Applications of Simulated Moving

Bed Chromatography in Bioindustry

Abhishek Choudhary, Ramratan,

Sambhav Mattoo, Tage Jigi
Principle of SMBC
● SMBC is a type of chromatography that enables substance
mixtures to be continuously separated and extracted in two
fractions. Its efficiency is significantly higher than batch
chromatography, through better utilization of the column
stationary phase.
● The basic concept of simulated moving bed chromatography is to
use multiple smaller columns containing the solid adsorbent beds
rather than one large column, and to achieve a countercurrent
flow by switching the inlet and outlet streams periodically, rather
than flowing fluids through one static bed.
● A “true” moving bed chromatography is practically not possible
because the stationary phase has to move with solvent which is
mechanically strenuous.
● Further, to overcome the problem of mixing of peaks when a
single column is used, we use multiple columns and can elute
from each.
● Feed is added at the junction. The junction provides a place to
connect valves which allows us to change inlet-outlet flow with
fluid direction.
● This can also be done in circular form where the direction of
solvent and the column are in opposite direction; it can also be
done by rotating the inlet and outlet.
Pros and Cons of SMBC
Advantages
● A continuous process with
higher yield than the batch
systems.
● Lower solvent utilization and
cost effective.
● High recovery and purity with
low labour.
● Economical for large scale
separations.
● Only partial separation of
solutes is required to obtain
high purity.
Disadvantages
● Only binary separations
possible.
● Complex transient and cyclic
steady state phenomena.
● Large number of system &
operating parameters to be
optimized.
● Equipment more complex than
batch versions, with few options
for multi-component
separations.
● There is more control of batch
identity and residence time.
History of SMBC
● Invented in the 1950’s by Broughton and colleagues at UOP for large-scale separation of n-paraffins.
● Originally developed for petrochemical separations which were not possible using distillation.
● First used in bioindustry for enantiomeric and chiral drug separation.
● Two companies in particular (UCB Pharma, Belgium; Daicel Chemical, Japan) have been operating production SMB units
since 1998, with a capacity of several tons per year of pure enantiomers from two different chiral drugs.
● Various other SMBC designs have been adapted for more complex separations such as for peptides and fermentation
broths.

