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ANALYTICAL AND
BIOANALYTICAL
TESTING
ANALYTICAL AND BIOANALYTICAL TESTING 2014
METHOD TRANSFER
4 Keys to Successful Method Transfer
EDITORIAL
Editorial Director Rita Peters [email protected]
Managing Editor Susan Haigney [email protected]
Science Editor Adeline Siew, PhD [email protected]
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Science Editor Randi Hernandez [email protected]
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Art Director Dan Ward
Contributing Editors Jill Wechsler [email protected]; Jim Miller info@
pharmsource.com; Hallie Forcinio [email protected]; Susan J. Schniepp
[email protected]; Eric Langer [email protected];
and Cynthia A. Challener, PhD [email protected]
Correspondents Hellen Berger (Latin/South America, [email protected]),
Sean Milmo (Europe, [email protected]), and Jane Wan (Asia, [email protected])
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Tel. 732.596.0276, Fax 732.647.1235, PharmTech.com
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Cynthia A. Challener
ANALYTICAL QUALITY BY DESIGN
11 AQbD Adds Rigor to Method Development
Paul Kippax
VIRAL CONTAMINATION
17 The Challenge of Finding the Unknown
Cynthia A. Challener
ELEMENTAL IMPURITIES
21 Rapid Screening for
Elemental Impurities using ICP-MS
Jonathan L. Sims and Fadi Abou-Shakra
ASSAY VALIDATION
26 Validation of a Multiplex
Bead-Based Assay
Rabia Hidi, Catherine Diot, Sebastien Melin,
Jiyhe Jang-Lee, and Alain Renoux
Joe Loggia, Chief Executive Officer Tom Ehardt, Executive Vice-President, Chief
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DATA MANAGEMENT
29 Standardizing Data Management
James M. Vergis and Dana E. Vanderwall
NEW TECHNOLOGY
31 Advances in Analytical Technology
Ashley Roberts
32 Ad Index
Issue Editor: Rita Peters.
On the Cover: Rafe Swan/Cultura/Getty Images; Dan Ward
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Method Transfer
Keys to Successful
Method Transfer
Cynthia A. Challener
Methods must be suitable
at each development
phase, robust, and effective
on multiple platforms.
M
ethod transfer during scale-up of the biopharmaceutical manufacturing process can be challenging.
Not only must methods be suitable at each phase of
the development process, they must be robust and
effective on multiple platforms. The skills and capabilities of the
technicians and quality control (QC) laboratory personnel (internal
or external) must also be considered. Most importantly, open effective communication between groups and clear, established protocols
are required, regardless of whether methods are being transferred
within the same organization or between contract manufacturers/
laboratories and biopharmaceutical companies.
Types of transfers
Analytical methods transfer exercises (AMTEs) occur throughout
the various phases of drug development and are almost unavoidable
at certain particular stages, such as when the process is transferred
from research to development for clinical trial material manufacturing and when scaling up for commercial production, according
to Roberto Rodriguez, an associate research fellow at Pfizer. Most
common are linear transfers (i.e., transferring from a laboratory that
supports early-stage drug development to one that supports latestage development) or for parallel use (i.e., multiple sites supporting
drug development).
Cynthia A. Challener is
a contributing editor to
Pharmaceutical Technology.
4
The most important aspect of analytical method transfer is ensuring that the methods are suitable for their intended purpose at the
various phases of the drug-development process and through the
product lifecycle, according to Mary Gasper, senior supervisor of an-
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
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Method Transfer
alytical development with SAFC. “In early stages,
that means using sound science in practice. Moving into phases I and II, it means looking ahead to
qualifying methods and preparing them for ICH
validation,” she explains.
Most methods begin as a
draft with loose specifications
that detail the general
expected results.
It is also important to recognize, Gasper notes,
that processes must deliver consistent and representative materials to move forward, and therefore,
methods cannot be locked down until the process
is locked down, which is a crucial factor for moving toward phase III and into commercialization.
“Our experience at Almac is that formal transfer
generally does not take place in the development
phase of a molecule, but only once the method
has been validated by the originating laboratory,”
agrees John Wood, analytical account manager
with Almac. At Pfizer, however, Rodriguez notes
that formal methods transfer is performed during
development, with methods validation appropriate
for the stage of development required.
The main goal of AMTEs, according to Rodriguez, is to achieve the same (or very similar)
method performance from one site to the other.
“Although it is tempting to look at AMTEs from
the technical standpoint only, performing an
assay in a second/new laboratory requires a lot
more than executing the method’s standard operating procedure (SOP). An extensive evaluation that includes stage of development (product
and method), method knowledge, type of method,
6
supporting systems (e.g., data collection, storage,
and retrieval), and cGMP requirements should
be performed prior to initiating an AMTE,” he
asserts. Once this information is collected from
the laboratories or sites involved, an analysis
should be performed with the goal of identifying the most appropriate strategy and uncovering
potential issues.
Transfer strategies
Most methods begin as a draft with loose specifications that detail the general expected results.
The transfer of these methods is a scientific exercise that gets stricter the further along in development, including more formal specifications and
limitations, according to Gasper. Rodriguez adds
that setting meaningful transfer acceptance criteria is easier in later stages of development when
the method has been optimized and there is less
variability. “The transferring strategy and requirements are dictated by the product stage of development, partly due to regulatory expectations, but
largely because method knowledge is much less
early in development than when fully validated,
and the success of the transfer depends on the
readiness of the method and all that is involved
on its execution,” he says.
Three of the most common strategies for method
transfer include transfer by scientific rationale,
transfer by performance, and transfer by co-validation, according to Rodriguez. AMTEs by “scientific
rationale” (i.e., paper exercises without wet analytical work) are used sparingly, because they are not
favored by regulatory agencies unless a strong justification is provided, and generally only when the
receiving lab has experience with a closely related
method used for the same or similar product.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
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Method Transfer
Transfer by performance requires a protocol
with AMTE acceptance criteria and execution of
the method in the receiving lab to generate data,
including repetition of some method validation
steps, such as precision and linearity that can be
compared to the transferring of laboratory data
for the same materials. “The second laboratory
must ensure that it can perform the analysis and
obtain the same results as the originating laboratory,” comments Wood. “The whole process should
be carried out according to a transfer protocol
generated by the method sending laboratory and
agreed by the receiving laboratory that establishes
the specific acceptance criteria for the transfer to
be successful.”
Transfer by co-validation commonly involves
the second laboratory in the execution of methods validation and demonstration that the pooled
data from both laboratories meet method validation acceptance criteria. This strategy would be appropriate if it is not possible to conduct comparative testing—such as when the sending laboratory
has previously outsourced the testing to another
laboratory and this laboratory is no longer in a position to carry out its part of the testing, according to Wood. AMTE by co-validation can be used
anytime the two exercises can be combined or are
close together, adds Rodriguez.
In all cases, the method-acceptance criteria should
be established before the drug is moved toward
phase III, so the focus can shift to validation. “It is
also important that the materials supply is locked in
and that the transfer methods are well documented.
QC methods need to have staying power for the life
of the drug. For instance, if the drug is produced
for 15 years, methodologies need to remain valid
through this life span,” Gasper asserts.
8
In addition, because most transfers (should) generate data, Rodriguez recommends the inclusion of
a statistician in the design and evaluation of the
transfer. He also notes that, just like method validation, AMTEs usually represent a “point in time”
or a “snapshot” that should be complemented with
continuous performance verification.
Need for cross-functionality
Once validated, a method should be effective for
any suitable instrument, although its performance
should be demonstrated as part of the method validation or during the transfer, according to Wood.
