Anal Bioanal Chem (2010) 398:137–154
DOI 10.1007/s00216-010-3781-x
REVIEW
Process analytical technology (PAT) for biopharmaceutical
products
A. S. Rathore & R. Bhambure & V. Ghare
Received: 26 February 2010 / Revised: 20 April 2010 / Accepted: 23 April 2010 / Published online: 18 May 2010
# Springer-Verlag 2010
Abstract The “Pharmaceutical Current Good Manufactur-
ing Practices (CGMPs) for the 21st Century—A Risk Based
Approach” initiative announced by the FDA in August
2002 to improve and modernize pharmaceutical manufac-
turing facilitated adoption of process analytical technology
(PAT) by the pharmaceutical industry. The potential for
improved operational control and compliance resulting
from continuous real-time quality assurance was highlight-
ed as a likely benefit that would result from PAT
implementation. A considerable amount of work has been
done on this topic by academic and industrial contributors
in the last decade. In this paper, we will start with a brief
overview of evolution of PAT concepts and a review of
their application in the wider pharmaceutical industry. The
rest of the paper focuses on PAT applications for biotech
processes with emphasis on developments in the last five
years. It is our observation that while significant advances
have been accomplished with regard to our ability to
analyze/monitor key process and quality attributes in the
biotech industry, much more needs to be done with regard
to utilizing the collected data for subsequent control of the
process, to achieve optimum yield and product quality. The
latter is necessary to achieve the benefits that will result
from PAT implementation.
Keywords Process analytical technology, PAT .
Bioprocessing . Quality by design
A. S. Rathore (*) : R. Bhambure : V. Ghare
Department of Chemical Engineering,
Indian Institute of Technology,
Hauz Khas, New Delhi 110016, India
e-mail: asrathore@biotechcmz.com
Url: www.biotechcmz.com
Regulatory framework
Process analytical technology (PAT) is a key element of
the “Pharmaceutical Current Good Manufacturing Practi-
ces (CGMPs) for the 21st Century—a Risk Based
Approach” initiative announced by the FDA in August
2002 to improve and modernize pharmaceutical manu-
facturing [1]. The PAT initiative was first proposed by the
United States Food and Drug Administration’s (FDA)
Center for Drug Evaluation and Research (CDER) with
the objective of achieving significant health and economic
benefits by application of modern process control and tests
in pharmaceutical manufacturing [2, 3]. Shortly thereafter,
with an endorsement from the FDA’s Science Board, the
PAT subcommittee under the Advisory Committee for
Pharmaceutical Science (ACPS) was formed in November
2001 and consisted of the following four working groups
with representatives from the FDA, industrial experts, and
academic representatives [3–5]
& PAT applications working group;
& PAT products and the process development working
group;
& PAT process and analytical validation working group;
and
& PAT chemometric working group.
The key objectives of the ACPS subcommittee were to
[6]:
& Identify and define the technology and regulatory
hurdles, possible solutions, and strategies for successful
implementation of PAT in pharmaceutical development
and manufacturing;
& Discuss the general principles of the regulatory appli-
cation of process analytical technology including the
138 A.S. Rathore et al.

