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BiOZEEN operates a state-of-the-art cGMP compliant pilot plant dedicated exclusively for training led by a team of industry professionals. Our curriculum is dynamic, interfaced with industrial experience, with the strong emphasis on bridging the gap between academia and industry. BiOZEEN provides an opportunity through its unique model of training which enables the participants to acquire the skills required to excel in biopharma industry in a much shorter duration of 6 months and to join a production team without any cooling period.
The training is oriented in upstream and downstream processes of microbial and animal cell culture fermentation. The standard programs are detailed below
Short term / Summer training
Long term / Advanced training
Project Work
| Short term / Summer training |
Duration: 6 weeks |
| Introduction (1 week) |
- Biopharmaceutical Industry
- Market Share
- Products
- Prospects
- Drug Development Processes
- Steps in Pharma-Biotech Production
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| Microbial Fermentation (1 week) |
- Fermentor principles & design
- Aeration & agitation and KLa estimation
- Inoculum development
- Microbial growth
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- Inoculum build up and fermentor inoculation
- Fermentor sampling
- Growth rate estimation
- Fermentor harvest and cleaning
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| Animal Cell Culture (1 week) |
- Good laboratory and cell culture practices
- Biosafety in the cell culture lab
- Primary cultures to established cell lines
- Nutritional requirements of cultured cells
- Cryo preservation principles and cell banking
- Contamination control in cell culture
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- Aseptic techniques in cell culture
- Cell quantization and examination
- Sub-culturing of monolayer cultures
- Establishment and monitoring of suspension culture
- Cryo preservation and revival of frozen cells
- Contamination studies
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| Downstream Processing (1 week) |
- Principles of centrifugation
- Principles of tangential flow filtration
- Protein quantification methods
- Chromatography basics
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- Clarification of cell broth
- Protein concentration
- Protein purification
- Protein quantification
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| Cleaning, Sterilization & Filtration (1 week) |
- Principles of heat sterilization
- Principles of cleaning in place (CIP)
- Cold sterilization by filtration
- Filter integrity testing
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- Operation of sterilizers
- CIP and SIP of process vessel
- Filtration of media
- IT of hydrophobic and hydrophilic filters
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| Compilation of results & analysis / Report writing / Interaction with HODs (1 week) |
| Long term / Advanced training |
Duration: 1 to 6 months |
| Introduction to biopharmaceutical processes (1 month) |
| Refer summer training program |
| Microbial Fermentation (1 month) |
- Prospects of microbial and animal cell culture derived biopharmaceuticals
- Understanding microbes
- Introduction to industrial microbiology
- Fermentor principles and design
- Designing media for industrial fermentation
- Media sterilization in fermentation & concept of SIP
- Microbial growth
- Inoculum development
- Maintenance & calibration of sensors used in fermentation
- Aeration and agitation
- Dynamic method of KLa estimation
- Growth kinetics of batch fermentation processes
- Fermentor sterility, containment & decontamination
- Qualification of fermentor
- Regulatory aspects of microbial testing & sterility testing
- Scale-up & Scale-down of fermentation
- Preservation of microbes ‐ fermentation economics
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At Shake Flask:
- Media preparation and autoclaving
- Shake flask optimization (media / pH / volume / inoculum percentage)
- Inoculum preparation
At 5L Lab Fermentor:
- Inoculum preparation
- Preparation of fermentation medium
- Calibration of pH / DO probes
- Set up of lab fermentor and autoclaving
- Inoculation of lab fermentor
- Dynamic way of KLa estimation
- Monitoring & maintenance of process condition (Agitation / Aeration / Temperature / pH / DO)
- Sampling and analysis
- Harvest and cleaning of lab fermentor
At 40L Pilot Fermentor:
- Setting up of pilot fermentor
- ESIP (Empty Sterilization In Place)
- Media preparation and charging
- Calibration of pH probe
- Mechanical seal sterilization
- FSIP (Full Vessel Sterilization In Place)
- Filter sterilization
- Foam control
- Setting up aeration & agitation
- Calibration of DO probe
- Aseptic inoculation
- Dynamic way of KLa estimation
- Monitoring & maintenance of process condition (Agitation / Aeration / Temperature / pH / DO / Back pressure)
- Sterile sampling and processing
- Monitoring growth and calculating growth rates
- Feed pump calibration
- Fed batch nutrient shot dosing
- Broth harvest
- CIP (Cleaning In Place)
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| Animal Cell Culture (1 month) |
| Good Cell Culture Practices |
- Aseptic techniques and prevention of contamination
- Sources, detection and eradication of contamination
- Safety and regulatory issues in the cell culture lab
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- Aseptic techniques in biosafety cabinet Type IIA
- Validation of Biosafety cabinet by settle plate method
- Staining of cells and examination of cell biology
-Mono & Polychromatic Contamination Studies
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| Cell growth and propagation |
- In vitro cell growth behavior
- Culture of adherent and suspension cells
- Microcarriers technology
- Feeding, subculture and propagation of established cell lines
- Cell line characterization
