Embed Size (px)
Citation preview
Name of the Trainee : Siddeshwar.S
Name of the Company : Biozeen, Bangalore
Name of the Supervisor/Guide : Mr.Sumanth chaubey
Title of Report : Overview of Biopharmaceutical Production
and Bioprocess Engineering
Field of Training : Production
Area of the project : Bioprocess Engineering
Abstract:
The “Biopharmaceuticals” refers to drugs that have unique qualities in the
way that they are derived and manufactured as opposed to traditional drug products.
Biopharmaceuticals are protein-based and may either be derived from genetically altered bacteria
or fungi or may come from blood and blood plasma products (referred as biologics).The main
objective of this training is to get experience about current industrial trends in biopharmaceutical
production which involves upstream (fermentation and cell culture) and downstream processing.
Introduction:
All the techniques used in biopharmaceutical industry are performed during the training
programme,this report contains all the experiments covered during the programme, four different
modules like animal cell culture, microbial fermentation, Downstream processing and
sterilization and filtration are performed.
ANIMAL CELL CULTURE
Experiments covered:
1. DETERMINATION OF BIOBURDEN OF THE ROOM & VALIDATION OF
BIOSAFETY CABINET (class II type A2)
Objective: To determine the bioburden in working area and BSC by settle plate test
Methodology : Settle plate method is under passive monitoring, the particles present in the
atmosphere will settle down due to gravity in the surface of petri plates over the time of
exposure. On incubation colonies are formed which will be proportional to the area of working
atmosphere.
Result: The bioburden of the room was calculated after 24 and 48 hours and was found to be 4.7
× 104 cfu and 6.42 × 104 cfu respectively whereas the biosafety cabinet was observed to be at
maximum sterility as the bioburden inside the cabinet was found to be 0, hence verifying that the
biosafety cabinet is sterile for work.
2. SUBCULTURING OF ADHERENT BHK21 CELL LINE
Objective: When the cell density (cells/cm2 substrate) reaches a level such that all of the
available substrate is occupied (When the confluence is achieved).To maintain the viability of
the cells in an actively growing log phase.
Procedure:
Results: The cell count for the first passage was found to be 1.04*106 c/ml and the subsequent
counts were found and tabulated as above. The given culture flask was subcultured in a T- flask
and incubated at 37°C for appropriate growth.
3. CELL CULTURE BASED STUDIES
3.1 Growth curve studies
Objective: To establish standard growth curve of BHK 21 cell line.
Figure: Growth curve studies in BHK21 cell line
Result :The growth curve of BHK 21 cell line was plotted on a semi log graph. A distinct log
phase, stationary phase and death phase was observed but no distinct lag phase was observed.
Graph:Standard growth curve of BHK 21 cell line
3.2 Serum studies
Objective: To optimize the serum concentration for cell attachment (BHK-21 cells)
Observation:
Lag
Chambers 0.5% 2% 5% 10% 15%
Top left 01 5 40 80 71
Top right 04 3 49 73 72
Bottom Right 04 4 38 47 50
Bottom left 00 5 57 58
Cell Count 0.045*106
cells/ml
0.075*106
cells/ml
0.885*106
cells/ml
1.28*106
cells/ml
1.25*106
cells/ml
Result: Based on the different serum concentrations 10% shows more confluency and 15% also
showing more confluency but in clumps hence 10% serum concentration is the optimum serum
concentration for cell attachment.
3.3 MTT assay
Objective: To determine cell viability and cell proliferation of BHK21 cells by MTT assay.
