Despite the strong impetus towards accelerating novel biotherapeutics in clinical trials, the development of cell culture processes remains a labour-intensive and time-consuming procedure. High-throughput methods are showing huge potential in the mission to reduce both time and costs This article is taken from European Biopharmaceutical Review July 2016, pages 36-40. © Samedan Ltd View the publication here: http://www.samedanltd.com/magazine/current/12

1. Cell Culture Development
Up to Speed
Despite the strong impetus towards accelerating novel
biotherapeutics in clinical trials, the development of cell culture
processes remains a labour-intensive and time-consuming
procedure. High-throughput methods are showing huge potential
in the mission to reduce both time and costs
36 July 2016
IND
Preclinical
Process
development
Manufacturing
for
tox
Clinical
manufacturing
Commercial
process
development
Process
characterisation
Process
validation
BLA
and
PAI
Process monitoring
and
improvement
FIH process
ambrTM
Commercial process
Tox Phase 1 Phase 2 Phase 3
Submission Lifecycle
managementand
approval
POBA
Discovery
support
Cell line
development
Design space
and
optimisation
Manufacturing
First in human (FIH) process
• Deliver clinical process quickly
• Platform process
• Clinical supply
Commercial process
• Deliver manufacturing process for
registrational trials and market
• Design keeping large-scale
manufacturing
• Improve productivity, efficiency,
robustness, manufacturability,
cost of goods (CoGs)
Process characterisation and validation
•Developin-processcontrol(IPC)strategythrough
understandingofprocessinputsandoutputs(designspace)
•Scale-downcharacterisationandvalidationstudies
•Large-scaleprocessvalidationtodemonstrate
processconsistency
•Biologicslicenseapplication(BLA)preparation
•Supportingdocumentsforlicensureinspections
•Post-commercialprocessimprovement(CI)
•Post-commercialprocessmonitoring
With the growing prominence of biopharmaceuticals in the clinic
(which currently amounts to more than 900), and a steady increase
in approvals totalling more than $100 billion annually, there is
a strong impetus within the industry to implement strategies
that will accelerate clinical entry (1). In the current regulatory
landscape, it often takes 10 years and billions of dollars to bring
a drug candidate from development to the shelves (2).
While it is typically desired to keep chemistry, manufacturing and
controls (CMC) activities off the critical path for drug development,
this situation cannot be avoided prior to clinical entry. Hence, the
industry has shown increased interest in pursuing methodologies
that can shorten the window for both process development
and manufacturing. Some of these have arisen in the form of
platform processes, high-throughput methods and single-use
manufacturing technologies (3-5). In this article, we will focus on
increasing experimental throughput in process development
utilising high-throughput methodologies.
Understanding the Processes
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
Shahid Rameez
and Abhinav A Shukla
at KBI Biopharma
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

2. www.samedanltd.com38
of various parameters on procedure and product quality
outcomes. Biosimilar developments 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 obstacle in development is finding
the right conditions to produce a molecule which matches the
product quality attributes of 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 methods typically have the longest experimental
duration, with inoculum seed train and production culture
stretching between four and six weeks. In order to test critical
process parameters such as pH, dissolved oxygen and agitation,
bioreactors must be used as shake flasks lack the necessary control
capabilities. During the optimisation of a typical cell culture
procedure, at least three to four 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 phase a key bottleneck stage
during process development. More importantly, to develop a
robust cell culture process that ensures batch to batch product
quality consistency, design of experiment based studies must be
implemented in order to reveal the effect of cell culture changes
on homogeneity, purity and post-translational modifications.
These studies provide for a comprehensive process
understanding, which subsequently aids the production of more
consistent batches. However, employing this approach produces
a large number of bioreactor runs and a vast amount of samples.
This, in turn, can exceed the resources and capacity of cell culture
and analytical labs, which primarily depend on conventional
small-scale glass bioreactors (1-15L in size) and high-performance
liquid chromatography (HPLC) and capillary electrophoresis-based
separations to monitor protein quantification and product quality.