“Methods that can only be run on a single platform or using an instrument from a single vendor
are not robust,” states Gasper. Alternative materials are shown to be suitable when using various sources and lot numbers provides the same
(equivalent) results. Demonstrating effectives on
different instruments, however, may require more
extensive work, according to Rodriguez. “Some instrument differences are easier to overcome than
others. For example, for HPLC [high-performance
liquid chromatography], retention time bias due
to dead volume can be addressed using a correction factor or having specific criteria within the
method for each model or brand. Detector differences such as sensitivity, on the other hand, result
in discrepancies for some validation parameters
(e.g., linearity, quantitation limit, and detection
limit) that may lead to a preference for one instrument,” he says.
There has been some movement in the industry
towards the harmonization of platforms, according
to Gasper, but at this point she believes it is still
important to run methods on different platforms
to understand the implications.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
Communication and trust
A significant consideration, no matter the phase,
is the intended location of manufacturing for the
drug, and it makes a difference whether it will be
manufactured at an internal GMP lab, pilot plant,
CMO, or even an internal manufacturing facility,
according to Gasper. In general, she notes that internal transfers are often much smoother because
the platforms for manufacturing are already in
place. Regardless of location, the capabilities and
skills of the receiving lab must be assessed, and
the greatest element of success is communication.
“Open, honest, face-to-face (if possible) discussions about the project background, methods, and
process transfer are imperative in order for the receiving lab to be fully invested in the success of the
transfer,” she observes. Wood adds that the receiving laboratory should perform a familiarization
exercise prior to the actual transfer and have any
questions addressed prior to commencement of the
transfer. He also notes that many sending laboratories send an analyst familiar with the method to
the receiving lab to aid in the familiarization stage.
There are additional considerations that must be
taken into account when transferring to external
laboratories. Potential issues can be uncovered if
the needs for each transferred method are clearly
defined, according to Rodriguez, including not
only the equipment, materials, personnel training
and experience, but also the data management
systems, document requirements for quality assurance (QA) compliance, and lifecycle management agreements (e.g., change approval processes
to avoid “assay drift”).
Pfizer has learned that differences may arise due
to subtle general laboratory practices not addressed
by documents used during the AMTE. “The art of
writing an SOP is a fine balance between providing
enough (and accurate) information and writing a
protocol the length of a novel. Many details that
may affect assay performance are often left out
because the writer considers them to be ‘general
knowledge’ for someone skillful in the execution
of the test,” Rodriguez explains. In one example,
an approximate 5% bias between laboratories was
traced to the general practice of “blowing” the
sample off the pipette tip used to dispense the sample in one laboratory but not the other. “This issue
demonstrates how an AMTE can be complicated
or derailed by the smallest details,” he comments.
In addition, when transferring to external laboratories, the transfer plan should include agreement to a strategy and timing that satisfies requirements at both companies. “AMTEs are by
no means standardized across the industry, and
contract laboratories may have internal requirements that are different than those of the transferring company. The challenge is to identify the best
approach to fill those gaps,” he says.
In fact, successful external method transfers
require a great deal of trust, according to Gasper.
CMOs need details about the full R&D data package to understand the chemistry or biology of the
drug product, but there can be hesitance to provide this information from an intellectual property
(IP) standpoint, she notes. “Even with the support
of nondisclosure agreements, drug manufacturers may be hesitant to share their IP. This challenge must be overcome so that the receiving lab
procures sufficient background on the drug and
critical information for the transfer. A template
for transfer can act as a guide, and it is additionally helpful for all methods to be qualified prior to
going into the relationship,” says Gasper.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
9
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The industry has reacted to these needs with
the development of more strategic alliances and
partnerships between companies to maximize
specific technical and functional areas of research, Gasper observes. “Collaboration allows
companies to focus on what they are good at,
which creates ample flexibility and cost savings.
There is also the benefit of already knowing that
the GMP and quality processes are in place, so
there can be faster turnaround,” she says.
Added complexity for biopharmaceuticals
In principle, there should be little or no difference in the transfer processes for small molecules
and biologics, according to Wood. “The goal for
both is to demonstrate that the methods can be
operated successfully and reliably in the second
laboratory,” he says. Biologics are, however, more
complex than small-molecule drugs, and there
is generally less historical data available. As a result, it is more difficult to understand the downstream fates of biotherapeutics and the impact
of impurities, according to Gasper. In addition,
she notes that biopharmaceutical production
processes are much more sensitive to processing changes and parameter variability, and there
is also often greater variability in raw materials
from lot-to-lot. “As a result, more sophisticated
analytical tools and resources are required to
characterize the science,” she notes. Wood agrees
that the acceptance criteria for method transfer
for biopharmaceuticals may be looser, but they
still must reflect the specification for the product
and the capabilities of the method. It may also be
necessary to take extra steps to ensure that the
receiving laboratory has the knowledge required
to run the methods reliably. PT
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
Analytical Quality by Design
AQbD Adds Rigor to
Method Development
Paul Kippax
MARCELLO BORTOLINO/GETTY IMAGES
Analytical procedures
and method validation
should be developed with
a structured and rigorous
approach. Analytical quality
by design (AQbD) is a means
of ensuring that rigor.
Paul Kippax is product group
manager, Malvern Instruments.
Q
uality by design (QbD) itself is not a regulatory requirement; however, the approach has now been
widely adopted and is encouraged by FDA. This
is particularly true within the generic drug sector,
since regulatory bodies, most especially FDA, have indicated
that they would look more favorably upon abbreviated new drug
application (ANDA) submissions that demonstrate adherence
to QbD principles. This regulatory landscape is promoting increasing acceptance of QbD and encouraging its application to
other activities. Analytical method development is high on the
list of potential beneficiaries.
Like QbD, analytical quality by design (AQbD) is not a regulatory
requirement, but there are strong motivating factors for its adoption
including commentary by FDA (1). AQbD extends the knowledgeled approach promoted by QbD to the development of robust analytical methodologies and similarly holds out the prize of improved
flexibility and control. This prize is achieved through a process of
systematic risk assessment and the implementation of appropriate
controls for all critical aspects of an analytical method. The application of AQbD principles ensures that an analytical method will consistently deliver accurate and precise data throughout the lifecycle of
a pharmaceutical product. It also brings benefits such as flexibility
within the defined design space and in-depth assessment of the impact that various analytical parameters have on the validity of the
results. Together, these benefits assist the ability of the method to
continue to perform through production scale ups and the resulting
method transfers between different laboratories at the same site or
between different sites. In addition to this, it also aids day-to-day
troubleshooting and any out-of-specification investigations.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
11
Analytical Quality by Design
A CQA is required to
be measured and controlled by the application of an appropriate
analytical method. For
instance, a CQA that influences dissolution rate
of an API is its particle
size distribution. Controlling particle size to
deliver the QTPP relies
on employing a suitable
analy tical technique.
This is where AQbD
comes in. AQbD begins
with the identification
of an analytical target
profile (ATP)—a definition of what the method
is required to do—such
as measuring API particle size in a way that is
meaningful to control of
dissolution and delivers a certain level of reproducibility and accuracy. A detailed consideration
of both the ATP and the range of analytical techniques available to measure the targeted CQA—
along with their basic principles, limitations, and
any potential sources of errors associated with delivering the required data—forms an integral part
of the AQbD process.
Once an analytical technique has been chosen,
AQbD follows the same process as QbD: identification of the critical method attributes that impact
the results generated by the analysis; systematic
assessment of any associated risks and variability;
and implementation of a system of control. The
AQbD and QbD processes are intrinsically linked,
and their respective workflows parallel one another
(Figure 1). The conventional QbD workflow begins
with the identification of performance targets that
define how a product will deliver the required
clinical efficacy, the quality target product profile
(QTPP). The QTTP usually relates to a defined
pharmacological or physical feature, such as the
dissolution profile for an oral solid dosage form.
The variables that must be controlled to deliver the
QTTP are then identified as critical quality attributes (CQAs). Subsequent steps involve identifying
the best way to implement control over these CQAs
which directly affect product performance.
