Name of the Trainee Name of the Company Name of the Supervisor/Guide Title of Report

: Siddeshwar.S : Biozeen, Bangalore : Mr.Sumanth chaubey : Overview of Biopharmaceutical Production

and Bioprocess Engineering

Field of Training Area of the project

: Production : 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 37C 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.

Lag

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:

Chambers Top left Top right Bottom Right Bottom left Cell Count

0.5% 01 04 04 00 0.045*10 cells/ml

6

2% 5 3 4 5 0.075*10 cells/ml
6

5% 40 49 38
6

10% 80 73 47 57
6

15% 71 72 50 58 1.25*106 cells/ml

0.885*10 cells/ml

1.28*10

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 -196C 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 Temperature pH DO Aeration Agitation Results Age (in Hrs) 1 2 3 23 24 25 26 pH 6.50 6.40 6.35 6.30 6.40 7.00 6.90 Temp (C) 30.0 29.7 29.7 29.7 29.8 30.0 29.9 Flow rate (LPM) 3 3 3 3 3 3 3 RPM 200 200 200 200 200 200 200 OD (at 600 nm) 0.39 0.67 0.56 20.39 22.98 23.86 24.66 Set point 30 C 6.50 > 40% 3 LPM 200 rpm

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) pH Temp (C) Flow rate (LPM) RPM OD (10 dil.) 1 2 21 22 23 24 25 45 7.1 7.2 7.3 7.6 7.2 7.2 7.1 7.3 37 37 37 37 37 37 37 37 20 25 30 30 30 30 30 30 200 250 250 300 300 300 300 300 0.114 0.118 0.271 0.543 0.585 0.799 0.819 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) 1 2 21 22 24 25 26 45 46

DO (%) 96.3 89.6 96.6 96.6 53.8 51.3 50.2 60.0 46.3

pH 6.5 6.5 6.7 6.6 6.5 6.3 5.9 6.4 6.4

Temp (C) 30.0 30.1 30.0 30.0 30.0 29.9 30.0 30.0 30.0

Flow rate (LPM) 20 20 20 20 20 20 20 20 25

RPM 150 150 150 150 250 300 300 350 350

OD (at 600 nm) 0.83 1.05 6.9 6.5 7.26 7.6 9.47 0.927 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 Load elution OD (280 nm) 3.75 0.268 Conc 5.68 0.406 Volume 1.5 18 Total protein 8.5 7.308 % of protein 100 % of recovery 87.5%

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 Load OD 0.464 Concentration Volume Total protein % of protein 0.703 25 ml 17.5 100

Flowthrough

0.006

0.009

13.5 ml 16.9 ml

0.0121 0.0963

36.1(loss)

Unbound wash 0.0038 0.0057 Elution 100 200 300 400 500 0.024 0.327 0.029 0.0318 0.495 0.044

24 ml 24 ml 12ml 9 ml 12 ml

0.763 11.88 0.531 0.55 0.104

63.9 (recovered)

0.0406 0.0616 0.0057 0.0087

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 Load Flowthrough OD 0.897 0.358 Concentration Volume Total protein % of protein 0.703 0.009 0.4079 10ml 9 ml 12 ml 13.59 4.88 4.89 100 (loss) 71.89%

Unbound wash 0.269 Elution 1M 0.051

0.077

15 ml

1.159

(recovered)

0.75M 0.50M 0.25M

0.009

0.0136

6 ml 15ml 9 ml

0.0818 0.3909 0.068

12.5%

0.0172 0.026 0.005 0.075

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: Concentration of protein (mg/ml) 6 10 Volume(ml) Total protein (mg) 60

Sample

Optical density(280nM)

Load Flow through Unbound wash Elution

0.064

0.097

10

0.97

0.069

0.104

15

1.56

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
HEAT PENETRATION STUDY IN STANDARD LOAD

LBB INSIDE THE 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

HEAT PENETRATION STUDY INPOROUS CYCLE
TEMPERATURE(C)

Drain Load Top Load Center

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

HEAT PENETRATION STUDY IN LIQUID CYCLE

Temperature(C)

Drain Inside the Liquid Above the Liquid

Time(min)

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.

HEAT PENETRATION STUDY ON STD LOAD

LBF

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.