A Rational, Step-Wise Approach to Process Characterization

Aug 01, 2003

By James E. Seely, PhD [1], Robert J. Seely [2]
BioPharm International
Volume 16, Issue 8
As we scale up manufacturing processes, increase production rates, and strive to minimize batch
failures in an increasingly stringent regulatory environment, it is valuable to characterize processes
at the levels appropriate for each product development stage (preclinical through phase 3 and
commercial manufacturing). Amgen currently characterizes several aspects of the manufacturing
process comparable to that performed by other biotech companies. The increased understanding
of each process that results from these characterizations can vary - from minimal knowledge,
during development and non-GMP runs, to detailed knowledge of each unit operation or group of
unit operations in late clinical development. Predictably, in-depth process characterization requires
a significant commitment of resources and time.
The overall goal of adequate process characterization for commercial manufacturing processes is
to ensure efficient and successful validation and the assurance of consistent process performance.
More specifically, the characterization should provide:
 an understanding of the role of each process step, such as where impurities are cleared
during a particular purification step
 an awareness of the effect of process inputs (operating parameters) on process outputs
(performance parameters) and identification of key operating and performance parameters
 assurance that the process delivers consistent product yields and purity in all operating
ranges
 acceptance parameters for in-process performance parameters.
This article describes a characterization strategy that is cost-effective and consistent.
Compliance and Business Drivers Process characterization has often been viewed as a "check the
box" activity or something that was completed but which required little scientific rigor. This was
most likely a result of cost cutting. Thorough process characterization requires a significant
resource from process development and analytical departments. Many companies now recognize
the benefits of sound process characterization - and, unfortunately, the cost of incompletely
understood processes (1).
For compliance, process characterization needs to identify key operating and performance
parameters, justify operating ranges and acceptance criteria, recognize interactions between key
variables, and ensure that the process delivers a product with reproducible yields and purity. This
is the complete and rational approach to process validation. Costly registration delays resulting
from poorly understood processes and failed validation batches can cost a company tens of
millions of dollars.
From a business standpoint, process characterization can improve run success rates and lead to
incremental, but significant, yield improvements. Better process understanding can also minimize
- and aid in the investigation of - process deviations.
Timing of the Studies To conserve resources, waiting until after phase 2 is the best time to start
"intensive" process characterization work. At that point, the commercial process should be
developed, and you know whether or not the product is likely to make it to market. Unfortunately,
the driver for process characterization timing is the start of conformance or validation lots.
Therefore the work should be completed by that point so that information gained from it can be
used to support operating ranges and acceptance criteria for validation protocols.
At least 12 to 15 months should be allowed for process characterization studies; therefore, they
should start no later than 15 months before the conformance runs (see Figure 1). Sometimes phase
2 studies are complete by this point; but if phase 2 data are still pending, process characterization
resources may have to be spent "at risk" to ensure completion of the work before the process
validation runs. Fortunately, characterization studies can sometimes be ramped up slowly during
the first few months, which may provide the time to complete the phase 2 studies, conserving
resources until a clearer picture of the product's viability is obtained. Figure 2 shows the "process
flow" involved in characterizing the manufacturing steps leading up to the validation studies.
Precharacterization Work Three steps are involved in precharacterization:
 mining data and assessing risk
 qualifying a scale-down model
 developing process characterization protocols.
Step 1: Data mining and risk assessment. Data mining - a retrospective review of historical data
- and risk assessment analyses determine what operating parameters need to be examined
experimentally during process characterization. Data from lab notebooks, technical reports,
process histories, run summaries, and manufacturing records are combined with a list of the
operating parameters and provisional operating ranges for each unit operation. The data are then
compiled and reviewed by the team that developed the process to be characterized. Frequently,
information on operating ranges from tests done during process development can help identify the
parameters most likely to affect a process (2).
Once data mining is complete, risk should be assessed for each unit operation on the effect and
likelihood of an excursion from operating ranges. Hazard analysis and critical control points
(HACCP) (3), failure mode and effects analysis (FMEA) (4-7), cause-and-effect diagrams (8), and
other risk assessment tools can be used for risk analyses. FMEA assigns a numerical rating
(typically 1 to 10) to the severity of an excursion from operating parameter ranges, the probability
of an excursion, and the likelihood of detecting an excursion before it has an effect on the product
(4-7). The combined risk factor - the risk priority number (RPN) - is a multiple of the three
variables, rating each parameter from 1 to 1,000 (4-7).
