ system we have
integrated two high throughput analytical
technologies to create a high throughput
process development (HTPD) platform
where the effect of media, feeds, feeding
frequency and process parameters on
various product quality attributes are
studied right from the early phases of cell
culture process development. The two high
throughput analytical technologies are
ForteBio's Octet® for rapid and accurate
analysis of antibody concentrations and
LabChip® separation system that utilizes
reusable micro-fluidic chips for rapidly
screening molecular weight, N-glycan and
protein charge profiles. Octet utilizes
biolayer interferometry (BLI) based
biosensors for antibody quantification.
These biosensors are coated with a
biocompatible matrix to analyze specific

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High-throughput Cell Culture Process Development
biomolecular interactions. Both these
analytical technologies provide particular
value in applications where existing
methods such as HPLC, ELISA, SDS-PAGE
and Capillary Electrophoresis, have
limitations in throughput, performance,
workflow, and ease of use. Figure 2 shows a
schematic for the HTPD approach which
utilizes high throughput microbioreactors
and high throughput analytics to accelerate
product development. HTPD approach can
be utilized all the way starting from
selection of a clone during the cell line
development. Due to limitation of time and
resources relatively few top clones (top 1 - 4
clones) are evaluated in conventional
bioreactors which decreases the chance of
identifying a high producing clone with
desired quality attributes. HTPD overcomes
this limitation of time and resources while
offering capability of evaluating a larger
number of clones (top 24 – 48 clones) in
parallel under representative stirred tank
bioreactor conditions. In particular, this
broader screening benefits biosimilar
programs in which the desire is to identify a
clone that is capable of producing specific
product quality attributes. In addition,
during the cell culture process development
phase, HTPD enables the investigation of
factors like pH, temperature, dissolved
oxygen, nutrients in media and feeds,
glucose, ammonia, salt and other
metabolites that have shown to affect the
productivity and product quality of proteins.
The ambr™ system when operated under
fed-batch conditions with appropriate pH,
DO and feed controls can successfully
simulate bioreactor culture conditions with
highly reproducible results between the
replicates. Cell growth, process capabilities,
and product titer and product quality
profiles are comparable to classical
bioreactors of various scales, 3, 15 and 200L
and found to be within 10-15% of mean
values. The 24-48 single use vessels provide
flexibility to run larger experimental designs
in parallel to evaluate feeding regimes,
process operating limits and interactions
between various operating parameters.
Overall, the reproducibility of key
observations and scalability of key results
with the system has been demonstrated to
be adequate to utilize this system for cell
culture process development.7
A typical optimization of a cell culture
process, which requires at least 3-4 rounds
of 10-12 bioreactor runs, it takes 3-4
months. This is due to duration of 2-3 weeks
for the production bioreactor step with
additional 1-2 weeks on the seed cultures.
The same optimization can be achieved in
ambrTM system (48 bioreactors) in a month
with experiments run in parallel. In
addition, the classical reactors require
cleaning, set up and autoclaving prior to

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High-throughput Cell Culture Process Development
Figure 2: Utilization of high throughput cell culture development and high throughput analytics (HTPD
approach) in accelerating product development during the first in human (FIH) phase of the
biopharmaceutical development lifecycle.
Figure 3: Comparison of time courses for viable cell growth for recombinant CHO cell lines in ambr™
vessel and other scales classical bioreactors; 3L and/or 15L glass bioreactors and 200L single-use
bioreactor for (A) mAb and (B) non-mAb. The experimental data for ambr™ shows an average of 3 and 2
vessels in figures A and B respectively.
their use in studies. The single use pre
calibrated bioreactor vessels in the ambrTM
system overcome this limitation and provide
significantly faster turnaround times while
significantly reducing time, cost and labor.

