Download Metabolic Process Engineering

B
i o
P
r o c e s s Technical
Metabolic Process Engineering
A Novel Technology Platform Applied to
Industrial Cell Culture Production Processes
Bernhard M. Schilling, Susan Abu-Absi, and Patrick Thompson
M
etabolic process engineering
(MPE) was developed at
Bristol-Myers Squibb
Company as a tool to
effectively control and optimize
industrial cell culture processes used
for production of biological drugs. A
fundamental need was identified to
introduce manipulations to the
metabolism of production cell lines
without genetic engineering.
Optimization goals for production cell
line performance include, for example,
volumetric productivity, control of
product quality attributes and
by-product formation, and improved
process scalability. With MPE, we
could achieve targeted changes to
cellular metabolism through timed
addition of chemicals to a production
process.
Here we describe the MPE concept
and provide examples of its use. We
made two further observations when
applying it: Well-understood,
scientifically based changes to the
production process had the highest
chance of meeting product
Product Focus: Proteins
Process Focus: Production
Who Should Read: Process
development, analytical, QA/QC, and
manufacturing
Keywords: Fed-batch, culture media
supplementation, glycosylation,
viability, lactate, optimization
Level: Intermediate
42 BioProcess International
10(1)
January 2012
A seed reactor at Bristol-Myers Squibb in Devens, MA
comparability needs, thus facilitating
regulatory approval. And poorly
understood, empirical approaches
carried the risk of introducing
undesirable variability into process
performance and changes to product
quality attributes.
In early product development, a
wide array of activities are ongoing to
define a cell culture production
process: e.g., host cell choice,
expression vector design, cell line
development, clone screening and
selection, media development, process
development, and process scalability
studies. In media development,
improvements in the design of
chemically defined media, taking
advantage of spent media analysis,
have advanced analytical capabilities
bristol-myers squibb company (www.bms.com)
and high-throughput technologies (1).
However, empirical nutrient
optimization is also continued, along
with the use of complex raw materials
(2).
The opportunity to use the
techniques described herein narrows
when entering the commercialization
phase of a biological drug.
Furthermore, regulatory expectations
are constantly increasing with regard
to the level of expected process
understanding, especially the required
knowledge behind proposed changes.
Our company therefore determined
that a tool needed to be developed
that would provide intrinsic process
understanding while assisting process
developers in meeting their desired
optimization goals.
Table 1: Examples of chemicals added to cell culture or microbial production processes
Chemical and Reference
Sodium butyrate (4)
Cell Line
CHO-K1
Sodium butyrate (5)
Vanadate (6)
CHO
CHO, Vero, BHK, HeLa,
COS, 293
CHO-K1, HEK-293
CHO
Peptide-based chemical caspase inhibitors (7)
Small-molecule enhancers: aromatic
carboxylic acids, hydroxamic acids including
hexanohydroxamic acid and acetamides (8)
Rapamycin (9)
Hybridoma
Dimethyl sulfoxide (10)
Dimethyl sulfoxide (11)
Hybridoma
CHO
Pentanoic acid (12)
Hydroxyurea (13)
CHO
Saccharomyces cerevisiae
Conceptual
MPE has similar goals as the wellknown concept of metabolic
engineering (3), but it achieves them
through timed addition of chemicals
to a production process rather than
the use of genetic engineering. Figure
1 presents the concept step by step.
First, identify your goal for a desired
production cell line performance
change. Collect and analyze
knowledge from key metabolic
pathways, metabolic flux analysis, and
scientific literature (including medical
publications) to form a hypothesis for
a potential mode of action. Next,
attempt a targeted manipulation of
your production cell line’s metabolism
through addition of selected candidate
chemicals to the production process.
•
MPE has similar
goals as the
well-known concept
of metabolic
engineering, but it
achieves them
through timed
addition of chemicals
rather than the use
of genetic
engineering.