12
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
ALL FIGURES ARE COURTESY OF THE AUTHOR.
Figure 1: The quality by design (left) and analytical quality by design workflows (right) are
analogous and share the strategy of controlling risk by rigorously understanding the potential
impact of all sources of variability.
systematic study of risk factors may be supported by design of experiments (DOE) or
multi-variate analysis (MVA)
tools and leads to the scoping of the design space or
method operable design region (MODR) for the analytical method. This is the operating area within which the
ATP is consistently met. As
with QbD, the entire AQbD
workflow is held within a system of lifecycle management,
ensuring a process of continuous improvement.
Applying AQbD to
particle size analysis
Figure 2: A pressure titration with a high energy disperser suggests that stable particle
size measurement is achieved at pressures in excess of 2.5 bar.
Figure 3: A pressure titration for the same material as in Figure 2, carried out using a less
energetic disperser, reveals a broader operating range for robust measurement.
Rather than identifying a
single set of measurement parameters to produce analytical data, an AQbD approach
involves the development of a
comprehensive understanding of how all influential factors impact the output from
an analytical method. The
AQbD approach would therefore prompt a more extensive
experimental program during
method development than the
conventional approach.
Laser diffraction is a method of choice for particle size measurement for many pharmaceutical
products and can, therefore, be helpful in providing some insight into the practicalities of the
AQbD approach to method development. Potential
sources of error in laser diffraction particle size
measurements can be classified as relating to instrumentation, sampling, and dispersion.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
13
Analytical Quality by Design
Laser diffraction is a mature technology and
instrument design has been refined to a high
degree of automation and accuracy. The errors
associated with instrumentation, therefore, tend
to be small, even at the extremes of the measurement range of the technique, which runs from
0.01-3500 µm.
Sampling is potentially a more
significant source of error,
especially for larger particles.
Sampling is potentially a more significant
source of error, especially for larger particles.
Sample size often has to be increased when measuring larger particles to ensure that a sufficient
number of particles are measured to achieve the
required accuracy.
Conversely, dispersion tends to be a greater
source of error for finer particles. Appropriate
dispersion ahead of measurement underpins the
generation of particle size distribution data for
primary particles in the sample, rather than any
aggregates present. The strength of inter-particle
forces increases with decreasing particle size,
making complete dispersion more difficult for
fine particles.
The two most commonly used techniques for
dispersing a sample prior to a laser diffraction
measurement are to either disperse the sample as
a dry powder or to disperse it within an appropriate liquid. In case of dry dispersion, the sample
is entrained in a pressurized air flow. Parameters
that need to be considered in controlling the state
of dispersion for dry powders include selection
of the dispersion air pressure and selection of an
14
appropriate disperser geometry. Higher air pressures tend to lead to more energy being available for dispersion, but also increase the risk of
particle damage occurring during the dispersion
process. The risk of particle damage can be mitigated by lowering the air pressure or by selecting
a different disperser geometry.
Figure 2 shows data from a dry powder dispersion pressure titration, in this case using a
disperser geometry design to deliver a high dispersion energy. A pressure titration is a plot of
measured particle size as a function of the pressure of air flow and therefore tracks the effectiveness of dispersion as energy input is increased.
This plot shows that stable particle size measurement is achieved at a pressure in excess of
2.5 bar, suggesting that these conditions may be
appropriate for analysis. However, comparison
with an orthogonal method (wet sample dispersion) indicates that realistic particle size data are
reported at a dispersion pressure of just 0.2 bar.
This suggests that application of a high pressure
causes milling of the sample. A possible decision
from these experiments would be to measure the
sample at a dispersion pressure of 0.2 bar and a
conventional approach would incorporate this in
a standard operating procedure (SOP).
An AQbD approach would, in contrast, focus
attention on the fact that at 0.2 bar the gradient of
this plot is noticeably steep, suggesting that with
this set-up the operating range, the MODR, is very
narrow. This in turn indicates that the method is
unlikely to be inherently robust; it is associated
with an intrinsically high level of risk.
Figure 3 shows a pressure titration using a
disperser geometry designed to deliver a lower
dispersion energy, and exemplifies the type of
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
extended investigation
Figure 4: The Mastersizer 3000 SOP player function allowed the user to build measurement
sequences to efficiently test the impact of parameters such as stirrer speed using existing SOPs.
prompted by a more
r i g o r o u s AQ bD a p proach. Here robust results are reported over a
relatively wide pressure
range, between 0–1.0
bar. The less energetic
disperser design is associated with a wider
MODR and a reduced
requirement to closely
control pressure. Here
then, a broader experimental program, coupled with some intelligent interpretation of
the experimental data,
delivers a fundamentally more robust method that is more likely to
Figure 4 shows an SOP sequence for stirrer speed
perform well over the long term. This is the exact on the Mastersizer 3000 laser diffraction analyzer
intention of AQbD.
(Malvern Instruments) software, which could be
used to efficiently conduct a stirrer speed titration
Analytical instrumentation and AQbD
as part of scoping the MODR for a laser diffracInstrument makers are increasingly aware of tion measurement based on wet dispersion. Such
the benefits of AQbD for the pharmaceutical sequences, which are easily saved and recalled,
industry and the need to lighten the associated are also useful during validation.
analytical workload through innovation. Recent
Other instrument features enable real-time asadvances in instrumentation and software help sessment of the impact of data analysis factors on
reduce some of the workload required to stream- analytical output. For example, the optical propline the implementation of AQbD. For example, erty optimizer (OPO) for the Mastersizer 3000
certain analytical instruments have the function- enables analysts to explore the impact of optical
ality to automatically run through a sequence of properties on reported particle size. In combinaoperating conditions. Such functionality allows tion with the SOP player, this functionality helps
analysts to build measurement sequences to fa- users to efficiently implement a DOE approach
cilitate rapid experimentation and automate both to comprehensively understand all aspects of the
method development and validation.
analysis.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
15
Analytical Quality by Design
Modern instruments also offer tools that provide
feedback on the quality of data being generated, enabling researchers to critically assess measurement
data and results during an analysis. These features
can provide advice relating to the measurement
process, helping to address the issue of control,
which is such an important aspect of AQbD. Software for automated data review is also available to
similarly provide verification that methods have
been used correctly, by enabling the systematic
cross-comparison of parameters and results.
Lifecycle management of analytical procedures
In going beyond simple SOP definition to create an
MODR, AQbD facilitates a responsive approach to the
variability encountered in day-to-day analysis. The
greater control and flexibility this provides ensures
that analytical methods remain robust, reliable, and
relevant throughout the lifetime of the product. AQbD
also has the potential to reduce the risks involved in
analytical method transfer, from the laboratory or
pilot scale to commercial production. The root cause
of failure of method transfer usually stems from insufficient consideration of the operating environment
and a failure to capture and transfer the information
needed to deliver robust measurement. The periodic
assessment of the method’s performance coupled with
in-depth understanding of the MODR forms the basis
of continuous improvement of an analytical method
throughout the lifecycle of a pharmaceutical product.
References
1. S. Chatterjee, “QbD Considerations for Analytical Methods—FDA Perspective,” IFPAC Annual
Meeting (Baltimore, MD, Jan. 25, 2013). PT
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Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
e2
Viral Contamination
The Challenge of
Finding the Unknown
Cynthia A. Challener
Advanced analytical
methods are speeding up
the targeted evaluation of
potential viral contaminants.
U
nlike with blood and plasma for which possible viral contaminants are generally known, because the raw materials used in the manufacture of biologic drugs can come
from animal, plant, and manmade sources, it is not possible to predict all potential viral contaminants. Analytical methods,
therefore, must be able to detect both known and unknown viruses.