Variations of SMBC
● Classical SMBC is operated in isocratic mode and utilizes true countercurrent separation. It is characterized by continuous
flow of a single solvent between separation zones defined by Feed and Desorbent inlets and Extract and Raffinate outlets.
This mode is extremely effective at separating closely-related molecules, such as glucose from fructose, with high purity
and yield.
● Step mode is characterized by operation of multiple distinct zones having different solvent conditions in a “bind-wash-elute-
clean-equilibrate”, or step gradient sequence, which requires more pumps, inlets and outlets than isocratic mode and
enables easier control over the flow rates in each zone. Step mode is especially effective in biopharmaceutical applications,
such as downstream processing of monoclonal antibodies, recombinant proteins, and vaccine components.
● A variation of the simulated-moving-bed (SMB) operation, is called Enriched Extract SMB (EE-SMB). In this technique portion
of the extract product is concentrated and the resulting enriched stream is re-injected into the SMB. This operation has
been recently patented.
Application 1: Protein Purification
● Proteins have, to date, only rarely been purified by SMB.
The first attempt was made by Huang et al. in 1986 to
isolate trypsin from porcine pancreas extracts using an
SMB made of only six columns, showing that a system with
a limited number of columns can be efficient.
● A separation of myoglobin and lysozyme has been
presented by Nicoud. This purification was performed on
SMB containing 8 columns using ACA 54 (Biosepra,
France) as support. Very pure extracts (>98%) and
raffinates (>98%) were obtained from a 50–50 mixture.
● One of the most difficult biochemical separations to after it. These two fractions are then processed in SMB
perform is associated with obtaining cyclosporine from systems packed with reversed-phase material or with
fermentation broth. This purification is tough because of normal silica. This coupling and the choice of the
the number of products closely related to cyclosporine A stationary phase, the retention orders as well as the
and because of significant kinetics limitations due to the composition of fractions enables cyclosporine A to be
size of the molecule. A silica based batch system is used obtained at the raffinate. Both SMB steps are performed
to make two pre-purified fractions, the first contains on a LICOSEP 8 X 50 (NOVA-SEP) equipped with eight
cyclosporine A and impurities that elute before it, and the axial compression columns (100m length X 50mm i.d.).
second fraction contains the same, and those that elute
Case Study: Whey protein concentrate
● Whey protein concentrate (WPC) is a whey product, produced by
treating whey by filtration to remove fat particles and membrane
filtration to remove water and lactose.
● Lactoperoxidase and lactoferrin are two individual whey proteins with
interesting biological properties, considered having commercial
potential as value added products.
● Chromatography columns (Ace Glass, USA), 10 ml column volume, 11
mm I.D. packed with Streamline-SP were used for the SMB runs.
 Case Study: Insulin Purification
● An early successful protein-separation by SMB is the purification of HSA using
two SMB-systems connected in series. The first SMB was used for removing the
less strongly retained components and the second one for removing the more
strongly retained components of the sample matrix.
● A similar technique is used in applying SMB for insulin purification, for the
separation of insulin from two impurities, high-molecular-weight proteins
(HMWPs) and ZnCl2.
● Insulin can be separated either from HMWPs or from ZnCl2 in the first ring.
Because HMWPs are unstable, they should be removed in the first ring.
● Comparison with column based method shows a 48% raise in productivity, a 4.3
times decrease in buffer consumption and 6.5 times raise in target protein
concentration.
Case Study: Monoclonal Antibodies
● The chromatographic support used for immobilization
was Cyanogen bromide-activated Sepharose 4 Fast
Flow from Pharmacia (Uppsala, Sweden).
● Several monoclonal antibodies (mouse IgGl ) were
prepared against penicillin amidase (PA) from E. coli
culture. One was found to be suitable for the bioaffinity
chromatography of PA.
● Affinity chromatography is used in SMBC. It is a highly
selective separation method, the solid phase adsorbs
the desired protein specifically.
● Since adsorption and desorption of the desired
product has to be performed under different
conditions, the SMB device has to consist of at least
two zones (adsorption and desorption zone). zones between adsorption/desorption steps could be introduced to
● Most of the impurities were eluted within the first increase the column yield.
minutes of each switching interval. A higher specific ● By adding two purge steps to the SMB system monoclonal
activity was achieved by wasting the extract eluted antibodies could be isolated directly from cell culture
within the first min of each switching period. This supernatant with a yield of >-90%.
resulted in a decreased product recovery. Two purge
Application 2: Desalting (Separation of
NaCl from Glucose)
● Desalting is another simple and interesting application of SMB in biotechnology.
● It occurs when buffer salts and other small molecules are removed from a sample. It provides fast
and simple way to purify biomolecules (proteins, nucleic acid etc.) away from salts and small
molecules.
● Glucose and NaCl have been separated from feed mixtures containing same amount of Glucose
and NaCl using a retardation 11 A-8 resin in four-zone moving-bed adsorber.
● Low molecular weight ions (here NaCl) are retarded by 11 A-8 resin by interaction with pairs of
adjacent fixed anion and cation exchange site.
