INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

PHARMACEUTICAL
BIOTECHNOLOGY
LAB REPORT

Member:

1. L Ngc Mai BTBTIU13104

2. V on Vn Trang BTBTIU13209

3. Phan Th Mai Uyn BTBTIU13262

4. V Ngc Lam Phng BTBTIU13143

TABLE OF CONTENT
Chapter I: Production of proteins of pharmaceutical
biotechnology

Lab 1: Upstream processing

Lab 2: Cell extraction

Lab 3: Concentration and Dialysis

Lab 4: SDS-PAGE Electrophoresis analysis

Chapter II: Formulation and preparation for

pharmaceutical products

Lab 5: Formulation and preparation for pharmaceutical

products

Chapter III: Preparation of killed and live attenuated

microorganism
 CHAPTER 1:
PRODUCTION OF PROTEINS OF
PHARMACEUTICAL
BIPTECHNOLOGY
Lab 1: Upstream processing
(bioprocess technology)
I. Introduction
Production of pharmaceutical protein includes upstream and
downstream processing. Upstream processing prefers to generation of
initial product. Downstream processing refers to purification and
generation of final product. In this lab, we produce the therapeutic
protein by fermentation of E.Coli transformed with gene coding for the
target protein.
The gene coding for target protein is cloned into pET expression vector
and is located downstream of T7 promoter. The vector is then
transformed into E.Coli host cell. The taget gene is expressed by host
cell T7 polymerase. Selection of host cell containing the vector with
target gene is based on color of colonies. E.Coli is cultured in antibiotic
(Ampicillin) medium. E.Coli cells that are not transformed do not
possess antibiotic resistant ability (the antibiotic resistant gene is
located on pET vector). E.Coli cells that receive vector not containing
target gene form colonies with blue color while E.Coli cells with vector
carrying target form colonies with white color.

II. Procedure
Material

IPTG 1M, LB broth/agar, E.Coli BL1 (DE3) target, E.Coli BL1 (DE3)
target, Spectrophotometer, Incubator, Centrifuge machine, Falcon
50mL.

Method

E.Coli cells transformed with vector carrying target gene was cultured
in LB medium overnight to obtain saturated culture (cells are in
stationary phase), which had OD600 value about 1 to 2 (about 109
cell/ml). This was our working cell bank. We created a cell bank system
including master cell bank and working cell bank in order to ensure
consistent quality of therapeutic protein production, prevent
contamination of master cell bank, and check for cell viability prior to
production.
From working cell bank, 50 l was used to inoculate 5 ml of LB medium
containing 50 M of Ampicilin. The culture was incubated in 2 to 3
hours at 37C until OD600 is at 0.5 to 0.6 (cell density is about 5x108).
We inoculated fresh medium with stationary phase cells from working
 cell bank for the cells to easily enter log phase and perform metabolic
activity.
Induction was performed with addition of 1mM IPTG. IPTG mimics the
structure of lactose, which binds to lac repressor protein allowing E.Coli
polymerase to bind to lac promoter of host cell. E.Coli polymerase then
transcribes T7 polymerase gene, which is downstream of host lac
promoter. T7 polymerase protein binds to T7 promoter and transcribes
target gene. Finally, translation creates the therapeutic protein. IPTG
cannot be broken down by lactase of E.Coli, hence, its concentration
remains during cell replication. Protein expression was induced with
OD600 at 0.5 to 0.6 because host cells are in log phase. In log phase,
bacterial cells are growing and dividing rapidly. Therefore, medium is
exploited at maximum rate, which results in bacteria consuming IPTG
from culture medium and target protein is effectively expressed. The
culture was incubated at 28C with aeration (E.Coli is falcutative
anaerobe, which refers utilization of oxygen to generate more ATP for
growing) for 2 hours (Length of time for incubation may vary among 30
minutes, 1 hour, and 2 hours. Optimal length of time for E.Coli to grow
and express target protein can be study by incubating 3 tubes each in
different duration and selecting the tube with OD value closest to 0.5-
0.6)
After induction, the culture was centrifuged at 10000 rpm in 15
minutes at 25C. Supernatant and pellet were collected and stored
separately to perform SDS-PAGE.