Traditional cell-based assays are effective but have limitations, including an extensive assay procedure time. Newer nucleic acid-based
methods are much more rapid, but those implemented to date are
generally designed for specific viral targets. Both biopharmaceutical
companies and FDA, however, believe that new sequencing techniques capable of identifying multiple viruses can be adopted by the
industry in the future.
GIPHOTOSTOCK/CULTURA/GETTY IMAGES
No safety concerns
Cynthia A. Challener is
a contributing editor to
Pharmaceutical Technology.
First, it must be stressed that the interest in new viral detection
methods does not stem from any issues or problem cases related
to the safety of biotherapeutics. “Biotechnology drugs are very safe
from a viral contamination standpoint. The international standard
ICH Q5A, which was promulgated in 1998, assures viral safety by
requiring both testing of cell banks, raw materials, and bioreactor
harvests and viral clearance using downstream purification processes,” says Kurt Brorson, a staff scientist for monoclonal antibodies
with the Center for Drug Evaluation and Research (CDER) at FDA.
Regardless of the analytical method, adds Ivar Kljavin, director of
adventitious agent management with Genentech, because testing is
trying to not only detect unknown viruses, but also do the impossible and prove a negative result, it is not sufficient by itself. “It is
very important to realize that testing is crucial but only one part of
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
17
Viral Contamination
an effective solution for viral contamination prevention. It is absolutely imperative that steps be
taken to minimize the risk of viral infection, such
as heat treatment, viral filtration, and the tracking
and tracing of all raw materials to their original
sources in order to be able to identify potential
risks,” he asserts.
Cell-based assays are
effective when performed in
conjunction with other riskmitigating steps like viral
clearance validation.
Current methods effective but with limitations
The traditional test method is the in-vitro adventitious virus assay, which uses various indicator
cell lines for detection of viruses for in-process or
lot-release testing of the bioreactor at the end of
the run, according to Dominick Vacante, scientific
director for virology with Janssen Pharmaceutical
R&D. “The assay is very sensitive and can potentially detect one infectious virus particle. The limitation is that the virus must replicate in at least one
of the indicator cell types and produce some type
of effect that is detected by the readouts of the assays, which are visual for cytopathic effects and/or
hemadsorption or hemagglutination of specific red
blood cells,” he explains. Problems do arise when
a virus replicates but causes no signs of cytopathic
effects, or is infectious but does not replicate in
the cell types selected for the assay, which in both
cases yields a false-negative result.
In some cases, the test article may interfere with
the ability of a virus to infect the cells or display
signs of infection, according to Kljavin. Cell-based
18
methods can also be variable; a virus that is detected in one assay may not be found when the
test is repeated. “Equally importantly, it takes time
for the viruses to replicate and grow, which leads
to a very long test time of 14–28 days. A significant amount of product can be produced and sent
downstream during that time, and if found to be
infected must be disposed. The entire production
facility, not just cell-culture areas, must be decontaminated, which can lead to a disruption in the
supply of the drug to patients, a situation that is
unacceptable,” Kljavin states.
He again stresses, though, that cell-based assays
are effective when performed in conjunction with
other risk-mitigating steps like viral clearance validation. The current issues revolve around development of new assays to test for viruses at the various
sampling points that are more rapid, more sensitive, and broader. Gaining a greater understanding
of how purification unit operations clear viruses,
how robust they are, and how to best validate the
clearance of different viruses are other measures
that mitigate gaps in testing, according to Brorson.
Evolving technology
While ICH Q5A serves the industry and regulators
well, Brorson notes that technology has evolved
since 1998, leading to the development of different approaches, such as modular validation for robust unit operations and the introduction of new
assays for cell-line characterization, like genome
sequencing-based methods. “ICH Q5A is silent on
these new technologies since it was written before
their introduction, and could stand to be updated
with developments of the past 15 years,” he says.
FDA has been following new pan virus detection
methods/deep-sequencing methods for cell-line
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
characterization, which are at the current time,
according to Brorson, great for investigations and
broad surveys of cell lines or raw materials. “What
isn’t clear yet, though, is whether they have reached
a state where they can be used in a routine QC
setting.” FDA expects, however, that the situation
could change over the next dozen years or so.
Massive parallel sequencing
is attracting a lot of
interest because it can
be used to detect multiple
DNA sequences from
different viruses.
Nucleic acid-based methods
More simple and specific polymerase chain reaction (PCR) methods are currently used by many
biopharmaceutical manufacturers. Each PCR test
accurately detects a DNA sequence from a specific
virus and is completed rapidly compared to cellbased assays (e.g., within one day vs. three to four
weeks). Presently, however, PCR is not applicable
for the detection of multiple viruses at once. Massive parallel sequencing, or deep sequencing, on
the other hand, is attracting a lot of interest because it can be used to detect multiple DNA sequences from different viruses.
In addition to deep sequencing, PCR combined
with mass spectrometry (MS) and microarrays are
considered advanced technologies for virus detection. PCR with degenerate probes may also be included as an advancement of PCR, according to
Vacante. “These technologies may improve virus
detection by enabling the detection of a broad
range of viruses, either in an unbiased manner as
with deep sequencing or through the detection of
consensus sequences of many or all viruses in a
virus family. The assays also by their nature provide information on the virus detected and taxonomy, including the virus family, subfamily, and
genus, which may be helpful for quickly determining where a contamination may have originated,”
Vacante observes.
It is important to remember, however, that with
these methods, only viral DNA sequences are detected, and not actual live particles, according to
Kljavin. “These tests do not indicate if any live
virus is present, only that a part of the DNA of
the virus is present. The challenge then becomes
how to respond if a positive result is obtained,” he
explains. It is necessary to work backward and run
other tests to determine if there is an actual infection. “If no infection is found, then a decision must
be made regarding how to proceed. Both industry
and FDA will have to figure out how to go forward
in such a situation,” Kljavin adds.
Real advantages
Both Genentech and Janssen have employed these
nucleic-acid techniques when dealing with possible
viral contamination. In 1993 and 1994, Genentech
experienced contamination by the rodent parvovirus, or minute virus of mice (MVM). In 1993, the
infection was detected using cell-based assays, but
only after production continued for three to four
weeks, thus the facility was contaminated and significant clean-up and production delays resulted.
By 1994, a PCR method was developed, and the
virus was detected before harvest from the bioreactor. “In this latter case, production was halted
before further downstream processing progressed,
and decontamination efforts were limited to the
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
19
Viral Contamination
impacted bioreactor, and other normal operations
in the facility continued,” notes Kljavin.
A few years ago at Janssen, in response to a suspected viral contamination, deep sequencing, and
DNA amplification followed by MS, previously
referred to as TIGER and now termed PLEX-ID,
was used to show that there was no contamination, according to Vacante. “Analysis of the effect
observed in cell culture and extensive screening
using the advanced tests provided convincing evi-
CDER has been involved with
industry and other players
in directed research on
standardization and improved
methods for validating viral
clearance unit operations.
dence for the lack of a viral contamination or other
biological agent,” Vacante adds. Using established
standard operating procedures, the impacted lots
were then retested and found to be actually negative. The initial test result was then assigned “falsepositive status,” and the product was ultimately
released to the market (1).
says. He also notes that a PDA interest group was
formed to exchange ideas and practices, evaluate
these technologies, and possibly develop best practices and standards or standardized approaches for
comparison of methods across different laboratories. FDA has been involved with both the white
paper and the interest group, and Arifa Khan at
the Center for Biologics Evaluation and Research
has provided leadership regarding the evaluation
of these new technologies for virus detection, according to Vacante.
CDER has also been involved with industry and
other players in directed research on standardization and improved methods for validating viral
clearance unit operations. “For example, CDER
performed crucial lab work that contributed to
the development of the first ever standard nomenclature for virus retentive filters promulgated in
PDA’s TR41, Virus Retentive Filters. We also laid
part of the groundwork for ASTM E2888 Standard
Practice for Process for Inactivation of Rodent Retrovirus by pH, and participated in the committee
writing the standard. A challenge for industry is to
implement these new standards, assays, and validation approaches,” observes Brorson.