44 BioProcess International
10(1)
January 2012
Purpose/Observation
Controlling sialic acid content of glycoproteins and cell-specific
productivity
Increasing synthesis of secreted proteins
Enhancing cell survival so that recovery of a polypeptide of interest
produced in the cells increases
Increasing integrated cell densities and monoclonal antibody titers
Increasing monoclonal antibody production
Keeping cell viabilities high and delaying cell death; attaining higher
maximum viable-cell densities and enhancing monoclonal antibody
production
Increasing specific monoclonal antibody and overall production
Inducing growth arrest to reduce cell growth rate and achieve a
high overall specific productivity
Enhancing protein biosynthesis
Controlling growth rate and cell division
Finally, verify successful results
through measurement of metabolic
parameters using, for example,
enzymatic or chemical analysis,
metabolomics, and/or transcriptomics.
Multiple published case studies
report successful addition of chemicals
to cell culture and microbial processes
for the purpose of manipulating process
performance. Table 1 lists some.
Application
Production cell lines usually have
weaknesses that affect overall
process performance. However, such
cell lines have been used in
manufacturing, where they define
the biochemical and physicochemical
characteristics of a biological drug.
The goals for a change in cell-line
performance may be
• volumetric productivity (peak
viable cell density, cell viability profile,
cell-specific productivity)
• product quality attributes (posttranslational modification, product
isoforms, product aggregation)
• by-product formation (lactate,
ammonia, carbon dioxide),
• process scalability.
Areas that can be researched for
potential targets of cell metabolism
include bioenergetics, cell signaling,
target protein expression, stress
reduction, membrane renewal, and
others. Enzymes, receptors, signaling
compounds, and metabolic flux are all
potential metabolic targets.
Candidate chemicals may serve as
effectors, precursors, traps, or
Figure 1: Step-by-step description of
metabolic process engineering (MPE)
Identification of desired change
in metabolic performance
Analysis of relevant
metabolic pathways
(e.g., flux analysis, metabolomics,
or transcriptomics)
(large amount of information available
in the public domain on mammalian
cell metabolism, and from medical
research literature re: cells,
tissues, organs, organisms)
Hypothesis formed on a possible
metabolic target and mechanism
Experiments with chemical agents
executed for proof of concept
If successful, verification of
mechanism at metabolic level
receptor blockers. Those chosen for
evaluation need to fulfill certain
criteria for use in industrial cell
culture processes. They must not be
nutrients, proteins or peptides, or
animal derived. They must be
nontoxic and economical in pricing.
And they must not lead to
performance trade-offs. Such tradeoffs are observed, for example, in the
use of sodium butyrate (14), with
which a higher observed cell specific
productivity is tied to reduced cell
growth.
For MPE, high-throughput
screening experiments are set up to
evaluate candidate chemicals.
Concentration and timing of
additions to the culture also play a
crucial role. To achieve a desired
process performance change, several
chemicals affecting different
metabolic targets may be combined
to improve overall effectiveness.
Some Examples
Here we present four examples of the
MPE in application. These examples
Figure 2: Impact of bioreactor scale on culture lactate formation and remetabolization
address process optimization goals in
the areas of
• control of by-product formation
• control of a product quality
attribute,
• volumetric productivity through
cell-specific productivity, and
• volumetric productivity based on
cell viability profile.
Example 1 — Control of By-Product
Formation: Lactate formation is
Lactate
5,000 L
75 L
5L
generally regarded as detrimental to
mammalian cell culture (14, 15), in
which increases in culture osmolality
can lead to cell death. We observed
higher lactate formation was observed
during scale-up of cell culture
Figure 3: Activity in key-enzyme in electron
transfer chain (ETC) increased after addition of
chemical A (arrow marks time of addition).