III. Result

Result of measurement optical density at 600 nm

Mean OD value of sample C (control) after 2 hours of incubation
(identified by computer as V) is 0.592.
Mean OD value of sample D (transformed) after 2 hours of incubation
(identified by computer as V*) is 0.081
 The result of SDS-PAGE is showed and analyzed in lab 5 (SDS-PAGE)

IV. Discussion and conclusion

The OD value has quantitative relation to the concentration of cell in

the culture. The OD value of control sample is in desire range of 0.5 to
0.6, which indicates that the cell culture has reached desire
concentration we can add IPTG to the culture. The OD value of
transformed sample has not reach the desire value, which indicates
that the cell may be still adapting to the new medium and may have
not reach the exponential phase. The culture should be incubated more
for the cell to reach the desire concentration. We should expect the
transformed cell to grow slower than the control cell as they need to
gather more material for growth and reproduction. The OD600 of our
group is measureed with blank medium that is different from our initial
medium. Therefore, the OD value is not accurate.
Lab 2: Cell Extraction
I. Introduction

Cell extraction is the first steps in the progress of therapeutic protein

production. In cell extraction, the cell membrane will be lysed and
disrupted by detergents then we can collect the crude protein, which is
followed by concentration and dialysis process, purification and SDS-
PAGE electrophoresis analysis to check the quality of the proteins.

Purpose

The purpose of this lab is to learn how to extract the intracellular

products of the host cell, which are crude proteins after upstream
processing.

II. Procedure
Material

Centrifuge machine, PBS buffer (pH7.4, contains lysozyme), glass

beads, eppendorf tubes, ice.

Method

First, the pellets of both samples (C and D) from previous lab were
collected and washed by 5mL of PBS buffer, which contains lysozyme,
to lyse the cell. These samples were centrifuged at 13,000 rpm for 10
minutes and the supernatants were removed.
Next, the pellets were resuspended by 1mL of PBS buffer and glass
beads were added into these eppendorfs for grinding to disrupt the cell
membrane. The grinding step was performed in the ice pack for 15
minutes. Then, the samples were weighed to ensure equal mass, if not,
PBS buffer was added to achieve the equal mass between eppendorfs,
and centrifuged at 12,000 rpm for 15 minutes at 4oC.
Finally, the supernatants of these samples were collected, 50L of
sample extraction (SE) was transferred to new eppendorfs and stored
for the function as the sample in SDS-PAGE, the remaining will be
prepared for salting out (precipitation process).

III. Results

The result of this process is related to the salting out process of the
samples and running SDS-PAGE.
IV. Discussion and Conclusion

The PBS buffer contains the lysozyme to make the cell lysis occurred
more easily.
Adding glass beads and screening to disrupt the cell membrane to
collect the crude intracellular protein.
Lab 3: Concentration and Dialysis
I. Introduction

Solubility of protein is affected by presence of salt in solvent. Salting

out is the effect observed at high salt concentration. Protein has
different charges based on the functional group, it contains hydrophilic
patch, which is negatively or positively charged and dissolved in water,
and hydrophobic patch, which is neutral and forms precipitates when
the patches link together. As salt dissolves in water, it dissociates into
positive and negative ions. The ions interact with opposite charged site
in hydrophilic patch of protein, hence exposing the hydrophobic patch
on the surface of protein. The hydrophobic patches on the protein
surface interact together and protein molecules aggregate and form
precipitation (salting out).

At low concentration of the salt, the effect showed is salting in. The
ions from the salt associate with the protein surface that leading to the
free of water molecules, which means less water molecules are needed
to interact with protein surface, hence, increasing solubility of protein.

There are two types of proteins that may present in our cell
extraction:native proteins, which are maintained the structure in 4
levels, and denatured proteins which are denatured by the
chemical/detergent to the primary or secondary structure.
 Purpose
In this lab session, the crude proteins got from the previous lab of cell
extraction, will be precipitated by salting-out. We also use two
concentration of Ammonium Sulfate (20% and 80%) to observe the
pellets formation at different concentration of Ammonium Sulfate.
To determine the mass of Ammonium Sulfate we need to add to the
sample to achieve 20% and 80% Ammonium Sulfate solution, based on
the following formula:
519.9 ( S 2S1 )
w=
1000.3 S2

In which
S1 and S2 are the initial and final concentration of Ammonium
Sulfate solution.
519.9 is the coefficient at 4oC, if the experiment was performed
at room temperature (25oC), the coefficient will be 523.3.