Reference
Industry and agency activities
To help evaluate the current status of analytical
technology with this goal in mind, a task force of
members of the Parenteral Drug Association (PDA)
that includes industry, government, and academia
are preparing a white paper on emerging methods
for virus detection. “This white paper describes
both convention
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ANALYTICAL AND BIOANALYTICAL TESTING 2014
METHOD TRANSFER
4 Keys to Successful Method Transfer
EDITORIAL
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and Cynthia A. Challener, PhD [email protected]
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Cynthia A. Challener
ANALYTICAL QUALITY BY DESIGN
11 AQbD Adds Rigor to Method Development
Paul Kippax
VIRAL CONTAMINATION
17 The Challenge of Finding the Unknown
Cynthia A. Challener
ELEMENTAL IMPURITIES
21 Rapid Screening for
Elemental Impurities using ICP-MS
Jonathan L. Sims and Fadi Abou-Shakra
ASSAY VALIDATION
26 Validation of a Multiplex
Bead-Based Assay
Rabia Hidi, Catherine Diot, Sebastien Melin,
Jiyhe Jang-Lee, and Alain Renoux
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DATA MANAGEMENT
29 Standardizing Data Management
James M. Vergis and Dana E. Vanderwall
NEW TECHNOLOGY
31 Advances in Analytical Technology
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32 Ad Index
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Method Transfer
Keys to Successful
Method Transfer
Cynthia A. Challener
Methods must be suitable
at each development
phase, robust, and effective
on multiple platforms.
M
ethod transfer during scale-up of the biopharmaceutical manufacturing process can be challenging.
Not only must methods be suitable at each phase of
the development process, they must be robust and
effective on multiple platforms. The skills and capabilities of the
technicians and quality control (QC) laboratory personnel (internal
or external) must also be considered. Most importantly, open effective communication between groups and clear, established protocols
are required, regardless of whether methods are being transferred
within the same organization or between contract manufacturers/
laboratories and biopharmaceutical companies.
Types of transfers
Analytical methods transfer exercises (AMTEs) occur throughout
the various phases of drug development and are almost unavoidable
at certain particular stages, such as when the process is transferred
from research to development for clinical trial material manufacturing and when scaling up for commercial production, according
to Roberto Rodriguez, an associate research fellow at Pfizer. Most
common are linear transfers (i.e., transferring from a laboratory that
supports early-stage drug development to one that supports latestage development) or for parallel use (i.e., multiple sites supporting
drug development).
Cynthia A. Challener is
a contributing editor to
Pharmaceutical Technology.
4
The most important aspect of analytical method transfer is ensuring that the methods are suitable for their intended purpose at the
various phases of the drug-development process and through the
product lifecycle, according to Mary Gasper, senior supervisor of an-
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
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Method Transfer
alytical development with SAFC. “In early stages,
that means using sound science in practice. Moving into phases I and II, it means looking ahead to
qualifying methods and preparing them for ICH
validation,” she explains.
Most methods begin as a
draft with loose specifications
that detail the general
expected results.
It is also important to recognize, Gasper notes,
that processes must deliver consistent and representative materials to move forward, and therefore,
methods cannot be locked down until the process
is locked down, which is a crucial factor for moving toward phase III and into commercialization.
“Our experience at Almac is that formal transfer
generally does not take place in the development
phase of a molecule, but only once the method
has been validated by the originating laboratory,”
agrees John Wood, analytical account manager
with Almac. At Pfizer, however, Rodriguez notes
that formal methods transfer is performed during
development, with methods validation appropriate
for the stage of development required.
The main goal of AMTEs, according to Rodriguez, is to achieve the same (or very similar)
method performance from one site to the other.
“Although it is tempting to look at AMTEs from
the technical standpoint only, performing an
assay in a second/new laboratory requires a lot
more than executing the method’s standard operating procedure (SOP). An extensive evaluation that includes stage of development (product
and method), method knowledge, type of method,
6
supporting systems (e.g., data collection, storage,
and retrieval), and cGMP requirements should
be performed prior to initiating an AMTE,” he
asserts. Once this information is collected from
the laboratories or sites involved, an analysis
should be performed with the goal of identifying the most appropriate strategy and uncovering
potential issues.
Transfer strategies
Most methods begin as a draft with loose specifications that detail the general expected results.
The transfer of these methods is a scientific exercise that gets stricter the further along in development, including more formal specifications and
limitations, according to Gasper. Rodriguez adds
that setting meaningful transfer acceptance criteria is easier in later stages of development when
the method has been optimized and there is less
variability. “The transferring strategy and requirements are dictated by the product stage of development, partly due to regulatory expectations, but
largely because method knowledge is much less
early in development than when fully validated,
and the success of the transfer depends on the
readiness of the method and all that is involved
on its execution,” he says.
Three of the most common strategies for method
transfer include transfer by scientific rationale,
transfer by performance, and transfer by co-validation, according to Rodriguez. AMTEs by “scientific
rationale” (i.e., paper exercises without wet analytical work) are used sparingly, because they are not
favored by regulatory agencies unless a strong justification is provided, and generally only when the
receiving lab has experience with a closely related
method used for the same or similar product.
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Method Transfer
Transfer by performance requires a protocol
with AMTE acceptance criteria and execution of
the method in the receiving lab to generate data,
including repetition of some method validation
steps, such as precision and linearity that can be
compared to the transferring of laboratory data
for the same materials. “The second laboratory
must ensure that it can perform the analysis and
obtain the same results as the originating laboratory,” comments Wood. “The whole process should
be carried out according to a transfer protocol
generated by the method sending laboratory and
agreed by the receiving laboratory that establishes
the specific acceptance criteria for the transfer to
be successful.”
Transfer by co-validation commonly involves
the second laboratory in the execution of methods validation and demonstration that the pooled
data from both laboratories meet method validation acceptance criteria. This strategy would be appropriate if it is not possible to conduct comparative testing—such as when the sending laboratory
has previously outsourced the testing to another
laboratory and this laboratory is no longer in a position to carry out its part of the testing, according to Wood. AMTE by co-validation can be used
anytime the two exercises can be combined or are
close together, adds Rodriguez.
In all cases, the method-acceptance criteria should
be established before the drug is moved toward
phase III, so the focus can shift to validation. “It is
also important that the materials supply is locked in
and that the transfer methods are well documented.
QC methods need to have staying power for the life
of the drug. For instance, if the drug is produced
for 15 years, methodologies need to remain valid
through this life span,” Gasper asserts.
8
In addition, because most transfers (should) generate data, Rodriguez recommends the inclusion of
a statistician in the design and evaluation of the
transfer. He also notes that, just like method validation, AMTEs usually represent a “point in time”
or a “snapshot” that should be complemented with
continuous performance verification.
Need for cross-functionality
Once validated, a method should be effective for
any suitable instrument, although its performance
should be demonstrated as part of the method validation or during the transfer, according to Wood.
“Methods that can only be run on a single platform or using an instrument from a single vendor
are not robust,” states Gasper. Alternative materials are shown to be suitable when using various sources and lot numbers provides the same
(equivalent) results. Demonstrating effectives on
different instruments, however, may require more
extensive work, according to Rodriguez. “Some instrument differences are easier to overcome than
others. For example, for HPLC [high-performance
liquid chromatography], retention time bias due
to dead volume can be addressed using a correction factor or having specific criteria within the
method for each model or brand. Detector differences such as sensitivity, on the other hand, result
in discrepancies for some validation parameters
(e.g., linearity, quantitation limit, and detection
limit) that may lead to a preference for one instrument,” he says.
