Scaling Down
of Biopharmaceutical
Unit Operations –
Part 2: Chromatography
and Filtration
Constraints associated with equipment can make
scale-down a challenging exercise.
C
reation and qualification
of scale-down models is
essential for performing
several critical activities
that support process validation and
commercial manufacturing. Part 1
covered fermentation. In this seg-
ment, we present some guidelines
and examples for scale-down of
common downstream unit opera-
tions used in biotech processes —
chromatography and filtration.
100 | BioPharm International
Anurag Rathore,
Raj Krishnan,
Stephanie Tozer,
Dave Smiley,
Steve Rausch, and
Jim Seely
2005;18(4):58–60,62,64
Chromatography Scale-down
In theory, chromatography is one of
the more straightforward unit opera-
tions to scale down. Factors at the
large scale such as column-header
design and packing can be difficult
to control and may result in reduced
accuracy of the scale-down model.
This section presents a review of gen-
eral guidelines for scaling down.
Use representative feed streams,
preferably from full-scale manufactur-
ing. First perform a complete analy-
sis of product concentration, quality
attributes, and other feed stream
characteristics (such as pH and con-
ductivity) to ensure a good match. If
possible, use feed streams from differ-
ent lots at small scale to learn about
the effect of feed stream quality on
the performance. In addition, acquire
some knowledge of the storage and
stability of the feed stream material.
Storage-related degradation of the
inventory could affect the outcome
of the small-scale runs.
Use a representative chromatogra-
phy resin. It would be ideal to have
resin from the lot being used at full
scale. If you can’t obtain GMP-released
resin, then make a match based on the
vendor’s certificate of analysis.
Buffers and other solutions should
be made up and released by man-
ufacturing. If it is not possible to
obtain these, mix solutions with
GMP-released raw materials, or mate-
rial of an equivalent grade from an
approved vendor. Follow exactly the
solution recipes from manufacturing.
The pH, temperature, and conductiv-
ity probes that check buffer properties
must be comparable with those used
at the large scale. Measure pH and
conductivity at the same temperature
spelled out in the recipe. The pH and
conductivity standards should also be
comparable (preferably identical) to
those used in manufacturing.
Hardware
Our experience has been that an
automated small-scale chromatog-
raphy system, such as the ÄKTA
Explorer, excels at small-scale stud-
Elements of Biopharmaceutical Production Series | 101
ies. It offers precise, accurate, and
reproducible control of flow rates
and gradients. However, you still
have to check and calibrate the accu-
racy and precision of in-line detec-
tors (pH, conductivity, temperature)
by using off-line probes.
The type of column is generally
not a major issue; however, when-
ever possible use the same bed sup-
port material. We prefer to not use
column diameters of less than 1.0
cm since the ratio of system dead
volume to column volume can result
in pool concentrations significantly
lower than those seen at large scale.
The packing procedures and solu-
tions should be similar to those used
at large scale.
Controlling temperature at small
scale to mimic that of cold rooms
or cold-boxes can be a challenge.
The use of water baths with col-
umn jacketing can provide precise
temperature control, but the tub-
ing leading up to the column can
frequently act as a mini heat
exchanger, making it difficult to
control the actual inlet temperature.
Using heat exchangers on the inlet
tubing and column jacketing may
be necessary for accurate, consistent,
and precise temperature control.
Operational Equivalence
The general approach towards quali-
fication of a scale-down model is
to run all operational parameters
(inputs) at the center of the operat-
ing range used at large scale (clini-
cal or commercial manufacturing).
The performance parameters (out-
puts) such as step yields, pool purity,
impurity clearance, elution volume
and retention time should be similar should be shown to be representa-
across scales. Some of the significant tive of commercial scale manu-
operational parameters include: facturing. If this is not possible
1

• Column loading (g protein/L resin) and the bed height is different at

should be the same at both scales. small scale, the linear flow velocity
In size-exclusion chromatography, should be adjusted appropriately to
the loading should be based on a keep the residence time constant. 2