II. Procedure
Material

Ammonium sulfate, protein extraction (from previous lab), PBS (pH7.4),

ice, eppendorf

Method

The volume of protein extraction was measured to calculate the

amount of Ammonium sulfate for concentration of 20%. Calculation of
Ammonium amount can be performed using encorbio Ammonium
sulfate calculator or use the formula mentioned in introduction. Volume
of sample C was 400 l so the amount of Ammonium sulfate needed
was 0.04 g. Volume of sample D was 600 l so the amount of
Ammonium sulfate needed was 0.07 g. The mixtures were stirred well
for the salt to dissolve. The solutions were centrifuged at 12000 rpm
for 15 minutes at 4C. Supernatants were collected and stored for
analysis on SDS-PAGE. Ammonium sulfate amounts were then
calculated for 80% concentration. The amount of salt for 350 l of
sample C was 0.14 g and that for 550 l of sample D was 0.22g. Well
stirring and centrifugation steps were repeated. The pellets were
resuspended in PBS buffer (pH 7.4) for SDS-PAGE analysis. Protein
precipitation is performed step wise with increased concentration of
salt (20% to 80%) because different proteins with different solubility
precipitate at different salt concentration. Step wise salting out
provides high specificity of precipitation.
Salt is separated to area with lower concentration. Crude protein is
placed inside dialysis membrane. Dialysis is secured with string and
 submerged in PBS buffer pH 7.4 for 16 hours. PBS buffer has lower
concentration of Ammonium sulfate, therefore, Ammonium sulfate
migrates across the membrane into the buffer. The protein is unable to
pass the membrane as the pores of the membrane have cut off value,
which means the pores have smaller size than the size of protein.
There are different types of dialysis membrane that have different cut
off values available in the market. During the whole process, sample
should be kept at 4C.

III. Result

The protein products we got from this lab will be checked when
performing SDS-PAGE electrophoresis.

IV. Discussion and Conclusion

Ammonium sulfate is commonly used for salting out for its high
solubility that allows solution for high ionic strength and ability to
stabilize protein struture. Hofmeister series is the order of ability of
cations and anions to precipiate protein:

According to the order, sulfate (SO42-) and ammonium ions (NH4+) have
the strongest ability to precipitate protein and stabilize protein
structure.
After salting out with 20% Ammonium sulfate, sample C and sample D
both have pellets, which indicate protein precipitation has occurred.
After salting out with 80% Ammonium sulfate, sample C has little pellet
while sample D has significant precipitation. We can suspect that most
proteins in C have been separated in previous steps.
Lab 4: SDS-PAGE Electrophoresis
analysis
I. Introduction
Purpose:

To check the quality of the product after purification

Principle:

SDS-PAGE (Sodium Dodecyl Sulfate Polyacryl Amide

Gel Electrophoresis) is used to analyze protein mixtures qualitatively as
well as quantitatively. SDS-PAGE is a denaturing electrophoresis where
proteins are denatured to their primary structure (polypeptide chain)
and all other structures are lost. It can also be used to determine the
molecular weight of the protein. SDS-PAGE is based on the separation
of proteins according to their molecular size.

II. Procedure
Materials and Chemical Reagents

Acrylaminde; Bis-acrylamide; Glycerol; Tris 0.75 M (pH 8.8); Tris 0.25 M

(pH 6.8); TEMED; Bromophenol blue; Beta-mercaptoethanol;
Ammonium per sulfate; Gycin; SDS; Trizma; Tricin; Methanol; Acetic
Acid; Coomasssie Blue; SDS-PAGE electrophoresis apparatus; Water
bath.

Methods

Preparing resolving gel

1. Resolving gel and stacking gel were prepared by TA/instructor.

Concentration of resolving gel was 10% and concentration of stacking
gel was 4%.
2. Add resolving gel into the gap between the glass plates. Then water or
isopropanol solution was added on the top of the resolving gel to make
resolving gel horizontal. Wait 1.5 to 2 hours for the resolving gel
polymerize completely then discard the water/isopropanol added.

Preparing stacking gel

3. Add stacking gel into the gap, and it was on top of the resolving gel.
 4. Insert the comb immediately without forming air under the teeth. Wait
for 30 min to let the stacking gel polymerize.
5. Take out the comb when the stacking gel polymerized completely.
6. Take the glass plates out of the casting frame and set them in the cell
buffer dam.
7. Add the running buffer into the inner chamber until the buffer surface
reached the required level in the outer chamber.

Preparing the samples:

8. Mix samples with sample buffer. Then heat them for 5 at 95C.
9. Load prepared samples into wells and not to overflow.

Ladder S20 P20 S80 P80

10. Then cover the top and connect the anodes. 100V and 80A were
used for this experiment.
11. Stop SDS-PAGE running when the protein marker almost reached
the foot line of the glass plate.