As a result, there is a compelling demand for a high-throughput
process development (HTPD) platform which facilitates key
decisions during the early process development phase.
Case in Point
The ambr™ system was employed to make crucial process
decisions during the development of a biopharmaceutical
manufacturing process (6).The capability to fine-tune process
controls with 24-48 single-use miniature bioreactor vessels
provides a platform to use fractional factorial and minimum-run
designs, enabling identification of key process parameters and
interactions of those parameters. Moreover, the reproducibility
and scalability of the system allows for high-throughput
experiments in cell culture process development during the FIH
phase of biopharma drug development, offering a significant
possibility of decreasing the development timeframe prior to
clinical entry (see Figure 1, page 36). In addition to this, two further
high-throughput analytical technologies were integrated to
enhance performance, workflow and ease of use.
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 (normalised), cell-maximum growth rate (1/d) and
cell-specific productivity (pg/cell/d) for a mAb and a non-mAb.
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 2: 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 parts A
and B respectively
Viablecellcount(106
cells/mL)
Viablecellcount(106
cells/mL)
0 02 24 46 68
Days
mAbA B
ambr ambr3L 15L 15L200L 200L
non-mAb
Days
810 1012 1214 1416 16
Bioreactor
system
Viability at
harvest (%)
Titer (normalised to
200L titer values)
Cell maximum
growth rateh
(1/d)
mAb
ambra
90.27 ± 0.14 0.96 0.37
3-Lb
98.70 1.06 0.37
15-Lc
91.38 ± 2.19 0.88 0.34
200-Ld
90.20 1.00 0.34
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

3. www.samedanltd.com 39
The HTPD approach utilises high-throughput
microbioreactors and analytics to accelerate product
development. The HTPD method can be deployed throughout
the entire process, starting with the selection of a clone
during the cell line development. However, due to limitations
in time and resources, relatively few top clones (the top 1-4)
are evaluated in conventional bioreactors; this decreases the
chance of identifying a high-producing clone with desired
quality attributes.
HTPD overcomes this restriction, while offering the capability
to evaluate a larger number of clones (the top 24-48) in parallel
under representative stirred tank bioreactor conditions. In
particular, this broader screening benefits biosimilar programmes
in which the objective is to identify a clone that is capable of
producing specific product quality features. Furthermore, during
the cell culture process development phase, HTPD allows the
investigation of factors including 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.
In Figure 4 (page 40), data is shown from two case studies
demonstrating the HTPD approach during cell culture process
development for a biosimilar.The aim of case study 1 (part A) was
to evaluate eight different feeds for Chinese hamster ovary (CHO)
cell line producing a biosimilar.This was followed by case study 2
(part B), which was a DOE.
As an example, Figure 4 shows one specific glycan structure
(G0F) from these case studies – a critical quality attribute in this
biosimilar – and the change it undergoes during various tested
process conditions. Based on these results, the conditions that
do not allow the G0F to remain within the value ± (more than,
or equal to the) variability of the originator molecule were
not carried forward. Therefore, feeds 3, 7 and 8 (see Figure 4,
part A) were not evaluated further. Moreover, the selected
feed showed strong interaction with respect to process pH
Figure 3: Comparison of (A and B): mAb X and mAb Y concentrations in eight 3L glass bioreactors using Octet™ and Protein A HPLC methods; (C and D): mAb
C charge variants using LabChip™ and conventional cation exchange chromatography (CEX) method; (E and F): mAb C N-glycan species using LabChip™ and
conventional fluorescence-based hydrophilic interaction liquid chromatography (HILIC) method. The variability between HTPD and conventional methods was less