principles of the method validation, specifications, use,
and validation of the chemometric tools; and
& Discuss the need for general FDA guidance to facilitate
the implementation of the PAT.
Based on the comments and recommendations suggested
by the ACPS PAT subcommittee, the FDA announced the
draft version of the PAT guidance “Guidance for Industry
PAT—A Framework for Innovative Pharmaceutical Manu-
facturing and Quality Assurance” in September 2003 [7].
The final version of the PAT Guidance was released in
September 2004 with the help of the Center for the
Veterinary Medicine (CVM) and the Office of Regulatory
Affairs (ORA) [8]. The guidance was the result of
collaborative work amongst several FDA offices includ-
ing the CDER, CVM, and ORA. The underlying vision
was that of utilizing innovation, fundamental scientific
and engineering knowledge, and quality-management
principles for efficient pharmaceutical manufacturing.
The PAT initiative has also been supported by the
European Medicines Agency (EMA), the Japanese
Ministry of Health, Labor, and Welfare (MHLW), and
by the International Conference on Harmonization (ICH)
guidelines [9].
The more recently launched initiative “Quality by
Design” (QbD) is also aligned with the PAT concepts [9–
14]. Quality by Design has been defined in the ICH Q8
guideline as “a systematic approach to development that
begins with predefined objectives and emphasizes product
and process understanding and process control, based on
sound science and quality risk management” [9]. The
approach involves identification of the product attributes
that are of significant importance to the product’s safety
and/or efficacy, design of the process to deliver these
attributes, a robust control strategy to ensure consistent
process performance, validation and filing of the process
demonstrating the effectiveness of the control strategy, and,
finally, ongoing monitoring to ensure robust process
performance over the life cycle of the product. PAT plays
a key role in creation of a robust control strategy for the
process and for ongoing process monitoring [11].
What is PAT?
Process analytical technology (PAT) has been defined as “a
system for designing, analyzing, and controlling manufac-
turing through timely measurements (i.e., during process-
ing) of critical quality and performance attributes of raw
and in-process materials and processes, with the goal of
ensuring final product quality” [8]. A desired goal of the
PAT framework is to design and develop well-understood
processes that will consistently ensure a predefined quality
at the end of the manufacturing process. A process is
generally considered well understood when:
1. All critical sources of variability are identified and
explained;
2. Variability is managed by the process; and
3. Product-quality attributes can be accurately and reliably
predicted over the design space established for materi-
als used, process conditions, manufacturing, environ-
mental, and other conditions.
The objective for PAT implementation could be one or
more of the following [8, 15–19]:
& Better process understanding
& Improved yield because of prevention of the scrap,
rejects, and reprocessing
& Reduction in the production cycle time by using on-
line/at-line or in-line measurements and control
& Decrease in the energy consumption and improved
efficiency from conversion of the batch process into a
continuous process
& Cost reduction because of reduced waste and reduced
energy consumption
& Real-time release of the batches
From an implementation perspective, perhaps, PAT can
be visualized as the three-step process illustrated in Fig. 1
[18, 19]. The design phase starts early in process
development when the given unit operation is being
designed and then later optimized and characterized [12,
20]. In this phase, the critical quality attributes (CQA) that
are being affected by the process step are identified along
with the critical process parameters (CPP) that have been
determined to affect the CQA. This process understanding
is the essence of PAT and critical for the next two phases. In
ANALYZE
Ability to
analyze for the
pertinent CQA
as well as
DESIGN to monitor
Knowing the the CPP
CQA pertinent
for the process
step and the
CPP that impact
them
CONTROL
Using process
understanding to
control CPP so as
to achieve
consistent
CQA
Fig. 1 The three steps that must be taken for PAT implementation,
and the objective(s) of each step
Process analytical technology (PAT) for biopharmaceutical products
the analyze phase, a suitable analyzer is identified for
monitoring of the CQA and the CPP. PAT application can
be at-line (sample removed, isolated from and analyzed
close to process stream), on-line (sample removed for
analysis from process stream and returned to process stream
again), in-line (sample not removed but analyzed in place),
and off-line (sample removed and analyzed away from
process stream) [8, 18, 19]. For a PAT application, it is
necessary for the analytical results to be available in the
time-frame necessary to facilitate real-time decision mak-
ing. Finally, the control phase involves designing a control
scheme based on the process understanding such that the
data from the analyzer can be utilized for making real-time
process decisions, and consistent process performance and
product quality can be achieved.
PAT is more than just analysis [18, 19]. This is further
illustrated in Fig. 2, which shows pooling of fractions from
a process chromatography column on the basis of UV
absorbance at 280 nm, an application that is very
commonly utilized in biotech processing. Figure 2a illus-
trates an example of what could be a PAT application. In
this case, the process chromatographic column is separating
the product from host-cell proteins (HCP). The two species
are well resolved on the column, as is evident from the
baseline separation. Thus, even though UV absorbance at
280 nm cannot distinguish between the product and HCP,
the baseline separation enables its use for consistent pooling
A spectroscopy, acoustic sensor, nuclear magnetic resonance
UV Absorbance at 280 nm
Product
Host cell Pooling cuts based
proteins
p on UV absorbance
Process time
UV Absorbance at 280 nm
Product
B content, and distillation temperatures) and the performance
Product
related
impurity Pooling cuts based
on UV absorbance
Process time
Fig. 2 Use of UV absorbance at 280 nm for pooling of fractions from
a process chromatography column separating a protein product from
other host cell proteins. a. Baseline separation of product from HCP.
b. Incomplete separation of product from HCP
139
of the product and separation from HCP, which is a CQA.
Thus, use of UV absorbance based pooling in this case is a
PAT application. Figure 2b illustrates a case where use of the
same analytical tool (UV absorbance at 280 nm) is not a PAT
application. In this case also the column separates the
product from HCP. As mentioned above, the UV absorbance
at 280 nm cannot differentiate between the protein and HCP
and, because the two species are not baseline separated,
implementation of this pooling scheme is likely to result in
inconsistent product quality with respect to the CQA
(variable HCP levels in the pool). Hence, unlike the case
illustrated in Fig. 2a, use of UV absorbance at 280 nm is not
the correct tool for PAT application for this case.
PAT applications in the chemical industry
The concept of PAT has been applied in the chemical
industry for several decades and has been the subject of
several reviews and books [21–29]. Over this time, the
chemical industry has moved towards implementation of
process analyzers and process modeling approaches that
allow not only optimization of productivity and product
quality but also provide real-time assurance that the process
is in control and in case of any deviations, suggest steps that
would bring the process back in control. Table 1 presents a
summary of some of the recent applications that have been
published on this topic [30–36]. Near infrared (NIR)
(NMR), Raman spectroscopy, attenuated total reflectance
(ATR), and Fourier transform infrared spectroscopy (FTIR)
have been used as process analyzers in these applications.
Process component analysis (PCA), partial least square
(PLS), and soft independent modeling of the class analogy
(SIMCA) are some of the statistical approaches that have
been used to enable analysis and modeling of the abundant
data that is generally provided by the above mentioned
process analyzers. Near infrared (NIR) and mid infrared
(MIR) spectroscopy have been proposed as tools for
predicting quality of diesel/biodiesel blends (density, sulfur
has been shown to match that of conventional approaches
that are much more cumbersome with regard to analysis and
portability [30]. NIR has also been used as an analyzer to
determine the effect of several operating conditions on
recovery, selectivity, and productivity for production of
methyl isobutyl ketone (MIBK) [31]. Use of this PAT
approach enabled the authors to perform the necessary
experiments in a time-efficient fashion and resulted in 30%
improved productivity of MIBK. NIR, in combination with a
suitable statistical tool, has also been used in other
applications such as raw material analysis [33], measurement
of product quality [34], and process monitoring [36].
140 A.S. Rathore et al.

Table 1 Examples of PAT applications in the chemical industry

Application Process analyzer Statistical tool Observation

Prediction of the properties
of the diesel or biodiesel
Catalysis reaction involving
conversion of acetone to
methyl isobutyl ketone
(MIBK)
Industrial process for
granulation of urea during
fertilizer production
Raw material identification
and quality control
Analysis of the organic
content of waste water
Simultaneous determination
of methanol and ethanol
in gasoline
Simultaneous monitoring of
solute concentration and
polymorphic state of the
crystal
Near infrared (NIR) and mid
infrared (MIR) spectroscopy
In-line NIR
Acoustic sensor with high-
temperature microphone probe
NIR
Nuclear magnetic resonance
(NMR) spectroscopy
NIR
Raman spectroscopy and
attenuated total reflectance
(ATR) Fourier transform
infrared (FTIR) spectroscopy
Partial least-squares (PLS) NIR and MIR spectra have been shown to
calibration help in predicting distillation temperature
and sulfur content of diesel or biodiesel [30]
Design of experiments PAT application helped in determination of
(DOE), Principle- the factors affecting the productivity,
components analysis (PCA), selectivity, and yield of the MIBK and
PLS, and cluster analysis thereby leading to improved productivity
for MIBK [31]
PCA and PLS PAT approach used to predict fluidization
airflow, reflux of fines to the reactor,
granule moisture content, and granule
size [32]
K nearest neighbor (KNN) Fast and cost-effective method for raw
and soft independent material analysis [33].
modeling of the class
analogy (SIMCA)
PLS Less time-consuming and the cost-effective
method for analysis of organic content [33]
PLS Non-destructive and non-polluting method
of analysis enables faster detection of
methanol adulteration of the
gasoline [34]
PLS PAT approach was utilized to understand
how the feeding strategy for the reactant
affects the polymorph composition of
L-glutamic acid [35]

On-line determination and NIR PLS with distributed On-line process control led to significant
control of the water content control system improvement in yield [36]
of a continuous conversion
reactor

PAT applications in the pharmaceutical industry
Innovations in the process analytical chemistry (process
analyzers) and in our ability to capture and analyze large
amounts of data have served as the key drivers for adoption
of PAT in the pharmaceutical industry [18, 19]. The key
feature of PAT is that quality is built into the product, rather
than being tested before release of product [8]. The PAT
framework comprises risk management, at/on-line sensors
that assist in monitoring/controlling/designing of the pro-

Fig. 3 The different unit

operations that comprise a
typical pharmaceutical process. NIR T NIR PSD NIR
Each step can potentially benefit
from implementation of one or
more PAT applications. Adapted
from Ref. [17]. Copyright Dispensing Blending Unit Op 1 Milling Blending Compressing Coating
permission from Advanstar
Communications. PSD: Particle
size distribution; T: Tempera-
ture; DW: Dry weight; PI: DW PI DW PI PI PI DW PI DW PI
Product impurity