Staining and Destaining

12. Take the gel out of the gap gently, allow water flow into the gap
to take it out and make sure not to ruin the gel.
13. Stain the gel with Coomassie Blue.
14. Destain the gel to remove dye.

Staining Destaining

Methanol 40% 20%

Acetic Acid 10% 10%

Water 50% 70%

Coomassive Blue 0.1% 0%

III. Result

The gel is loaded with the samples of group 1

and group 2. From right to left of the upper gel:
ladder; SE of G1; S20 sample of G1; P20 sample
of G1; S80 sample of G1; P80 sample of G1; S20
sample of G2; P20 sample of G2; S80 sample of
G2; P80 sample of G2. Our samples are C
samples. SE is the sample after grinding with
glass beads. Samples S20 and P20 are
supernatant and pellet, respectively whose
proteins are already precipitated by using
ammonium sulfate 20%. And the S80 and P80
samples are proteins (of S20) precipitated by
using ammonium sulfate 80%.

IV. Conclusion
From the picture above, proteins in these samples are not present on
the gel. There is no band at all.
I suggests that the technique when we extracted the cell is wrong,
which leads to no result in cell extraction.
Or wrong technique when collected the pellets and resuspended cells,
leading to the loss of proteins along with PBS solution.
V. Discussion
- Function of resolving gel and stacking gel?
Stacking gel: has a lower concentration of acrylamide (e.g., 7% for
larger pore size), lower pH (e.g., 6.8) and a different ionic content.
This allows the proteins in a loaded sample to be concentrated into a
tight band during the first few minutes of electrophoresis before
entering the resolving portion of a gel.
Resolving gel is small pore (3 - 30%) which is prepared using Tris
buffer used is of pH 8.8. In this gel, macro molecules separate
 according to their size. Normally in experiment, 8% gel for separate
24 205 kDa proteins, 10% gel for separate 14-205 kDa proteins and
12% gel for separate 14-66 kDa proteins.

- 2 different pH in stacking gel and resolving gel ( pH 6.8 and 8.8)?

At pH 6.8, chloride has negatively charge while glycine has no charge
at all; so it moved slower than chloride. Protein was being sandwiched
between Glycine and Chloride ion; so protein was pushed to move
down the loading well. Proteins deposited at the top of the resolving
gel as a narrow band.
At pH 8.8, chloride has negatively charge as well as glycine; so
glycine moved likely at the same speed with chloride. When glycine
migrates much faster than protein due to higher charge/mass ratio.
Now proteins are the main carrier of current and separate according
to their molecular mass by the sieving effect of pores in gel.

- Functions of each chemicals in preparing the gels?

Acrylamide: when dissolved in water, slow, spontaneous
autopolymerization of acrylamide takes place, joining molecules
together by head on tail fashion to form long single-chain polymers. A
solution of these polymer chains becomes viscous but does not form a
gel, because the chains simply slide over one another. Gel formation
requires linking various chains together.
Bisacrylamide (N,N'-Methylenebisacrylamide): is the most frequently
used cross linking agent for polyacrylamide gels. Chemically it can be
thought of as two acrylamide molecules coupled head to head at their
non-reactive ends. Bisacrylamide can crosslink two polyacrylamide
chains to one another, thereby resulting in a gel.
10% Sodium Dodecyl Sulfate (SDS): SDS is a strong detergent
agent used to denature native proteins to unfolded, individual
polypeptides. When a protein mixture is heated to 100 C in presence
of SDS, the detergent wraps around the polypeptide backbone. In this
process, the intrinsic charges of polypeptides become negligible when
compared to the negative charges contributed by SDS. Thus
polypeptides after treatment become rod-like structures possessing
equal mass to charge ratio so as to allow the protein to move
constantly through the gel based on their molecular weight.
10% Ammonium persulfate (APS): It is used with TEMED to
catalyze the polymerization of acrylamide and bisacrylamide based on
a free radical mechanism. APS is a source of free radicals and is often
used as an initiator for gel formation.
Tetramethylethylenediamine (TEMED): Stabilizer reacts with the
APS splitting the APS into the sulfate free radical which then initiates
the polymerization of the Acrylamide and Bisacrylamide. The rate of
polymerization and the properties of the resulting gel depend on the
concentrations of free radicals.
 CHAPTER II:
FORMULATION AND
PREPARATION FOR
PHARMACEUTICAL PRODUCTS
Lab 5: Formulation and
preparation for Pharmaceutical
products
I. Introduction
Purpose:

To formulate and compounding Hyaluronidase cream (topical uses).

To understand the pharmaceutical formulation.