than 5%
mAb X ProA HPLC
3L bioreactor number
3.50
A B
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
3.00
2.50
2.00
1.50
1.00
0.50
0.00
B01 B01B02 B02B03 B03B04 B04B05 B05B06 B06B07 B07B08 B08
3L bioreactor number
ProA HPLCOctet Octet
Antibodyconcentration(g/L)
Antibodyconcentration(g/L)
mAb Y
Fluorescence
600
500
400
300
200
100
0
30 35
Time (sec)
Free Dye
Basic
50
10
0
20
40
30
60
70
80
Microchip CZE (mAb C)
Percentage
CEX (mAb C)
Main Acidic
Main
AcidicBasic
40 45
Fluorescence
4000
3000
2000
1000
0
8 9 10 11 12
Multiplate overlay electropherogram
(mAb C triplicate measurements)
Man5
50
10
0
20
40
30
60
70
MicroChip N-glycan
Percentage
HILIC N-glycan
Man5G2G0G2FG1FG0F
G0
G0F
G1F
G1F
G2F
Size (CGU)
C
E
D
F

4. Abhinav A Shukla is Senior Vice President
for Development and Manufacturing at
KBI Biopharma, a rapidly growing CDMO
organisation. Abhinav has over 18 years of
experience in developing biopharmaceuticals
from early development through
commercialisation launch. Most recently, he was Director
of Manufacturing Sciences andTechnology at Bristol-Myers
Squibb, where he led the successful commercialisation of
Yervoy®
, Nulojix®
and Orencia®
, in addition to other late-stage
programmes. He also led the creation of a platform approach
for mAbs at Amgen. Abhinav is a known industry expert in
biopharmaceutical development and has over 40 publications
in this area. He serves on the editorial boards of several
journals, including Biotechnology and Applied Biochemistry
and Bioprocess International. He has a PhD in Chemical and
Biochemical Engineering from Rensselaer Polytechnic Institute.
Email: ashukla@kbibiopharma.com
Shahid Rameez, PhD, is a Principal Scientist
for Process Development at KBI Biopharma
Inc. Shahid has experience in CMC activities
responsible for design, development and
implementation of efficient and robust cell
culture processes for small- and large-scale
manufacturing using various expression systems. Prior
to joining KBI in 2012, Shahid completed his doctoral and
postdoctoral studies at Ohio State University on designing
cellular protein therapeutics. He has over 10 publications in
the area of process development and protein therapeutics.
Email: srameez@kbibiopharma.com
About the authors
40
to control the critical quality attribute in this biosimilar (see
Figure 4, part B). Both these studies helped to assess product
quality metrics from cell culture process development, while
identifying the right conditions to produce the molecule with
matching product quality attributes to the innovator.
Method Analysis
The multi-stage nature of process development and the long
duration of mammalian cell culture experiments make it a time-
and resource-intensive procedure. Of the approaches tested,
the HTPD method offers a realistic possibility of decreasing
the timeline for process development experimentation, which,
in turn, decreases the timeframe for manufacturing clinical
material prior to clinical entry. In addition, material needs and
other resources are minimised, and thus a larger number of drug
candidates can be advanced into the clinic faster, in order to
address unmet clinical needs.
Acknowledgements
The authors would like to thank Joe McMahon, Chief
Executive Officer 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.
We would also like to thank Srivatsan Gopalakrishnan,
Carl Zhang, Jaspreet Notey, Christopher Miller, Derek Ryan,
Nathan Oien, James Smedley and Sigma Mostafa for
their help with this article.
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Figure 4: Percentage (normalised to innovator value) of specific glycan structure (GoF) in case studies 1 (A) and 2 (B), a critical quality attribute in the biosimilar, and change
it undergoes under various tested process conditions. Based on these results, the conditions which do not allow the GoF to remain within the value ± vavariability (blue
region) of the originator molecule were not carried forward
120 120
A B
110 110
100 100
90 90
80 80
70 70
60 60
1
Feeds
%GoFofinnovatorproduct
%GoFofinnovatorproduct
2 3 4 5 6 7 8 7.00 ± 0.2
FDSA FDSB FDSC FDSD
7.00 ± 0.1 7.00 ± 0.05
www.samedanltd.com
pH range