Adsorptive and
PROCESSING
chromatographic
separations of life
science molecules.
Part II – Process
design and scale-up *
T
he exercise of designing a
chromatographic separation
technology essentially involves
three steps:
– Selection of media and scouting for
best protocol;
– Optimizing the protocol for purity and
productivity; and
– Scaling up and design of the process
and equipment.
In Part I of this two paper series we
discussed some basic aspects pertaining to
adsorbent media properties and their
bearing on selection for a given application. large diameter short height columns.
Optimizing an operation at
laboratory or semi- Table I – Maximum permissible linear flow velocity
preparatory scale involves for various matrices
optimizing the operation
with respect to flow rate,
pressure drop,
productivity, and
resolution. A high
inter-phase mass transfer
rate is desirable at the
lowest possible pressure
drop. Unfortunately these
two objectives work
against each other. Solute
mass transfer rate from
mobile to stationary phase
is inversely proportional to
square of particle
diameter, and so is the
bed pressure drop. Hence
any attempt to reduce particle size to
improve mass transfer results in increasing
pressure drop and reduction of throughput.
Process design approach to overcome
the above mentioned catch has evolved
several solutions like expanded bed
adsorption, monolithic columns, super
porous beaded adsorbents and
* Part I – This journal – 2005, October,
p. 52-55
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membrane columns (1). The basic idea
is provision of low pressure system that ARVIND LALI 1
at the same time affords large number of SANDEEP KALE 1
UMESH INGLE 1
theoretical plates per unit column height. SILVIA NICOTRA 2
Maximum allowable pressure drop PAOLO CAIMI 2
determines the limiting linear flow MORENO DAMINATI 2
velocity through an adsorbent bed. The MATTEO VILLA 3
larger this velocity the better is the 1. Chemical Engineering Division
adsorbent from a throughput standpoint. Institute of Chemical Technology
Soft gel matrices like those based on Matunga, Mumbai, India 400 019
agarose, cellulose or dextran permit low aml@udct.org
velocities due to their compressible
2. Resindion Srl
nature and hence scale-up results in Via Roma, 55
20082 Binasco (MI), Italy
marketing@resindion.com
3. Hydro Air Research SpA
Strada Provinciale 181, 11
26833 Merlino (LO), Italy
Rigid adsorbents on the other hand,
permit high velocity and thus higher
throughput. Table I gives the comparison
of maximum permissible linear velocities
on different types of resins used in Part I – Selection
preparative scale chromatography. of adsorbent
Solute mass transfer rate is reflected
Dynamics of adsorption -
in HETP, lower HETP signifying faster
Thermodynamics and
rates of mass transfer. Low HETP bed will
fluid dynamics
not only give sharp breakthrough profiles
and higher dynamic binding capacity, but Sepabeads® media
also result in sharper peaks and Media selection
increased resolutions. However, as Summary
NOVEMBER/DECEMBER 2005 37
pointed above, EXPANDED adopts a combination of both approaches
HETP is directly BED since using mere one of the two
related to particle ADSORPTION approaches has been observed to cause
diameter, and also bed instabilities leading to deterioration of
increases with flow Sepabeads® EB bed performance. Figure 3 gives the bed
rate. Figure 1 is a range of expansion characteristics of
typical variation of adsorbents Sepabeads® EB in comparison with other
HETP with linear (Figure 2) have available matrices.
mobile phase been developed in It generally appears that EBA
velocity. It is particle sizes that technology has not achieved the
important to note make them useful potential that was once considered
that an increase in for both packed possible. A number of reasons may be
linear velocity does Figure 1 – Variation of HETP with flow velocity. bed and given. One reason seems to be the lack
not result in For a given CV/hour it is always safe to increase of variety of adsorbents intended for
expanded bed
linear velocity for scale-up and still have
increased HETP to operations. EBA EBA. An EBA adsorbent is given to far
reasonable number of N
the same degree. (Expanded Bed rougher environment than a packed bed
This implies that a column can be Adsorption) is useful when adsorption can adsorbent. Thus a rigid adsorbent may
operated at increased velocity for higher be possible directly from the particulate be preferred. Secondly, it appears that
throughput with lesser corresponding loss matter containing crudes without considerable care needs to be exercised
on number of plates. Hence, an intervening solid-liquid separation steps to ensure stable bed throughout the
important scale-up sequence of operations.
