Advancing Biopharmaceutical Process Control

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Advancing Biopharmaceutical Process Control
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Introduction
*
Deploying Smart Process Sensors
Sensibly
CLICK HERE p. 3

Multivariate Data Analysis for
Biotechnology and Bioprocessing
CLICK HERE p. 9

Seeking Innovation to Solve
Persistent Problems

CLICK HERE p. 16

Bioreactor Operational Excellence:
Best Practices from Scale-up to
Control
CLICK HERE p. 23

Additional Resources
CLICK HERE p. 31

CONTENTS
In biopharmaceutical development, scaleup and scaledown are critical to
developing a robust process and maintaining product quality. Technology
must be correctly transferred within the context of a multi-disciplinary
process that requires knowledge of real world materials and equipment,
potential sources of variability, and critical quality attributes for the product.
The Pharmaceutical Quality by Design framework, ICH Q10, FDAs 2004
Process Analytical Technology (PAT) Guidance and 2011 Process Validation
Guidance can help manufacturers develop a strategy for process and
analytical method transfer, validation and continuous quality improvement.
However, for any biopharmaceutical process, the foundation for all of this
work is frequent and accurate process measurement, typically using dissolved
oxygen, pH and conductivity sensors.
This e-resource summarizes best practices for biopharmaceutical process
scaleup, as well as technology and analytical method transfer from experts
at pharmaceutical manufacturing and engineering companies, while it also
highlights the role that DO, pH and conductivity sensors play in ensuring
process robustness.
INTRODUCTION
TODAYS DEMANDING biopharmaceutical
manufacturing operations require
careful control of process conditions,
whether during cell culture, purication
or drug product formulation. Process
sensors play a critical role in enabling
high-performance manufacturing by
providing real-time visibility into how a
process attribute is changing as well as
the ability to correlate that change with
the stage of the process.
In the pharmaceutical industry, it
is extremely valuable to see how
an attribute changes with time and
correlate that change with part of the
process, says L. Harry Lam, Ph.D.,
a biopharmaceutical manufacturing
industry expert. It is imperative to
have efective equipment that provides
reliable measurements.
With the critical role that process
sensors play in bioprocesses, selecting
the best sensors for the job is the key
to process success and understanding.
To successfully deploy sensors, pharma
manufacturers should consider following
these principles:
Defne objectives, and fnd a sensor
technology that provides the
information needed to accomplish
your goals.
The sensible choice depends on the
situation; for example, the size and
age of the manufacturing operation
and relevant regulatory guidelines help
determine what is sensible in addition
to the particular product being
manufactured.
Successful sensor deployment requires
follow-through to implement the
system correctly.
MATCHING SENSOR TECHNOLOGY
WITH GOALS
The right choice of sensor begins with
the particular manufacturing challenge
being addressed. Diferent products
require diferent controls and possibly
diferent sensors. The rst principle for
successfully deploying sensors is that
a manufacturer must clearly dene the
problem to be solved and identify the
sensor technology that provides the
data required to solve it.
You must know what is important
about the process, and then you can
pick the sensor, says Tina Larson, a
Deploying Smart Process Sensors Sensibly
Advancing sensor technologies improve choices and reliability
BY AMBER RATCLIFF, ANALYTICAL SENSORS MARKET SEGMENT MANAGER, HAMILTON COMPANY
ADVANCING BIOPHARMACEUTICAL PROCESS CONTROL
www.pharmamanufacturing.com
bioprocess industry expert who
heads up manufacturing operations
and engineering for a major
pharmaceutical company.
This principle is especially important
to remember for organizations
scouting new technologies. Although
new technology is exciting and
touted for its promise, focus must be
maintained on identifying the best
suited sensor technology to measure
the target critical process parameter.
The key question is, warns Larson,
do the data give you what you need
to solve your problem? There is no
shortage of challenges that drive
manufacturing operations to invest in
new sensor technology. Investment
in new sensor technology is driven
by those manufacturing challenges
where there is the greatest amount of
uncontrolled variability, says Larson.
In biopharmaceutical manufacturing,
that is the cell culture stage.
Accurately measuring cell mass
in cell cultures, for example, is
an unsolved problem that still
eludes efective measurement.
Another area of uncontrolled
variability is glycosylation in cell
cultures; researchers need a better
understanding of the biological
control of glycosylation to develop
proper sensors for this complex
problem.
SUCCESS DEPENDS ON
THE SITUATION
In addition to seeking a sensor
technology that provides the
data needed to manage a critical
process attribute, regulatory and
business considerations also impact
bioprocess sensor implementation
decisions.
The recent U.S. Food and Drug
Administration Process Analytical
Technology (PAT) initiative is
driving a closer look at bioprocess
analytics by pharmaceutical
manufacturers. The PAT guidelines
urge manufacturers to update their
sensor technology as needed so
Figure 1. Hamilton Arc Sensor operation (left) with the wireless hand held (right) at the main 750 liter
fermenter of a microbial fermentation plant. The hand held eliminates the need for multiple transmitters,
handling data from as many as 31 sensors at a time.
that they have data on those process parameters that
afect the quality of their products. For example, antibody
production is a core process in biopharmaceutical
manufacturing. Methods for purifying antibodies and
producing the antibody protein are well established, and
bioprocess sensors such as pH and DO are standard.
These sensors allow operators to manage critical process
parameters in real time. Further, having routinely collected
the data, they are now available for use in analyzing
issues when they do occur, perhaps even allowing
manufacturers to spot success trends not previously
recognized.
The PAT guidelines point to best practices that ensure
critical attributes are monitored and that provide
insurance against process failures. Other factors to be
considered in making a new sensor deployment decision
are the size and age of the manufacturing plant, which
afect the cost of making the change. There must be an
important business case for going through the change
process, states Larson.
Process sensors measure a range of attributes, from
traditional pH and dissolved oxygen (DO) to cutting-edge
attributes whose relevance is still being proven. Whether a
new sensor is state-of-the-art, a workow innovation and/
or cutting edge afects the implementation decision.
State-of-the-art sensors are newer versions of well-
established sensors. They measure critical attributes
that facilitate high-performance plant operation and
biopharmaceutical production. State-of-the-art might
OPTICAL DO SENSORS
Optical DO sensors replace traditional Clark cells, which
were based on electrochemical measurement within the
sensor itself. In the classical amperometric (electrochemical)
procedure described by Clark, oxygen diffuses through a
membrane and induces a chemical reaction with the elec-
trolyte behind it, creating a voltage differential propor-
tional to the amount of oxygen present. Optical measure-
ment, in contrast, is based on the uorescent quenching
of oxygen on a luminophore. This optical system is located
inside the membra ne cap and is thus not affected by fac-
tors such as turbidity.
Because of pressure effects on the uid-lled membrane
cap, classical amperometric sensors can give inaccurate
readings when exposed to pressure and temperature
changes. This makes it difcult to assess the status of the
culture and triggers alarm systems unnecessarily, disrupt-
ing daily operations. In contrast, optical DO sensors have a
more rugged sensor cap that easily handles broad perfor-
mance ranges, accommodating measurement temperatures
from -10C to 80C, pressures up to 12 bar/174 psi, and pres-
sure spikes up to 80 bar.
Optical DO sensors are also more robust for modern
biopharmaceutical processes that require sterilization-in-
place (SIP), cleaning-in-place (CIP) and autoclaving. Com-
pared to optical sensors that do not rely on electrolytes,
amperometric sensors recover slowly after cleaning, and
their signal quality deteriorates with repeated sterilization
or long run times. Slow recovery extends downtime and
can lead to waste if sensors are not fully responsive before
restarting the process. Optical sensors have no electrolytes
that require polarization time and can therefore remain
stable over entire production runs and repeated steriliza-
tion cycles.
mean more accurate, more stable,
or easier to calibrate. Two examples
of state-of-the-art sensors are
optical DO sensors (see Technology
Spotlight 1) and smart sensors (see
Technology Spotlight 2).
A manufacturing plants size and
age are key factors in the decision
to deploy up-to-date sensor
technologies. A small company
building its rst or second plant
will use state-of-the-art technology
so they dont have to worry about
obsolescence, says Larson. On
the other hand, a manufacturing
operation with several plants and a
need to align operations globally will
only incorporate such upgrades as
part of planned obsolescence.
Workow innovation sensors do
just what their name implies: they
improve workfow efciency. Smart
sensors, now state-of-the-art,
began as a workow innovation to
reduce human intervention. In place
of a potentially wasteful preset
maintenance schedule, the smart
sensor noties the operator when its
performance is declining, resulting
in a better optimized maintenance
schedule. At line analytics, wireless
sensors, and single-use sensors are
other workow innovations that
are becoming more common in
biopharmaceutical manufacturing.
With at line analytics, also called
QC on the oor, a test sample is
analyzed right on the plant oor
3
.
At line analytics are useful for non-
critical parameters or for cases in
which a technology is not available in
online format yet. The PAT guidelines
indicate that critical process
attributes should be measured online
whenever possible. At line analytics
allow manufacturers to test new
or non-critical attributes without
making breaks in the sterile barrier or
impacting the design of the vessel or
other capital equipment.
Wireless technology is a workow
innovation that impacts virtually
every aspect of our lives. In 2010,
wireless technology reached
the biopharmaceutical process
sensor industry when Hamilton
Company introduced its wireless
technology platform. The direct
connectivity eliminates the need
to send information through costly
transmitters, the signals are more
robust and reliable than the low
currents produced in traditional
measurement systems, and wireless
communication to a handheld device
enables users to move around the
facility and readily monitor data
from multiple sensors at a time, says
Hamilton Companys Jahir Kololli.
Beyond the advantages of
convenience, exibility, cleanliness
and elimination of wiring costs,
removing intrusive wiring ensures
quality by reducing penetrations into
the system, explains Lindsay Leveen,
a bioprocess industry strategist.
Like wireless technology, single-
use or disposable technology
is well known in the modern
world. Certain applications lend
themselves well to disposable
technology. In biopharmaceutical
manufacturing, single-use
bioreactors were introduced in
1996
4
, and implementation has
become more sophisticated over
time
5
. Single-use sensors are also
taking advantage of disposable
technology in their designs. Although
many still have their limitations, new
sensing technology has allowed for innovations in DO
and pH measurement specically, where the expensive
electronics, which do not need sterilization, are not
single use.
Cutting-edge bioprocess sensor technology brings
novel and unproven approaches to solving problems.
This might be a new engineering approach, a new assay,
or a new chemical measurement. Solid-state and near-
infrared (NIR) sensors are two examples of cutting-edge
technology for the biopharmaceutical industry. Solid-
state technology is an exciting, promising innovation
for bioprocess sensor engineering because of the
potential for low-cost manufacturing and low power
consumption. Near-infrared data processing, although
not a new technology in itself, is experiencing renewed
interest, driven by advances in NIR data processing that
allow its use in aqueous situations. With these advances,
NIR sensors are now being reconsidered for bioprocess
monitoring. To what extent each type of cutting-edge
technology will be useful in bioprocess sensors remains
to be seen.