Raw Material NIR Temperature Control Particle Size Distribution Average Tablet Weight
20.5˚C

20.3˚C

20.1˚C

19.9˚C

19.7˚C
Process analytical technology (PAT) for biopharmaceutical products 141

cess and prediction of process performance [37]. A variety
of analytical techniques have been used in the pharmaceu-
tical industry, including Fourier transform infra-red spec-
troscopy (FTIR), UV-spectroscopy, gas chromatography,
high performance liquid chromatography (HPLC), X-ray
diffraction spectroscopy, and NIR spectroscopy.
Figure 3 illustrates a typical tablet manufacturing process
in a pharmaceutical process [17]. It is seen that PAT
approaches could be applied to the various unit operations
in the process: dispensing, blending, milling, compression,
and tablet coating. NIR provides a means of quick and
reliable testing of raw material quality such that only those
raw material lots that meet the required specifications
would be used during processing. In-line temperature
monitoring of the performance of the extrusion step could
be used to control the step via a feedback loop to the
heating/cooling system that controls the temperature.
Further, particle-size distribution will be continuously
monitored during milling for process consistency and
controlled via feedback or feed forward control. The
weight, thickness, potency, and hardness will be tested at-
line at the tablet press for continuous quality verification

Table 2 Examples of PAT applications in the pharmaceutical industry

Application Process analyzer Statistical tool Observation

Rapid and accurate tablet
identification
Active determination of content
of uncoated pharmaceutical
pellets
Mechanical property determination
of the drug tablet
Analysis of sustained-release tablet
film coatings using terahertz
pulsed imaging (TPI)
Roller compaction process of dry
granulation
Evaluation of content uniformity
for low-dose tablets
Powder flow characterization
NIR measurement of the potency
of an API
Active drug identification and
content determination
Monitoring capsule manufacturing
at small-scale level
Quantification of the active
ingredient in pharmaceutical
injectable formulations
Acoustic resonance Principle-components A fast and non-destructive method for on-line analysis
spectroscopy analysis (PCA) and label comparison before shipping, to avoid
mislabeling of drug [38]
NIR Partial least-squares NIR method was developed and validated for
(PLS) analysis determination of active content ranging from
80-120% of the usual active content of the
uncoated pharmaceutical pellets [39]
Air-coupled excitation Iterative computational Examination of the vibrational resonance frequencies
and laser interferometric technique can be directly correlated with the mechanical
detection properties of the tablet providing a non- destructive
technique for physical characterization of the
tablet [40]
Terahertz pulsed – Tablet coating thickness, coating reproducibility,
spectroscopy (TPS) distribution, and uniformity can be easily
determined. The method was validated against
optical microscopy imaging [41]
Thermal effusivity – Effusivity measurement were used to monitor the
measurement using the roller compaction process [42]
effusivity sensor
NIR PCA NIR/PCA was used to predict content uniformity of
low-dose tablets manufactured by a direct
compression process [43]
NIR PLS Real time information on the flowing cohesive powder
mixture was used to avoid powder segregation or
agglomeration and thus to maintain product
quality [44]
NIR PLS Potency of heparin active pharmaceutical ingredient
was determined by this non-destructive
method [45]
NIR PLS NIR method was used for qualitative and quantitative
determination of ranitidine in granules for
compression, cores, and final tablet [46]
NIR PLS PAT was utilized for testing of identity and quality
of raw materials, for blend uniformity analysis,
and for final content analysis of busulfan pediatric
capsules [47]
NIR and UV–visible PLS More economical and less time-consuming method
spectroscopy for quantification of the lysine clonixinate [48]

Prediction of dissolution for a
sustained-release dosage form
Analysis of liquid formulations
containing sodium chloride
NIR PLS This method was used to identify differences in the
composition of the coating polymer used for a
tablet and thus assist with prediction of dissolution
behavior [49]
Laser-induced breakdown – Method does not need any sample preparation and is
spectroscopy (LIBS) less time-consuming [50]
142 A.S. Rathore et al.

Vial Shake Seed Production Harvest via Chromatography Viral lnactivation

Flasks Expansion Bioreactor centrifugation, Column 1 (for Mab product)
depth filtration,
membrane filtration

Drug Product Bulk Ultrafiltration/ Chromatography Viral Filtration Chromatography

Processing/ Filling Filtration Diafiltration Column 3 (for Mab product) Column 2
(UF/DF)

Fig. 4 A typical production process for a biotech product

and feedback control of compression. This approach will
enable both increased process understanding and process
control.
Table 2 reviews some recent PAT applications in the
pharmaceutical industry [38–50]. With regard to process
analyzers, NIR is the most popular and is used in a diverse
set of applications including estimation of active content
[39, 43, 45, 46, 48], powder-flow characterization [44], raw
material analysis [47], and dissolution rate [49]. Some other
analyzers that have been reportedly used include acoustic
resonance spectroscopy [38], air-coupled excitation and
laser interferometric detection [40], terahertz pulsed spec-
troscopy [41], effusivity sensor [42], and laser-induced
breakdown spectroscopy [50]. It is evident from the
diversity of the analytical tools that are available and their
capabilities that a part of the vision outlined in Fig. 3 can be
realized, i.e. the process can be designed such that at the
end of each step, assurance can be provided that the step
performed its function in a satisfactory manner. However,
examples of using the information from the analyzers to

Table 3 Illustration of the challenges of executing PAT for biotech processes

Process Major process steps1 Typical process Typical decision Quality Attribute (QA) Analytical Typical analysis, Ratio
segment time, tp (hrs)2 time, td (hrs)3 or Process Attribute (PA)4 Method5 ta (hrs)6 (td/ta)

Upstream Microbial fermentation 24 2 Misincorporation HPLC 1 2

(production)
Mammalian cell culture 240 10 Glycosylation Oligosaccharide 1 10
(production) profile
Harvest Centrifugation 8 1 Recovery HPLC 1 1
Downstream Refolding 20 2 Misfolds HPLC 0.5 4
Chromatography 8 0.5 Aggregation HPLC 1 0.5
Drug product Formulation 6 0.5 Protein concentration A280 0.1 5
processing Lyophilization 24 1 Moisture NIR 0.1 10
Filling 12 2 Foreign particles Visible particles 0.5 4