Enzyme Activity
(mU/10 6 cells)
20,000 L
With addition
Control
Time
Time
e le
nc ilab
e
er a
nf e Av !
o
C ur ne
h
li
oc on
br
April 16-18, 2012 • JW Marriott desert ridge resort • phoenix, arizona
The Best Content in the Industry
Conference Highlights Include:
• Two Great Opening Plenary Topics:
• Future Benefits for Patients: From Discovery to Commercial
Products, Cellular and Gene Therapies, David Shanahan,
President, Mary Crowley Research Center and President,
CEO and Founder, Gradalis
• The Future of Personalized Medicine – Challenges Ahead,
Ted Love, MD, Executive Vice President, R&D and Technical
Operations, Onyx Pharmaceuticals
• Plenary Session Two:
• The Future of the Biopharmaceutical Industry,
David Urdal, Chief Scientific Officer, Dendreon
• The Future of the Biopharmaceutical
Regulatory Perspective,
FDA Representative Invited
• Student Call for Posters –
Abstracts Due February 6, 2012
• Closing Plenary Topics:
Also:
• Manufacturing Opportunities and
Post-Conference
Challenges in the Next 10-20 Years,
Workshop
Matt Croughan, Professor, Keck Graduate
on Single Use
Institute of Applied Life Sciences
Systems!
• Emerging Regulatory Expectations,
Emily Shacter, PhD, Chief, Laboratory
of Biochemistry, CDER, FDA
• NEW: Breakfast Sessions on: Career Development
Strategies and the Quality and Regulatory Job Market Outlook 2012
• Networking Receptions & Events like the 6th Annual PDA Golf
Tournament at the Wildfire Golf Club & the PDA 6th Annual
Walk/Run (benefiting the Phoenix Children’s Hospital)
• Post-Conference Workshop: PDA Single Use Systems Workshop on
April 18-19 and eight courses hosted by PDA TRI on April 19-20
• Hotel activities for the entire family!
www.pda.org/annual2012
Exhibition: April 16-17 | CArEEr FAir: April 16-17 | Post-ConFErEnCE WorKshoP: April 18-19 | CoursEs: April 19-20
Lactate
Figure 4: Culture lactate levels decreased
after addition of chemical A (arrow marks time
of addition).
Control
Concentration 1
Concentration 2
efficiency of the pyruvate f lux and
cellular energy charge.
We hypothesized and confirmed
that lactate can be remetabolized
through LDH activity with
achievement of a higher flux of
Figure 5: Intracellular UDP-nucleotide
concentration increased with addition of
chemical B.
Control
Time
Time
Table 2: Decrease of monosaccharide #3 and associated reduction of serum half-life was observed
during process scale-up; addition of chemical B restored monosaccharide #3 level and
pharmacokinetic properties.
Monosaccharides (normalized molar ratio)
#1
#2
#3
#4
#5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.6
1.0
0.9
1.1
1.0
0.9
1.0
1.1
Development scale
Industrial scale
Industrial scale with
addition of chemical B
pyruvate into the tricarboxylic acid
(TCA) cycle. We accomplished this
by increasing the activity of a key
enzyme in the electron transport
chain (ETC). Figure 3 illustrates the
increased activity of an ETC key
enzyme when chemical A was added.
Figure 4 describes the immediate
reaction of cells to timed addition of
chemical A and the resultant decrease
in culture lactate levels.
Example 2 — Control of Product
Quality Attributes: A further
With addition
Concentration
processes to industrial production
scale (Figure 2). We considered an
obvious metabolic target for
manipulation: lactate dehydrogenase
(LDH) activity. However,
suppressing such activity involved
significant performance trade-offs.
So we focused instead on overall
Serum half-life
(normalized)
1.0
0.7
1.0
challenge during scale-up of highly
glycosylated biological drugs is
control of protein glycosylation
characteristics (14). We observed a
decrease of the content of a specific
monosaccharide in the glycosylation
pattern during scale-up to industrial
production scale. This change
affected the pharmacokinetic
properties of the protein product.