FOLLOW-THROUGH
It is hard to get new sensors into manufacturing at a
large pharmaceutical company, says Larson. You need
a lot of data to make the case that the new sensor will
be benecial.
We test new sensor technology in our pilot plants,
she explains. Once we have experience with it, and
it proves robust and solves the problem that we are trying
SMART SENSORS
Bioprocess operational performance has also been hindered
by the difculty of transmitting information to and from sen-
sors. The introduction of smart sensors makes sensor and data
management easier. Smart sensors contain a memory chip
embedded in the electronic circuitry to store identication
and calibration information. The advantages of smart sensors
include:
Eliminating the need to calibrate sensors in line, reducing
system downtime;
Reducing incorrect estimations of sensor life, reducing batch
failure due to sensor failure;
Eliminating extensive manual documentation of sensor
performance to meet quality system reporting requirements,
reducing administration costs.
Smart sensors self-monitor measurement performance; an
intrinsic quality indicator predicts a failing sensor before a run
starts, reducing the risk of in-process failure and maximizing
useful life instead of requiring scheduled maintenance. Out-of-
range results are reported immediately, triggering alarms and
prompting action as appropriate.
Because smart sensors store calibration data, pre-calibration
and conguration can be done right in the lab. Installation and
downtime costs are reduced by more than half with
smart sensors.
Smart sensors improve time to market, allowing less complex
setup and faster qualication, says Lam.
Finally, smart sensors report identication and calibration
information electronically, resulting in better quality system
adherence while saving time previously spent on manual
documentation.
to solve, then we send the data
to manufacturing operations for
deployment consideration. For sensor
design engineers wondering how to
get started with PAT, Larson advises a
sensible approach. The technologist
designing a sensor must understand the
manufacturing problem.
Once the decision is made, the
deployment itself requires careful
attention to ensure proper setup and
conguration. Thoroughly planning
implementation ensures that the
benets of the new sensor technology
are realized, while helping to minimize
organizational and validation-related
questions. Training, data collection,
data analysis, integration into third-
party equipment, version management
and reporting are just a few of the
considerations that require careful
planning before implementation begins.
Another important question to answer
is how data acquisition technology will
be used to maintain control of all the
new data.
With the goal of any process
improvement being to initiate a new
approach, it is important to have
support, both internally and externally.
Partnerships with sensor vendors
extend beyond ensuring successful
deployment. Pharmaceutical
manufacturers get involved in sensor
design by surveying the landscape for
new sensor technology and disposables
and partnering with vendors in two
basic situations: when they see a
technology that they think is worth
developing, and when a vendor is close
to bringing to market a new technology
that is of interest to the company,
says Larson.
Regardless of the type of innovation
being introduced, successful
deployment of bioprocess sensors
requires that implementation of a new
technology follow PAT guidelines,
be suited for the manufacturing
process, answer the question it is
meant to address, and add enough
value to justify making a change. The
right choice varies with the situation;
diferent products require diferent
controls and possibly diferent
sensors, and the cost-benet ratio
depends on the size and age of the
manufacturing plant. Adoption of new
technology often results in unexpected
improvements in process understanding
and performance.
ACKNOWLEDGEMENTS
The authors would like to thank the following
industry experts:
Tina Larson is a bioprocess engineer
who has spent her career in biochemical
manufacturing operations for major
pharmaceutical corporations. In her
current role, she is the head of technical
development operations and engineering
for a large biopharmaceutical manufacturing
organization.
L. Harry Lam, Ph.D., is a bioprocess
engineer who has spent his career in
biochemical manufacturing operations for
major pharmaceutical corporations, where
he has been responsible for technology
implementation and many successful
development projects and manufacturing
campaigns. Currently, Dr. Lam works at
Shire Pharmaceuticals in La Jolla, Calif.
Lindsay Leveen, currently a consultant, is a
manufacturing process industry strategist
and early technology adopter focused
on sustainable development. He led
manufacturing technology implementation
for biopharmaceuticals, where he was an
early proponent of single-use methods.