There has been some movement in the industry
towards the harmonization of platforms, according
to Gasper, but at this point she believes it is still
important to run methods on different platforms
to understand the implications.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
Communication and trust
A significant consideration, no matter the phase,
is the intended location of manufacturing for the
drug, and it makes a difference whether it will be
manufactured at an internal GMP lab, pilot plant,
CMO, or even an internal manufacturing facility,
according to Gasper. In general, she notes that internal transfers are often much smoother because
the platforms for manufacturing are already in
place. Regardless of location, the capabilities and
skills of the receiving lab must be assessed, and
the greatest element of success is communication.
“Open, honest, face-to-face (if possible) discussions about the project background, methods, and
process transfer are imperative in order for the receiving lab to be fully invested in the success of the
transfer,” she observes. Wood adds that the receiving laboratory should perform a familiarization
exercise prior to the actual transfer and have any
questions addressed prior to commencement of the
transfer. He also notes that many sending laboratories send an analyst familiar with the method to
the receiving lab to aid in the familiarization stage.
There are additional considerations that must be
taken into account when transferring to external
laboratories. Potential issues can be uncovered if
the needs for each transferred method are clearly
defined, according to Rodriguez, including not
only the equipment, materials, personnel training
and experience, but also the data management
systems, document requirements for quality assurance (QA) compliance, and lifecycle management agreements (e.g., change approval processes
to avoid “assay drift”).
Pfizer has learned that differences may arise due
to subtle general laboratory practices not addressed
by documents used during the AMTE. “The art of
writing an SOP is a fine balance between providing
enough (and accurate) information and writing a
protocol the length of a novel. Many details that
may affect assay performance are often left out
because the writer considers them to be ‘general
knowledge’ for someone skillful in the execution
of the test,” Rodriguez explains. In one example,
an approximate 5% bias between laboratories was
traced to the general practice of “blowing” the
sample off the pipette tip used to dispense the sample in one laboratory but not the other. “This issue
demonstrates how an AMTE can be complicated
or derailed by the smallest details,” he comments.
In addition, when transferring to external laboratories, the transfer plan should include agreement to a strategy and timing that satisfies requirements at both companies. “AMTEs are by
no means standardized across the industry, and
contract laboratories may have internal requirements that are different than those of the transferring company. The challenge is to identify the best
approach to fill those gaps,” he says.
In fact, successful external method transfers
require a great deal of trust, according to Gasper.
CMOs need details about the full R&D data package to understand the chemistry or biology of the
drug product, but there can be hesitance to provide this information from an intellectual property
(IP) standpoint, she notes. “Even with the support
of nondisclosure agreements, drug manufacturers may be hesitant to share their IP. This challenge must be overcome so that the receiving lab
procures sufficient background on the drug and
critical information for the transfer. A template
for transfer can act as a guide, and it is additionally helpful for all methods to be qualified prior to
going into the relationship,” says Gasper.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
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The industry has reacted to these needs with
the development of more strategic alliances and
partnerships between companies to maximize
specific technical and functional areas of research, Gasper observes. “Collaboration allows
companies to focus on what they are good at,
which creates ample flexibility and cost savings.
There is also the benefit of already knowing that
the GMP and quality processes are in place, so
there can be faster turnaround,” she says.
Added complexity for biopharmaceuticals
In principle, there should be little or no difference in the transfer processes for small molecules
and biologics, according to Wood. “The goal for
both is to demonstrate that the methods can be
operated successfully and reliably in the second
laboratory,” he says. Biologics are, however, more
complex than small-molecule drugs, and there
is generally less historical data available. As a result, it is more difficult to understand the downstream fates of biotherapeutics and the impact
of impurities, according to Gasper. In addition,
she notes that biopharmaceutical production
processes are much more sensitive to processing changes and parameter variability, and there
is also often greater variability in raw materials
from lot-to-lot. “As a result, more sophisticated
analytical tools and resources are required to
characterize the science,” she notes. Wood agrees
that the acceptance criteria for method transfer
for biopharmaceuticals may be looser, but they
still must reflect the specification for the product
and the capabilities of the method. It may also be
necessary to take extra steps to ensure that the
receiving laboratory has the knowledge required
to run the methods reliably. PT
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
Analytical Quality by Design
AQbD Adds Rigor to
Method Development
Paul Kippax
MARCELLO BORTOLINO/GETTY IMAGES
Analytical procedures
and method validation
should be developed with
a structured and rigorous
approach. Analytical quality
by design (AQbD) is a means
of ensuring that rigor.
Paul Kippax is product group
manager, Malvern Instruments.
Q
uality by design (QbD) itself is not a regulatory requirement; however, the approach has now been
widely adopted and is encouraged by FDA. This
is particularly true within the generic drug sector,
since regulatory bodies, most especially FDA, have indicated
that they would look more favorably upon abbreviated new drug
application (ANDA) submissions that demonstrate adherence
to QbD principles. This regulatory landscape is promoting increasing acceptance of QbD and encouraging its application to
other activities. Analytical method development is high on the
list of potential beneficiaries.
Like QbD, analytical quality by design (AQbD) is not a regulatory
requirement, but there are strong motivating factors for its adoption
including commentary by FDA (1). AQbD extends the knowledgeled approach promoted by QbD to the development of robust analytical methodologies and similarly holds out the prize of improved
flexibility and control. This prize is achieved through a process of
systematic risk assessment and the implementation of appropriate
controls for all critical aspects of an analytical method. The application of AQbD principles ensures that an analytical method will consistently deliver accurate and precise data throughout the lifecycle of
a pharmaceutical product. It also brings benefits such as flexibility
within the defined design space and in-depth assessment of the impact that various analytical parameters have on the validity of the
results. Together, these benefits assist the ability of the method to
continue to perform through production scale ups and the resulting
method transfers between different laboratories at the same site or
between different sites. In addition to this, it also aids day-to-day
troubleshooting and any out-of-specification investigations.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
11
Analytical Quality by Design
A CQA is required to
be measured and controlled by the application of an appropriate
analytical method. For
instance, a CQA that influences dissolution rate
of an API is its particle
size distribution. Controlling particle size to
deliver the QTPP relies
on employing a suitable
analy tical technique.
This is where AQbD
comes in. AQbD begins
with the identification
of an analytical target
profile (ATP)—a definition of what the method
is required to do—such
as measuring API particle size in a way that is
meaningful to control of
dissolution and delivers a certain level of reproducibility and accuracy. A detailed consideration
of both the ATP and the range of analytical techniques available to measure the targeted CQA—
along with their basic principles, limitations, and
any potential sources of errors associated with delivering the required data—forms an integral part
of the AQbD process.
Once an analytical technique has been chosen,
AQbD follows the same process as QbD: identification of the critical method attributes that impact
the results generated by the analysis; systematic
assessment of any associated risks and variability;
and implementation of a system of control. The
AQbD and QbD processes are intrinsically linked,
and their respective workflows parallel one another
(Figure 1). The conventional QbD workflow begins
with the identification of performance targets that
define how a product will deliver the required
clinical efficacy, the quality target product profile
(QTPP). The QTTP usually relates to a defined
pharmacological or physical feature, such as the
dissolution profile for an oral solid dosage form.
The variables that must be controlled to deliver the
QTTP are then identified as critical quality attributes (CQAs). Subsequent steps involve identifying
the best way to implement control over these CQAs
which directly affect product performance.
12
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
ALL FIGURES ARE COURTESY OF THE AUTHOR.
Figure 1: The quality by design (left) and analytical quality by design workflows (right) are
analogous and share the strategy of controlling risk by rigorously understanding the potential
impact of all sources of variability.
systematic study of risk factors may be supported by design of experiments (DOE) or
multi-variate analysis (MVA)
tools and leads to the scoping of the design space or
method operable design region (MODR) for the analytical method. This is the operating area within which the
ATP is consistently met. As
with QbD, the entire AQbD
workflow is held within a system of lifecycle management,
ensuring a process of continuous improvement.