Assumptions: 1. Biotech processes are recognized as complex processes that utilize a variety of unit operations. In this analysis, we are discussing
only a few major unit operations to illustrate PAT applications. 2. Typical times for a step can greatly vary from the nature of the molecule (fusion
protein vs. monoclonal antibody product, microbial cell line vs. mammalian cell line etc) and from product to product within the same class. Here,
we use typical values to illustrate the PAT concept. 3. Typical time that is available to make a decision before moving to the next process step. 4.
Every step in a biotech process may have multiple quality attributes (QA) or process attributes (PA) associated to it. Here we are just picking a
single attribute to illustrate the PAT concept. 5. Every QA or PA of a biotech product may be analyzed by multiple analytical methods, often
orthogonal to each other. Here we are just picking one assay for illustration purpose. 6. The analysis time for a given analytical method can greatly
vary. Here we are using a typical value for the sake of illustration.
Process analytical technology (PAT) for biopharmaceutical products 143

change the operating conditions and bring the process back
in control (control step of Fig. 1) are not too common and
creation of such control schemes is likely to be the focus of
the pharmaceutical industry in the future.
PAT applications in the biotechnology industry
Figure 4 illustrates a typical process for a biotech product. It
shows some of the significant unit operations that together
form the process. While the above mentioned discussion on
implementation of PAT in the chemical and pharmaceutical
industries is relevant to applications in the biotech industry
also, there are some unique considerations that come into
play with biotech processes and products [15].
1. Proteins are large, complex and heterogeneous molecules.
2. Biotech processes are more complex than a typical
chemical or small-molecule drug-manufacturing pro-
cesses with regard to number of batch records, number
of product quality tests, number of critical process
steps, and the amount and complexity of process data
that is generated [51].
3. Protein products are extremely sensitive to the manufac-
turing process used for their production. Lot to lot
variability in product quality is commonly observed even
when manufacturing has been performed using exactly
the same process, and variability of source material has
also been known to affect product quality [15].
4. Raw materials used in manufacturing of biotech products
can be complex, with lot to lot variability [52].
5. Our understanding of the link between each product
quality attribute and the clinical safety and efficacy of
the drug is generally limited [15, 51].
As a result of these differences, implementation of PAT
for biotech processes is bit more challenging [11, 18, 19,
53]. The challenges can be understood from the information
presented in Table 3 and Fig. 5. Table 3 shows the process
time for some of the key biotech unit operations defined as
the typical time that is available for making decision for a
step before proceeding to the next unit operation; quality or
process attribute that is typically important for the step;
analytical method used for measuring the attribute; time
required for analysis; and the ratio of the process time
available for decision making to the time needed for
analysis. Figure 5 illustrates how the ratio changes for
different unit operations. It is evident that the ratio can be
used as an indicator of the ease of implementation of PAT
for a given unit operation. Process steps (for example
mammalian cell culture) where the time window for
decision making is wide and sample analysis for deciding
the course of action can be conveniently performed,
implementation of PAT is relatively straightforward. How-
ever, in the case of unit operations such as process
chromatography, where the time available for taking a
decision is smaller than the time for analysis, implementing
a PAT application successfully requires changes to process,
equipment, and analytical methods [11, 54–56].
In this review, we will divide the process into four parts:
upstream, harvest, downstream, and formulation. In the
following sections, we will review PAT applications in each

Fig. 5 Ease of PAT implemen-

tation for some of the commonly
used unit operations in biotech
processes

PAT implementation is relatively easier

Ease of PAT implementation

12.8

6.4

3.2

1.6

0.8
PAT implementation is relatively difficult

0.4

0.2
Bioprocessing unit operations
144 A.S. Rathore et al.

Table 4 Examples of PAT applications in microbial fermentation unit operation

Analytical tool PAT Status Target of PAT application

Luminescence-based pH optrode On-line pH control in fermentation [59]

Nicotinamide adenine dinucleotide (NADH)
fluorescence (enzyme assay)
Fourier transform infrared (FTIR) with MVDA
Optic turbidimetric sensors
CO2 sensors
Flow-injection analysis (FIA) coupled
with biosensors
NIR with dynamic orthogonal projection
NIR and MIR
Galvanic decoupled sensors for measuring
electrical impedance
Flow-injection system (FIS) with on-line sensors
Flow-injection system coupled with biosensors
FTIR analysis with multiple linear
regression (MLR)
Electrochemical analysis (amperometric sensors)
Mass spectroscopy
Sequential injection on-line analysis system
Mass spectroscopy membrane sensor
2D fluorescence spectroscopy
Transflective embedded NIR (TENIR)
Automatic membrane inlet mass
spectroscopy (MIMS)
Electronic nose NIR with standard
bioreactor probe
Flow-injection analysis with enzyme sensor
Glucose sensors (enzyme electrodes)
Thermal biosensor with FIA system
Ion chromatography with double chamber
acoustic wave detector
Ion chromatography with piezoelectric
quartz crystal
HPLC
On-line Biomass measurement and control of physiology of cell in industrial
fermentation [60]
On-line Concentration of components such as glucose, fructose, and ethanol [61]
On-line Biomass/cell concentration and automatic regulation of substrate
and nutrient feed rate [62]
On-line Dissolved CO2 concentration and control of medium feed rate [63]
On-line Sucrose, glucose monitoring and fed batch fermentation control [64]
On-line Alcohol-content measurement for ensuring robustness of calibration
model applied on-line for a fermentation process [65]
On-line Monitoring of broth concentrations in an industrial fermentation
process [66]
On-line Metabolic monitoring of growth curve and measurement of electrical
property of cell solution [67]
On-line CO2 and O2 gas analysis, monitoring of ammonia control of fed
batch penicillin fermentation and control of feed addition rate [68]
On-line Glucose concentration measurement and control of cell growth [69]
On-line Measurement of concentrations of proteins and polysaccharides for
solid-state fermentations [70]
On-line Viable cell measurement and control on metabolic activity of cell [71]
On-line Gas analysis (CO2, O2, CH4) and control of metabolic rate [72]
On-line D-Lactic acid measurement and control of degree of acidification in
fermentation process [73]
On-line Monitoring of acetone, butanol, and ethanol concentrations and control
of fed batch fermentation of acetone–butanol fermentation [74]
On-line Monitoring of chemometric model and control of metabolic changes in
fermentation [75]
Semi-on-line Measurement of concentrations of glycerol and volatile fatty acids for
control of anaerobic fermentation [76]
On-line Aromatic compound analysis and monitoring of changes for a
two-phase process [77]
On-line Monitoring of pH, lactate, glucose, and galactose for control of yogurt
fermentation [78]
On-line Monitoring of glucose concentrations [79]
On-line Glucose monitoring for development of new feedback-control strategy [80]
On-line Monitoring and control for penicillin V production [81]
In-line Measurement of cations (Na+, K+, Ca2+, NH4+) for control of cell
growth [82]
On-line Lactic acid monitoring for control of production rate [83]
At-line Analysis of metabolites for control of product formation [84]

of these areas. We will also address PAT applications in the
field of chemometrics.
PAT applications in upstream unit operations
Application of PAT in upstream unit operations of microbial
fermentation or mammalian cell culture is complicated by
the fact that these unit operations are encumbered by use of
several complex raw materials that have undergone limited
characterization, and the large number of process conditions
that can affect the performance of these steps [18, 57]. It
should be emphasized that perhaps this part of the process
is the most critical because this is where product and
product related impurities are produced. The latter may
include product-related variants that closely resemble the
desired product and have an equivalent potency and safety
profile (e.g. minor post-translational modifications such as
C-terminal processing, N-terminal variants, deamidation)
and product-related impurities that do not resemble the
Process analytical technology (PAT) for biopharmaceutical products 145