II. Materials and Methods

Materials and Chemical Reagents

Therapeutic protein; Vaseline; Tween 80; Distilled Water; Glass rods;

Watch glasses; Motar and pestle; 100mL Beaker.

Methods

Mix 1
- Melt 0.5g Vaseline completely and pour into the watch glass contains
0.5g of Tween 80, mix well.
- Following the equal amount principle:
Melt 1g Vaseline completely and add into the mixture.
Melt another 2g Vaseline completely and add into the mixture.
Mix 2
- Slowly add 500l DW into 0.5g of TP.
Combination
- Combine Mix1 and Mix 2, mix well.
- Check the quality of the preparation and perform SDS-PAGE,
homogeneity, microbial contamination.

III. Result
Our group cannot create cream. The mixture did not homogenize.
Vaseline and water could not combine together.

I. Discussion
- Function of Tween 80?
 It is an emusifier, one of the primary stabilizer to assisit the
homogenizing between oil (Vaseline) and water ( contain solubled
protein).
 CHAPTER III:
PREPARATION OF KILLED AND
LIVE ATTENUATED
MICROORGANISMS FOR
VACCINE
Lab 6: Preparation of
killed/attenuated microorganisms
I. Introduction
Purpose:
This supplies the primary steps for preparation of killed or live
attenuated vaccine.

II. Materials and Methods

Materials and Chemical Reagents

Pathogen microorganism (Streptococcus); Lysogeny broth (LB); LB

agar; Formalin; PBS (pH 7.4); Centrifugator; Test tubes; Incubator; Petri
dises; Pipet set.

Methods

1. Streptococcus was prepared before class. It was grown to nearly

stationary phase (optical density = 0.80 at wavelength of 600 nm).
2. Pipet 100l of the culture into an eppendorf, which contained 900l of
LB medium, to dilute the culture 10 times, mixes well.
3. Pipet 100l of 10 times diluted culture into an eppendorf (contained
900l of LB medium) to dilute the culture 102 times, mixes well.
4. Following the previous steps to make 103 times and 104 times dilute
culture.
5. Transfer 50l in diluted eppendorf into a petri disk contaning LB agar
and culture overnight in 370C. Count the CFU the next day.
6. Pipet 200l of the stock culture to another eppendorf, centrifuge in 10
mins with 12000rpm at room temperature and collect the pellets,
discard the supernatant
7. Wash the pellets with 200l of PBS (pH = 7.4) and then resuspend in
PBS, centrifuge in 10 mins with with 12000rpm at RT, and collect the
pellets. Repeat this step 2 times.
8. Pipet 200l of PBS to the pellets. Then add Formalin 0.07%.
9. Incubate at room temperature for 1 hour.
10. Centrifuge in 10 mins with 12000rpm at room temperature and
collect the pellets, discard the supernatant.
11. Wash the pellets with 200l of PBS (pH = 7.4) and then
resuspend in PBS, centrifuge in 10 mins with with 12000rpm at RT, and
collect the pellets. Repeat this step 2 times.
12. Pipet 200l of PBS to the eppendorf, resuspend the pellet with
PBS.
13. Transfer 100l into a petri disk contaning LB agar and culture
overnight in 300C - 370C. Count the CFU the next day.
III. Result

Figure 6.1: Cultures of Streptococcus are diluted 10, 102, 103

and 104 times.
 Figure 6.2: Cultures of Streptococcus incubated for 1 hour with
formalin 0.07%.

II. Conclusion
Figure 6.1: Cultures of Streptococcus are diluted 10, 102, 103 and 104
times.
No observation of different color no contamination in all 4
dishes.
All of 4 diluted samples, colonies formed were too dense to be
counted Streptococcus was alive and grows strongly.
The culture of Streptococcus should be diluted 105 to 107 times
fewer number of colonies (less dense) CFU can be calculated.
Figure 6.2: Cultures of Streptococcus incubated for 1 hour with formalin
0.07%.
Colonies of Streptococcus were formed but less dense than the
diluted sample formalin work effectively but not enough to
inactivate all the bacteria.
I suppose that we could increase formalin concentration or increase
incubation-time.
III. Discussion
Function of Formalin: It is used to inactivate microorganisms so that
they don't cause and to detoxify bacterial toxins, such as the toxin
used to make vaccine.
CFU (colony-forming unit) is a unit used to estimate the number of
viable bacteria or fungal cells in a sample. Viable is defined as the
ability to multiply via binary fission under the controlled conditions.
CFU = (no. of colonies x dilution factor) / volume of culture plate.

Colonie