Editors Note: For the complete list of refer-
ences associated with this article, visit:

THE MODERN biopharmaceutical/
biotechnology manufacturing facility
contains many sophisticated control,
data logging and data archiving
systems. Massive amounts of data are
collected from sources such as raw
materials analysis, process outputs
and nal quality assessments, which
are stored in data
warehouses.
The sheer volume of
data contained in these
warehouses makes it a
near impossible task to
extract the information
using simple charting and
univariate methods of
analysis. Such complex
data requires methods
of analysis that can cope
with multiple variables
simultaneously that not
only reveal inuential
variables, but also reveal
the relationship such
variables have with
each other. This is where Multivariate
Analysis (MVA) is nding a much
greater role in the analysis of complex
bioprocess data.
Much more efort is being put into
the discovery and development
of biotherapies and personalized
medicines. Biopharmaceutical and
biotechnology companies are looking
for ways to accelerate drug discovery,
and through quality and compliance
initiatives such as the Food and Drug
Administrations (FDA) current Good
Manufacturing Practice (cGMP) and
Quality by Design (QbD) principles, and
Data Driven Knowledge
Discovery, reduce the
regulatory approval
time and be rst to
market. This means
that data collected
throughout the entire
product lifecycle
must be analyzed and
interpreted in order
to gain extensive
product and process
understanding. This, in
turn, leads to improved
quality, greater
condence in the
market for a companys
products and ultimately
market capitalization.
Multivariate Data Analysis for Biotechnology, Bio-processing
Powerful MVA and DoE methods are giving biotech companies greater insights from complex data

By Brad Swarbrick, Vice President Business Development
It is estimated that the time it takes to bring a new drug
or therapy to market is approximately 12 years. This
usually involves three phases: discovery, clinical trials
and registration.
Coupled with these phases is the development of a suitable
manufacturing process that can consistently produce the
highest quality product and be compliant with FDA current
Good Manufacturing Practice (cGMP) guidance. This
includes the development of a formulation that is robust
under processing conditions, scale up considerations and
technology transfer from facility to facility or even between
diferent types of manufacturing equipment. Each of these
phases can be improved and accelerated through the use of
the tools of MVA and Design of Experiments (DoE).
Even before data is analyzed, one of the biggest challenges
facing the industry is getting this data into a format that is
amenable to MVA. Many data collection and agglomeration
systems are commercially available for compiling various
forms of data and these can be seamlessly integrated
into MVA packages so that the vast array of graphical
and analytical approaches can be applied to reveal the
information it contains. The general statement is data is
only data until the information is extracted from it, and
from there, information leads to knowledge.
THE APPLICATION OF MVA AND DOE IN THE BIOTECH PRODUCT LIFECYCLE.
Figure 1: Biotechnology companies can realize signicant benets using MVA and DoE from product development through to manufacturing and quality control.
MVA for isolating
candidate therapies
DOE for formulation
MVA for assisting
with the
interpretation of
clinical statistics &
demographics
DOE for technology
transfer and scale-up
MVA for process
control and early
event detection
DOE for optimizing
analytical
procedures
MVA for developing
robust quantitive
models
Faster time to market
Reduced development time
frames and costs
Improved process under-
standing
Increased product quality
Development &
Discovery
Clinical Trials Quality Control
Manufacture &
Control
Business benefts
MVA IN THE PRODUCT LIFECYCLE
Unlike small molecule drug product development,
biotherapies are fundamentally more complex in terms
of structure and application and sufer greatly from
natural biological variability. For example, isolating and
selecting cell cultures or bacterial strains to further
develop into future products is aided greatly by the tools
of MVA, including the monitoring of the processes (e.g.,
fermentation reactions) used to produce them. From
there, the tools of DoE can be used to devise formulations
that stabilize the active component(s) during manufacture
and are also useful in product scale-up studies.
Once the candidate therapy (cell cultures, antibody, virus
strain, etc.) has been formulated into a stable matrix,
MVA can be used to assist in the interpretation of clinical
trial data and can even lead to accelerating the lengthy
process through a much more comprehensive and overall
approach to data analysis, especially when combined with
the principles of adaptive designs and the Critical Path
Initiative endorsed by FDA.
When the candidate therapy has been approved for
market release, the tools of MVA are useful for assessing
the success of technology transfer from R&D to
production, or from one manufacturing facility to another.
In the production environment, MVA is useful for assessing
incoming or internally produced raw material quality and
characteristics. Combined with rapid spectroscopic (or
other characterization methods) control strategies for the
real-time monitoring and adjustment of processes within
the so-called design space can be devised so that
proactive quality control can be realized. DoE and MVA
are then used in developing robust analytical methods
for stability studies and other post-production analyses.
Data collected over time from a manufacturing facility
can be modeled to assess batch-to-batch consistency
and facilitate continuous improvement (CI) and preventive
maintenance and corrective action (CAPA) programs.
The entire process is summarized in Figure 1.
Candidate Therapy Discovery: During the initial
development of new therapies, there is usually much
information available on candidate cultures, antibodies,
etc., in respect to their chemical, biological and
toxicological properties. Combined with information
from origin and other background information, the
method of Principal Component Analysis (PCA) provides
a key data mining tool for the development scientist
to not only classify candidates of similar properties
and characteristics, but also to discover unique classes
that may be better suited to the treatment of specic
conditions.
PCA provides a visual map of the sample groupings,
allowing for the more efcient selection of real candidate
therapies, but it also provides a map of the input variables
and their relationships that cause the samples to group
the way they do. Figure 2 provides an example of the
outputs of a PCA in the form of the scores and loadings
plots. The scores provide a map of the samples and the
loadings provide a map of the input variables.
PCA (or more generally MVA) applied to this kind of data
is sometimes referred to as Quantitative Structure Activity
Relationships (QSAR) and has helped some companies to
signicantly reduce the time and efort required to isolate
suitable candidates for further development.
Formulation of suitable products: Stabilizing the candidate
into a suitable matrix for manufacturing and delivery is
best approached using DoE and, in particular, excipient
screening and mixture designs. Excipient screening
designs allow the formulation scientist to select the best
components that will preserve the nature of the candidate,
while mixture designs allow for the development of the
best combination that will not only stabilize the candidate,
but also protect it during subsequent manufacturing
processes.
CLINICAL TRIALS
Clinical trials have traditionally been the domain of
univariate statistical approaches (in particular clinical
statistics) where statistical signicance is assessed for
parameters such as efcacy and major side efects. The
tools of MVA can be used to compliment the ndings
generated by clinical trial statistics to further conrm and
accelerate key ndings through this phase of product
development.
The ability to incorporate demographic, age, sex and
patient history into predictive or exploratory models is
a unique feature of the MVA method, and approaches
such as the L-PLS model can provide an overall picture
of the patient groups, disease markers and the candidate
properties to better assess the efect of the therapy on
specic patient groups. Figure 3 provides an example of
the L-PLS model structure and an example output.
Through the use of MVA tools for monitoring and
controlling bioprocesses, manufacturers worldwide have
realized signicant cost savings through proactive quality
control. During the scale up and technology transfer
of a process from R&D to full scale manufacturing, the
use of DoE is a critical strategy for assessing the efect
SCORES AND LOADINGS PLOTS FOR A CANDIDATE SELECTION STUDY
Figure 2: In this example, Source 1 samples have high amounts of impurities
whereas Source 3 samples have the highest cell count. As a rule of thumb,
variables located outside the inner ellipse are regarded as being important in
interpretation of clusters in the Scores plot.
of changing process and equipment variables. This
allows the defnition of the Design Space, which defnes
the most efective control strategy for the process.
Multivariate Statistical Process Control (MSPC) uses
multivariate exploratory and predictive models and
integrates them into the entire data collection and
process control system.
This allows manufacturers to be more innovative in their
approach to quality combining in-line process analytics
into single or holistic process models that better assess
the quality of production than single measurements in
isolation. Two particular processes that are commonly
used in biotherapy manufacture are fermentation and
lyophilization. Some applications of MVA to these are
discussed in the following sections.
MVA FOR FERMENTATION MONITORING
For many years manufacturers have been challenged
with the development of suitable models for monitoring
the progress of batch processes, fermentation being
one such process. These batch models aim to establish
a process trajectory and associated limits around the
trajectory that dene the bounds of acceptable
product quality.
Methods exist that unfold batch data and use so-called
maturity indices to model the process. However,
the major drawback of these methods is that they
assume linear relationships in the processes, which are
fundamentally incorrect and have only partially solved
the batch problem. Other approaches use time warping
to distort the time scale and align batch trajectories.
Again, these approaches also sufer fundamentally as
they distort the chemistry or biology of the system and
hence do not describe the true state of the process.
Relative Time Mapping (RTM) addresses the
shortcomings of the previously dened methods by
keeping the chemistry/biology of the system intact,
while at the same time, providing the usual batch
trajectory plots and associated diagnostics that have
become synonymous with this type of analysis.
Whether batch models or traditional Statistical Process
Control (SPC) charts are used to assess the progress
of a bioprocess, there are many diagnostics available in
multivariate models that can be used to determine the
onset of process failure.