Table 5 Examples of PAT applications for mammalian cell culture unit operation

Process variable Analytical tool Target of PAT application

Glucose concentration
Biomass concentration
Biomass concentration
Oxygen uptake rate (OUR) and
specific OUR
Biomass, glucose, and acetate
concentrations
Cell size, biomass concentration
Glucose and biomass concentrations
Gas and hydrocarbons
Heat fluxes
Metabolic flux analysis
Continuous FIA with chemiluminescence
assay
Conductance and capacitance-based
biomass probe
In-situ microscopes with image processors
and computer control
Mass-balance technique for measuring kLa,
dissolved O2 concentration, gas
compositions
NIR, spectroscopy and electronic nose
instrument
Dielectric spectroscopy
Multiwavelength fluorescence (MVF),
software sensors and MVDA
Gas sensor array as an electronic nose
Flow micro calorimeter, dielectric
spectroscopy
C-Glucose and 2D NMR spectroscopy
(isotope tracer method)
Control volume mixing pattern and residence time
monitored to indicate metabolic condition to prevent
cell starvation [89]
Specific growth rate and product formation monitored
to determine onset of stationary phase and
cell lysis [90–93]
Specific growth rate used to control product formation
in agitated cell broth cultivation [92, 94]
Control of physiological state of cells and cellular
activity [57]
Control of fed batch cultivation and monitoring of the
quality of the culture [95]
Closed-loop control of medium feed rate in fluidized
bed fermenters [96–98]
Control of specific growth rate and product
quality [99]
Detection of early infections in mammalian cell
cultures [100]
Monitoring of specific growth rate and control of
utilization of media components [101]
Control of metabolic activity of Chinese hamster
ovary (CHO) cells in high-density perfusion
cultures [102]