Several metabolic targets were
identified. As Figure 5 shows, the
timed addition of chemical B
increased the level of a key nucleotide
in the intracellular pool. That higher
level then enabled comparability of
BioProcess
TM
I
N
T
E
R
N
A
T
I
O
N
A
L
POSTER HALL 2011-2012
A CompilAtion of proCess teChnologies And expertise presented in poster formAt
AvAilAble At: www.bioprocessintl.com/posters
Figure 6: Key-enzyme activity increased after
addition of chemical C (level of control at 1.0).
Figure 8: DNA laddering decreased with the addition of chemical D (bioreactor profiles from days
3 to 11; agarose gel after ethidium bromide staining).
Enzyme Activity
(mU/106 cells)
2
Control
1 Kb
D3
With Addition
D5 D7 D9 D11 D3 D5 D7 D9 D11
D4 D6 D8 D10
D4 D6 D8 D10
1 Kb
1
0
Time
Cell Specific Productivity
Figure 7: Cell-specific productivity increased
after addition of chemical C (blue = control;
red = with addition of chemical C)
the protein glycosylation pattern from
development to production scale
(Table 2).
Example 3 — Volumetric
Productivity Increase through
Cell-Specific Productivity: The
physiological limit of a cell to
produce, for example, monoclonal
antibodies (MAbs) (qPmax) was
estimated to be 175 pg per cell each
day, based on an assumed ribosomal
speed (RS max) of 8,000 MAbs/cell
each second synthesized (16). We
calculated qPmax by assuming an
antibody molecular weight (MW) of
150 kDa and using the formula
qPmax = RS max × MW ÷ Avogadro’s
Number. Currently reported cellspecific productivities are below that
theoretical maximum (17), so we
focused on evaluating metabolic
pathways that could improve target
protein production. We identified a
critical pathway connecting
glycolysis and TCA and screened a
number of candidate chemicals for
their effectiveness. Figure 6 shows
how a key enzyme connecting the
two central metabolic pathways was
elevated in its activity with chemical
C present. And Figure 7
demonstrates a significant increase
in cell-specific productivity when
48 BioProcess International
10(1)
January 2012
Cell Viability
Figure 9: Cell viability profile improved with
addition of chemical D (arrow marks time of
addition).
With addition
Control
Time
chemical C was added to the
production process.
Example 4 — Volumetric
Productivity Increase Based on Cell
Viability Profile: One of the easiest
ways to increase volumetric
productivity of a cell culture is to
extend the viability of productproducing cells already present. This
strategy offers other benefits,
particularly in regard to maintaining
similar viral and impurity levels at
harvest as well as comparability of
product quality attributes. We
evaluated a number of candidate
chemicals for their ability to delay
cell apoptosis. Figure 8 demonstrates
that timed addition of chemical D
reduced the onset of DNA
laddering, a measure of the apoptotic
state of cultured cells (18). Figure 9
illustrates how timed addition of
chemical D to the process
significantly improved the cell
viability profile.
We have found MPE to be an
effective tool for solving many typical
process challenges encountered
during late-stage and postcommercial
product phases of development.
Acknowledgments
The authors thank Abhinav Shukla, PhD (KBI
Biopharma); Siddhartha Jain, PhD, and
Christoph Joosten PhD (Novartis AG); Jeff
Savard, PhD (Merck); and Nancy Mackin,
PhD, Tara Stanko, and Momina Andrabi
(BMS) for their support.
References
1 Zhang M, et al. Rapid Development
and Optimization of Cell Culture Media.
BioPharm Int. 21, 2008: 60–68.
2 Lu C, et al. A T-flask Based Screening
Platform for Evaluating and Identifying Plant
Hydrolysates for a Fed-batch Cell Culture
Process. Cytotechnol. 55, 2007: 15–29.
3 Stephanopoulos GN, Aristidou AA,
Nielsen J. Metabolic Engineering: Principles and
Methodologies, 1st Edition. Academic Press: San
Diego, CA, 1998.
4 Etcheverry T, et al. Process for Controlling
Sialylation of Proteins Produced by Mammalian
Cell Culture. Patent WO/1996/039488.