The FMEA exercise should include not only the scientists who developed the process but also
quality personnel and plant and process engineers because they bring insight into the likelihood of
a process excursion and the difficulty in detecting it. Significant differences in processes may exist
between commercial manufacturing of a product and the first-in-human process, so there may be
little historical data at commercial levels. Risk analyses may have to draw on development and
historical data from both clinical and commercial scales.
Developing an FMEA rating system that is not totally subjective - that is consistent and agreed on
by everyone - can be challenging. Table 1 (9) provides an example of how parameters in the FMEA
rating system can be defined. The resulting data - the RPNs - are usually presented in a Pareto
chart (Figure 3). RPNs that fall below a predetermined threshold are not considered key parameters
and are not examined during the characterization experiments. A report summarizing FMEA study
findings should include the rationale for determining that some parameters do not need to be
characterized.
Step 2: Scale-down model qualification. Process characterizations require that a representative
scale-down model be qualified. Some unit operations, such as homogenization or centrifugation
steps, are more difficult to scale down and typically have to be qualified at pilot-plant scale. Other
operations such as chromatography, ultrafiltration, and some cell culture and fermentation
operations can normally be qualified at bench scale.
In general, scale-down model parameters should be qualified at the center points of clinical or
large-scale operating ranges. If the process has not yet been run for clinical manufacturing, the
operating parameters should mirror those of the largest pilot-scale runs. If no appropriate large-
scale data are available, data from an earlier manufacturing stage of the product can be used.
However, the scale-down model has to provide data that are consistent with large-scale runs.
 For fermentation and cell cultures, the operating parameters that need to remain consistent
include pH, temperature, oxygen transfer and CO2 sweeping rates, feed rates, the order in
which media components are added, seed strategies, and antifoam strategies.
  For chromatography, operating parameters such as load factors, pH, temperature,
conductivity, linear flow rates, bed height, and gradient and wash volumes in columns
should be the same as those run at large scale.
 For ultrafiltration steps, membrane loading, transmembrane pressures, and cross-flow rates
should be the same as those at large scale.
Key performance parameters from small-scale studies should be within the historical range of
those observed at large scale.
 For fermentation and cell culture steps, performance parameters might include titers,
growth rates, final optical density (OD), nutrient use, and qualitative assessments of the
product.
 For purification steps, performance parameters might include product-related impurity
levels and profiles, host cell proteins, and step yields.
 Additional performance parameters such as pool volumes and concentrations may be
difficult to mimic because of hold-up volumes and other factors. These limitations to the
scale-down model are acceptable as long as they are understood and identified up front.
Scale-down models also ensure that various operating parameters are measured accurately and
represent what happens at large scales. Because operating parameters vary during process
characterization studies, pH, conductivity, temperature, flow rate, pressure, and other
measurements need to be calibrated and read accurately and with adequate precision. Flow cells
for bench-scale chromatography should provide the same absorbance readings at large scale,
particularly at higher absorbances.
Scale-down experiments should use feedstock representative of that to be used at commercial
scale. Storage conditions and feedstock stability should be evaluated at each step to ensure the
material used during characterization does not degrade during the course of the study. Doing so
will also identify processes that need a constantly replenished supply of representative feedstock.
Although not a requirement, released CGMP raw materials, such as buffer salts, media, and resins,
should be used when possible.
Step 3: Protocols. Although not absolutely necessary during pre-characterization, having written
protocols preapproved for individual unit operations is valuable in planning, executing, and
documenting process characterization procedures. Protocols should indicate the operating
parameters to be tested and the operating ranges to be examined. Protocols should also indicate
which performance parameters will be monitored and what assays will be run. Acceptance criteria
are not necessary in the characterization protocols because the outcome of many of the experiments
may be unknown.
Process development scientists and the analytical groups who run assays and validations should
review and approve the protocols. Process characterization studies draw heavily on the resources
of the analytical department, so its personnel need to have the appropriate resources to qualify the
assays before the start of characterization work. Protocol approval, at this stage, by the validation
group ensures the process characterization package delivered to them includes everything they
need for writing validation protocols. For more complex studies, the protocols should be reviewed
by statisticians to ensure the studies deliver meaningful results without using excess resources.
Process Characterization Studies
Process characterizations require five steps:
 characterizing the process performance
 screening experiments
 identifying interactions between key parameters
 process redundancy experiments
 Developing unit operation reports.
Step 1: Characterizing process performance or "process fingerprinting" data may be available