volume percentage of the column • Pooling criteria should be the same

bed volume.
• Gradient lengths (in number of col-
umn volumes) and slopes (increase
in percent limit buffer per unit
time, or similar measurement)
should be the same for all steps of a
chromatography procedure such as
equilibration, wash, and elution.
• Temperature, pH, and conductiv-
ity should be identical for all buf-
fers and feed streams. Differences
in temperature across scales can
lead to significant changes in con-
ductivity of some buffers that may
impact step performance.
• Bed height of the chromatography
column and the linear flow velocity
are generally kept constant across
scales. The ICH guideline on viral
safety states that the validity of the
scaling down should be demon-
strated, and that column bed height
as in manufacturing. They can be
online (conductivity or absorbance)
or offline (gels or HPLC). In our
experience, if the pooling criteria
are UV-based, it is a good practice to
ensure that the UV detectors of the
small-scale systems correlate well
with those in the large-scale system.
UV detectors may be inaccurate or
non-linear over the optical density
(O.D.) ranges required. If the absor-
bance range is non-linear at 280
nm, it may be prudent to measure
the on-line O.D. at 300 nm or some
other wavelength that has a lower
extinction coefficient.
Model Qualification
Pre-determine the acceptance criteria
prior to the scale-down runs. These
can be based on a statistical compari-
son of the small-scale data against the

Table 1. Comparison of Scale-down Model to Large-scale Cation-exchange Column.3

Input material roughly 98 percent antibody, 3 ppm DNA, 3,000 ppm CHOP and <7.8
ppm Protein A. (Reference 3 shows intermediate cycles.)

SCALE Cycle # Yield, % CHOP,* DNA, ppm Protein A,

ppm ppm

Small-scale 1 88.7 75 < 0.006 < 7.8

51 88.3 34 0.01 < 7.8

Manufacturing 1 84.6 116 < 0.07 < 7.8

Scale 50 81.1 72 < 0.03 < 7.8

*Chinese hamster ovary proteins (host-cell proteins)

102 | BioPharm International

full-scale data using a t-test, tolerance
intervals, or other statistical tests.
Alternatively, it may be demonstrated
that the small-scale data are within
the full-scale historical range. The
approach taken may depend on the
inherent variability of the assays used
to determine quality attributes or the
amount of large-scale data available.
Sometimes there may be no large-
scale data available at this stage of
the project and the qualification of
the small-scale model may have to be
done retrospectively after data from
large scale are available. It is a good
practice to perform several small-scale
runs (at least three), to better under-
stand the inherent variability of the
process, particularly when there are
limited amounts of large-scale data
available for comparison.
The model should achieve the
same level of separation as at large
scale. Aim for a similar elution
profile. Deviations that cannot be
avoided should be analyzed care-
fully with regard to influence on the
results. Step yields and the quality
attributes of the product (host cell
proteins, host cell contaminants,
product related variants, and aggre-
gates) should be comparable to full-
scale. Height-equivalent theoretical
plates (HETP) and asymmetry values
should also be within the accept-
able range of that for the large-scale
columns (although not necessarily
identical).
Take data and evaluate the
importance of other parameters
that indicate column performance,
including pool volume, peak reten-
tion time, and peak shape. System
dead volume and unavoidable scale
Elements of Biopharmaceutical Production Series | 103
differences such as header effects
and pressure vs. flow characteristics
may result in anomalies with regard
to these parameters. You must be
able to explain these scale-depen-
dent differences.
If significant differences exist
between the two scales, make a care-
ful determination of the impact of
these differences and if necessary,
redesign the small-scale model. In
any case, having a qualified scale-
down model prior to performing
small-scale cycling studies is neces-
sary, and using a scale-down model
that is flawed will lead to unreliable
lifespan study results and a waste of
time and resources.
Table 1 presents an example of
a successful scale-down of a cat-
ion-exchange chromatography step
and its use for measuring column
lifespan. This column is perform-
3
ing purification in production of a
monoclonal antibody. The input val-
ues are roughly 3,000 ppm Chinese
Hamster Ovary Proteins (CHOP), 3
ppm DNA, and <7.8 ppm Protein
A. At both scales the step yields are
comparable and the key functions of
the column step, which are reducing
CHOP and DNA and clearing Protein
A, are equivalent at both scales.
Figure 1 presents another compari-
son of the chromatographic profiles
at small and large scale. The pro-
file (absorbance) and the resolution
between the product and the dif-
ferent impurities are similar across
scales. Impurity concentrations were
higher in the feed material for the
small-scale run due to under-perfor-
mance of the preceding step.
Figure 2 illustrates an example of
 Main Peak - LS (percent) Main Peak-SS (percent)
Absorbance - LS (mAU) Absorbance-SS (mAU)

100 3
90
80 2.5
Purity, Percent

70 2

Absorbance
60
50 1.5
40
30 1
20 0.5
10
0 0
0 10 20 30 40
Sequential Sample Number

Absorbance - LS (mAU) Absorbance - SS (mAU)

