Department of Chemical & Biomolecular Engineering

National University of Singapore

CN2102 Chemical Engineering Principles and Practice II

Studio No: #3
Product Purification

Group No.: BT3 Group 3

Group Members
Name Matric No
An Soobin A0245996H
Chattopadhyay Aditi A0245902H
James Clement Lim A0245631J
Lim Yee Hao A0240875Y
Phia Kai Jie Kenyesse A0240205Y
Objectives: Purification is an essential process used to remove contaminants from the desired product
of interest. Examples of purification techniques include filtration, crystallization and chromatography. In
this studio, the method of affinity chromatography is used, and the aim of this experiment is to assess
its effectiveness in extracting and purifying Green Fluorescent Protein (GFP) at 25OC and 37oC by
comparing the binding and separation efficiencies of the process at different stages. By doing so, its
application in bioprocesses will be analyzed considering factors such as yield, purity, time, and cost.
Results and Discussion:
Affinity chromatography is a protein purification method based on specific binding interaction between
an immobilized ligand and its binding partner. In this studio, the ligand selectively binds GFP (product of
interest) which are His-tagged. Table 1 below compares the advantages and disadvantages of other
possible methods of protein purification.
Table 1: Advantages and Disadvantages of Various Separation Methods

There are various factors to be considered when choosing the appropriate separation method, for
example, the degree of product purity, available budgets (costing) and raw materials needed during the
process. To measure the level of purification, specific enzyme activity can be measured (Földesi, 2019).
This is calculated by dividing the total enzyme activity by the total quantity of protein present. A pure
protein will give a value of 1. Furthermore, high percentage yield may not mean high percentage purity.
To ensure that the method is efficient and effective, a balance must be reached. An optimal balance
between reagent and operating cost, and yield can also be determined by calculating the cost of
purification per percentage yield. This allows large-scale processes to remain cost-efficient.

To evaluate the yield of the purification, the fluorescence intensity was measured over various stages of
the experiment. The supernatant was first drained through the Biorad column containing activated
HisPur Ni-NTA resin. The activated resin due to the binding buffer contained nickel ions (Ni2+) which
retained the histidine tag protein in the column. Since other proteins with similar property also bind and
remain in the resin, an elution buffer of imidazole was utilised – so that only the pure GFP protein is
displaced while leaving other proteins behind in the column. Hence, the column was once again
centrifuged to drain the elution buffer through the column to elute the bound protein. The previous
step was repeated to elute the remnant left in the column.

The evaluative data were calculated using the following equations:

- Protein concentration (nM) = Fluorescence (AU)
-
 -

-
Table 2: Evaluation data of raw results collected of samples at various stages of experiment
Supernatant Flow-through 1st Pass 2nd Pass
Label A B C D E F G
Temperature (°C) 25 25 37 25 37 25 37
Volume (μL) 300 1200 1200 600 600 600 600
Fluorescence (AU) 9282 3405 5069 8431 6500 2048 1458
Concentration Of GFP (nM) 116.03 42.56 63.36 105.39 81.25 25.6 18.23
Mol of GFP (10-11) 3.48* 5.11 7.60 6.32 4.88 1.54 1.09
Binding (blue)/ Separation (green) efficiency (%) N/A 63.3 45.4 71.7 77.2 17.5 17.2
Overall Pass Separation Efficiency (%) T1(25°C) = 89.22, T2(37°C) = 94.46
Overall Yield (%) T1(25°C) = 56.47, T2(37°C) = 42.89
*The number of moles in A were standardized to the other samples due to the volume difference.
Hence, the number of moles was multiplied by 4 (13.92 mol) to make the mole values comparable to
those with volumes of 1200μL.