EARLY EVENT DETECTION
The term Early Event Detection (EED) is being
increasingly used to describe the application of
Multivariate Statistical Process Control for the detection
of process faults. The diagnostics from these models
can be fed back into the manufacturing control systems
using protocols such as OPC to automate process
adjustments and therefore maximize the quality of the
nal product.
An extension of MSPC is the use of Hierarchical Models
(HM). These models provide an excellent way of
classifying the state of discrete phases of processes
such as fermentation and adapt to changing conditions
as they occur. HMs can be set up
as Classication Classication,
Classication Prediction
and Projection Prediction
models which can be adapted to
applications such as analysis of
raw materials, process monitoring
and quality-control applications.
Near Infrared (NIR) spectroscopy
has been used for many years
with multivariate predictive and
exploratory models for the rapid,
non-destructive assessment of
product quality. One common
application of the NIR method
is the quantitative analysis of
residual moisture in lyophilized
products.
Lyophilization is a common
method used in the manufacture
of biopharmaceutical products
as it uses low temperatures to
remove residual moisture, thus
preserving the structure of the
active components and allowing
their storage at room temperature.
The traditional method of analysis
for residual moisture in lyophilized
product is Karl Fischer (KF)
titration which is a destructive test
and can only be applied to a small
number of samples.
Replacement of the KF method
with NIR not only results in non-
destructive testing, but also allows
for 100% inspection systems to
be put in place. These systems
use MVA predictive models to
transform the NIR spectrum into a
single value for residual moisture
(or other properties) and are used
to accept and reject product as it
is being manufactured.
In one case, a biopharma
manufacturer saved about $1
million by using the NIR method
combined with PCA to validate
the performance of a new freeze
THE L-PLS MODEL AND ITS POTENTIAL FOR CLINICAL TRIAL DATA ANALYSIS.
Figure 3: In this example, the variables in green describe the background information of the patients, the
variables in blue are the side efects of the formulations (the actual formulations in light blue) and the red
dots indicate patient groups. This combined plot is the most informative way of displaying the relationship
between the three data tables depicted in the frame above.
Factor - 1
F
a
c
t
o
r

-

2
Correlation loadings from L-PLS regression
dryer. They also developed a quantitative Partial Least
Squares Regression (PLSR) model to replace the KF
method in the laboratory. This method saves them
$1,000 per sample and provides more condence when
releasing the batch to market.
QUALITY CONTROL
Although initiatives such as Process Analytical
Technology (PAT) have been used by many
manufacturers globally to assess product and
process quality at the point of manufacture, not every
process measurement can be replaced at the point
of manufacture. Quality Control (QC) operations are
still vital in the nal release stage of some, if not all,
products.
Due to the high variability in many biological assays,
DoE and MVA can be used to design and rene the
analytical methods used in the QC laboratory and
has been successfully applied to the optimization of
chromatographic methods, the renement of sampling
procedures and the analysis of complex data produced
by mass spectrometers.
Another advantage of combining spectroscopic
analysis with MVA methods is in stability studies. Since
the NIR method is non-destructive and is sensitive to
changes in the product and its matrix, the same sample
can be assessed over the entire timeframe of the
study. Where applicable, this avoids the destruction of
product, and the results are completely representative
as the same sample is being assessed each time.
MVA and DoE are fast becoming essential tools for
all process development and monitoring applications.
Bioprocesses provide an excellent but challenging
application area. Modern manufacturing execution
systems and control platforms produce a massive
amount of data that requires the tools of MVA to fully
data mine the most important information and make
real-time quality decisions.
From raw material analysis to nal product release,
MVA models can be integrated into the total Quality
Management System (QMS), allowing manufacturers
to realize the benets of the Quality by Design (QbD)
initiative.
Multivariate data analysis and DoE are powerful
tools ideally suited for understanding the complex
behavior and relationships in biological systems. These
methods can be used across the full biotech product
lifecycle, from discovery and development, to scale up,
production and quality control. Todays leading MVA
and DoE solutions can be seamlessly integrated with
other systems including process equipment, laboratory
and spectroscopy instruments, enabling faster and
more informed decision making.
Leading biotechnology companies that implement
and exploit the power of MVA and DoE can realize
substantial benets including lower development
and production costs, improved product quality and
compliance, technology transfer, faster time to market
and ultimately increased business value.
ITS PROBABLY fair to say that
innovation in the biopharmaceutical
manufacturing industry is a slow
cycle. But that would ignore the many
changes in biomanufacturing over the
past 5 to 10 years: Better expression
systems, widespread adoption of novel
single-use applications, re-emergence
of perfusion technologies, new modular
and exible facilities, better sensors,
control systems and downstream
technologies.
The industry now nds itself very aware
of the promise new technologies carry
in terms of optimizing, and in some
cases revolutionizing existing processes.
Results from our 10th Annual Report
and Survey of Biopharmaceutical
Manufacturers
1
, in which we surveyed
238 biomanufacturers, indicate that
end-users are still actively looking for
a range of new technologies to solve
persistent problems.
MORE FOR LESS
The driving factors today in
bioprocessing innovation, according
to our study, involve improving efciency
and productivity. This equates to getting
more out of existing processes for less
money. For example, roughly two-thirds
of the industry attributes improvements
in manufacturing performance to single-
use systems and applications. But most
of the recognized benets involve
improved efciency, especially in clinical-
scale processes. Single-use devices
shorten the time getting facilities up and
running and reduce capital investments
necessary for new plants. New facilities
ofer more exibility, and modular
approaches along with faster campaign
turnaround times and lower annual
maintenance costs.
Similarly, the industry is demanding
better downstream processes
demands that are generally focused
on cheaper, equally efective
chromatography, protein-A and
purication steps. Again, innovations
need to be about cost efectiveness. So
as innovators and suppliers develop new
products, theyll need to demonstrate
their technologies are actually better
than current approaches.
PRODUCTIVITY INNOVATION 2013
In a separate survey Bioplan Associates
ran late last year, among the more than
450 global subject matter experts and
senior participants who make up our
Biotechnology Industry Council,
2
the
study found consistent expectations
regarding improvements in productivity.
Again, improvements in downstream
processing and single-use technologies
ranked as the top 3 trends for 2013,
these were followed by demands for
Seeking Innovation to Solve Persistent Problems
Study reveals biopharmas growing demand for efective technologies to drive efciency and
productivity in and cost out of biopharmaceutical processing