7. 7
High-throughput Cell Culture Process Development
Table 1: Cell culture performance comparison between bioreactor systems (ambrTM, 3 and/or 15L Glass
bioreactors and 200L single-use bioreactor) for Viability at harvest (%), Titer (Normalized), Cell-
maximum growth rate (1/d) and Cell-specific productivity (pg/cell/d) for a mAb and a non-mAb.
Bioreactor
System
Viability at
Harvest
(%)
Titer
(Normalized to 200L
titer values)
Cell Maximum
Growth Rateh
(1/d)
Cell-specific
Productivity
(pg/cell/d)
mAb
ambra 90.27 ± 0.14 0.96 0.37 16.20
3-Lb 98.70 1.06 0.37 10.60
15-Lc 91.38 ± 2.19 0.88 0.34 10.80
200-Ld 90.20 1.00 0.34 11.70
non-mAb
ambre 81.20 0.99 0.46
15-Lf 61.40 0.94 0.51
200-Lg 84.20 1.00 0.47
a: n = 3, b: n = 1, c: n = 4, d: n = 1, e: n = 2, f: n = 1, g: n = 1, h: Measured from days 0-8 for mAb
and from days 0-7 for non-mAb .
Figure 4: Comparison of two mAbs (X and Y) concentrations in eight 3L glass bioreactors using OctetTM
and Protein A HPLC methods. The results between the two methods are comparable. For most of the
samples the variability between two methods was less than 5%. Figure 3A shows the experimental data for
Octet™ as an average of 3 measurements. Reproducible results are obtained between replicates in
Octet™. The titers are within ±1% of each other. In addition, the % CV was less than 3%.
We present data from two case studies
demonstrating HTPD approach employed
during cell culture process development for
a Biosimilar. Case study I aimed at
evaluating 8 different feeds for CHO cell
line producing a Biosimilar. This was
followed by case study II which was a DOE
study evaluating the effect of process pH
and four different feeding frequencies (FDS
A, B, C and D) for the selected feed on the
Biosimilar. We monitored the productivity
and product quality attributes (charge and
N-glycan) and compared them to the
innovator drug product.

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High-throughput Cell Culture Process Development
Figure 5: Multiple overlay electropherogram for a mAb C showing different charge species (left figure).
Comparison of mAb C charge variants using LabChipTM and conventional cation exchange
chromatography (CEX) method (right figure). Comparable results were obtained between two methods
for different charge variants. The variability between two methods was less than 5%.
Figure 6: Multiple overlay electropherogram for a mAb C showing different charge species (left figure).
Comparison of mAb C charge variants using LabChipTM and conventional cation exchange
chromatography (CEX) method (right figure). Comparable results were obtained between two methods
for different charge variants. The variability between two methods was less than 5%.
As an example, Figure 7 A and B show one
specific glycan structure (G0F) from these
case studies, a critical quality attribute in
this Biosimilar, and show the change it
undergoes under various tested process
conditions. Based on the results, the
conditions which do not allow the G0F to
remains within the value ± variability of the
originator molecule were not carried
forward. Thus feeds 3, 7 and 8 (Figure 7A)
were not evaluated further. Moreover, the
selected feed showed strong interaction with
respect to process pH to control the critical
quality attribute in this Biosimilar (Figure
7B). Both these studies helped assess
product quality metrics from cell culture
process development and identify right
conditions to produce the molecule with
matching product quality attributes to the
innovator.

9. 9
High-throughput Cell Culture Process Development
Figure 7: Percentage (normalized to innovator
value) of specific glycan structure (G0F) in case
studies I and II, a critical quality attribute in the
Biosimilar, and change it undergoes under
various tested process conditions. Based on the
results, the conditions which do not allow the
G0F to remains within the value ± variability
(blue region) of the originator molecule were not
carried forward.
Conclusions
The multi-stage nature of process
development and the long duration of
mammalian cell culture experiments makes
it time and resource intensive. HTPD
approach offers realistic possibility of
decreasing the timeline for process
development experimentation. This in turn
decreases the timeframe to manufacturing
clinical material prior to clinical entry. In
addition, material needs and other
resources are minimized and thus a larger
number of drug candidates can be advanced
into the clinic faster to address the unmet
clinical needs.
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High-throughput Cell Culture Process Development
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Acknowledgements
We thank Joe McMahon, CEO of KBI Biopharma Inc., for his support for this work. Members
of the process development, analytical development and formulation sciences teams at KBI
Biopharma Inc. are thanked for providing support during the pursuit of process development
programs.