Scaling Down
of Biopharmaceutical
Unit Operations –
Part 1: Fermentation
The fermentation process can be challenging
to scale down and several factors must be
evaluated for each step.
C
reation and qualification
of scale-down models are
essential for performing sev-
eral critical activities that
support process validation and com-
mercial manufacturing. As shown in
Figure 1, these activities include process
characterization and production sup-
port studies that are performed to eval-
uate column and membrane lifetimes,
demonstrate clearance of host-cell
impurities and viruses, and trouble-
shoot manufacturing issues. While the
underlying fundamentals are relatively
14 | BioPharm International
Anurag Rathore,
Raj Krishnan,
Stephanie Tozer,
Dave Smiley,
Steve Rausch, and
Jim Seely
2005;18(3):60,62,64,67-68
the same as those when scaling up,
some unique considerations should
be taken when scaling unit operations
down.1-4 The goal when scaling down
is to create a small-scale or lab-scale
system that mimics the performance
of its large-scale (pilot or manufac-
turing) counterpart, when both the
process parameters are varied within
their operating ranges and also when
a process parameter deviates outside
its operating range. Before it can be
used for lab studies, the scale-down
model needs to be qualified and its
Figure 1. Scale-down Models are Best Utilized for Process Characterization
and Production Support
Phase I
Clinical
Timeline
Process Development
Commercial Phase III
Process
Development
Activities
equivalence to large-scale examined.
Data from an inaccurate scale-down
model could result in conclusions
that may not be applicable to large-
scale, resulting in an unsuccessful
process-validation campaign or con-
tinued lot failures in a manufactur-
ing campaign.
This article is divided into two seg-
ments. The first part focuses on an
upstream unit operation — fermen-
tation. The next segment will cover
two downstream unit operations —
chromatography and filtration.
Hardware Scale-down
Guidelines
Fermentation processes often
involve several scales of operation,
encompassing inoculum develop-
ment, seed expansion, and produc-
tion fermentation. The differences
in volumes between the steps in a
single fermentation process can be
10X to 100X for the pilot scale, and
1,000 to 100,000X for the produc-
Approval
& Launch
Phase II Phase III
Clinical Production
Process
Characterization
Process Validation & Production Support
Launch Supplies Production
tion scale. This may cause the fer-
mentation processes to be challeng-
ing to scale down and the specific
process parameters, vessel geome-
tries, and operational control strate-
gies must be evaluated for each step.
Some general guidelines to consider
in developing a representative scale-
down model follow.
Practitioners use the terms “simi-
lar reactor” or “similar vessel geom-
etries” to describe optimal conditions
for a scale-down strategy. However,
similarity in vessel geometry does not
necessarily imply identical systems,
although this would be the most
attractive option. Instead, geomet-
ric similarity means that the overall
aspect ratios of each vessel (small vs.
large) are close enough to not impact
performance. More importantly, the
impeller and sparger designs and
placements within the vessel are
nearly identical.
Methods for assessing process
performance should be identi-
Elements of Biopharmaceutical Production Series | 15
 Lot-to-lot variations
between raw materials
can greatly impact
process performance.
cal between scales. These include down process, as long as the sam-
sample-dilution schemes and mea- pling does not occur at a crucial time
surement times for calculating such as at the start or finish time of
culture optical densities, wet and feed delivery.
dry cell-weights, media metabolite Maintenance of equivalent oxy-
levels, and protein expression. For gen transfer (kLα) and control of dis-
example, if spectrophotometers are solved oxygen across process scales
used to make culture optical-density is the most important requirement
(OD) measurements, use the same for most fermentation scale-down
model in the scaled-down process. strategies (see Table 1 for equations).
If the same model is not available, However, the efficiency of oxygen
use a spectrophotometer with the transfer and control in a production-
same path length and instrument scale fermentor (10,000 to 100,000
precision (relative to one used in L) is often significantly lower than
manufacturing). To verify, use sev- in laboratory-scale (1 to 100 L). The
eral solution standards of known overall oxygen-control strategy —
optical density. including sparger design, calibration,
At the small scale, minimize reac- and placement within the reactor
tor sampling volumes and times as — should be identical to the large-
much as possible. Since each sam- scale process. If the sparger design is
ple that is taken in the scale-down different between scales, then agita-
process represents a larger percent- tion, aeration, and oxygen enrich-
age of the total process volume, ment may need to be adjusted to
the percentage of loss at the small- provide equivalent oxygen transfer
scale must be minimized to prevent in the scaled-down process.
depletion of culture broth beyond For inoculum development and
acceptable levels. If the sample size expansion, conserve the vessel geom-
cannot be reduced, then adjust the etries, incubation conditions, and
frequency of sampling. For example, working volumes at each step when-
optical-density or wet cell-weight ever possible. Therefore, if 2-L baffled
measurements performed every two shake-flasks are used for seed expan-
hr at the manufacturing scale could sion in the full-scale process, use the
be done every ten hr in the scale- same volume and flask type in the