5 Dorner AJ, et al. Increased Synthesis of
Secreted Proteins Induces Expression of
Glucose-Regulated Proteins in ButyrateTreated Chinese Hamster Ovary Cells. J. Biol.
Chem. 264(34) 1989: 20602–20607.
6 McGrew JT. Cell Culture Performance
with Vanadate. US PTO 6,974,681.
7 Arden N, et al. Chemical Caspase
Inhibitors Enhance Cell Culture Viabilities and
Protein Titer. Biotechnol. Prog. 23, 2008: 506–
511.
8 Allen M, et al. Identification of Novel
Small Molecule Enhancers of Protein
Production by Cultured Mammalian Cells.
Biotechnol. Bioeng. 100, 2008: 1193–1204.
9 Balcarcel RR, Stephanopoulos G.
Rapamycin Reduces Hybridoma Cell Death
and Enhances Monoclonal Antibody
Production. Biotechnol. Bioeng. 76, 2001: 1–10.
Continued from page 25
10 Ling WL, et al. Improvement of
Monoclonal Antibody Production in
Hybridoma Cells by Dimethyl Sulfoxide.
Biotechnol Prog. 19, 2003: 158–62.
11 Liu CH, Chen LH. Promotion of
Recombinant Macrophage Colony Stimulating
Factor Production by Dimethyl Sulfoxide
Addition in Chinese Hamster Ovary Cells. J.
Biosci. Bioeng. 103, 2007: 45–49.
12 Liu CC, Hwang S. Pentanoic Acid, a
Novel Protein Synthesis Stimulant for Chinese
Hamster Ovary (CHO) Cells. J. Biosci. Bioeng.
91, 2001: 71–75.
13 Jagadish MN, Carter BLA. Effects of
Temperature and Nutritional Conditions on
the Mitotic Cell Cycle of Saccaromyces
Cerevisiae. J. Cell Sci. 31, 1978: 71–71.
14 Sharfstein S. Advances in Cell Culture
Process Development: Tools and Techniques
for Improving Cell Line Development and
Process Optimization. Biotechnol. Prog. 24,
2008: 727–734.
15 Ozturk SS, Riley MR, Palsson BO.
Effect of Ammonia and Lactate on Hybridoma
Growth, Metabolism, and Antibody
Production. Biotechnol. Bioeng. 39, 1992: 418–
431.
16 Savinell JM, Lee GM, Palsson BO. On
the Orders of Magnitude of Epigenic
Dynamics and Monocolonal Antibody
Production. BioProcess Eng. 4, 1989: 231–234.
17 Wurm FM. Production of Recombinant
Protein Therapeutics in Cultivated
Mammalian Cells. Nat. Biotechnol. 22, 2004:
1393–1398.
18 Thrift J, et al. Chapter One:
Characterization of Apoptosis in a CHO Cell
Line Cultivated in Batch and Continuous
Culture: Effect of Medium, Specific Perfusion
Rate and Chemical Inhibitors. Animal Cell
Technology Meets Genomics: ESACT 2005
Proceedings, Volume 2. Springer Netherlands,
2005: 125–128. •
Corresponding author Bernhard M.
Schilling, PhD, is head of biologics
technology transfer, Susan Abu-Absi,
PhD, is head of biologics upstream
process characterization, and Patrick
Thompson is a scientist in biologics
upstream process characterization at
Bristol-Myers Squibb Company, 6000
Thompson Road, East Syracuse, NY 13057;
[email protected].
To order reprints of this article, contact
Rhonda Brown ([email protected])
1-800-382-0808. Download a low-resolution
PDF online at www.bioprocessintl.com.
Effective PMP System
LEX System technology offers several
advantages over other systems (Figure
4). It minimizes the risk of accidental
genetic release of product or
contamination of food sources.
Although nontoxic and edible, Lemna
is not commonly used as a food
source. A further advantage is that no
pesticides, herbicides, or antibiotics
are used in Lemna cultures. In
addition, the single-use aspect of this
production system has also nearly
eliminated cleaning validation
activities from upstream processes.