desired product with respect to safety and/or efficacy (e.g.
aggregates and highly truncated forms). In several cases the
impurity may not be affected by subsequent steps, thus
making upstream unit operation the sole process step for
controlling the level of the impurity. Many off-line and on-
line techniques are available that can either directly monitor
actual product formation and/or biomass concentration or
indirectly monitor variables which serve as markers for
product formation. On-line monitoring has the advantage
that it reduces the frequency of sampling and thereby the
potential for microbial contamination [58].
Use of PAT for controlling microbial fermentation
processes has been a topic of interest for several decades.
Table 4 presents a summary of the several PAT applications
that have targeted microbial fermentation. Microporous
ultrafiltration membranes integrated with chromatographic
systems have been used for in-line monitoring of fermen-
tation [85]. Different variables, for example biomass
production, specific growth rate, product concentration,
and byproduct formation during batch and fed batch
fermentations have been monitored for control of the
fermentation process [37]. A combination of NIR spectros-
copy and multivariate data analysis (MVDA) has been
successfully used for screening many basal medium
powders used in a mammalian cell culture process [86].
The ability to fingerprint the raw material enabled the
authors to differentiate between good and poor performing
raw material lots. In high cell-density fermentations, NIR
spectroscopy has also been used for at-line control and fault
analysis in fermentation processes [37, 87]. Gnoth et al.
have demonstrated the usefulness of mechanistic modeling
in obtaining consistent total biomass and the product
concentration in an E. coli fermentation [88].
Several applications have also been published for mam-
malian cell culture (Table 5). Use of liquid chromatography
to quantify the titer of the antibody in the cell culture and,
thus, indicate the time for harvesting, has been reported [58].
The on-line system is likely to reduce the potential for
microbial contamination as repeated sampling for titer
measurement is not necessary. Kussow et al. monitored
growth rate of mammalian cells by utilizing the stoichio-
metric correlation between base addition for pH adjustment
and nutrient consumption (glucose). The perfusion rate was
monitored on-line via this stoichiometric correlation, and
nutrient concentration was controlled within a set range
[103]. The authors suggested that this approach could be
useful in timing of virus infection and result in a higher
virus concentration. Reinecke et al. have developed an
automated immunoassay technique instead of the com-
monly used enzyme-linked immunosorbent assay (ELISA)
for determination of Immunoglobulin G (IgG) production
in mammalian cell culture monitored via fluorescence. Their
system utilized surface plasma resonance to detect antigen–
antibody complexes, thus offering a rapid and sensitive assay
146
that could be used for process design and optimization [104].
Different enzyme sensors for detection of glucose, lactate,
and glutamine connected via flow-injection analysis (FIA)
devices have been used, although their large-scale adoption
is hindered by issues regarding their stability, calibration, and
validation [105–107]. On-line cell concentration measure-
ments via in-situ microscopy (ISM) have been shown to be
useful in capturing images with image processors and
providing estimates of hybridoma cell concentration in
mammalian cell cultures [108].
More recently, use of radio-frequency impedance for on-
line monitoring of viable cell density in cell culture manufac-
turing processes as an accurate and reliable method has been
suggested [109]. Monitoring of metabolic activity of mam-
malian cells and thus estimation of specific growth rate has
been made possible by use of techniques such as NMR, mass
spectroscopy, and radioactive tracers [110]. Automated flow
cytometry has been used for monitoring culture homogeneity
over time, thus enabling understanding of cell death kinetics
and making the technique useful during scale-up of cell-
culture steps [111]. In situ 2D fluorimetry, in combination
with chemometrics, has been suggested as an approach for
monitoring of key bioprocess variables for mammalian cell-
culture processes [112]. Viable cell density and recombinant
protein concentrations could be actively monitored, leading
to potential PAT applications utilizing this tool.
In summary, the biotech industry has made significant
advancements in developing analyzers and modeling
approaches that enable us to monitor key process and quality
attributes in real-time or near-real-time. However, only a few
of these references meet the true expectations of PAT quality
[58, 86, 88], i.e. not only process understanding but also
process control (Fig. 1) to result in consistency of process
performance and product. Because upstream unit operations
have a critical effect on product quality and also typically
Fig. 6 Use of an ultrafiltration
membrane in combination with
analytical chromatography for
on-line monitoring of a harvest
process
PREPERATIVE
AFFINITY COLUMN
ELUENT
A.S. Rathore et al.
allow for more time to analyze samples and make process
decisions (Fig. 5); we believe we will see more “true” PAT
applications in the future for this part of the process.
PAT applications in harvest unit operations
As can be seen in Fig. 4, microfiltration, depth filtration,
homogenization, centrifugation, and precipitation are some
of the most commonly used unit operations for harvest of
biotech products. The objective for this part of the process is
generally to make the output from the upstream step
(fermentation broth) ready for further purification by the
downstream unit operations. This is achieved via clarification
of the process stream in a manner that maximizes product
recovery across these process steps and avoids any degrada-
tion of the product during the processing. It should be noted
that the literature on this part of the process is relatively
sparse in comparison with that on upstream unit operations.
For depth filtration, the possibility of using a PAT
application for monitoring separation of host cell proteins
(HCP) by utilizing absorbance at 410 nm has been suggested
[58]. Host cell proteins (HCP) absorb at 410 nm and thus, by
monitoring conditions including transmembrane pressure,
flow rate, optical density (OD) of permeate at 280 nm and
OD of permeate at 410 nm, blockage of the depth filter can
be assessed [113]. This can be used to create a control
scheme for the depth filtration step such that HCP level in
the filtrate is maintained at or below the target level.
Ultrafiltration units for clarification, integrated with col-
umn liquid chromatographic systems, have been used for on-
line monitoring of harvest and fermentation processes [58,
114]. Zhang et al. have used a hollow-fiber membrane for
extraction of Mab produced during fermentation. As shown
in Fig. 6, the permeate obtained from the hollow-fiber
HOLLOW FIBRE
PUMP
MEMBRANE
FERMENTER
ANALYTICAL
COLUMN
COMPUTER CONTROL
Process analytical technology (PAT) for biopharmaceutical products
module was directed either to a chromatographic harvesting
column or to an analytical chromatographic column (protein
G-specific). Data obtained from analysis were used to monitor
the harvest process [58]. PAT has also been used for on-line
monitoring of biomass-based power plant effluents during
microfiltration. By monitoring fouling behavior by turbid-
ometry, a suitable regeneration strategy can be applied
during microfiltration [115]. To obtain constant flux,
membrane retention, and turbidity reduction, intermittent
back flushing with compressed nitrogen gas can help to
avoid cake formation and membrane fouling. This strategy
has been shown to result in product retention of more than
99.5% and turbidity reduction of greater than 95% [116].
Use of near infrared (NIR) spectroscopy for measure-
ment of sedimentation velocity in a centrifuge has been
suggested [117]. The authors used a particle-separation
analyzer for measurement of separation/sedimentation
kinetics of bentonite/xanthan gum mixtures during centri-
fugation. They used an analytical centrifuge with an opto-
electronic sensor system which measures NIR transmission
profiles of horizontally inserted samples tubes. Thus,
clarification data enabled optimization of concentration of
excipients to be added to the final formulation. In another
application of NIR, a method has been developed for
monitoring the active pharmaceutical ingredient (API)
concentration in the different process intermediates during
harvest steps [118]. The method had an accuracy of 0.01%
(w/w) for an API operational range between 0.20 and
0.45% (w/w). In summary, the harvest steps have not seen
as much interest in the development of PAT applications as
the other parts of the process. This could be because of the
limited effect harvest steps typically have on final product
quality. Hence, the benefits of implementing PAT are not as
significant as they may be for the upstream or the
downstream steps of the process. There may be applica-
tions, though, where, because of unique drivers (such as
product stability) PAT applications such as those reviewed
above may be of interest to the industry.
PAT applications in downstream unit operations
Commonly used unit operations for downstream processing
of biotech products include refolding, precipitation, filtration
(depth filtration, microfiltration, ultrafiltration/diafiltration,
nanofiltration etc), and liquid chromatography. The objective
of purification is to isolate the product of interest from the
process stream that comes from the harvest unit operations
and contains a variety of impurities besides the product of
interest. These impurities include product-related variants that
closely resemble the desired product and have an equivalent
potency and safety profile (e.g. minor post-translational
modifications such as C-terminal processing, N-terminal
147
variants, deamidation), product-related impurities that do not
resemble the desired product in respect of safety and/or
efficacy (e.g. aggregates and highly truncated forms), and
process-related impurities (e.g. host-cell DNA, host-cell
proteins, and raw materials from the process). Isolating the
product of interest from these myriad of species to achieve the
high purity level expected of a biotech product today is a
difficult task. As a result, biotech processes consist of several
unit operations, with each unit operation serving a defined
purpose. For some of the product quality attributes, a certain
level of redundancy in the process is expected. The
interdependencies between the performance and expectations
of each unit operation are particularly relevant to downstream
processing. The need for appropriate control of each step and
continuous assessment of its performance is also of great
importance. In the following text we review PAT applications
in some of the most commonly used downstream unit
operations.
HPLC has been used as a monitoring tool for a refold
step [53]. Product purity and the levels of the different
product-related variants and impurities were monitored as a
function of time. The data suggested that it is feasible to
implement a PAT-based control scheme that enables ending
of refold on the basis of product quality data. This would
ensure consist product quality of the refold end pool and
improve operational efficiency by keeping refold time to
what is really needed.
Protein crystallization has also been of some interest for