Impurity 1 - LS (percent) Impurity 2 - LS (percent)
Impurity 3 - LS (percent) Impurity 1 - SS (percent)
Impurity 2 - SS (percent) Impurity 3 - SS (percent)
3 14

2.5 12
Impurity, Percent

10
Absorbance

2
8
1.5
6
1
4
0.5 2

0 0
0 10 20 30 40
Sequential Sample Number

Figure 1. Chromatographic Profiles of Small- and Large-scale

Columns. Raw material is a proprietary aqueous solution that
has undergone one step of preliminary purification. The small
column (green data) is 1.6 cm dia by 12 cm. The large column
(black data) is 63 cm dia by 12 cm.

104 | BioPharm International

a successful scale-up from lab-scale
to pilot-scale facility as the column
performance improved both in
terms of step yield and pool purity.4
However, if one were to evaluate the
performance in the lab with respect to
qualifying the scale-down model, dif-
ferences in step yield and pool purity
would necessitate modification of the
scale-down model.
Filtration Scale-down
The unit operation of filtration is per-
haps the most common unit opera-
tion that is used in bioprocesses. It is
used as depth filtration for separation
of particulates (cell debris) from the
process stream; as normal flow filtra-
tion (NFF) for separation of impuri-
ties (viruses, host cell proteins, DNA)
from the process stream; as ultrafiltra-
tion (UF) for concentration of the feed
stream; and as diafiltration (DF) for
buffer exchange. Here is a review of
general guidelines to consider while
scaling down a filtration step. Many of
these guidelines apply to all formats,
but some may be specific for a certain
format. It is up to the reader’s discre-
tion to apply these appropriately.
As with the chromatography steps,
use feedstock from large-scale GMP
manufacturing runs (or equivalent).
Buffers should either be made in
GMP manufacturing or according to
the manufacturing recipes.
The retentate path-length of the
equipment should be the same at
both scales. The Millipore Pellicon
XL format scales from 50 cm 2 to
2.5 m2 and the Pall Centramate scales
from 0.1 to 50 ft2. The type of pump
used should be of the same shear
characteristics. If a low-shear rotary-
Elements of Biopharmaceutical Production Series | 105
lobe pump is used at large-scale, do
not use a high-shear gear pump at
small scale.
Select membranes from a lot that
is representative of full-scale produc-
tion. For new membranes, it is a good
practice to utilize normalized water
permeability (NWP) as a benchmark
for their integrity. Before running the
system, run a baseline of the permeate
at 280 nm and a pressure-hold test to
ensure the system is not leaking.
When making tests, run all opera-
tional parameters at the center point
of the operating range used for the
large-scale filtration. These include:
retentate cross-flow rate, transmem-
brane pressure (TMP), both inlet
and outlet pressures, temperature,
number of diafiltration volumes,
and concentration factor. Crossflow
and transmembrane pressure are the
two main drivers for removal of low
molecular weight solutes and concen-
tration of the protein. Also make sure
the membrane loading (g product/m2
membrane) is the same across scales.
Adequate stirring of the retentate res-
ervoir is important to ensure the dia-
filtration is as complete as possible.
Model Qualification
Pre-determine the acceptance criteria
prior to the scale-down runs. These
can be based on a statistical compari-
son of the small-scale data against the
full-scale data. Be cautious — as the
system dead volume is almost always
larger at the small scale and will result
in lower pool concentrations or lower
step yields. Deviations such as these
should be kept under consideration
when evaluating results from the
scale-down model and also when set-
 LAB SCALE
Dimensions: 1.6 cm x 5 cm (10 mL)
Pool purity: 94% (by AE-HPLC)
Yield: 51%

100

Prepeak Impurity
80
AE-HPLC Purity %

Product
Postpeak Impurity
60

40

20

0
2 6 10 14 18 22 26
Column Fraction

PILOT SCALE
Dimensions: 20 cm x 20 cm (6.3 L)
Pool purity: 99% (by AE-HPLC)
Yield: 61%

100
90
80 Product
AE-HPLC Purity %

70 Impurity
Impurity*
60
50
40
30
20
10
0
1 6 11 16 21

Column Fraction

Figure 2. Example of a Successful Scale-up but an Unsuccessful

Scale-down for a Chromatography Step.4 Feedstock is an aqueous
solution. AE-HPLC = Anion exchange high-pressure liquid chromatography

106 | BioPharm International

Figure 3. Comparison of Performance of a Depth Filtration
Step at Small-scale and Large-scale.5

