CHOptimizer®: Design Space Estimation

Factorial design as a basis to estimate design space

and robust setpoint conditions in ambr®15 bioreactors

Background
This DOE exercise is a sequel to the DOE exercise called CHOptimizer®: Media Optimization. You may
want to read parts of CHOptimizer®: Media Optimization to get an introduction to the scope of the
current exercise. Briefly, in the previous exercise, mixture design was used to induce systematic
changes in four base media mixed together to make up the final cell culture formulation. Based on the
results obtained three mixture formulations were identified:

- One formulation was optimized for titer production

- One formulation was optimized for doubling time
- One formulation was optimized for titer production, doubling time and VCD weighted equally

The relevance of the three chosen formulations was further scrutinized in a second phase DOE
exploring how changes in temperature, pH and media composition affect critical cell culture responses.
We will review the second DOE conducted in this exercise.

Objective
The objective of this exercise is to:
 investigate how factorial design can be used to encode systematic alterations in reactor
environments in ambr®15 bioreactors, and
 understand how such data can be used to define appropriate design space and robust setpoint
conditions.

In order to do so, we will:

 Learn how to generate a factorial design using MODDE and its Design Wizard
 Analyze DOE data using the Analysis Wizard of the MODDE software
 Understand how changes in factors correlate with the responses
 Use response contour plots for model visualization and interpretation
 Use SweetSpot and Design space plots to propose suitable operating conditions where
specifications on the responses are fulfilled.
Factors
The influence of three factors will be explored. The first factor is a qualitative factor in three settings
representing the three optimized media defined in the first exercise. The other two factors are
quantitative: Temperature and pH.

Responses
Three responses relating to titer, viable cell count (VCC) and percentage viability were measured. All
three should be maximized and the relevant settings for minimum and target values are given below.

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Tasks
Task 1
The first task is to create a MODDE project and reproduce the experimental design that was used.
Task 2
Use the Analysis Wizard to model the three responses.
Task 3
Use contour plots to visualize how changes in the factors correlate with changes in titer, VCC and
viability.
Task 4
Use SweetSpot and Design space plots to investigate if there exists a region in factor space in which all
goals for the responses are met.
Task 5
Use the Optimizer to make more rigorous design space calculations and to search for a robust setpoint.

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Tasks
Task 1
The first task is to create a MODDE project and reproduce the experimental design that was used. The
basic design used is a Full Factorial Mixed design in 12 runs (3*2*2) without centerpoints. This design
was replicated one time to get 24 runs (2*12). In the ambr® bioreactor each set of 12 reactors was set
to use the same temperature. This means the run order suggestion in the standard MODDE worksheet is
irrelevant in this case. Hence, the DOE protocol used in reality deviates a little from the one proposed
by MODDE. We will therefore copy and paste the real data from an Excel-file. But first we need to
specify the design framework.
Use the Design Wizard to create a new MODDE project:

Define the factors and responses according to the information given above. Select objective Screening,
select the Full Fac (Mixed) design. Set the number of center-points to 0 and the number of replicates
to 1. Click Finish.

Upon exiting the Design Wizard a preliminary worksheet with 24 rows (24 experiments) is created. The
run order suggestion in this worksheet is irrelevant as all will be run simultaneously.

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Open the file DOE data Sartorius internal course.xlsx and copy/paste the contents into the MODDE
worksheet. Note that the column denoted Exp Name has been purposely edited to reflect sample and
replicate belongings. The real worksheet is seen below and your worksheet should be identical.

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Task 2
Use the Analysis Wizard to model the three responses.

For each response judge the replicate experiments quality, response normal distribution, model quality
etc.

Are there any deviators?

Which factors have the highest / lowest influence?

Are the investigated factors influencing the three responses in the same way?

Which factor setting is favorable for maximizing titer? Maximizing VCC? Maximizing viability?

Use the One-click analysis functionality to optimize the various models.

Task 3
Use contour plots to visualize how changes in the factors correlate with changes in titer, VCC and
viability.
How should you adjust the factors to accomplish the specifications on the responses?

Task 4
Use SweetSpot and Design space plots to investigate if there exists a region in factor space in which all
goals for the responses are met.
What is the risk of failing to comply with the response specifications?

Task 5
Use the Optimizer to make more rigorous design space calculations and to search for a robust setpoint.
Is it possible to define a robust setpoint?

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Solutions
Task 2
We here present the results for one response at a time.
Titer
The plots of the Analysis Wizard arising from calculating the initial interaction model are seen below.