BY ERIC S. LANGER, PRESIDENT AND MANAGING PARTNER, BIOPLAN ASSOCIATES INC.
better analytical methods. New analytical
methods are required for better process
monitoring and process improvements.
In addition, to develop biosimilars, the
industry needs better characterization
techniques, and better processes.
Otherwise, even if similarity with a
reference biologics could be shown,
the cost of producing a new biosimilar
might not be much lower than the
original; this could dramatically reduce
the attractiveness of any such high-cost
generic version.
Returning to the attractiveness of single-
use technologies, we found in our annual
survey that respondents today estimate
35% of their upstream clinical production
operations to be single-use. This compares
with 25% of respondents that said more
than 80% of their downstream clinical
production steps are now single-use.
The number is 16% of downstream
commercial-scale production. This wasnt
surprising to see that the lowest use was
for downstream commercial production,
which remains mostly xed stainless-steel
equipment. It was also fairly consistent
to see the highest adoption rate be for
downstream clinical production, likely
due to broader use of disposable tubing
and lters, bufer containers, etc.
FOCUS SUPPLIERS, FOCUS
Researchers asked respondents to
consider new product and services
developed by suppliers and to identify
the top areas they want suppliers to
focus their development eforts on. This
year, of the 21 areas the study listed,
the areas highlighted in prior years, and
other studies continue to occupy the
top position. Specifcally:
Disposable products, including bags,
connectors and other devices
Better probes and sensors
Process development services
(up-and down-stream)
Chromatography products
Biopharmaceutical manufacturers are seeking new modular and exible facilities with better sensors, control
systems and downstream technologies.
Some of the specifc new product development areas
being demanded by biopharmas operational managers are
included in Figure 1. Identication of these areas by end-users
is not a reection of their need for technical advances to
produce drugs that otherwise wouldnt be possible. Rather
it mirrors their requirements to produce more efciently and
less expensively. This shift in focus on manufacturing is a
maturation process that has been growing over the past
10 years.
TRENDS OVER TIME
Looking at responses from the past four years, the study
shows that interest in more innovative approaches to
bioprocessing, again, centers on process improvements. For
example, demand for better upstream process development
services has increased ve percentage points over the past
four years, while interest in downstream PD has decreased
from a high of 35% in 2008, down to 26% this year. Similar
declines in interest in new chromatography products are
evident. This suggests the acute bottlenecks created around
downstream operations have been abating, while the chronic
need for improved productivity has not.
Better disposable devices, including bags and connectors,
has increased by about ve percentage points from 2008.
Demand for disposable probes and sensors is up by roughly
10 percentage points, while interest in new bioreactors and
purication products has remained steady during the period.
Predictably, interest in various new technologies varies by
business model and even geography and the studys
results ofer a window into how diferent parts of the
Disposable products, bags, connectors, etc.
Disposable product: probes, sensors, etc.
Disposable product: bioreactors
Disposable product: purication
Process development (upstream) services
Analytical assays
Process development (downstream) services
Analytical development
Cell culture media
0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%
43.8%
39.6%
34.0%
34.0%
29.9%
27.1%
26.4%
26.4%
25.0%
25.0%
Use of high capacity resins
Single use lters
Buffer Dilution systems/skids
In-line Buffer dilution systems
Single use disposable TFF membranes
SELECTED NEW PRODUCT DEVELOPMENT FOCUS AREAS
ADOPTING NEW DSP TECHNOLOGIES 2010 VS 2013
Downstream Purication (DSP) technologies being considered
54.0%
41.2%
48.2%
43.5%
44.4%
36.8%
43.0%
28.7%
38.1%
42.6%
36.8%
35.7%
36.5%
39.7%
47.4%
45.2%
34.9%
38.2%
37.7%
27.8%
Source: 10th Annual Report and Survey, Biopharmaceutical Manufacturing and
Capacity, www.BioPlanAssociates.com, April 2013
Figure 1
Figure 2
Disposable products, bags, connectors, etc.
Disposable product: probes, sensors, etc.
Disposable product: bioreactors
Disposable product: purication
Process development (upstream) services
Analytical assays
Process development (downstream) services
Analytical development
Cell culture media
0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%
43.8%
39.6%
34.0%
34.0%
29.9%
27.1%
26.4%
26.4%
25.0%
25.0%
Use of high capacity resins
Single use lters
Buffer Dilution systems/skids
In-line Buffer dilution systems
Single use disposable TFF membranes
SELECTED NEW PRODUCT DEVELOPMENT FOCUS AREAS
ADOPTING NEW DSP TECHNOLOGIES 2010 VS 2013
Downstream Purication (DSP) technologies being considered
54.0%
41.2%
48.2%
43.5%
44.4%
36.8%
43.0%
28.7%
38.1%
42.6%
36.8%
35.7%
36.5%
39.7%
47.4%
45.2%
34.9%
38.2%
37.7%
27.8%
industry are looking at innovation.
When comparing biomanufacturing
developers to contract manufacturing
service providers (CMOs), for example,
the study found that while the former
were more interested in disposable
probes and sensors (41.3% vs. 27.8%),
the latter were twice as interested in
disposable purication products (61.1%
vs. 30.2%). Again, this is likely the result
of CMOs need for efciency to remain
competitive.
THE INNOVATION PENDULUM
Although bioprocessing industry growth
isnt as radical as in semiconductors
(where Moores Law proposed that
the number of transistors on a chip
doubles every 18 months), demand for
innovation that improves efciency
is similar in respect to how new
product developments swing from
one bottleneck to the next. This year,
single-use applications are clearly
the subject of much interest when it
comes to innovation; in prior years, and
likely in the future, other technology
areas will bounce back. Downstream
purication, new and better analytical
tools, and improved services oferings
for example are likely to re-emerge as
urgent problems as the industry resolves
current, more acute, issues.
For example, separately in our study
we asked respondents which of 21
diferent, novel downstream purication
(DSP) technologies they were actively
considering to address bioprocessing
problems (see Figure 2). The responses
are indicative of potential future
adoption and do not take into account
respondents already having adopted these
technologies or those who are considering
but not actively pursuing them.
Topping the list of new downstream
processing solutions being considered
Among the technologies biopharmaceutical manufacturers want suppliers to focus innovation and
development on are single-use technologies including bags, connectors and other devices.
this year are high capacity resins, by
54% of respondents. Following are
single-use lters (44.4%), bufer dilution
systems/skids (38.1%), in-line bufer
dilution systems (36.5%) and single-use
disposable TFF membranes (34.9%).
Compared with years past, we see
a greater interest in the use of high
capacity resins (this years 54% being up
from 41% in 2010) and single-use lters
(44.4%, up from 28.7% in 2010). Some
downstream areas are showing a trend
toward decreased consideration. For
example, the 36% actively considering
in-line bufer dilution systems is down
from 48% a couple of years ago. These
declines may also reect greater adoption
of these technologies in the past few
years, with fewer respondents falling
into the actively considering column
as a result of following through on those
considerations. It may also be that
incremental improvements in processes,
elimination of purication steps and a
diminished interest in alternatives for
current technologies (such as Protein A
alternatives) have weakened the urgency
for new solutions.
Our analysis nds that CMOs may be a
leading-edge indicator regarding plans
for adoption of alternative downstream
processing technologies. Results ofer
some insight into which markets are
likely to expand in the coming months