Elements of Biopharmaceutical Production Series | 107

Table 2. Comparison of Small-scale Disc Format with Large-scale Cartridge Format.6
Aqueous solution feed for a NFF step for viral clearance
Discs
Lot 1 Lot 2
Average Process Flux 75.3 70.6
(L/(m2-hr)
Protein Recovery, percent 98 97
øX174 LRV 4.0 4.2
ting the acceptance criteria.
TMP vs. flux curves are useful to
compare the decay in flux over pro-
cessing time. In cases where the UF
or DF step affects product quality,
a measurement of these key quality
attributes should be included.
Diafiltration efficiency is impor-
tant for DF applications and should
include measurements of diafiltration
volumes vs. conductivity and pH. A
measurement of excipient concen-
trations may be appropriate for final
formulation steps.
Figure 3 illustrates a scale-down
of a depth filtration step. The 23-
cm 2 model closely mimics the
performance at 1.8 m2 in the operat-
ing range with respect to the pres-
sure drop and the filtrate turbidity
vs. throughput.5 Table 2 shows data
supporting the use of filter discs for
scale-down modeling of large filter
cartridges using the øX174 bacterio-
phage model.6 Flux, yield, and viral
clearance are shown to be comparable
across the scales.
Summary
Creation of scale-down mod-
els that can meet the qualification
108 | BioPharm International
Cartridges
Lot 3 Lot 1 Lot 2
74.6 75.1 74.9
98 100 100
4.5 4.0 4.0
requirements can be challenging.
In general, downstream unit opera-
tions are easier to scale down than
upstream ones. Exceptions to this
are protein-refolding unit operations
that may require more extensive
modeling efforts.7 These models are
useful for performing experimental
studies in an efficient and economi-
cal fashion.
However, there are consider-
ations unique to each unit opera-
tion that apply during scale-down.
Con-straints associated with equip-
ment or methodology may prevent
a “true” scale-down of a unit opera-
tion. Dead volumes and hold times
are typically greater at manufacturing
scale. Path lengths of detectors typi-
cally vary between instruments at
the lab and the manufacturing plant.
Wetted materials (gaskets, valves,
and pumps) are not likely to be the
same at two scales. Differences in cas-
sette design between two scales may
impact the outcome of lifetime stud-
ies of UF and DF membranes.
Scale-down studies certainly
increase the chances for a successful
validation campaign, but they only
provide “guidance.” The actual “con-
firmation” must be made at large
scale. The only exception to this is
the viral-clearance validation studies
that are performed at small scale. ◆
References
1. ICH, Quality of biotechnological prod-
ucts: Viral safety evaluation of biotech-
nology products derived from cell lines
of human or animal origin. International
Conference on Harmonization Step 4
Endorsement 1997 March. Available at:
www.fda.gov/cder/guideance/Q5A-fnl.pdf.
2. Yamamoto S, Nomura M, Sano Y,
Resolution of proteins in linear gradi-
ent elution ion exchange and hydro-
phobic interaction chromatography.
J. Chromatogr. 1987:409:101-110.
O’Leary RM, Feuerheim D, Peers D,
Xu Y, Blank GS. Determining the use-
ful lifetime of chromatography resins,
Biopharm Int. 2001 Sept.; 14 (9):10-
18.4.Rathore AS, Velayudhan A. Scale-
up guidelines for preparative chroma-
tography, Biopharm Int’l. 2003 Jan.; 16
(1):34-42.
Originally published in the April 2005 issue of BioPharm International.
Elements of Biopharmaceutical Production Series | 109
3. O’Leary RM, Feuerheim D, Peers D,
Xu Y, Blank GS. Determining the use-
ful lifetime of chromatography resins,
Biopharm Int’l. 2001 Sept.; 14 (9):10-
18.4.
4. Rathore AS, Velayudhan A. Scale-up
guidelines for preparative chromatog-
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(1):34-42.
5. Rathore AS, Wang A, Menon M, Riske
F, Campbell J, Goodrich E, Martin J.
Optimization, scale-up and validation
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August; 17 (8):50-58.
6. Lutz H, Siwak M, Carter J. Viral
clearance performance and regula-
tory requirements, Poster, Biophex/
Interphex, 2002 October 23-24; Santa
Clara, CA.
7. Rathore AS, Sharma A, Zhang RJ, Chilin
D. Scale-down modeling and character-
ization of a protein refolding step, Oral
presentation, 227th American Chemical
Society National Meeting, 2004 March
28-April 1; Anaheim, CA.