PAT applications, because of the increase in structure-based
drug design fuelled by the recent developments in genomics
and proteomics [119, 120]. In-situ microscopy has been
used to monitor the progress of crystallization processes
and to differentiate among the various crystal forms [119].
Microfluidics-based high-throughput approaches have been
used for optimization of conditions for crystal formation
[121–123]. This is necessary because in order to be used for
structural analysis, the protein has to be of very high purity.
Large crystals of some of the model proteins (lysozyme and
aprotinin) have been produced by controlling the supersatu-
ration level by changing the temperature or the ionic strength
of the solution [124, 125]. This strategy or a more advanced
control strategy could be used in combination with high-
throughput techniques to improve protein crystal growth. At
this time, protein crystallization remains a technique for
production of protein in small quantities. In the future it is
possible that advances in our understanding of factors that
affect protein crystallization may lead to utilization of this
procedure for large-scale production of proteins [126]. PAT
is likely to play a key role in this evolution.
Rathore et al. have used a PAT-based control scheme for the
diafiltration step in an ultrafiltration/diafiltration (UF/DF)
process. The concentration of each species in the product
stream was measured after each diafiltration volume to assess
148
the performance of the DF step as a function of number of
diafiltration volumes. On the basis of the results, monitoring
of the pH during diafiltration was proposed as a PAT tool for
indicating completion of the DF process [53]. This approach
was suggested to be particularly attractive for cases when an
expensive diafiltration buffer is being used or when the
diafiltration time must be minimized because of product-
stability concerns, because in both of these cases minimizing
the number of diafiltration volumes and process time would
be beneficial.
Use of high-performance liquid chromatography (HPLC)
as an analytical tool for process monitoring has been proposed
in the literature [127, 128]. Use of on-line reversed phase
HPLC for separating recombinant human insulin-like growth
factor-I (IGF) and IGF aggregates to facilitate real time
pooling based on product quality has been successfully
demonstrated [129]. A similar application using chromato-
graphic separation with protein A-immobilized media has
been shown to reliably control antibody loading on a protein
A column in a CHO-derived recombinant antibody purifica-
tion process [130]. Recently, an approach utilizing a
combination of on-line size-exclusion HPLC, differential
refractometry, and multi-angle laser light scattering analysis
(MALLS) has been shown to be useful for real-time
estimation of product quality of a mutant form of the human
immunodeficiency virus (HIV) [131]. The accuracy of the
approach was found to be >89% and intra and inter-day
precision were found to be 0.9 and 3.6% RSD, respectively.
More recently, Rathore et al. have published a series of
case studies demonstrating the utility of a variety of
analytical tools for performing analysis that would facilitate
pooling of large-scale chromatography columns. The
analytical tools that have been utilized include HPLC [55,
56], ultra-performance liquid chromatography (UPLC) [54],
and fluorescence [132]. These applications are based on the
premise that for the cases where the chromatographic
separation is high-resolution and part of the peak is being
collected to pool the product and exclude the impurity/
impurities, these approaches offer a way of performing
analysis in the required time frame and enable a decision
based on product quality. This is in contrast with the often
used practice of pooling based on UV absorbance, such as
absorbance at 280 nm, which though an operationally
simpler approach to implement, suffers from the fact that
absorbance at 280 nm cannot differentiate between the
product and other product-related impurities. In one case,
use of on-line HPLC resulted in the pool purity varying
between 90.2 and 91.6% even though the purity of the feed
material varied from 62.8–81.6% [56]. Utilization of
absorbance-based pooling resulted in variability of pool
quality from 81.3–95.4%. This case study clearly highlights
the effect of using PAT-based controls on the consistency of
product quality.
A.S. Rathore et al.
In summary, the downstream process steps, in particular
process chromatography, have a lot of gain from imple-
mentation of PAT. As is evident from this section, this topic
is also relatively unexplored when we think of the potential
opportunities it presents [53–56, 130]. However, imple-
menting PAT for this part of the process will be challenging
because of the short process times typical of these unit
operations (Fig. 5). We expect that we will see more
development and implementation of “true” PAT applica-
tions in the future for downstream processing.
PAT applications in drug product unit operations
In this section we have purposely included some applica-
tions to traditional small molecules, because the tools and
approaches are likely to be extended to their biotech
counterparts also.
In the formulation step of drug product processing,
different chemical substances including excipients with
active drug substance are combined to produce the final
medicinal product. Raman spectroscopy with wide-area
illumination (WAI) has been suggested for monitoring of
active pharmaceutical ingredient (povidone) in the eyewash
formulation, directly through plastic bottles [133]. The
Raman data were modeled using PLS for quantification of
active pharmaceutical ingredient (API) content. A similar
application used Raman spectroscopy with WAI and PLS for
determination of acetaminophen in vials containing suspen-
sion [134]. Laser-induced breakdown spectroscopy (LIBS)
has been used for direct and rapid analysis of pharmaceutical
liquid formulations. In this technique, the atomic line from
sodium is used to quantify NaCl in formulations such as
injectables, nasal solutions, and bactericidal injections [50].
NIR spectroscopy has been used for quantification of lysine
clonixinate in an intravenous injection formulation [48].
Bench-top NMR and MRI (magnetic resonance imaging)
have been used for monitoring of emulsions, lipid ingre-
dients, and their adsorption characteristics. This technique
enables non-invasive monitoring of drug-delivery processes
of these formulations [135]. NIR has also been used during
mixing of to illustrate differences in the mixing character-
istics of aspirin, citric acid, aspartame, or povidone with
avicel, and thus to elucidate the different cohesive properties
of the particles [136]. The approach was used for under-
standing the dynamics of powder mixing and to make
quantitative measurements and monitor possible changes in
particle size during blending.
Lyophilization is one of the most commonly used unit
operations in drug product processing of biotech products.
In this step, water and other solvents are removed first by
freezing the product and then removal of frozen ice by
sublimation. In primary drying, frozen ice is sublimated and
Process analytical technology (PAT) for biopharmaceutical products
in secondary drying, residual water is removed from the
porous matrix. Because of the great effect of this process
step on final product quality and the sensitivity of the step
to various environmental conditions, lyophilization has
been a focus of numerous PAT applications. As seen in
Figure 7, on-line monitoring of residual water content
during drying, by use of moisture sensors that measure
water vapor pressure, has been used to predict sublimation
end point [137]. NIR spectroscopy and Raman spectrosco-
py are most commonly used to determine residual water
during drying. NIR spectroscopy has been used for
determination of surface and bound water during drying,
and, with the model proposed, used to accurately predict
the end point for drying [138]. Use of Kalman filter-based
observers with mathematical modeling has been suggested
as an approach that enables in-line monitoring of primary
drying phase in vials [139]. Tunable diode laser absorption
spectroscopy (TDLAS) has been applied as a non-invasive
technique to monitor water vapor concentration and vapor
flow velocity during freeze drying, which enables measure-
ment of the total amount of water removed [140]. Another
recent publication has demonstrated how, by integrating the
various devices (pressure gauge, mass spectrometer, ther-
mometer, moisture sensor), an innovative model can be
developed to enable monitoring of sublimating interface
temperature and heat and mass transfer coefficients during
freeze drying in vials [141]. A mono-dimensional model
capable of giving mass and energy balances in dried layers
during primary drying in vials has been validated using
experimental data obtained in a pilot scale freeze dryer and
used for on-line monitoring [139].
In summary, although, similar to upstream processing,
many advances have enabled us to monitor key process and
Pressure Vaccum chamber
sensor
Vials
NIR Probe Heating/Cooling Shelf
NIR S
Spectrometer
Temperature
Control system
Condenser
Computer
Fig. 7 On-line monitoring, by NIR spectroscopy, of moisture content
(both unbound and bound water) during freeze drying in vials
149
quality attributes in real or near-real time, these data have
rarely been used for process control (Fig. 1) [138]. Because
most drug product unit operations allow more time to
analyze samples and make process decisions (Fig. 5), we
believe the industry will start implementing “true” PAT
applications in the future for this part of the process.
PAT applications in chemometrics
Chemometrics, the science of extracting relevant informa-
tion from chemical processes, has had major effect on
process analytical chemistry. For complex systems, for
example biotech manufacturing, analytical data from on-
line monitoring instruments is typically not amenable to
direct interpretation. So chemometric methods are essential
for effective data analysis [86, 142, 143]. In addition,
chemometrics makes it possible to obtain real-time informa-
tion from data. Fast, precise, accurate, and non-destructive
process analyzers in combination with chemometrics are
suitable for process analysis and optimization leading to
improved product quality, productivity, and efficiency. Che-
mometric methods help in the development of an empirical
model based on the collected data that can be used for the
predictive estimation of the properties of a chemical
process and hence help in process analysis, optimization,
and control.
As can be seen in Fig. 8, developing and implementing a
chemometric approach is often a multistep process, neces-
sitated by the huge datasets that are to be analyzed and the
sophisticated statistical tools that are used.
Preprocessing of the data
Pretreatment of the data by approaches such as standard
normal variate (SNV), orthogonal signal correction (OSC),
and multiplicative scatter correction (MSC) may be a
critical step in creating an accurate chemometric model in
some applications [144, 145].
Data reduction
Reduction of the multidimensional data can be achieved by
use of regression methods such as the PCA. This enables us
to use the principle components to quantify the interactions
among the variables by calculating the matrix of the
correlations for the whole data in the form of scores and
loadings matrix [8, 86, 146].
Discrimination of data
Discrimination methods help in the grouping of the data
according to the specific properties of the data [130].
150 A.S. Rathore et al.