MTT Assay set after seeding and after incubation respectively
Result: The MTT assay was performed and the concentrations of the unknown samples were
found to be: Unknown I: 3.44 x 10 5 cells/ml,Unknown II: 5.90 x 10 5 cells/ml
4.BIOREACTOR STUDIES
4.1 Sterility check
Objective: A mock run with water is performed to check the sterility of the process
4.2 Monitoring
Objective: Glucose estimation is done by using Glucometer, simply by placing a drop of sample
on the strip.Cell count is done by heamocytometer in phase contrast microscope Ammonia
estimation is done by Ammonia Assay (Kit based method )
4.3 Scale-up
Objective: To scale up the cells from T25 flask to 8*108 cells and also to estimate the glucose,
NH3, and cell count in the bioreactor vessel.
4.4 Perfusion
Objective: Spin filter is used to separate the spent media and cells from the reactor and new
media added simultaneously to balance the volume in the reactor
5.CRYOPRESERVATION
Objective: To cryopreserve cells and to obtain maximum survivability upon thawing.
Result and Discussion: The given monolayer culture was successfully cryopreserved at -196°C
and on revival, was found to have a percentage Survivability of 38 %.The cells can be stored for
longer time by cryopreservation technique using liquid nitrogen at -1960 C. The cells can be
reused simply by thawing the vial. It is the one most common method to preserve the cells for
future use without affecting any metabolism change in the cell.
6.KARYOTYPING
Objective: Karyotyping is a test to examine chromosomes in a sample of cells, which can help
identify genetic problems as the cause of a disorder or disease. This is performed by using
Giemsa banding technique.
Observation: The chromosomes of BHK 21 monolayer cells at metaphase were observed.
MICROBIAL FERMENTATION
Experiments covered:
1.OPTIMIZATION STUDIES AT SHAKE FLASK LEVEL
Objective:To optimize various growth parameters at shake flask level for Pichia pastoris and E. coli
Parameters of optimization: Media 3 types of media are used. They include Tryptone Soya Broth, Luria Bertani, and Nutrient Broth.pH conditions: Pichia pastoris– 5.5 and 6.5 , E. coli – 6.0 and 7.0 Working volume - 75ml.Inoculum size - 7% of the medium volume. (5.25ml of the respective Inoculum)
Results and discussion:The media best suited for growth for both E. coli and Pichia pastoris
was optimized to be TSB.pH for E. coli was optimized to be 7 and for Pichia pastoris was
optimized to be 6.5.Working volume for E. coli - 75 ml in 500 ml flask; Pichia pastoris-125 ml
in 500ml flask.Inoculum size for E. coli - 9%; Pichia pastoris - 7%
2. SCALE UP OF PICHIA PASTORIS IN 5L FERMENTOR
Objective: To study the growth curve of Pichia pastoris in a 5L lab-scale fermentor
Parameters Set point
Temperature 30º C
pH 6.50
DO > 40%
Aeration 3 LPM
Agitation 200 rpm
Results
Age (in Hrs)
pHTemp (ºC)
Flow rate (LPM)
RPMOD (at 600
nm)1 6.50 30.0 3 200 0.392 6.40 29.7 3 200 0.673 6.35 29.7 3 200 0.5623 6.30 29.7 3 200 20.3924 6.40 29.8 3 200 22.9825 7.00 30.0 3 200 23.8626 6.90 29.9 3 200 24.66
Figure:5l Fermentor
3.SCALE UP OF E. COLI IN 40L FERMENTOR- BATCH FERMENTATION
PREPARATION
Objective: Scale up of E.coli from the parameters optimized in 5l fermentor to 40 l fermentor
Results:
Age (in Hrs)
pHTemp (ºC)
Flow rate (LPM) RPM OD (10 dil.)