As seen from the results in Table 2, a higher temperature results in a greater separation efficiency.
However, at higher temperatures, the binding efficiency and overall yield decreases. Furthermore, the
separation efficiency significantly decreases between Pass 1 and Pass 2. Samples B and C are the values
generated from the flow through of the HisPur Ni-NTA resin, hence some of the GFP protein were bound
to the resin – resulting in a lower protein concentration than A. Samples D and E are eluted from the
first pass, where most of the GFP-binded to the resin is released. Therefore, samples F and G are not
able to elute as much as protein compared to samples D and E, as shown by the significantly smaller
separation efficiency for Pass 2. The opposing trend between temperature-efficiency and temperature-
yield implies the importance of the balance between the three components. Moreover, while the yield
inevitably increases with greater number of runs of passes, factors such as purity, time, and cost cannot
be ignored. For example, the high percentage yield in protein may lead to low purity due to the dilution
from buffer solutions. In addition, if the experiment was to be rescaled to match industrial processes,
the amount of energy and time required to elude the protein may not be economically desirable due to
the decreasing marginal yield per successive elusion.

Besides the yield of the purification, it is important to consider the purity of the protein which can be
calculated using the SDS-PAGE system which allows the separation of protein by its weight. However, a
high percentage yield does not guarantee a high percentage purity. In the pharmaceutical industry, high
purity is critical as a contaminated drug may harm patients. In addition, a high protein functionality is
important for a product’s efficacy. This can be measured by comparing viscosity, emulsification,
foaming, whipping, and many more properties. In addition, there are other important components for
instance, time, cost, and safety which also should be considered when choosing the separation
technique. Time can be quantified by the product’s processing efficiency throughout the experiment.
Cost is quantified by fixed costs of equipment, manpower, and profits from the product. Lastly, possible
contaminants that are present in the product should be dealt thoroughly to minimize the dangerous
effects to humans and the environment.
References

1
Amersham Pharmaceia Biotech. (n.d.). Hydrophobic Interaction Chromatography. Principles
and Methods. Available at: https://jcsmr.anu.edu.au/files/hic_handbook.pdf [Accessed
February 15, 2022].
2
Aryal, S. (2019, April 29). Gel Permeation Chromatography. Microbe Notes. Available at:
https://microbenotes.com/affinity-chromatography/#limitations-of-affinity-
chromatography [Accessed February 15, 2022]

3
Aryal, S. (2021, November 3). Affinity Chromatography- Definition, Principle, Components,
Steps, Applications. Microbe Notes. Available at: https://microbenotes.com/affinity-
chromatography/#limitations-of-affinity-chromatography [Accessed February 15, 2022]

4
Bio-Rad Laboratories. (n.d.). Ion Exchange Chromatography. Available at: https://www.bio-
rad.com/en-nl/applications-technologies/ion-exchange-
chromatography?ID=MWHAY9ESH [Accessed February 15, 2022]

5
Földesi, B. (2019, March 11). Guide to Enzyme Unit Definitions and Assay Design. Biomol
GmbH - Life Science Shop. Available at: https://www.biomol.com/resources/biomol-
blog/guide-to-enzyme-unit-definitions-and-assay-design [Accessed on February 15, 2022]

6
Gottschalk, U. (2011). Overview of Downstream Processing in the Biomanufacturing Industry.
Comprehensive Biotechnology, 669–682. https://doi.org/10.1016/b978-0-08-088504-
9.00238-5

7
Merck. (2021). Performing a Purity and Homogeneity Check. Available at:
https://www.sigmaaldrich.com/SG/en/technical-documents/technical-article/protein-
biology/protein-purification/purity-homogeneity-check [Accessed February 15, 2022].

8
What is HPLC. (2020). Advantages and Disadvantages of Affinity Chromatography. Available
at: https://whatishplc.com/hplc-basics/advantages-and-disadvantages-of-affinity-
chromatography/ [Accessed on February 15, 2022]

9
Xiong, Youling. (1997). Protein Denaturation and Functionality Losses. Available at:
https://link.springer.com/chapter/10.1007/978-1-4615-5975-7_8 [Accessed February 15,
2022].