Applying AQbD to
particle size analysis
Figure 2: A pressure titration with a high energy disperser suggests that stable particle
size measurement is achieved at pressures in excess of 2.5 bar.
Figure 3: A pressure titration for the same material as in Figure 2, carried out using a less
energetic disperser, reveals a broader operating range for robust measurement.
Rather than identifying a
single set of measurement parameters to produce analytical data, an AQbD approach
involves the development of a
comprehensive understanding of how all influential factors impact the output from
an analytical method. The
AQbD approach would therefore prompt a more extensive
experimental program during
method development than the
conventional approach.
Laser diffraction is a method of choice for particle size measurement for many pharmaceutical
products and can, therefore, be helpful in providing some insight into the practicalities of the
AQbD approach to method development. Potential
sources of error in laser diffraction particle size
measurements can be classified as relating to instrumentation, sampling, and dispersion.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
13
Analytical Quality by Design
Laser diffraction is a mature technology and
instrument design has been refined to a high
degree of automation and accuracy. The errors
associated with instrumentation, therefore, tend
to be small, even at the extremes of the measurement range of the technique, which runs from
0.01-3500 µm.
Sampling is potentially a more
significant source of error,
especially for larger particles.
Sampling is potentially a more significant
source of error, especially for larger particles.
Sample size often has to be increased when measuring larger particles to ensure that a sufficient
number of particles are measured to achieve the
required accuracy.
Conversely, dispersion tends to be a greater
source of error for finer particles. Appropriate
dispersion ahead of measurement underpins the
generation of particle size distribution data for
primary particles in the sample, rather than any
aggregates present. The strength of inter-particle
forces increases with decreasing particle size,
making complete dispersion more difficult for
fine particles.
The two most commonly used techniques for
dispersing a sample prior to a laser diffraction
measurement are to either disperse the sample as
a dry powder or to disperse it within an appropriate liquid. In case of dry dispersion, the sample
is entrained in a pressurized air flow. Parameters
that need to be considered in controlling the state
of dispersion for dry powders include selection
of the dispersion air pressure and selection of an
14
appropriate disperser geometry. Higher air pressures tend to lead to more energy being available for dispersion, but also increase the risk of
particle damage occurring during the dispersion
process. The risk of particle damage can be mitigated by lowering the air pressure or by selecting
a different disperser geometry.
Figure 2 shows data from a dry powder dispersion pressure titration, in this case using a
disperser geometry design to deliver a high dispersion energy. A pressure titration is a plot of
measured particle size as a function of the pressure of air flow and therefore tracks the effectiveness of dispersion as energy input is increased.
This plot shows that stable particle size measurement is achieved at a pressure in excess of
2.5 bar, suggesting that these conditions may be
appropriate for analysis. However, comparison
with an orthogonal method (wet sample dispersion) indicates that realistic particle size data are
reported at a dispersion pressure of just 0.2 bar.
This suggests that application of a high pressure
causes milling of the sample. A possible decision
from these experiments would be to measure the
sample at a dispersion pressure of 0.2 bar and a
conventional approach would incorporate this in
a standard operating procedure (SOP).
An AQbD approach would, in contrast, focus
attention on the fact that at 0.2 bar the gradient of
this plot is noticeably steep, suggesting that with
this set-up the operating range, the MODR, is very
narrow. This in turn indicates that the method is
unlikely to be inherently robust; it is associated
with an intrinsically high level of risk.
Figure 3 shows a pressure titration using a
disperser geometry designed to deliver a lower
dispersion energy, and exemplifies the type of
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
extended investigation
Figure 4: The Mastersizer 3000 SOP player function allowed the user to build measurement
sequences to efficiently test the impact of parameters such as stirrer speed using existing SOPs.
prompted by a more
r i g o r o u s AQ bD a p proach. Here robust results are reported over a
relatively wide pressure
range, between 0–1.0
bar. The less energetic
disperser design is associated with a wider
MODR and a reduced
requirement to closely
control pressure. Here
then, a broader experimental program, coupled with some intelligent interpretation of
the experimental data,
delivers a fundamentally more robust method that is more likely to
Figure 4 shows an SOP sequence for stirrer speed
perform well over the long term. This is the exact on the Mastersizer 3000 laser diffraction analyzer
intention of AQbD.
(Malvern Instruments) software, which could be
used to efficiently conduct a stirrer speed titration
Analytical instrumentation and AQbD
as part of scoping the MODR for a laser diffracInstrument makers are increasingly aware of tion measurement based on wet dispersion. Such
the benefits of AQbD for the pharmaceutical sequences, which are easily saved and recalled,
industry and the need to lighten the associated are also useful during validation.
analytical workload through innovation. Recent
Other instrument features enable real-time asadvances in instrumentation and software help sessment of the impact of data analysis factors on
reduce some of the workload required to stream- analytical output. For example, the optical propline the implementation of AQbD. For example, erty optimizer (OPO) for the Mastersizer 3000
certain analytical instruments have the function- enables analysts to explore the impact of optical
ality to automatically run through a sequence of properties on reported particle size. In combinaoperating conditions. Such functionality allows tion with the SOP player, this functionality helps
analysts to build measurement sequences to fa- users to efficiently implement a DOE approach
cilitate rapid experimentation and automate both to comprehensively understand all aspects of the
method development and validation.
analysis.
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
15
Analytical Quality by Design
Modern instruments also offer tools that provide
feedback on the quality of data being generated, enabling researchers to critically assess measurement
data and results during an analysis. These features
can provide advice relating to the measurement
process, helping to address the issue of control,
which is such an important aspect of AQbD. Software for automated data review is also available to
similarly provide verification that methods have
been used correctly, by enabling the systematic
cross-comparison of parameters and results.
Lifecycle management of analytical procedures
In going beyond simple SOP definition to create an
MODR, AQbD facilitates a responsive approach to the
variability encountered in day-to-day analysis. The
greater control and flexibility this provides ensures
that analytical methods remain robust, reliable, and
relevant throughout the lifetime of the product. AQbD
also has the potential to reduce the risks involved in
analytical method transfer, from the laboratory or
pilot scale to commercial production. The root cause
of failure of method transfer usually stems from insufficient consideration of the operating environment
and a failure to capture and transfer the information
needed to deliver robust measurement. The periodic
assessment of the method’s performance coupled with
in-depth understanding of the MODR forms the basis
of continuous improvement of an analytical method
throughout the lifecycle of a pharmaceutical product.
References
1. S. Chatterjee, “QbD Considerations for Analytical Methods—FDA Perspective,” IFPAC Annual
Meeting (Baltimore, MD, Jan. 25, 2013). PT
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Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
e2
Viral Contamination
The Challenge of
Finding the Unknown
Cynthia A. Challener
Advanced analytical
methods are speeding up
the targeted evaluation of
potential viral contaminants.
U
nlike with blood and plasma for which possible viral contaminants are generally known, because the raw materials used in the manufacture of biologic drugs can come
from animal, plant, and manmade sources, it is not possible to predict all potential viral contaminants. Analytical methods,
therefore, must be able to detect both known and unknown viruses.
Traditional cell-based assays are effective but have limitations, including an extensive assay procedure time. Newer nucleic acid-based
methods are much more rapid, but those implemented to date are
generally designed for specific viral targets. Both biopharmaceutical
companies and FDA, however, believe that new sequencing techniques capable of identifying multiple viruses can be adopted by the
industry in the future.
GIPHOTOSTOCK/CULTURA/GETTY IMAGES
No safety concerns
Cynthia A. Challener is
a contributing editor to
Pharmaceutical Technology.