1 7.1 37 20 200 0.1142 7.2 37 25 250 0.11821 7.3 37 30 250 0.27122 7.6 37 30 300 0.54323 7.2 37 30 300 0.58524 7.2 37 30 300 0.79925 7.1 37 30 300 0.81945 7.3 37 30 300 0.833
4. SCALE UP OF PICHIA PASTORIS IN 40L FERMENTOR-FED BATCH FERMENTATION
Objective: Scale up of pichia pastoris from the parameters optimized in 5l fermentor to 40 l fermentor
Results:
Age (Hrs) DO (%) pH Temp (ºC) Flow rate (LPM) RPM OD (at 600 nm)
1 96.3 6.5 30.0 20 150 0.832 89.6 6.5 30.1 20 150 1.0521 96.6 6.7 30.0 20 150 6.922 96.6 6.6 30.0 20 150 6.524 53.8 6.5 30.0 20 250 7.2625 51.3 6.3 29.9 20 300 7.626 50.2 5.9 30.0 20 300 9.4745 60.0 6.4 30.0 20 350 0.92746 46.3 6.4 30.0 25 350 1.232
DOWNSTREAM PROCESSING
1. OPTIMIZATION OF CFR AND TMP OF THE MICROFILTRATION FOR
BROTH CLARIFICATION
Objective: To optimize the CFR and TMP of the microfiltration for Broth clarification.
Procedure: Flushing with clean water,Optimization of CFR,Optimization of TMP,Clarification of
E. Coli broth,Flushing of microfiltration system,Cleaning-In-Place of Microfiltration system.
Result and discussion: The cross flow rate and transmembrane pressure of broth were
optimized at 0.8bar feed pressure and 0.2bar retentate pressure.The optimal cross flow rate of
broth in microfiltration was calculated by taking the highest difference of CFR between the feed
pressure of 0.6 and 0.8bar.The optimal TMP of broth was calculated by taking the highest CFR
value.
2. OPTIMIZATION OF THE CFR AND TMP OF ULTRAFILTER FOR
PROTEIN CONCENTRATION
Objective: To optimize the CFR and TMP of the ultrafilter using clarified broth of E. coli.Procedure: Flushing with clean water,Optimization of CFR,Optimization of TMP,Clarification of
E. Coli broth,Flushing of microfiltration system,Cleaning-In-Place of Microfiltration system.
Result and discussion: The cross flow rate and transmembrane pressure of broth were
optimized at 0.6bar feed pressure and 0.1bar retentate pressure. The optimal cross flow rate of
broth in ultrafiltration was calculated by taking the highest difference of CFR between the feed
pressure of 0.4 and 0.6bar. The optimal TMP of broth was calculated by taking the highest CFR
value.
3. GEL FILTRATION CHROMATOGRAPHY
Principle: Gel based separation or size exclusion chromatography facilitates the separation of
molecules or particle on the basis on their molecular size. To perform a separation, gel filtration
medium is packed into a column to form a packed bed. . It should be noted that samples are
eluted isocratically, i.e. there is no need to use different buffers during the separation.
Observation : Protein Recovery Chart:
Sample OD(280 nm)
Conc Volume Total protein
% of protein
Load 3.75 5.68 1.5 8.5 100
elution 0.268 0.406 18 7.308 % of recovery87.5%
SEC data showing the % recovery of the protein.
Result: The chromatogram was obtained shows distinct resolved peaks for protein elution as well as the salt elution during the end of elution step. About 87.5% of protein was recovered for the size exclusion chromatography.
4. ION EXCHANGE CHROMATOGRAPHY
Principle: Separation in ion exchange chromatography depends upon the reversible adsorption
of charged solute molecules to immobilized ion exchange groups of opposite charge. In our
procedure the matrix binds the negatively charged proteins and hence called anion exchanger.
However, the functional group or the ligand in each of the exchangers will have an opposite
charge to that of the protein i.e. anion exchanger will have a positively charged functional group
or ligand so that it can bind to the negatively charged proteins and hence vice-versa in the case of
a cation exchanger.
Observation and result:
Sample OD Concentratio
n
Volume Total protein % of protein
Load 0.464 0.703 25 ml 17.5 100
Flowthrough 0.006 0.009 13.5 ml 0.0121 36.1(loss)
Unbound wash 0.0038 0.0057 16.9 ml 0.0963
Elution
100 0.024 0.0318 24 ml 0.763 63.9 (recovered)
200 0.327 0.495 24 ml 11.88
300 0.029 0.044 12ml 0.531
400 0.0406 0.0616 9 ml 0.55
500 0.0057 0.0087 12 ml 0.104
Protein recovery chart for anion exchange
The given protein was eluted successfully by anion exchange chromatography.