and years. For example, CMOs are far
more likely to be considering single-use
disposable TFF membranes (50% vs.
32%) and disposable UF systems (40%
vs. 28%).
The biopharmaceutical manufacturing industry is actively considering and adopting a range of new
technologies for all facets of the manufacturing process. (Photo: Boehringer Ingelheim)
We also found signicant diferences on
a regional basis, with U.S. respondents
generally more likely than Western
European respondents to be considering
a range of technologies. For example,
use of high capacity resins (71% U.S.
vs. 33% W Europe), a 37.3 point gap;
and In-line bufer dilution systems (53%
U.S. vs. 11% W Europe). On the other
hand, Western Europeans demonstrated
signicantly more active consideration
for membrane technologies, alternatives
to chromatography and precipitation.
WHATS AHEAD FOR NEW
TECHNOLOGIES
The biopharmaceutical manufacturing
industry is actively considering and
adopting a range of new technologies
for all facets of the manufacturing
process. Single-use devices continue to
crop up in any conversation about new
technologies and a move towards leaner,
more exible and modular systems
seems likely in biopharmas future.
It is also important to note that
introduction of innovations is not at
all easy in this industry, and there are
many obstacles to new technology
introductions. Regulatory issues
force biomanufacturers to stabilize
bioprocessing systems early on, so
processes can remain largely unaltered
through a drug products lifetime. This
can make manufacturers less receptive
to improvements. Thus, manufacturing
strategy takes a long view when it
comes to adoption of innovation.
On the other side, vendors, larger ones
in particular, invest signicantly in R&D
and product lines. They have a vested
interest in evaluating where future
adoptions will be needed, and how
rapidly they will be taken up.
Economic conditions can also play a
role. While budgets are again expanding,
tighter conditions continue to
discourage the nancing and entrance
of smaller suppliers in the market,
reducing the pool of likely contributors
to innovation. Even so, biomanufacturers
are showing a renewed urgency to
improve productivity, reduce costs while
boosting quality. This is reected in
increasing budgets over the past four
years supporting activities focused on
production efciency.
Given the complexity, and the long
product development cycle, the only
way to ensure efcient process is
for industry suppliers and vendors
to continue to identify and meet the
demands of end-users. This will drive
future investing in the development
of new technologies. And supporting
this, on the suppliers side, we nd that
vendors R&D budgets for new product
development have also expanded over
the four years.
So, with increased budgets and interest
from both manufacturers and vendors
on innovation that boost efciency,
its easy to visualize a robust future
for the industry. For biomanufacturers
to truly push forward innovation and
remain competitive as cost pressures
increase, and biosimilars evolve, they
will continue to demand better ways
of evaluating new technologies to cut
downtime to market and streamline the
overall testing process.
Survey Methodology: The 2013
10th Annual Report and Survey of
Biopharmaceutical Manufacturing
Capacity and Production yields a
composite view and trend analysis
from 238 responsible individuals at
biopharmaceutical manufacturers
and contract manufacturing
organizations (CMOs) in 30 countries.
The methodology also included over 158 direct suppliers of
materials, services and equipment to this industry. This years
study covers such issues as: new product needs, facility
budget changes, current capacity, future capacity constraints,
expansions, use of disposables, trends and budgets in
disposables, trends in downstream purication, quality
management and control, hiring issues, and employment.
The quantitative trend analysis provides details and
comparisons of production by biotherapeutic developers
and CMOs. It also evaluates trends over time, and assesses
diferences in the worlds major markets in the U.S.
and Europe.
ABOUT THE AUTHOR
Eric S. Langer is president and managing partner at BioPlan Associates
Inc., a biotechnology and life sciences marketing research and publishing
rm established in Rockville, MD, in 1989. He is editor of numerous
studies, including Biopharmaceutical Technology in China, Advances in
Large-scale Biopharmaceutical Manufacturing, and many other industry
reports. Contact Eric at: elanger@bioplanassociates.com; 301-921-5979;
www.bioplanassociates.com.
REFERENCES
1
10th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and
Production: A Survey of Biotherapeutic Developers and Contract Manufacturing
Organizations, BioPlan Associates, April 2013.
2
BioPlan Associates 2013 Biotechnology Industry CouncilTM Trends Analysis
Study
Demand for disposable probes and sensors is up by roughly 10 percent-
age points, while interest in new bioreactors and purication products
has remained steady.
BIOREACTOR OPERATIONAL EXCELLENCE:
Best Practices from Scale-up to Control
In biomanufacturing, knowledge of scale-up, processing and control should be shared and
documented in order to achieve and sustain operational excellence.
BY BRIAN J. STAMPER AND CILLIAN MCCABE, BIOPROCESS RESEARCH AND DEVELOPMENT, ELI LILLY AND COMPANY
THE MODERN cell culture bioprocess
has been successfully scaled up to
volumes greater than 25,000L through
sound engineering fundamentals and
thorough process understanding. This
hasnt happened by accident, but rather
by bioprocessing professionals taking a
systematic approach to characterizing
the bioreactors capabilities and
tendencies, developing robust and
reliable scale-up procedures, and
establishing and maintaining proper
control criteria. Those manufacturers
that identify and document operational
best practices for a cGMP cell culture
plant also tend to be those that sustain
operational success and deliver high-
quality biopharmaceuticals to patients
in a timely and reliable manner.
Identifying and documenting bioreactor
operation best practices allows for
more robust processing by helping
to properly educate the operations,
engineering and technical staf who
oversee the bioreactor processes.
Shared learning helps to reduce
the amount of tribal knowledge
that exists within a group and to
maintain high levels of operational
excellence even in times of employee
turnover, with the end result being
a sustainable and reliable supply of
biopharmaceuticals.
With these ideas in mind, we have set
out to document best practices that
we have learned for bioprocessing,
most notably in the areas of equipment
design and overall process control.
BIOREACTOR DESIGN
Starting of with the proper bioreactor
design can resolve many process
issues before they arise. One key
bioreactor design issue that should not
be underestimated is the importance
of geometric similarity between
bioreactors: maintaining aspect ratios,
impeller sizing ratios, impeller spacing
ratios and bafe size and location will
greatly increase the probability of
success at scale. A properly designed
bioreactor can lead to reduced
qualication and process validation
timeframes, as well as increased apparent process
robustness and operational success.
Another key aspect to bioprocess scale-up success is the
design of the sparger. Often, two spargers are installed
in the production bioreactors while only one sparger is
used for the seed bioreactors. While various types of
spargers have been utilized within the industry, we have
successfully implemented the use of drilled pipes and
sparge stones. The drilled pipe yields large bubbles and
a lower kLa (which will be discussed below), while the
sparge stone yields small bubbles and a very high kLa
such that a greater amount of oxygen can be delivered
into the cell broth for the same gas ow rate. Figures
1 and 2 illustrate the sparger location and two sparger
types.
The ability of the bioreactor to deliver oxygen to the
cells is dened by the mass transfer relationship shown
in Equation 1. The change in oxygen concentration
is controlled by k
L
a, the average saturation oxygen
concentration of the bubbles,, dissolved oxygen
concentration ([O
2
]dissolved) and the oxygen uptake by
any cells present (OUR).