The current single-use process has
handed off materials and product
validation responsibility to vendors and
manufacturers of disposable products.
This has provided additional benefits
of reduced work-force requirements and
documentation burden.
Using disposable aseptic connectors
eliminates the need for classified areas
in upstream processes. This reduces
facility and operating costs and process
complexity. Aseptic connectors
provided for a process that can specify
and achieve low contamination levels,
well within process requirements. In
receiving a contaminant-free feed
stream, downstream processing
benefits as well.
LEX System technology has merged
plant-based pharmaceutical protein
production with single-use materials.
The benefits and advantages to this
approach have been demonstrated
through investor support and FDA
acknowledgement of successful clinical
trials. A crucial synergy has manifested
itself in the careful adaptation of
single-use products to the Lemna-based
PMP technology.
References
1 Office of the Press Secretary, The White
House. Fact Sheet: Obama Administration Takes
Action to Reduce Prescription Drug Shortages in the
US; 31 October 2011; www.whitehouse.gov/thepress-office/2011/10/31/fact-sheet-obamaadministration-takes-action-reduce-prescriptiondrug-sh.
2 Molecular Farming: Plant Bioreactors. Bio
Pro, Baden-Württemberg; www.bio-pro.de/
magazin/thema/00178/index.html?lang=en.
3 Thomson JA. Seeds for the Future: The
Impact of Genetically Modified Crops on the
Environment. Cornell University Press, Ithaca,
NY; 2006.
4 Gasdaska JR, Spencer D, Dickey L.
Advantages of Therapeutic Protein Production
in the Aquatic Plant Lemna. BioProcessing J
March–April 2003: 49–56.
5 Cox KM, et al. Glycan Optimization of
a Human Monoclonal Antibody in the Aquatic
Plant Lemna minor. Nature Biotech. 24 2006:
1591–1597.
6 Dzublyk I, et al. Phase 2a Study to
Evaluate the Safety and Tolerability and
Antiviral Effect of Four Doses of a Novel,
Controlled-Release Interferon alfa-2b
(LocteronTM) Given Every Two Weeks for 12
Weeks in Treatment-Naïve Patients with
Chronic Hepatitis C (Genotype 1). J.
Hepatology 46, 2007: 4S:232A.
7 Lawitz E, et al. Early Viral Response of
Controlled-Release Interferon alpha2b and
Ribavirin vs. Pegylated Interferon alpha 2b and
Ribavirin in Treatment-Naïve Genotype1
Hepatitis C: 12 Week Results (SELECT-2
Trial). J. Hepatology 52, 2010: S114.
8 Long WA, et al. Timing and Frequency
of Depression During HCV-Treatment with
Controlled Release INFa2b (CR2b) vs.
Pegylated IFNa2b (PEG2b): Results from
SELECT-2, a Randomized Open-Label
72-Week Comparison in 116 Treatment-Naïve
Patients with Genotype 1 HCV. J. Hepatology
54, 2011: S181–182.
9 Cox KM. Production of Antibodies in
Plants. An Z and Strohl W, Eds. Therapeutic
Antibodies: From Theory to Practice. Wiley, Inc:
New York, NY; September 2009.
10 Private communication between Biolex
and regulatory authorities
11 Biolex internal documents. •
Corresponding author Keith Everett is
director of process design and research at
Biolex Therapeutics, Inc, 158 Credle St.,
Pittsboro, NC 27312; 1-919-542-9901, ext.
2032; [email protected]. Lynn Dickey,
PhD is vice president of research and
technology development, John Parsons is
research scientist of plant biology, Rachel
Loranger is upstream research coordinator,
and Vincent Wingate is associate director
of upstream process development and plant
biology, all at Biolex Therapeutics.
To order reprints of this article, contact
Rhonda Brown ([email protected])
1-800-382-0808. Download a low-resolution
PDF online at www.bioprocessintl.com.
January 2012
10(1)
BioProcess International
49