criterion can simply be An expanded bed can
to use higher bed destabilize either due to
height at highest incorrect hydrodynamic
allowable linear flow design of the column,
rate for the adsorbent and/or due to
in use. Naturally, rigid operational
adsorbents thus can discontinuities. In
be used with higher collaboration with
bed heights giving Hydro Air Research and
required number of Resindion, we have
plates at higher been into development
throughput in column of a column design with
diameters lesser than associated
those required for soft instrumentation that not
gel adsorbents. Figure 2 – Adsorbent range from some of the manufacturers. only provides steady
Both column Sepabeads® (Resindion); Toyopearl (Tosoh Bioscience); EMD (Merck): Acrylic state bed stability, but
performance polymers; Sepharose (GE Healthcare): Crosslinked agarose; Streamline also ensures stability
parameters HETP and (GE Healthcare): Agarose coated on quartz (for expanded bed) against ‘disturbances’
dynamic binding [From Part I] arising from switching
capacity are seriously feeds into the column
affected on scale-up unless proper like centrifugation and/or membrane and those arising from variation in feed
guidelines are followed. As mentioned in filtrations besides permitting high flow properties and flow rates.
Part I, HETP is a strong function of pore rates without the problem of back pressure The evolved EBA equipment design
diffusion. Small molecules like antibiotics (2,3). An adsorbent intended to be used and adsorbents has enabled us to
diffuse at high rate even in adsorbent pores in expanded bed format needs to meet develop cost effective single step
compared to diffusion rates of some additional criteria in addition to purification strategies for low price bulk
macromolecules like proteins and DNA. those required for traditional preparative products like penicillin G and rifamycin
Thus scale-up for adsorption of small scale adsorbent matrices. Besides being directly from their respective
molecules, e.g. antibiotics, can be based on hydrophilic and rigid, adsorbents for EBA fermentation broths.
mean residence time in terms of column should be heavier
volumes per hour. On the other hand, so that the bed not
macromolecules move rather sluggishly only permits high
and have serious effect of linear velocity on flow rates without
HETP, DBC and resolution. Scale-up unduly large bed
procedure for macromolecular expansion, but also
chromatographic separations therefore allows the
follows the dictate of linear velocity suspended matter
similarity. in the feed to flow
Synthetic polymer based through without
Sepabeads® range of adsorbents forms a retention. An EBA
useful platform for developing separation matrix can be made
technologies in the form of rigid and heavier by either
robust adsorbent matrices. Available in employing larger
different particle sizes they are suited to size, or using
scale-up from litre level to thousands of denser matrix, or
litres level. Further, availability in three both. Figure 3 – Bed Expansion with linear superficial mobile phase
mean pore sizes also make them flexible Sepabeads® EB range velocity (distilled water) for Streamline DEAE (Amersham
for a variety of products from small of adsorbents Biosciences), Sepabeads® EB-DA2 and Sepabeads® EB-DA3
molecules to large proteins. designed for EBA (Resindion Srl)

38 NOVEMBER/DECEMBER 2005 DOWNSTREAM PROCESSING

Table II – HARSep Pilot Plant Unit Specifications with
HEBA G225 or S225 column
DESIGN AND SCALE-UP
OF PACKED BED
AND EXPANDED BED ADSORPTION
Ironically, scale-up of expanded bed is
more constrained compared to packed
bed on account of bed expansion and
linear velocity limitations. Within this
constraint, tall columns for small
molecules and large diameter short
columns for macromolecules are
accepted practice in plant scale
operations for both packed and
expanded bed operation. Flat flow
distribution of liquid or crude entering
the column bed is a very essential
requirement for good column
performance. In addition, uniformity of
packing throughout the bed diameter
and height is also necessary for fault
free operation. Rigid adsorbent matrices
again are useful in the respect that they
permit packing procedures that are easy
to perform and reproduce without
damage to the adsorbent. A number of
column designs are on the market from
different companies some of which also
have presence in the adsorbent market.
