Introduction

In this exercise, we had been asked to complete the process from previous exercise which is
purification of product from a fermenter in order to extract MAB crystal using EBA
chromatography and then followed by freeze dried.
Theory
Chromatography refers to a set of science techniques which used to distinguished different
compound based on their structure and /or composition. The word was first derived from Greek
chromatos (color) and graphein (to write). In short, chromatography means separating chemicals
and identifying them by color. It capable to separate a mixture into its components with great
precision for example it is used to separate between two very similar components like protein
that may be different only by a single amino acid and also with right materials and operating
conditions, it capable to purify any soluble or volatile substance. Chromatography comes in
many forms such as expanded bed chromatography and hydrophobic interaction
chromatography. expanded bed adsorption chromatography is a single pass operation where
desired proteins are purified from crude, particulate containing feed-stock without the need for
initial purification. An upward liquid flow is applied to column while streamline adsorbent is
expanded and equilibrated. Balance between particle sedimentation velocity and upward liquid
flow velocity is achieved when a stable fluidized bed is formed when adsorbent particles is
suspended in equilibrium. The column adaptor is positioned in the upper part of the column
during this phase. The feed is applied to expanded bed with the same upward flow as used during
expansion. This result in the target proteins are being bound to adsorbent while cell debris, cells,
particulates and contaminants pass through unhindered which then being washed out from
expanded bed using upward liquid flow. Adsorbent particles then quickly settle in the column.
The column adaptor is lowered to surface of sedimented bed. By using suitable buffer conditions,
captured proteins are eluted from sedimented bed. This happen when flow is reversed. The eluate
contain target protein, later then increased in concentration, clarified, partly purified and ready
for further purification by packed bed chromatography. Next, the bed is regenerated by washing
it with downward flow in sedimented bed mode using buffer specific for the type of
chromatographic principle applied. This result in more strongly bound proteins which are not
removed during the elution phase. Then cleaning-in place procedure is applied to remove non-
specifically bound or denatured substances from the bed and restore it to its original
performance. At this phase, a moderate upward flow isused with column adaptor at
approximately twice sedimented bed height.

Figure 1. Schematic presentation of the steps of expanded bed adsorption.
(Adapted : http://wolfson.huji.ac.il/purification/PDF/Expanded_Bed_Absorption/PHARMACIA_IntrodEBA.pdf)


Methodology
1. Firstly, mode of operation is set to batch so all stream flow are displayed on a per-batch
basis. Every unit procedure involved in this process are created like fermentation,
centrifugation, EBA Cromatography, Crystallizer and Freeze dryer. New components
involved are added such as urea, glycine and methanol at pure component in toolbar
Task.
2. Secondly each of the equipment, their operation data is registered. At each unit
procedure, all substance input and output are labeled correctly. For centrifugation, the
time for centrifugation is set for 6 hours for solids removal and the relative to another
operation in another procedure is set from P-1(in FR-101) and operation is set to
FERMENT-1.
3. Third, operation data for EBA Chromatography is registered which are EQUILIBRATE,
LOAD, ELUTE and WASH and filled with experimental data provided correctly. At
equilibrate, button of relative to another operation in another procedure in scheduling part
is clicked and P-2(in DS-101) and operation (CENTRIFUGE-1) is selected and last start
button is clicked. At operation condition, (In #1: acetone) is selected at inlet stream and
bed volume of glycine is set to 2 BV and output stream is zero and (Out #5 :waste 2) is
selected at outlet stream. Next operation button is clicked LOAD-1(EBA Column
Loading) appeared. At waste in operation condition part, outlet stream (Out #1: waste 1)
is selected. Next at resin binding part, 100% binding and 99% yield of MAB is set and
calculated.
4. Besides, at crystallizer part, TRANSFER-IN-1(Transfer in) is selected. Transfer in using
(In #1:S-103) is clicked. Button Set by Master-Slave Relationship is clicked and
operation WASH-1 and in Procedure P-3(in C-101) is chosen. At CHARGE-
1(Charge), (In #:S-105) is chosed and at amount part button Set by User is clicked and
0.01 kg mass of methanol is set. At REACT-1(Batch Stoich.Reaction), at thermal mode
part, button Set Final Temperature is clicked and 5C value of final temperature and
heating agent(NaCl Brine) is set.Sceduling tab of REACT-1, CHARGE-1 and END
button is clicked at reference part.
5. Then at TRANSFER-OUT-1(Transfer Out), (Out #9:S-104) of Transfer out Using is
selected to transfer 100% of product from crystallizer to freeze dryer. Sceduling tab of
Transfer Out, REACT-1 and END button is clicked which is correspond to Relative to
Previous Operation in this procedure at reference part. Set time to 12 hours for reaction to
occurred.
6. Next at freeze dryer part, Set by User button is clicked and Methanol is clicked at
volatile browse for 100% removal and OKbutton is clicked. Finally RUN button is
clicked for checking either the design is successful or not.

Result & Discussion
For the question one, EBA Chromatography needed for this production is one which is
economically. This is because it give higher levels of purity of product and capable to purify
volatile substance contained in this process which is 100% of methanol is able to removed
completely. For second question, MAB Crystal produced is 899.498543 g/L and it gives 100%
purity of product. Last question, for me to design new plant which is economically feasible and
production of desired product could be increased, I supposed that to increase chromatography
unit in my plant subsequently do not use centrifugation unit. This is because from my self-
research done previously, I found that EBA chromatography capable to separate and purify the
product themselves and more efficient and precise than centrifugation. However due expensive
price of a unit chromatography, I thought that I will include two unit of EBA chromatography in
my plant so that when I increase amount of feed enter at fermentation unit, I will be able to
obtain double amount of desired product at EBA chromatography. This is due to EBA
chromatography itself which plays important role in this purification process. Thus, I thought
this plant is economically feasible as by do not use centrifugation unit, the budget for purchasing
it can be used to purchase other more profitable unit operation like EBA chromatography and it
may increase our profit.

Figure 1 show new design for economically feasible plant.


Conclusion
Therefore, by using powerful software like Super Pro Designer, a complex process with a
multiple unit operation to operate, the process is possible to operate precisely and correctly.
With a good purification unit process like EBA chromatography used to purify the MAB
accompanied by other unit operation like fermentation, centrifuge, crystallizer and freeze dryer.

References
1. Expanded-Bed Adsorption.Randall Willis.@2000 American Chemical Society.Retrieved
at 6
th
April 2014 from
http://pubs.acs.org/subscribe/archive/tcaw/09/i11/html/11instru.html
2. Introduction to Expanded bed Adsorption.Retrieved at 6
th
April 2013 from
http://wolfson.huji.ac.il/purification/PDF/Expanded_Bed_Absorption/PHARMACIA_Int
rodEBA.pdf