before the process characterization studies begin. At the end of this step, however, your team needs
to understand what each process contributes, including its yield, impurity clearance, or in-process
pool quality. For cell cultures, this step may be a titer or a qualitative assessment of the product
(such as the degree of glycosylation or sialylation). For a chromatography process, this step would
delineate the impurities cleared and the point in the process that they are cleared (for instance, in
the wash step, before product elution, after product elution, or during the regeneration step). For
an ultrafiltration method, conductivity or pH after different turnover volumes might be examined.
The outcome of process fingerprinting should be the identification of key performance parameters
for each process step, which can then be used to design further characterization experiments. How
these performance parameters are affected by excursions from the operating ranges is the objective
of Step 2 characterization studies.
Step 2: Screening experiments. FMEA or risk assessments have, by this step, identified the
nonkey parameters - parameters with minimal effect on product quality or yield. Screening
experiments at this point are designed to eliminate additional parameters from further, more
rigorous, process characterization work. The design of experiments (DOE) can make screening a
variety of operating parameters easy (10,11). DOE software packages can aid in the design and
interpretation of these experiments (12,13).
Fractional factorial, Plackett Burman, and D-optimal DOE designs can be used for screening
(10,11). Table 2 shows an example of a simple Resolution III fractional factorial design for a
fermentation process (11). In this design, only nine experiments are required to screen six
operating parameters. Results can be analyzed using Pareto charts or regression analysis. Although
these screening experiments do not allow you to rule in or out any interactions between parameters,
they allow you to determine the main effects and identify the factors with the greatest effect on
key performance parameters.
Sometimes a "one-off" study - where only a single operating parameter is tested and all other
parameters are held at their center points - is simpler. Although this approach can require more
experiments, its results are more easily interpreted.
Typically, the performance response throughout the operating parameter range tested is linear,
therefore only two-level (a high and a low value) studies are used (Table 2). The two levels we
prefer to test are at about 1.5 to 2 times the preferred operating range during manufacturing. Table
3 shows several common operating parameters and their respective ranges for screening
experiments. This approach accomplishes several things:
 It enables a clear indication of the effect of an operating parameter on performance. The
range is wide enough to see an effect if it exists but not so wide that a performance failure
is inevitable. Therefore, the test yields information on process robustness, such as whether
the process can run outside the prescribed operating range. This can be important in closing
manufacturing incidents and excursions from set operating ranges.
 It suggests whether a particular operating range needs to be tightened. The information is
valuable during manufacturing because it delineates which parameters need tight control
and which require less attention (Table 3).
 It separates key from nonkey parameters. An operating parameter tested over this range
that has no significant effect on process performance can be designated a nonkey
parameter. However, even if excursions from operating parameters have no product effect,
they should still be monitored to ensure consistent process control. Those parameters that
 do have a significant effect are identified as key parameters and should be tested in the next
step of the process characterization studies.
Step 3: Interactions between key parameters. From screening experiments
results, the operating parameters tested during Step 3 are known to affect Table 4. Anion-
process performance. Therefore, these parameters are typically tested only to exchange
the edge of their normal operating range. As in the screening experiments, DOE chromatography
approaches are useful. Depending on the number of variables to be tested, full design of
factorial, fractional factorial, or another DOE can be used (10,11). In general, experiment
however, you want to use a design from which the effect of any suspected (DOE) in which
interaction can be determined. This means higher resolutions (IV or V) are operating
needed in the experimental design (10,11). Although performance response is parameters are
typically assumed to be linear throughout the operating range tested, the validity combined to
of that assumption needs to be judged. When nonlinearity is suspected, give maximum
multilevel experimental designs should be used. and minimum
An outcome of these experiments may be the identification of other weak spots retention times,
in the process resulting from interactions that were not observed during the as well as
initial screening experiments. In some instances, operating ranges may have to maximum and
be readjusted. The final information delivered by these experiments is an minimum pool
assurance that the process runs "as advertised" within the confines of the various volumes.
combinations of operating ranges and that there is no point where combinations
of two or more operating parameters, run to the edge of their respective ranges, cause the process
to fail.
Variables can sometimes be combined to more quickly explore the "operating space" of a given
unit operation (10), minimizing the amount of experimentation required. Combined variables,
however, may offer results that are difficult to interpret when a process fails, and combining
assumes there are additive effects between operating parameters, which may not always be the
case.