Considerable work at our Institute, and
then in collaboration with Resindion Srl
and Hydro Air Research SpA, has lead
us to design column hardware for both
packed bed and expanded bed format
of adsorption chromatography. Packed
bed and expanded bed purification
technologies have been developed for
molecules from penicillin G, rifamycin,
vitamin B12, and β-carotene to proteins
like lactoferrin, lactoperoxidase,
lysozyme, bromelain, insulin etc. Hydro
Air Research offers laboratory scale to
plant scale column chromatography
setups complete with column, pumps,
piping system and measurement and
controls. These ready to use set ups
have been helpful in developing the
technologies and scaling them for plant
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level operations. requirements. Figure 5 presents a picture of
Table II presents the peak profile that may be obtained using
details of a a non-binding tracer molecule. The method
standard pilot basically comprises injecting a finite pulse
scale expanded of a non-binding tracer into the mobile
bed set up that phase (normally the equilibrating buffer)
also serves well and recording the peak profile. Analysis of
for any packed peak profile can yield the following useful
bed operation in validating parameters (1,4):
both up flow – Mean Residence time (or peak time)
and down flow – Dimensionless Variance
modes. – HETP
Figure 4 is the – Peak skew ness
schematic flow – Peak asymmetry
diagram of the – Tailing factor
set up. Major Table III gives the formulae that can
issues with yield these parameters. A similar analysis
scaled-up of the product peak emerging from the
columns relate column in production cycle can also be
to their used as validating parameter. In addition,
hydrodynamic the Resolution parameter Rs (also given
performance in Table III) also quantifies the column
with respect to performance. Any variation in these
– Uniformity of packing density; parameters between runs can be
– Flow distribution (especially in large regarded as a matter of concern and
diameter columns); and result of faulty column performance. A
– Deterioration of packing over time. faulty column performance should lead
There are several testing procedures for to aborting the run and emptying,
estimating these parameters recommended cleaning and repacking the column.
by column or media manufacturers. The It is essential that a column when
final choice rests with the user and it is packed and operated gives a performance
useful to lay down SOPs for determination that is consistent over a large number of
of the above three aspects. Two simple and repeated cycles. There can be occasional
very useful methodologies are deterioration of the column performance
– Devising non-binding tracer method to which is unacceptable from regulatory
check column performance in between point of view. There is therefore a need to
runs; and adopt procedures that quantify column
– Devising an in built performance performance from time to time not only
evaluation method that quantifies each in terms of production related figures but
successive runs made on the column. also through the diagnostic parameters.
Both these can be built into the system The performance parameters listed in
data logs to comply with regulatory Table III are a good measure of column
Figure 4 – Schematic Flow Diagram of the HARSep-HEBA G225 Pilot Scale Expanded / Packed
Bed plant. Several on-off valves control the direction of the flow upward or downward through
the column and to different outlets
NOVEMBER/DECEMBER 2005 39
Table III – Parameters for chromatographic
peak analysis
behaviour during operation as well, and
can be used in analyzing product peak
profiles.
In large columns use of small particle
size adsorbent is more likely to result in
variation of performance between
subsequent packing. Hence, larger particle
size may be preferred even at the expense
of the lower efficiency in terms of HETP.
40 NOVEMBER/DECEMBER 2005
Typically, a 300 µm mean
particle size is considered
good for scale-up and can
give up to 500 plates/m
bed. Again, as mentioned
earlier larger bed depths
required for such
adsorbents necessitate
that the adsorbent matrix
is stable under its own
weight. Soft gel matrices
can cause more
problems
compared to rigid
beads like
Sepabeads®.
SUMMARY
Scale-up and
design of plant
scale column
chromatography
can follow different
Figure 5 – Peak profile obtained using a non-binding
guidelines for
tracer molecule (or during product elution)
small molecule
adsorption and
macromolecular
adsorption.
Macromolecule
adsorption is far more
diffusion limited than
small molecules, and
hence while small
molecule
chromatography can be
scaled up using simpler
mean residence time
criterion, macromolecule
chromatography required more careful
scale up. Larger the product molecule
(e.g. pDNA) larger is the effect of fluid
dynamics on binding capacity and peak
resolutions.
Production scale packed and
expanded bed adsorption columns
need specific attention to design details
and use of methods to check and
quantify column performances between
and during the runs. Distributor designs
and operation protocols with respect to
stream switching and changes in flow
rate need special attention to details.
Chromatographic assembly hardware
thus should not only be designed well,
but must also be supported by
adequate software to operate, control
and quantify its performance both
during a run and in between runs.
REFERENCES
1) LIGHTFOOT E.N., MOSCARELLO J.S., TEETERS
M.A., ROOT T.W. “Interaction of Mass
Transfer and Fluid Mechanics” in Scale-up
and Optimization in Preparative
Chromatography; Rathore A.S., Velayudhan
A. Eds.; Chromatographic Science Series,
Marcel Dekker, 2003
2) CHASE H.A. “Purification of Proteins by
Adsorption Chromatography in Expanded
Beds” Trends Biotechnol. 1994, 12,
296-303
3) LALI A.M. “Expanded Bed Affinity
Chromatography” in Methods for Affinity
based Separations of Enzymes and
Proteins; Gupta M.N. Ed.; Birkhauser Verlag:
Basel, 2002
4) RATHORE, A.S., KENNEDY R.M., O’DONNELL
J.K., BEMBERIS I., KALTENBRUNNER O.,
“Quantification of a Chromatographic Column,
Why and How to Do It” Biopharm
International 2003, March 30
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