16 | BioPharm International
Table 1. Scale-down Equations for a Typical Fermentation Process

• Constant volumetric oxygen-transfer coefficient (kLa):

kLa = C * (Pg/V)α * (Vg)ß, where C, α, and ß = proportional constants, Pg = total gassed power input,
V = working volume, and Vg = superficial air velocity.
• Constant tip speed (vT):
vT = π * N * Di, where N = process agitation and Di = impeller diameter.
• Constant Reynolds Number (NRE):
NRE = ρ * N * Di 2/μ, where ρ=culture density, N = process agitation, Di = impeller diameter,
and μ = culture viscosity.
• Constant ungassed power input per unit volume (P/V):
P/V = k * N3 * Di5 * ρ/V, where k = a proportionality constant, N = process agitation, Di = the
impeller diameter, P = the ungassed power input, V = the working volume, and ρ = solution density.

Units are not given because any consistent set that produces a dimensionless Reynolds
Number will suffice.
Gassed power is measured when the sparger is adding gases to the mixture. Ungassed power is
measured when the sparger is idle.

scale-down process. All process con-
trol setpoints and ranges (tempera-
ture, shaker speed, stroke length, pH,
and dissolved oxygen) should be the
same. If the same equipment is not
available, then vessels with different
volumes but similar geometries may
be used. However, the operational
control parameters may need to be
adjusted to account for different ves-
sel geometries.
Operational Scale-down
Guidelines
Set up and sterilize all process vessels
and tanks according to the current
manufacturing procedure for the
process. Sterilization temperatures,
procedures for probe and flowmeter
calibration, and post-use cleaning
protocols should be the same as the
large-scale process. If extended (or
multiple) sterilization cycles are nec-
essary for any piece of equipment,
assess the impact on process perfor-
mance and individual medium com-
ponent stability.
Use GMP-released raw materials
that are identical to those used for
the full-scale process. Lot-to-lot varia-
tions between raw materials, includ-
ing master or working cell-bank
vials, as well as all media compo-
nents, antifoam additions, and acid
and base stock solutions, can greatly
impact process performance. If the
same lot of raw materials is not avail-
able, have the vendor supply a repre-
sentative lot based on the certificate
of analysis. Similarly, prepare buffers
and all bulk media solutions accord-
ing to the GMP-approved manufac-
turing procedures for the full-scale
process. Match current manufactur-
ing procedures for the order of addi-
tion of media ingredients, mode of
sterilization and addition (filter, heat
or steam), mixing times and tem-