5.HYDROPHOBIC INTERACTION CHROMATOGRAPHY
Principle: HIC separates proteins according to the differences in their surface hydrophobicity by utilizing a reversible interaction between these proteins and the hydrophobic surface of a HIC medium. A high concentration of salt enhances the interaction while lowering the salt concentration weakens the interaction. Hydrophobic interactions are the strongest at high ionic strength. But as the ionic strength of the buffer is reduced the interaction is reversed and the protein with the lowest degree of hydrophobicity is eluted first.
Observation and result:
Sample OD Concentration Volume Total protein % of protein
Load 0.897 0.703 10ml 13.59 100
Flowthrough 0.358 0.009 9 ml 4.88 (loss)
71.89%Unbound
wash
0.269 0.4079 12 ml 4.89
Elution
1M 0.051 0.077 15 ml 1.159 (recovered)
12.5%0.75M 0.009 0.0136 6 ml 0.0818
0.50M 0.0172 0.026 15ml 0.3909
0.25M 0.005 0.075 9 ml 0.068
Protein recovery chart for HIC
Proteins were separated based on hydrophobic interaction successfully. In this experiment we
were able to recover only 12.5% of the load. Most of the protein of interest was lost in unbound
wash and flowthrough. This might be due to uneven packing of column and/or due to presence of
air bubble.
6.AFFINITY CHROMATOGRAPHY
Principle: The techniques require that the material to be isolated is capable of binding reversibly
to a specific ligand that is attached to an insoluble matrix
Macromolecule + Ligand ↔ Complex
Observation and result:
Sample
Optical
density(280nM
)
Concentration
of protein
(mg/ml)
Volume(ml)
Total
protein
(mg)
Load 4 6 10 60
Flow
through0.064 0.097 10 0.97
Unbound
wash0.069 0.104 15 1.56
Elution 1.188 1.8 30 54
By affinity chromatography we recovered 90 % of protein during 0.1M Glycine elution and a loss of 6% of protein.
7. PRODUCTION SCALE CHROMATOGRAPHY SYSTEMS
AKTA PROCESS
AKTAprocess is an automated liquid chromatography system built for process scale-up and
large-scale biopharmaceutical manufacturing. The built-in computer with UNICORN software
allows standalone operation or integration into any plant-wide control system.
Objective: AKTA Chromaflow column packing by Chromaflow packing station and check the
column efficiency.
Procedure: Column was packed by Packing in place method and priming was performed to
remove air bubbles. Efficiency of packed column (HETP & Asymmetry) was tested by Passing
1%Acetone
Result: Performed column packing procedure by AKTA chroma flow packing station and the
efficiency of column was tested by Height Equivalent of Theoretical Plates (HETP)3617
plates/meter(N/m) and Asymmetry is 0.90
STERILIZATION AND FILTRATION
1. AUTOCLAVE - HEAT PENETRATION STUDIES
1.1 Heat penetration study of standard load
Objective : To study the heat penetration in standard cycle.
Procedure:
1. Load was prepared.
2. Temperature sensors were placed inside the chamber as follows:
1: LHS top front
4: Load (LHS bottom back)
5: Drain
6: Outside of the load
3. Load was placed inside the chamber along with the temperature sensors.
4. Parameters for the cycle were set.
5. Cycle was run as per the parameters i.e. 20 minutes @ 121°.
Results:
Data logger graph
LBBINSIDE THE LOADDRAINTOP OF THE LOAD
HEAT PENETRATION STUDY IN STANDARD LOAD
1.2 HEAT PENETRATION STUDY OF POROUS LOAD
Objective: To study the penetration of heat in porous cycle/fabric cycle/HPHV cycle.