● Elution is carried out with water with relatively little dilution of high molecular weight solutes which
are not retarded.
● Retarded NaCl is collected from the extract stream and glucose is collected from the raffinate
stream.
For the separation of glucose and NaCl, four zone
moving bed adsorber is used. Direction of feed and
adsorbent is shown in the diagram. When feed of
glucose (less adsorptive) and NaCl (adsorptive)
mixture is applied, they separated by the moving bed
adsorber. In zone 1, glucose remaining in the fluid
stream and when it moving towards zone 2, it Concentration profile of NaCl and
moving out from raffinate point. In zone 1 & 2, NaCl glucose in SMB adsorber
remained tightly adsorb and it desorbs by desorbent
when it arrives at zone 4 and extracted out.
Application 3: Separation of Fructose
from Date Syrup
● The high fructose syrup is widely used as nutritional sweetener because of its high quality characteristics
such as sweetness, flavor enhancement etc.
● In most of the commercial methods, the high fructose syrup is produced from corn but it can also
produced by date using chromatographic separation process.
● Date syrup contains 39.63% glucose, 33.68% fructose and its sucrose was low.
● The separation is preferably performed on an ion-exchange resin (polystyrene based Dowex Monosphere
99 cation exchange resin in Ca2+ form has particle size of 300-350 um and heat tolerance upto 1200 C.),
and using warm water as eluent.
● The adsorptive separation of glucose and fructose depends on the selective complexing of fructose by
Ca2+ ions, which results in the retardation of fructose while glucose is carried away by mobile phase.
● In order to achieve separation, the rate at which the ports are advanced must be faster than the velocity
of more strongly adsorbed (fructose), but slower than the velocity of less strongly adsorbed (glucose).
● The strongly adsorbed component (fructose) is collected in the extract stream whereas a weaker
adsorbed component (glucose) is collected in the raffinate stream.
All experimental test parameters sugar concentration, flow
rate, temperature were found affect the fructose
concentration. Fructose % increased with increase in sugar
concentration. The higher separation performance is
obtained with date syrup concentrated at 50% which we
obtained a fructose concentration higher than 67%.
Application 4: Separation of Amino Acids
● Glutathione is a tripeptide compound consisting of glutamic acid
attached via its side chain to the N-terminus of cysteinylglycine.
● L-Glutathione is produced by yeast fermentation and serves as a
therapeutic in certain liver diseases.
● L-Glutathione of at least 99% purity is required in the final
crystallization step of the pharmaceutical production process.
● It is difficult to obtain an L-Glutathione of such high purity especially
with regard to the amino acids impurities present in the original
fermentation broth, the most challenging among them being the
separation of glutamic acid, which impedes crystallization.
● A cation exchange resin (Amberlite IR200C, 350-590 um) was used.
It was implemented in a 16 column - SMB.
● Pyrex glass columns were used, 20 cm in height, 1.05 cm I.D., with
HCl as a desorbent
● The Glutathione was obtained in the raffinate stream at 99% purity
with 99% yield.
Application 5: Separation of Chiral Drug
Molecules
● The separation of optical isomers is a very typical yet challenging two component separation encountered in the
pharmaceutical industry.
● Since the optical isomers are chemically equivalent, most chemical syntheses result in racemic mixtures. The biological
activities of the two isomers may differ drastically and an efficient separation is necessary.
● The two first references disclosing the use of SMB for performing optical isomers separation were published in 1992 by
Negawa on phenyl-ethyl alcohol and Fuchs on threonine.
● The first real industrial example has been made by Sandoz. The enantio-purification of the (±)- 1a, 2, 7, 7a -tetra hydro 3 -
methoxy napht ( 2 , 3 - b) -oxirane (SANDOZ Pharma) is important because this epoxyde is a valuable intermediate in
enantioselective synthesis. The separation was performed using microcrystalline cellulose triacetate (C.T.A.) from
MERCK KGaA (Darmstadt, Germany) as stationary phase and pure methanol as eluent.
● Particular examples for the separation of optical isomers in the (pharmaceutical) industry using SMB include
prazinquatel, b-blockers, thiadiazin and hetrazipine.
● The first demonstration of large scale separations of optical isomers with SMB was performed by SEPAREX
Chromatographie in 1994, on the LICOSEP 8-200, which led to the separation of 2 kg of racemic binaphthol per day on a
Pirkle type 3,5-DNBPG-Silica CSP (MERCK KGaA, Germany).
● Various companies such as Daciel, Nissan Chemicals and UCB Pharma now use SMB for the mass production of
undisclosed pharmaceutical products.
Future developments in SMBC and its
Applications
● SMB with Supercritical CO2 and Eluent Strength Modulation: A lot of organic solvents have been
disallowed for use in the pharmaceutical industry, due to toxicity and environmental concerns, and
CO2 is a good substitute, as it is cheap, non toxic, and its polarity, and thus eluent strength can be
changed by changing pressure. Combining SCFC with SMB would also allow us to have a spatial
gradient in the system.
● Integration of SMB systems with a Solvent Recycling System, in Pharmaceuticals (SANDOZ), so one
does not have to invest in buffer tanks.
● The coupling of SMB with enantioselective crystallization, as done on Chiral Esters with LICOSEP
systems.
● Use of computer simulations to optimize the complicated SMB systems.
● New testing methods to efficiently find the loading capacity of stationary phase, to optimize QC and
reproducibility.

…..among other general developments in SMB.

Thank You!
11 - 11 - 2019
BSE492A