Figure 4. An
example of
resetting The best time to combine variables is when you have confidence in your operating

column 1 ranges, and you need only confirmatory information that your process performs
impurity properly over those ranges. An example of this is shown in Table 4. The initial

acceptance screening experiments identify operating parameters that affect either pool

criteria based volume or peak retention time on an anion-exchange chromatography step. In our
on process example, we combined variables to determine maximum and minimum pool
robustness volumes and the earliest and latest retention times. (Another way to look at this is

studies. that we set up a full factorial experiment using pool volume and retention time as

Running input parameters.) The performance parameters (purity and yields) were within

column 1 historical ranges for this set of experiments. Therefore, by combining the six
under worst variables in the left-hand column in this way, we confirmed that the process
case performs as advertised throughout all of its prescribed operating ranges for these

conditions key parameters.

leads to high Step 4: Process redundancy experiments - using feed quality as a process
levels of input. Until this point, all experiments have been performed with representative
impurity, feed material. However, to test the process's "top-to-bottom" robustness, the effect

which are of feed quality on each unit operation should be tested. This provides an
subsequently understanding of the downstream sensitivity to upstream excursions.
cleared to  For fermentation, this might include different seed fermentor cell densities.

within  For cell harvesting, it might include different fermentation media OD or

historical viscosity.

ranges by  For chromatography steps, the purity of product from the previous step or

column 2. the load factor should be tested (although we usually run all process
characterization experiments at the upper end of the loading range).
All other operating parameters (pH, temperature, and the like) are run at the center of their
respective ranges, because the likelihood of having both an operating parameter excursion and a
feed quality excursion at the same time is remote.
Process redundancy experiments can be used to set acceptance criteria for performance parameters
for each unit operation. A unit operation can be run under conditions that cause it to fail to perform
adequately. The pool from the failed unit operation is used to determine if downstream operations
can make up for poor upstream performance, and thus keep the product within specifications.
An example of process redundancy experiments is shown in Figure 4. In this experiment, column
1 was run in such a way that a product-related variant was present at values higher than the
historical range. When the column 1 pool was processed through column 2, however, the amount
of this variant fell to within the historical range for the column 2 pool. Therefore, rather than setting
the acceptance criteria for the column 1 pool based on historical ranges for the product-related
variant, a wider acceptance criteria can be set because of the process redundancy between columns
1 and 2. There may be other instances in which a pool has to be processed through more than one
additional downstream step to determine the process redundancy, but the principle is the same.
Setting acceptance criteria by determining process redundancy is scientifically based and can give
a more realistic indication of what a process actually delivers than statistical analyses of historical
data (such as standard deviation or tolerance intervals) (14), particularly because at the time the
process characterization is carried out, there may be little or no historical data from which to set
statistically valid acceptance criteria. Setting acceptance criteria for process validation studies
based on what a process can actually deliver results in fewer validation failures from criteria being
set incorrectly. Of course, as the process matures and more lots are run, the acceptance criteria can
be more easily based on statistical analyses.
Step 5: Finishing up - reports and follow-up. Process characterization reports, which include
the results from the clearance, screening, interaction, and process redundancy studies, should be
written for each unit operation. Key operating parameters and respective ranges should be
identified, as should acceptance criteria for all key in-process performance parameters. A rationale
for why certain parameters are identified as nonkey should be included as well. Data from these
reports will be used to support validation studies, and the reports should be completed before
writing validation protocols.
After the characterization work is completed, it may be valuable to go back and repeat the FMEA
exercises. The severity factor for each operating parameter is known by this step, and repeating
the exercise could dramatically change the RPN. In this way, facility engineers can devote their
time to those unit operations and operating parameters that require the greatest control and
detection.
The benefits Process characterization requires a significant commitment of time and resources, but
the payoff is better process understanding, improved manufacturing success rates, and avoidance
of costly regulatory delays, which make the investment worthwhile. The process characterization
approach we have described will furnish you with information that can be used for setting
operating ranges and acceptance criteria for performance parameters, as well as defining the
overall robustness of your process. At Amgen and at other companies, however, our process
characterization strategies continue to evolve, and we fully expect even more efficient and
consistent approaches to be developed in the future.
Acknowledgments The authors wish to thank Tim Tressel, Dan Weese, Dave Smiley, and Mike
Covington for their suggestions and input.
References (1) Bobrowicz, G., "The Compliance Costs of Hasty Process
Development," BioPharm 12(8), 35 38 (August 1999).
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Process Variables," Pharmaceutical Process Validation, G. Sofer and D. Zabriskie, Eds. (Marcel
Dekker Press, New York, 1999, pp. 61 76).
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(4) Kieffer, R., et al., "Applications of Failure Mode and Effects Analysis in the Pharmaceutical
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(6) Sahni, A., "Using Failure Mode and Effects Analysis to Improve Manufacturing
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(10) Kelley, B., "Establishing Process Robustness Using Designed Experiments," Pharmaceutical
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(11) Haaland, P., Experimental Design in Biotechnology (Marcel Dekker, Inc., New York, 1989).
(12) Juran, J.M. and Godfry, A.B., Juran's Quality Handbook, 5th ed. (McGraw-Hill, New York,
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(13) Montgomery, D.C., Design and Analysis of Experiments, 5th ed. (John Wiley and Sons, New
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Figure 1. Timing of process characterization
studies; it's preferable for work to start at the
end of phase 2, but it needs to be completed
before the start of conformance or
validation lots.