The k
L
a can be mapped as a power law function of the
power/volume and supercial gas velocity. Understanding
of the k
L
a allows for estimation of the OUR capacity of the
FIGURE 1.
Illustration of the sparger location within a bioreactor
(not drawn to scale).
Figure 2. Comparison of bubble sizes erupting from sparge stone and drilled
pipe (reproduced from www.mottcorp.com).
SPARGE STONE DRILLED PIPE
bioreactor, prediction of required oxygen
ow rates and prediction of the time-
course prole of the dissolved carbon
dioxide levels.
Based on process needs and sparger
capabilities, the process engineer must
determine the preferred conguration for
the bioreactors. If process OUR needs are
sufciently low, the default conguration
can be the use of one drilled pipe in
the seed bioreactors, and two drilled
pipes in the production bioreactors. The
combination of a drilled pipe and sparge
stone may also be used, but the oxygen
transfer ability aforded by the sparge
stone is typically not necessary. Use of
the sparge stone should be avoided if
possible due to the increased operational
complexity associated with bioreactor
set-up, manual changes in gas ows
during a process to maintain dissolved
carbon dioxide levels, increased foaming
and potential cleaning concerns.
MIXING CHARACTERIZATION
When scaling up a free suspension
cell culture bioreactor, a thorough
understanding of the mixing
characteristics is essential. If the mixing
inside the bioreactor is appropriately
controlled, then the cells will experience
an environment very similar to that of the
bench-scale bioreactor and will therefore
be much more likely to behave as they
did in the scale-down bioreactors.
The literature shows that many methods of
scale-up have been considered, including
matching power/volume, impeller blade
tip speeds, bulk mixing Reynolds numbers
and bulk mixing times. Due to the nature of
these various parameters, it is not possible
to maintain them all during scale-up under
one set of conditions.
Experience has shown that maintaining
a similar power/volume (P/V) at the
various bioreactor sizes greatly increases
the probability that the mixing within
the bioreactor will be appropriate. P/V
is a function of the impeller geometry,
the agitation rate and working volume,
as shown in Equation 2, where is the
density, n is the number of impellers,
N
p
is the impeller type power number,
N is the agitation rate, D
i
is the impeller
diameter and V is the liquid volume.
To further characterize the mixing,
the bulk mixing time at various
agitation rates can be measured via
pH or conductivity, or calculated using
commercially available models. This
is performed to determine the length
of time required for the bulk liquid to
become 99% homogeneous with respect
to pH or conductivity.
Combining the results of the kLa
mapping, mixing time determination and
P/V calculations can lead the process
engineer to choose the appropriate
agitation setpoint to enable successful
scale-up. Once the agitation setpoint is
determined, the sparging scheme can
be designed to ensure the dissolved
oxygen in the bioreactor is maintained
while carbon dioxide is efectively
stripped from the bioreactor and foam
accumulation is kept to a minimum.
BIOREACTOR CONTROL AND
ALARMING
Mammalian cell culture based
bioprocesses require monitoring and
control of the bioreactor environment
to ensure consistent bioprocess
performance. The parameters requiring
control include temperature, agitation,
dissolved oxygen (DO) and pH. Other
parameters such as cell density,
nutrient concentration, desired and
undesirable culture by-products are
controlled indirectly via medium and
feed formulation and can be greatly
afected by the physical and chemical
parameters. Neglecting control of
these parameters could potentially
impact nal product quality, so
online measurements can be
employed to maintain the culture
in an optimal state. Bioreactor
control schemes entail a series of
steps as outlined below.
Parameter monitoring and control
requires the use of an appropriate
analytical device, an appropriate
sampling method and a control
system which can act appropriately
to the information it receives. Online
monitoring control systems are rapid,
non-invasive and minimize potential
for contaminant introduction and may
be performed inside or outside of the
bioreactor but must be connected
directly to the bioreactor interior.
Conventional parameters subject
to online analyses include pH, DO
concentrations, agitation, backpressure
and temperature. Measurements
of additional chemical parameters
including cell density and viability,
waste metabolites, nutrients and
product concentration have historically
been measured ofine, although many
technologies are becoming available
that enable online measurement.
Online control employs probes, each
of which have a sensor whose function
is to gather information relevant to
the biological state of the culture. This
information is then converted into an
electrical signal that can be amplied,
recorded and analyzed so as to drive
the applicable control scheme. In
light of this, it is important that this
information is pertinent to the current
or future state of the bioprocess and
be quickly generated and processed
with minimal manual intervention.
The sensors should be selected based
on the following criteria: potential to
cause contamination, robustness and
reliability of sensor elements, specicity
for parameter being measured and
insensitivity to the harsh environment of
the bioreactor. Maintaining reproducible
and acceptable product quality
and productivity, while minimizing
downtime, are the primary business
drivers behind an efective bioprocess
monitoring and control strategy.
Efective bioreactor control may entail
monitoring more than just the primary
control parameters. For example, we
once encountered a situation in which
the pH stayed within the control range,
but caustic was being fed to the tank
even though the base controller had an
output of zero such that the controller
was not trying to feed caustic. Our
investigation led to the discovery that
Measure of response variable
Comparison of measured value to process setpoint
Activation of control scheme
the tubing from the caustic vessel to the
bioreactor was not installed properly in
the peristaltic pump, and caustic was
leaking past the pump and into the
bioreactor.
Another problem we experienced
entailed hyperoxygenation of one of the
seed bioreactors. This led to decreased
growth, viability and increased specic
lactate production. Investigation of
the incident led to the discovery that
oxygen was leaking into the process air
line. The DO probes had been calibrated
per ticket instructions but the oxygen
leak led to false readings and improper
DO control of the bioreactor. The
only indicator, other than cell culture
performance, of these false readings
was the nano-Amp readings of the DO
probes, which were found to be much
higher than normal.
TEMPERATURE CONTROL
Temperature is a key parameter requiring
monitoring and control throughout
bioprocesses to ensure an actively
growing and productive mammalian
cell population. In general, an accuracy
of 0.5C is considered adequate
for cell culture, although transient
excursions may exceed that range with
no impact to product quality or cell
culture performance. Typical bioreactor
temperature measurement devices are
resistance temperature devices (RTDs),
which are highly accurate, reproducible
and only moderately expensive. The
response time of these devices is in
the order of several seconds. These
RTDs rely on the fact that the platinum
core wire conductance varies with
temperature to quantify temperature.
In the RTD temperature sensor control
scheme, the signal is amplied,
linearized and transmitted to a controller
whereupon it is compared to a setpoint.
Based on this continuous comparison,
the bioreactors temperature is regulated
by adjusting the temperature of the
jacket surrounding the bioreactor. If and
when temperature deltas are recorded,
the temperature of the jacket is adjusted
appropriately through use of heat
exchangers.
DISSOLVED OXYGEN CONTROL
Mammalian cell cultures require oxygen
for the production of energy from
organic carbon sources e.g., glucose.
Given oxygens poor solubility in water-
based solutions, the control of oxygen
ow is carefully regulated to ensure
it does not become a rate-limiting
factor in the process. In contrast, a
hyperoxygenated bioreactor air supply
can irreversibly and adversely impact
culture performance.
Due to uctuating cell concentrations
and the associated uctuating oxygen
consumption rate, the quantity of
dissolved oxygen (DO) in culture
medium is in a state of dynamic
equilibrium. At a constant temperature,
the DO concentration in the culture
media (CL) is proportional to the
amount of oxygen in the vapor phase
within the media (CG) in a manner that
is dependent on temperature and media
composition (represented by Henrys law
constant, H in the equation below).
CL = HCG
Amperometric DO probes are typically
used, which measure the reduction of
oxygen at a cathode and the formation
of silver chloride at the anode with an
electrolyte solution bridging the gap
between the nodes. Given the nature of
amperometric DO probes, it is necessary
for these probes to be allowed to
polarize prior to their use. A calibration
is then performed, and in the event that
the probe falls outside of the acceptable
calibration range, the probe membrane
body and electrolyte are replaced.
PH CONTROL
Along with temperature and dissolved
oxygen control, efective pH control
is vital to ensure process success
given the sensitivity and potential
cellular damage that may occur if pH
control remains unchecked. Although
cell culture media typically provides
substantial bufering of pH, mammalian
cell metabolism routinely decreases
the culture pH due to the production
of lactate and carbon dioxide, both of
which are acidic in nature. Excessive
hydrogen ion concentration may
alter normal cell metabolism and
proliferation by impairing substrate
uptake and product release. In addition,
it is possible that the bioactivities of
some secreted monoclonal antibodies
or therapeutic peptides could be pH
sensitive.
Typically, the pH probes on the
bioreactors are calibrated while
connected to the transmitters on the
bioreactor that is destined for use
and prior to installation into the tanks.
Typical calibrations are conducted
using two bufers, with a calibration
check performed in an intermediate
bufer. Failed calibrations are typically
due to damaged pH probes, but may
also be attributed to faulty cables or
transmitters.
Once calibrated, pH probes have
occasionally been observed to generate
incorrect readings, due to probe
drifting, slowed response time or
impaired sensitivity. These erroneous
readings are typically attributed to
sensor membrane alterations due
to extreme temperature swings
and fouling from media and cellular
components. As a result, a policy for
re-standardization of the probes
may need to be developed using an
orthogonal pH measurement method
as the gold standard.
Efective pH control can be achieved
through use of two separate PID
loops, where one is the acid controller
and one is the caustic controller. In
a bicarbonate-bufered system, the
acid controller controls the carbon
dioxide ow and is congured such
that the carbon dioxide ow ramps up
very quickly when the process value is
above set point and instantly turns of
when the acid controller set point is
reached. The liquid caustic controller
utilizes a pulse width modulator (PWM)
to control the amount of time the
caustic peristaltic pump is on or of.
The set point on the peristaltic pump
is set manually per manufacturing
ticket instructions and the controller
only turns the pump on and of. The
frequency of measurement and pulse
addition and duration can be altered
to efect varying levels of control by
tuning the control loop. Due to the
high pH of the caustic feed, it should
be fed into the bioreactor through a
sub-surface port to facilitate quick
dispersion into the culture.
DISSOLVED CARBON DIOXIDE
CONTROL
Dissolved and evolved (i.e., headspace)
carbon dioxide levels can be indicative of
cellular metabolism and are thus routinely
monitored as indicators of culture
performance. In general, the mammalian
cell cultures display sensitivities to
extremes of dissolved carbon dioxide
(pCO
2
) be they low or high. High pCO
2