Sample removal using Regression analysis of the data (multivariate data calibration)
Sampling technique
Sampling Techniques
and Process Analysis This step helps in correlating the data with quantifiable
Process Analyzer properties of the sample. PLS is the regression method most
commonly used for quantitative prediction of the dependent
Data collection variables [146, 147].

Validation of the chemometric model

Preprocessing of Data
This is a key step, because chemometric analyses often lack
Chemometrics and any mechanistic basis. Thus, this step enables accurate
Data Reduction Multivariate Data determination of the principle components affecting the
Analysis process and reliable estimation of the noise and error in the
analysis. While test-set validation may be used when a
Data Discrimination
large number of data is available, cross validation is often
used when the number of samples is limited. Model
Regression Analysis of Data performance is measured by use of correlation coefficients
which indicate relationships between the measured refer-
ence and predicted reference [142, 143, 148, 149].
Multivariate Data Calibration Application of chemometrics to experimental data in
process analytical technology has two key objectives.
First, qualitative data analysis using chemometrics aids
Validation of Chemometric identification of patterns in data, via trends analysis, and
Model helps to eliminate outliers. Second, quantitative data
Process Control
analysis using regression methods is useful for predicting
Process Control relationships between independent and dependent varia-
bles. Figure 9 illustrates some of the commonly used
analytical tools that together with the appropriate chemo-
Successful PAT Application
metric technique form the basis for a PAT application.
Process analyzers could be hardware sensors or soft
Fig. 8 The various steps that must be taken for development and sensors and may be implemented on-line, in-line, at-line,
implementation of a chemometry-based PAT application
or off-line. Of the hardware sensors, NIR is one of the most
commonly used analyzers in the pharmaceutical industry.
Other commonly used process analyzers include Raman
Unsupervised classification methods, for example the spectroscopy, image probe, mass spectroscopy, biosensors,
Gaussian mixture model and principle-components analysis dielectric spectroscopy, 2D fluorescence spectroscopy,
(PCA), and supervised classification methods, for example biosensors, and enzyme-linked immunosorbent assay
partial least-squares (PLS) analysis and soft independent (ELISA). Soft sensors are software that assist in the
modeling of class analogy (SIMCA), are commonly used in processing of several measurements. Digital signal pro-
biotech applications [52, 86, 147]. cessing is a key part of software sensors. Kalman filters

Fig. 9 The various combina-

tions of analyzers and statistical Statistical and mathematical methods
tools that together form a PAT
application
Analytical instrumentation
Software assistance
Applications
• Design of experiment
• Pattern recognition
• Multivariate calibration
• Near infrared spectroscopy
• Raman spectroscopy
• High performance liquid chromatography
• Matlab
• Excel
• SIMCA
• Process analysis and control
• Process optimization
Process analytical technology (PAT) for biopharmaceutical products
and fuzzy controllers are the most common examples of
soft sensors [146, 150].
Several applications involving use of chemometric
tools in biotech production have been published recently.
PCA has been applied to near-infrared spectral informa-
tion derived from scanning unprocessed culture fluid
samples from a complex antibiotic production process
for process diagnosis and rapid assessment of process
performance [152]. An integrated on-line multivariate
statistical process monitoring, quality prediction, and fault
diagnosis framework for batch processes has been applied
for simulated fed-batch penicillin fermentation [153]. The
approach enabled prediction of the values of the end-of-
batch quality during progress of the batch; this was useful
for anticipating effects of excursions on final quality, and
suggested control strategies. Use of robust statistical
methods to reduce or remove the effect of outlying data
points, in order to enable the good data to primarily
determine the result, has been suggested [154]. A success-
ful case study involving modeling of data from a
pharmaceutical process and using the model for making
predictions has been presented [151]. The authors empha-
sized the importance of using design of experiments (DOE)
during process development and then later using a well
managed system of data collection, data bases, data
analysis, and networks to connect these pieces.
More recently, Kirdar et al. have examined the feasibility of
using chemometrics for supporting key activities required for
successful manufacturing of biopharmaceutical products,
including scale-up, process comparability, process character-
ization and fault diagnosis [155]. Gunther et al. have
demonstrated application of a flexible process-monitoring
method to industrial pilot plant cell culture data for fault
detection and diagnosis [156]. In a more recent publication,
application of chemometrics as a diagnostic tool for
identifying the root cause of a problem and designing
experimental conditions to demonstrate and correct this root
cause has been published [86]. Another recent paper from
Kirdar et al. reports a case study involving use of a combined
approach of near-infrared (NIR) spectroscopy and chemo-
metrics for screening of lots of basal medium powders based
on their effect on process performance and product attributes
[52]. A combined NIR/chemometrics approach was shown
to make it possible to fingerprint the raw materials to
distinguish between good and poorly performing media lots.
In summary, chemometrics has become almost a
necessary counterpart to the process analyzer in any
successful PAT application for biotech processes. As the
industry moves, in general, from “analysis for monitoring”
to “analysis for monitoring followed by process control to
get to the desired end point”, we will see an even bigger
role of chemometrics in process modeling necessary for
connecting the analysis with process control.
151
Summary
The biotech community agrees that it is mutually beneficial
to the industry and to its regulators to use PAT applications
to improve control processes, to deliver more consistent
product quality, and, perhaps, to raise operational efficien-
cy. Implementing PAT, however, requires a higher level of
process understanding which results in an immediate need
for greater investment of time and resources. Hence, the
pros and cons of implementing a PAT application must be
analyzed before taking the decision to proceed [53].
In this paper, we hope we have captured the evolution of
the concepts of PAT and reviewed the major developments
that have occurred with regard to implementation of PAT in
biotech processes. Wold et al. have classified the PAT
applications into five levels as follows [136]:
PAT Level 1 Determination of the concentration and other
properties of component from the spectrum
PAT Level 2 Raw material quality check and the classifica-
tion from spectral data
PAT Level 3 Batch process monitoring by means of the
multivariate measurement
PAT Level 4 Using the batch monitoring data and process
information to predict the quality of the
product
PAT Level 5 Feedback control of the process to maintain
the quality of the product
It is our observation that although significant advances have
been accomplished with regard to our ability to analyze/
monitor key process and quality attributes in the biotech
industry (Levels 1–3), much more needs to be done with regard
to utilizing the collected data for subsequent control of the
process, to achieve optimum yield and product quality [4, 5].
Of all the references cited in this paper, only a handful achieve
the latter objective. Thus, more needs to be done to achieve the
benefits that will result from true PAT implementation.
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