Elements of Biopharmaceutical Production Series | 17

Table 2. Comparison of Performance of Fermentation Scale-down Model
with Large-scale Fermentation Process
Process Scale Final Culture
Optical Density
15 L 1.0 x O* ± 50
300 L O* ± 50
*O, S, T and G are constants that are used to compare performance at lab scale to
production scale. The factor 1.0 shows that the scale down matches.
peratures, media hold times, and the
preparation of mixing tanks.
After assembling a system with all
the similar geometries, it is time to
tune the controls. All volume-inde-
pendent operational control-param-
eter setpoints should be identical to
the center point of the operating
ranges of the large-scale fermenta-
tion process. The volume-indepen-
dent parameters include:
• process temperature
• pH
• inoculation percentages (v/v) for
each step
• schedule of feed-media additions.
If oxygen transfer rates are equiva-
lent, then the dissolved-oxygen con-
trol setpoint and vessel backpressure
should also be held constant.
Except for agitation, use a linear
adjustment for all the volume-depen-
dent operational control-parameter
setpoints. The scale factor should be
equivalent to the ratio of overall pro-
cess volumes. For example, if the pro-
cess volumes are 300 L and 15 L, the
scale factor (as a divisor) is 20.
The volume-dependent parameters
include:
• Pre and post-sterilization volumes of
growth media. The volumes of ini-
18 | BioPharm International
Final Percent Final Product Titer Specific Growth
Solids (%) (g/L) Rate (hr-1)
1.0 x S* ± 1 1.0 x T* ± 0.05 1.0 x G* ± 0.02
S* ± 1 T* ± 0.05 G* ± 0.01
tial growth media at the beginning
and end of sterilization should
be equal to the initial and final
volumes in the manufacturing pro-
cess divided by the scale factor.
Excessive water gain or loss during
sterilization can result in slightly
offset volumes, which may result
in product and cell mass dilution
and possibly an increase in overall
time (if the process target is based
on optical density). It is crucial to
know the extent of these volume
differences in order to assess the
accuracy of the scale-down model.
• Feed media delivery rates. Adjust all
feed rates based on the scale fac-
tor. The method of delivery (based
on unit weight or unit volume per
hour) and the control (flowmeter
vs. air pressure) should be the same
as the large-scale process; other-
wise it will be difficult to equate
the two processes.
• Total airflow. Scale down the total
airflow linearly to ensure similar
oxygen transfer and carbon diox-
ide stripping between the different
scales.
• Oxygen flow rate. In most cases,
the maximum oxygen flowrate,
whether using pure oxygen or
 Creation of scale-down models
that meet qualification requirements
can be challenging, particularly for
upstream unit operations.
oxygen-enriched air, should be
scaled-down linearly. However,
if there are profound differences
in the sparger or vessel geom-
etry between scales, then the total
flowrate of oxygen, relative to air,
may need to be changed to ensure
equivalency in oxygen transfer
rates. The oxygen flowrate should
be increased only if the agitation
has been increased to its maxi-
mum tolerance for the process, or
if the culture is extremely shear
sensitive.
Set agitation to provide either rep-
resentative oxygen transfer rate (kLa),
tip speed (vT), Reynolds Number (NRE),
or power-input per unit volume (P/
V), according to the equations listed
in Table 2. Assuming equivalent ves-
sel geometries and sparger design, the
best bet for agitation is to provide a
representative oxygen transfer rate
(kLa) between scales. If kLa data are not
available at the different scales, then
set agitation to provide an equivalent
power input per unit volume (P/V).
This should result in similar mixing
profiles across scales, and thus similar
oxygen transfer and dispersion. Scale
down by constant tip speed or con-
stant Reynolds Number has also been
reported.
Elements of Biopharmaceutical Production Series | 19
Qualification of the
Scale-down Model
The general approach to scale-down
model qualification is to run all oper-
ating parameters at the center of the
operating range of the manufacturing
process. Compare the specific perfor-
mance (output) parameters and pro-
cess control sensitivity at both scales.
Establish the acceptance criteria prior
to the scale-down runs, which can be
based on statistical analysis of histori-
cal large-scale data.
Culture growth is a critical perfor-
mance parameter for qualifying the
scale-down model. The assessment
measures are culture optical density,
percent solids accumulation, and wet
and dry cell-weights. Evaluate cul-
ture growth both qualitatively, based
on a comparison of growth profiles
(log and linear plots), and quantita-
tively, through a direct comparison
of specific growth rates. The equip-
ment and procedures used for these
measurements should be identical to
those used in the current manufac-
turing process. In a good qualifica-
tion, the overall specific growth rate
and final cell yield will approximate
the large-scale process.
Oxygen consumption is another
Figure 2. Comparison of Culture Growth Profiles in Small- and Large-scale
Fermentations
Full-scale Run #1
Full-scale Run #3
15 L Scale-down Run #2
Full-scale Run #4
Optical Density
Fermentation Time (hours)
important performance parameter
for scale-down qualification. Similar
trends in dissolved oxygen profiles,
and oxygen and airflow rates repre-
sent comparability in oxygen usage
by the cultures at each scale. In addi-
tion, by using a mass spectropho-
tometer, specific oxygen uptake rates
(OUR) can be calculated and com-
pared across scales through an analy-
sis of off-gas oxygen levels.
A key comparison involves final
product titer and quality, at a speific
rate of production, which is defined
as the amount of product expressed
per unit biomass weight (wet or dry).
The analytical assays and method-
ologies used to measure product titer
should be identical between scales.
Product quality may be difficult to
assess, because it requires the devel-
opment of a representative down-
20 | BioPharm International
Full-scale Run #2
15 L Scale-down Run #1
15 L Scale-down Run #3
stream scale-down model, including
any harvest procedures, which are
challenging to replicate at the small
scale. Data from large-scale runs
should be collected and analyzed
in order to understand the inherent
variability in the analytical assays
before using the assays for scale-
down model qualification.
Measure the rates of consumption
of nutrients (such as glucose) and
accumulation of specific metabo-
lites (ammonia, lactate, acetate) at
both scales to demonstrate process
similarity. Once again, due to the
variability in measurements of these
components, the equipment and
procedures employed for calculating
the rates must be identical to those
used in the manufacturing process.
Since cellular metabolism can be
influenced by slight shifts in culture
Figure 3. Comparison of Dissolved Oxygen Profiles in Small- and Large-scale
Fermentations