levels have been reported in the literature
as an inhibitor of growth and metabolism
and can impact product quality
characteristics such as glycosylation
of the protein product.
Several parameters can afect the
pCO
2
levels, including pH set point,
temperature, sodium bicarbonate
concentration, cellular metabolism,
caustic addition to the medium and
gas ows. Each of these parameters
must be considered carefully to
enable successful pCO
2
control.
pH, temperature and bicarbonate
concentrations are typically not
adjusted during a process to control
pCO
2
, but rather gas ows and caustic
addition are controlled to maintain the
pCO
2
within the desired target range.
The gas ows can be chosen to strip
out the desired amount of dissolved
carbon dioxide as experience has shown
the carbon dioxide levels are inuenced
more by total gas ow through the
bioreactor rather than kLa.
If the culture pH has drifted to the
acidic side of the dead band, increasing
the airow strips out carbon dioxide
potentially leading to an overall
reduction of caustic addition. The
reduced amount of caustic can lead to
a lower pCO
2
at the end of the culture
when lactate levels typically decrease.
However, if the pH is on the basic side
of the dead band such that the CO
2
is
being fed, increasing the airow will only
lead to increased CO
2
ow and will not
afect the pCO
2
.
BACKPRESSURE CONTROL
The stainless steel bioreactors are
maintained under positive pressure
to create an environment that is more
conducive to axenic operation. A
backpressure setpoint is generated by
maintaining a constant overlay process
air ow into the headspace of the
bioreactor. The backpressure can then
be controlled via a PID control loop
that operates a ow control valve on
the vent line. To avoid safety concerns
associated with over-pressurization,
rupture discs may be incorporated into
all pressurized stainless steel vessels
to act as pressure relief devices. In
addition to the bioreactor headspace,
positive pressure should be maintained
on all transfer lines within the sterile
boundary and any associated auxiliary
stainless steel vessels used for additions
to the bioreactors.
Backpressure can also be used as the
driving force to govern bioreactor-to-
bioreactor transfers and bioreactor-
to-primary recovery transfers. Care
should be given to ensure the transfer is
fast enough to not allow cells to settle
during the transfer, but not so fast as
to subject the cells to excessive shear.
The transfer time can be dictated by the
pressure drop and pipe dimensions.
ALARM STRATEGY
The alarm strategy should be
congured to alert the operators
that the process is deviating from its
acceptable range, but should also
provide early warnings such that the
operator can respond in time to prevent
loss of the batch. To this end, multiple
levels of alarming may be implemented.
The rst level of alarms can be set to
include the normal variability present
within a control loop such that if the
alarm is activated, operations can
assume that an unexpected excursion
has occurred but will have time to take
pre-emptive action before the process
is negatively impacted. The nal level
of alarms should be set to match the
acceptable ranges listed in the process
ow chart specic to a process. While
determining the alarm strategy, care
should be taken to apply alarms only to
the appropriate parameters. If excessive
alarming occurs, operators may begin
to not respond efectively to alarms to
the extent that important alarms may
be missed.
USE OF OFFLINE DATA
Additional information regarding
culture health and performance may be
obtained ofine using analysis of aseptic
samples of the culture. Typical ofine
testing will yield information regarding
cell numbers and cell viability using
an automated cell counter system. In
addition, a blood gas analyzer can be
used to determine the levels of relevant
parameters including lactate, glucose,
pCO
2
and pH. The ofine samples may be
used primarily for informational purposes
and not linked into automated response
systems to drive bioreactor control
changes. However, ofine measurements
may be used by technical services
to monitor the process and instruct
operators to, for example, restandardize
the pH probes should a drift from
setpoint be observed.
BIOREACTOR FEEDING STRATEGY
The basal medium may be added to the
bioreactor from a disposable bag via
peristaltic pump, or for larger volumes
from stainless steel media make-up
tanks via pressure transfer. For the tank
transfers, the media is typically sterilized
in-line via two hydrophilic lters, a pre-
lter followed by a sterilizing grade lter.
The lters are steam sterilized in line
simultaneously with the transfer path
and are cooled prior to media transfer.
Nutrient feeds are typically prepared
in disposable bags. The nutrient feed
typically consists of multiple stock
components that need to be well mixed
and may require pH adjustment. Upon
one nutrient feed into a bioreactor, a
high pH excursion was observed in the
bioreactor which was later attributed
to poor mixing of the nutrient feed
components. The nutrient feed bag was
subsequently re-designed to allow for
better mixing within the bag to avoid the
pH change in the bioreactor.
Nutrient feeds are delivered to the
bioreactors at pre-determined times
during the cell culture process. These
feeds are manually added to the
bioreactors, with very little automation
associated with them. In fact, the
automation is congured such that the
near-to block valve on the nutrient feed
line is always open during culture phase
to maintain positive pressure on the line.
Therefore, the peristaltic pump head is
the only block on the line, such that the
operator can initiate the feed simply
by turning on the pump. The nutrient
feeds may be slightly acidic, so a typical
concern associated with addition of the
feed is a change in pH in the bioreactor.
To account for this, the addition ow
rate of the feed is dictated, as well as
instructions to the operators to stop the
feed if the pH exceeds the acceptable
range.
ABOUT THE AUTHORS
Cillian McCabe has a rst-class honours degree
in Biotechnology from National University
of Ireland (NUI), Galway, and a PhD in Gene
Therapy Approaches to the Treatment of Type
1 Diabetes Melitus from the School of Medicine
at NUI. He joined Eli Lilly & Co. in 2007 and
has supported Bioprocess Development and
Manufacturing Operations both in the U.S. and
Ireland as part of Lillys Manufacturing Science
& Technology functional group.
Brian Stamper has a B.S. in Biochemistry from
Indiana University (Bloomington, Ind.) and an
M.S. in Biological Engineering from Purdue
University (West Lafayette, Ind.). He joined
Eli Lilly in 2001 as a development scientist
in Bioprocess Development and transitioned
to Bioprocess Operations in the Clinical
Trial Material Supply pilot plant in 2005 as a
manufacturing associate.
ABOUT HAMILTON COMPANY
For more than 30 years, the name Hamilton has been associated worldwide with
uncompromised quality in precision uid measuring and analytical products as
well as in fully automated analytical processes.

The same competence has led the Sensor Technology Group to design a line of
high quality pH, ORP, optical and amperometric Dissolved Oxygen, Conductivity
and Cell Density electrodes for process and laboratory measurement.
Hamilton Company
4970 Energy Way
Reno, NV 89502
Phone: 775-858-3000
www.HamiltonCompany.com
ADDITIONAL RESOURCES CLICK ON LINK
Brochure: Biopharmaceutical Sensor Solutions

http://www.hamiltoncompany.com/downloads/Hamilton_Sensors_Solutions_for_BioPharm_06.2012.pdf
Biopharmaceutical Process Sensor Selection Tool

http://www.hamiltoncompany.com/products/sensors/c/1008/
Article: Advances in Sensor Technology Improve
Biopharmaceutical Development
http://www.hamiltoncompany.com/downloads/Advances_in_Sensor_Technology.pdf
Article: In-Line Sensor Systems

http://www.hamiltoncompany.com/downloads/In-Line_Sensor_System.pdf

Advancing Biopharmaceutical Process Control

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