First, it must be stressed that the interest in new viral detection
methods does not stem from any issues or problem cases related
to the safety of biotherapeutics. “Biotechnology drugs are very safe
from a viral contamination standpoint. The international standard
ICH Q5A, which was promulgated in 1998, assures viral safety by
requiring both testing of cell banks, raw materials, and bioreactor
harvests and viral clearance using downstream purification processes,” says Kurt Brorson, a staff scientist for monoclonal antibodies
with the Center for Drug Evaluation and Research (CDER) at FDA.
Regardless of the analytical method, adds Ivar Kljavin, director of
adventitious agent management with Genentech, because testing is
trying to not only detect unknown viruses, but also do the impossible and prove a negative result, it is not sufficient by itself. “It is
very important to realize that testing is crucial but only one part of
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
17
Viral Contamination
an effective solution for viral contamination prevention. It is absolutely imperative that steps be
taken to minimize the risk of viral infection, such
as heat treatment, viral filtration, and the tracking
and tracing of all raw materials to their original
sources in order to be able to identify potential
risks,” he asserts.
Cell-based assays are
effective when performed in
conjunction with other riskmitigating steps like viral
clearance validation.
Current methods effective but with limitations
The traditional test method is the in-vitro adventitious virus assay, which uses various indicator
cell lines for detection of viruses for in-process or
lot-release testing of the bioreactor at the end of
the run, according to Dominick Vacante, scientific
director for virology with Janssen Pharmaceutical
R&D. “The assay is very sensitive and can potentially detect one infectious virus particle. The limitation is that the virus must replicate in at least one
of the indicator cell types and produce some type
of effect that is detected by the readouts of the assays, which are visual for cytopathic effects and/or
hemadsorption or hemagglutination of specific red
blood cells,” he explains. Problems do arise when
a virus replicates but causes no signs of cytopathic
effects, or is infectious but does not replicate in
the cell types selected for the assay, which in both
cases yields a false-negative result.
In some cases, the test article may interfere with
the ability of a virus to infect the cells or display
signs of infection, according to Kljavin. Cell-based
18
methods can also be variable; a virus that is detected in one assay may not be found when the
test is repeated. “Equally importantly, it takes time
for the viruses to replicate and grow, which leads
to a very long test time of 14–28 days. A significant amount of product can be produced and sent
downstream during that time, and if found to be
infected must be disposed. The entire production
facility, not just cell-culture areas, must be decontaminated, which can lead to a disruption in the
supply of the drug to patients, a situation that is
unacceptable,” Kljavin states.
He again stresses, though, that cell-based assays
are effective when performed in conjunction with
other risk-mitigating steps like viral clearance validation. The current issues revolve around development of new assays to test for viruses at the various
sampling points that are more rapid, more sensitive, and broader. Gaining a greater understanding
of how purification unit operations clear viruses,
how robust they are, and how to best validate the
clearance of different viruses are other measures
that mitigate gaps in testing, according to Brorson.
Evolving technology
While ICH Q5A serves the industry and regulators
well, Brorson notes that technology has evolved
since 1998, leading to the development of different approaches, such as modular validation for robust unit operations and the introduction of new
assays for cell-line characterization, like genome
sequencing-based methods. “ICH Q5A is silent on
these new technologies since it was written before
their introduction, and could stand to be updated
with developments of the past 15 years,” he says.
FDA has been following new pan virus detection
methods/deep-sequencing methods for cell-line
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING P h a r mTe c h . c o m
characterization, which are at the current time,
according to Brorson, great for investigations and
broad surveys of cell lines or raw materials. “What
isn’t clear yet, though, is whether they have reached
a state where they can be used in a routine QC
setting.” FDA expects, however, that the situation
could change over the next dozen years or so.
Massive parallel sequencing
is attracting a lot of
interest because it can
be used to detect multiple
DNA sequences from
different viruses.
Nucleic acid-based methods
More simple and specific polymerase chain reaction (PCR) methods are currently used by many
biopharmaceutical manufacturers. Each PCR test
accurately detects a DNA sequence from a specific
virus and is completed rapidly compared to cellbased assays (e.g., within one day vs. three to four
weeks). Presently, however, PCR is not applicable
for the detection of multiple viruses at once. Massive parallel sequencing, or deep sequencing, on
the other hand, is attracting a lot of interest because it can be used to detect multiple DNA sequences from different viruses.
In addition to deep sequencing, PCR combined
with mass spectrometry (MS) and microarrays are
considered advanced technologies for virus detection. PCR with degenerate probes may also be included as an advancement of PCR, according to
Vacante. “These technologies may improve virus
detection by enabling the detection of a broad
range of viruses, either in an unbiased manner as
with deep sequencing or through the detection of
consensus sequences of many or all viruses in a
virus family. The assays also by their nature provide information on the virus detected and taxonomy, including the virus family, subfamily, and
genus, which may be helpful for quickly determining where a contamination may have originated,”
Vacante observes.
It is important to remember, however, that with
these methods, only viral DNA sequences are detected, and not actual live particles, according to
Kljavin. “These tests do not indicate if any live
virus is present, only that a part of the DNA of
the virus is present. The challenge then becomes
how to respond if a positive result is obtained,” he
explains. It is necessary to work backward and run
other tests to determine if there is an actual infection. “If no infection is found, then a decision must
be made regarding how to proceed. Both industry
and FDA will have to figure out how to go forward
in such a situation,” Kljavin adds.
Real advantages
Both Genentech and Janssen have employed these
nucleic-acid techniques when dealing with possible
viral contamination. In 1993 and 1994, Genentech
experienced contamination by the rodent parvovirus, or minute virus of mice (MVM). In 1993, the
infection was detected using cell-based assays, but
only after production continued for three to four
weeks, thus the facility was contaminated and significant clean-up and production delays resulted.
By 1994, a PCR method was developed, and the
virus was detected before harvest from the bioreactor. “In this latter case, production was halted
before further downstream processing progressed,
and decontamination efforts were limited to the
Pharmaceutical Technology ANALYTICAL AND BIOANALYTICAL TESTING
19
Viral Contamination
impacted bioreactor, and other normal operations
in the facility continued,” notes Kljavin.
A few years ago at Janssen, in response to a suspected viral contamination, deep sequencing, and
DNA amplification followed by MS, previously
referred to as TIGER and now termed PLEX-ID,
was used to show that there was no contamination, according to Vacante. “Analysis of the effect
observed in cell culture and extensive screening
using the advanced tests provided convincing evi-
CDER has been involved with
industry and other players
in directed research on
standardization and improved
methods for validating viral
clearance unit operations.
dence for the lack of a viral contamination or other
biological agent,” Vacante adds. Using established
standard operating procedures, the impacted lots
were then retested and found to be actually negative. The initial test result was then assigned “falsepositive status,” and the product was ultimately
released to the market (1).
says. He also notes that a PDA interest group was
formed to exchange ideas and practices, evaluate
these technologies, and possibly develop best practices and standards or standardized approaches for
comparison of methods across different laboratories. FDA has been involved with both the white
paper and the interest group, and Arifa Khan at
the Center for Biologics Evaluation and Research
has provided leadership regarding the evaluation
of these new technologies for virus detection, according to Vacante.
CDER has also been involved with industry and
other players in directed research on standardization and improved methods for validating viral
clearance unit operations. “For example, CDER
performed crucial lab work that contributed to
the development of the first ever standard nomenclature for virus retentive filters promulgated in
PDA’s TR41, Virus Retentive Filters. We also laid
part of the groundwork for ASTM E2888 Standard
Practice for Process for Inactivation of Rodent Retrovirus by pH, and participated in the committee
writing the standard. A challenge for industry is to
implement these new standards, assays, and validation approaches,” observes Brorson.
Reference
Industry and agency activities
To help evaluate the current status of analytical
technology with this goal in mind, a task force of
members of the Parenteral Drug Association (PDA)
that includes industry, government, and academia
are preparing a white paper on emerging methods
for virus detection. “This white paper describes
both convention