The replicate plot shows there is fairly small variability among the replicates. The histogram plot
indicates that the response does not need a transformation and can be analyzed using the
untransformed metric. According to the coefficient plot, the temperature is the factor having the
strongest impact on titer. Running on low temperature coupled with Mix 1 yields most titer. The
summary of fit plot, the normal probability plot of residuals, and the observed versus predicted plot all
point to a very good model for the titer response.

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In order to optimize the model we used the One-click analysis functionality. The results of the
optimized model is shown below. The final model consists of the three main effects and the interaction
term between Temperature and pH. It is a very good model with high performance statistics.

One-click analysis results One-click analysis results

One-click analysis results One-click analysis results

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VCC
The plots of the Analysis Wizard arising from calculating the initial interaction model are seen below.

The replicate plot shows there is fairly small variability among the replicates. The histogram plot
suggests no need for any response transformation. The regression coefficient plot indicates the
temperature to be the strongest factor in affecting VCC. There is also significant influence from the
cross-term between temperature and media. This influence is easier to interpret using contour plots,
which is discussed in Task 3. Some non-linear structure to the points in the normal probability plot of
the residuals indicates that adjustments to the model are needed.

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In the next step, we used the One-click analysis functionality. The results of the refined model are seen
in the quartet of plots below. Overall, as with titer, we have obtained a very good model also for VCC.
No structure in the N-plot of residuals remain after revising the model.

One-click analysis results One-click analysis results

One-click analysis results One-click analysis results

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Viability
The plots of the Analysis Wizard arising from calculating the initial interaction model are seen below.

The replicate plot suggests a fairly small replicate error and the histogram is OK. Compared with the
results for the two foregoing responses, the modelling results are in this case more or less a two-point
correlation situation (see the observed versus predicted plot). The only factor that influences viability is
the temperature. When the temperature is high there is low viability, and vice versa, and this is what
causes the split of the data points seen in the observed versus predicted plot.

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With the One-click analysis functionality we get a revised model consisting only of the linear terms.
The temperature is by far the dominating factor. Low temperature combined with low pH and Mix 1 as
media maximize percentage viability.

One-click analysis results One-click analysis results

One-click analysis results One-click analysis results

The final stage of the Analysis Wizard is a summary page providing a detailed account of all the
changes done to the initial models.

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Task 3
Triplet of contour plots for Titer. Lower left-hand corner of leftmost plot gives highest titer.

Triplet of contour plots for VCC. Lower left-hand corner of rightmost plot gives highest VCC.

Triplet of contour plots for Viability. Lower left-hand corner of leftmost plot gives highest Viability.

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Task 4
The SweetSpot plots below demonstrate that SweetSpot areas exist both when using Mix 1 and Mix 2
as media, but not when using Mix 3. The largest SweetSpot region is found for Mix 1.

By creating a Design Space plot, configured in the same way as the SweetSpot plot, we realize that the
safe area according to the Design Space plot is much smaller than what is suggested by the SweetSpot
plot.

The settings used were:

- Select responses: VCC, Titer, Viability
- Uncertainty interval: Confidence
- Acceptance limit: 1%

Using low temperature, low pH and Mix 1 results in the most robust conditions and with smaller than
1% risk of failing to comply with the response specifications.

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Task 5
The Optimizer was launched and used to search for a robust setpoint, the details of which is seen
below.

In the next step, the Design Space Explorer tool was used to visualize the design space. The cross-hairs
symbol shows the location of the robust setpoint.

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Conclusions
This exercise shows how a factorial design can be used for studying the influence of temperature, pH
and cell culture media on important responses such as titer, cell count and viability. The conclusion
within the current set of experiments is that the factor temperature is by far the most influential one.
Running on low temperature is beneficial for all three responses.
Overall, very good regression models were obtained, indicating high-quality data underpinning this
DOE-exercise. The high-performance statistics of the three models imply high model reliabilities and
strong connections between the three factors and the three responses.
Because of the strong models, the Optimizer was launched and used to search for a robust setpoint
and indeed such a point existed. Using the visual capabilities of the Design Space Explorer tool it was
concluded that the settings
- Mix 1
- Temperature 32.3 ± 0.3
- pH 6.81 ± 0.01
correspond to cell culture conditions within which there is lower than 1% risk of failing to comply with
the response specifications (i.e., titer > 350 mg/l, VCC > 4 and viability > 70%).

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