Procedure:
The load was prepared. Then the temperature sensors were placed inside the chamber in the following positions :
1. Drain
4. Load-inside
5. Load-inside center
6. Load-inside bottom
Load was placed inside the chamber along with the temperature sensors
The door of the autoclave the closed. Parameters for the cycle were set in the program. Cycle was run as per the parameters i.e 20 minutes @ 121°.
Result:
Data logger graph
DrainLoad TopLoad CenterLoad Bottom
HEAT PENETRATION STUDY INPOROUS CYCLE
TE
MPE
RA
TU
RE
(C)
TIME(min)
1.3 HEAT PENETRATION STUDY OF LIQUID LOAD
Objective:To study the heat penetration in liquid cycle.
Procedure:
1. Load was prepared as above stated.Temperature sensors were placed inside the chamber as follows
1. Inside the liquid.
4. Above the liquid.
5. Drain.
6. LHS bottom back
2. Load was placed inside the chamber along with the temperature sensors. 3. Parameters for the cycle were set in the program.4. Then the cycle was run as per the parameters i.e 20 minutes @ 121°.
Result:
Data logger graph
DrainInside the LiquidAbove the LiquidTop of load
HEAT PENETRATION STUDY IN LIQUID CYCLE
Time(min)
Tem
pera
ture
(C)
2. HEAT PENETRATION - DHS
Objective: To study the heat penetration in a DHS.
STANDARD OPERATING PROCEDURE:
1. Load was prepared.
2. Temperature sensors were placed inside the chamber as follows:
CH01: Inside the canister
CH04: Left bottom fort of chamber
CH05: Top of the canister
CH06: Right bottom back of chamber
3. Load was placed inside the chamber along with the temperature sensors.
5. Parameters for the cycle were set.
6. Cycle was run as per the parameters i.e. 3hours @ 180°.
LBF
HEAT PENETRATION STUDY ON STD LOAD IN DHS_18.05.12
3.INTEGRITY TESTING OF FILTERS
3.1WATER INTRUSION TEST
Objective: To perform the integrity test of hydrophobic filter by water intrusion test
Procedure:
Water was collected in a vessel. The filter was connected to the vessel. Filter holder was connected to a source of regulated pressure. The pressure difference between two filters was maintained as 0.3 bar. The upstream of filter was filled with water and closed the valves. Then the integrity testing machine was connected to the system and the test was
performed automatically.
Result:-The water intrusion test gives net volume as 1.9ml/10min. The manufacturer recommended maximum value is 4ml/10min. So the test is passed indicating the filter is integral.
3.2 BUBBLE POINT TEST
Objective: To perform the integrity test of the hydrophilic filters by bubble point method.
Procedure:
Connect the filters to the collection tank.
Collect water in the collection tank.
Flush the filter with water and then drain.
Connect a piece of flexible tubing from the downstream port of the test filter into a beaker filled with water.
Connect the outlet fitting from the compressed air pressure regulator to the upstream side of the filters.
Starting from zero pressure, increase the pressure to 80 % of the minimum bubble point
From the set pressure, gradually increase the pressure to the test filter using the pressure regulator.
Observe the submerged end of the tubing for the production of bubbles as the upstream pressure is slowly increased in 0.10 mbar increments.
The bubble point of the test filter is reached when bubbles are produced from the tube continuously. Note the pressure and compare with the minimum bubble point pressure given by the manufacturer.
The same test can be performed by the integrity testing machine. Instead of manually pressurizing the filter, it is being done automatically by the IT machine.
Result: The minimum bubble point pressure is found to be
4.02 bar. The minimum bubble point pressure recommended
by manufacturer was 3.2 bar. So the test is passed. This
indicates that our filter has not lost its integrity.
Conclusion
The experiments mentioned above are performed successfully and the results are attached.