15 L Scale-down Run #1 15 L Scale-down Run #2

15 L Scale-down Run #3 Full-scale Run #1
Full-scale Run #2 Full-scale Run #3
Dissolved Oxygen (%)

Fermentation Time (hours)

conditions, the exact metabolite lev-
els may vary across scales. However,
the specific rates of consumption
and accumulation for each compo-
nent should be similar.
Process-control sensitivity for dis-
solved oxygen, pH, temperature,
agitation, and feed delivery must be
verified at the small-scale. For exam-
ple, feed delivery data must be col-
lected and analyzed to ensure the
flow controller accurately delivers
the feed at the specified target rate. If
equipment limitations cause a change
in feed rate, the performance of the
scale-down model will not be repre-
sentative of the large-scale process.
Other process control issues to
consider are maintenance of equiv-
alent heat transfer rates during
in-process temperature shifts, differ-
ences in heat transfer and genera-
tion at different scales, and overall
PID control loop response times. The
process control profiles do not need
to be identical to one another, with
respect to the fluctuations around a
given setpoint, although the trends
in the profiles for all the control
parameters should be comparable.
Additions of acid and base for pH
control should also be measured and
compared at each scale. However, it
is important to note that the addi-
tions of each component may not
scale linearly. In some cases, the base
that is used may also be a nitrogen
source for the culture. Therefore,
its usage is related to the growth of
the culture, as well as pH control.
In addition, slight shifts in cellular
metabolism (e.g. lactate production
and consumption), which are not
easily predicted or controlled, can

Elements of Biopharmaceutical Production Series | 21

result in changes in acid and base
delivery, in order to maintain culture
pH. In any case, variations in acid or
base delivery will affect process vol-
umes and media composition. As a
result, it is important to evaluate the
differences in acid and base addition
at each scale for any impact on cell
growth or production.
In general, perform at least three
small-scale runs in order to demon-
strate reproducibility and to deter-
mine the inherent variability in the
process. Since the scale-down mod-
els may also be used to evaluate pro-
cess deviations, further qualification
of the scale-down model — which
would include fermentation trials at
the edges of the operating parameter
ranges — may need to be performed
and compared to similar process data
acquired in the large-scale process.
Table 2 summarizes different per-
formance attributes for small and
large-scale fermentations including
a comparison of product titer, per-
cent solids accumulation, and specific
growth rate.
Figures 2 and 3 illustrate a suc-
cessful scale-down of a microbial fer-
mentation process based on culture
growth and dissolved oxygen profiles,
respectively. While the dissolved oxy-
gen profiles in Figure 3 were slightly
different, the rates of oxygen con-
sumption (excluding the fluctuation
at t=20 hours) were similar.
Originally published in the March 2005 issue of BioPharm International.
22 | BioPharm International
Summary
Creation of scale-down models that
meet qualification requirements can
be challenging, particularly for up-
stream unit operations. These mod-
els can be of great use when per-
forming experimental studies in an
efficient and economical fashion.
However, there are considerations
unique to each unit operation that
apply during scale-down. It must be
mentioned that while scale-down
studies certainly increase the chanc-
es of having a successful validation
campaign, they only provide guid-
ance. The actual confirmation must
be made at large-scale. ◆
References
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RL, and George HA. Development of
scale-down technique for investigation of
recombinant Escherichia coli fermenta-
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Prog.1993; 9:580-586.
2. Reynolds T, Boychyn M, Sanderson T,
Bulmer M, More J, and Hoare M. Scale-
down of continuous filtration for rapid
bioprocess design: Recovery and dewa-
tering of protein precipitate suspensions,
Biotech. Bioeng. 2003; 83:454-464.
3. Varga EG, Titchener-Hooker NJ, and
Dunhill P. Prediction of the pilot-scale
recovery of a recombinant yeast enzyme
using integrated models, Biotechnol.
Bioeng. 2001; 74:97-107.
4. Godavarti R, Petrone J, Robinson J, Wright
R, and Kelley BD. Scale-down models
for purification processes: Approaches
and applications, in process validation
in manufacturing of biopharmaceuticals,
eds. Rathore AS and Sofer G. Marcel
Dekker, New York 2005.