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- Cell enumeration
- Set up of monolayer cultures
- Sub-culturing and maintenance of adherent cultures
- Seeding suspension culture 100ml
- Scale up of adherent cultures using microcarriers
- Growth curve establishment and analysis
- Cytotoxicity assay - MTT
- Metaphase spread of chromosomes
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| Cell Banking |
- Principles of Cryo preservation
- Cell banking methods
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- Cryo preservation of cultures
- Thawing of cells and viability assessment
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| Nutritional requirement of cultured cells |
- Components of basal media and their importance
- Serum-free media formulations: implications for process development
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- Media preparation and filtration
- Effect of serum supplementation on cell proliferation
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| Animal cell culture in Bioreactor Design |
- Bioreactor design and scale up
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- Scale up of suspension cultures in a 5 L bioreactor
- Set up of 5 L lab fermentor and autoclaving
- Calibration of pH / DO Probes
- Inoculation of lab fermentor
- Monitoring & maintenance of process condition
- (Agitation / Temperature / pH / DO)
- Sampling and analysis ‐ harvest and cleaning of Lab fermentor
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| New trends |
- Large scale monoclonal antibody production
- Antibody fragment technology
- Gene introduction methods
- Stem Cell Technology
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| Downstream Processing (1 month) |
| General |
- Techniques in downstream process
- Methods for protein quantification
- Gel electrophoresis
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- Electrophoretic techniques
- Protein quantification techniques
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| Centrifugation |
- Centrifugation principles and applications
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- Functions and process optimization for centrifugation processes at variable speed and flow rate
- Continuous centrifugation for cell concentration or cell debris removal
- Cleaning and sterilization of continuous centrifuge
- Microbial concentration / separation analytical method by spectrometry and assay validation
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| Tangential Flow Filtration |
- Principles of TFF and functions of TFF
- Process optimization of TFF for product
- TFF membrane selection criteria
- Principles of diafiltration
- Cleaning and sterilization of TFF
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- Concepts of tangential flow filtration
- Optimization of cross flow rate and transmembrane pressure for optimal process
- Tangential flow filtration for microbial concentration
and recovery of extracellular product with 0.45mic
and 0.2mic polymeric membrane
- Protein concentration by ultra filtration with
appropriate molecular cut-off polymeric membrane
- Purification & salt removal from concentrated
protein by diafiltration
- CIP / SIP processes & CIP / SIP validation for tangential flow system
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| Chromatography |
- Ion exchange chromatography principles
and applications
- Gelfiltration chromatography principles
and applications
- Hydrophobic interaction chromatography
principles and applications
- Affinity chromatography principles and
applications
- Membrane chromatography
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- Optimization of Ion Exchange / Hydrophobic
Interaction / Gelfiltration / Affinity chromatography
for purification
- Column packing procedures and efficiency
assessment
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| Cleaning, Sterilization & Filtration (1 month) |
| Cleaning-In-Place |
- Cleaning-In-Place process (CIP)
- Design & engineering of CIP station for
efficient cleaning
- Definition of residue & acceptance criteria
- Validation of CIP process
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- Familiarization with process vessel
- CIP processes
- Generation of P& ID and valve matrix for CIP processes
- OQ of process vessel: spray ball coverage test with riboflavin
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| Sterilization-In-Place |
- Sterilization-In-Place process (SIP)
- Design & engineering considerations (dead
legs, steam traps, valve position…) for
efficient sterilization
- Validation of SIP process
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- SIP processes
- Generation of P&I D and valve matrix for SIP processes
- SIP PV: Heat penetration study for ESIP and FSIP using temperature probes / biological / chemical Indicator
- Correlation between thermal death time and Fo
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| Sterilization by Filtration |
- Fundamentals of gas & liquids sterilization by filtration
- Selection of filter types and pore sizes, filterability studies
- Design and engineering considerations of filtration systems for efficient sterilization
- Operation, maintenance, cleaning and sterilization of gas & liquid filtration systems
- Integrity testing of liquid and gas sterilizing grade filters
- Validation of sterilizing grade filters for process compatibility
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- Familiarization with filtration system
- Generation of P&I D and valve matrix for all processes
- SIP of filtration system and media transfer line
- Integrity test of filters (Bubble point, Diffusion and Water Intrusions tests)
- Media filtration (maintenance of Flow rate, Differential for increasing filter service life)
- CIP of filtration system and media transfer line
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| Sterilization-Out-of-Place |
- Overview of moist and dry heat sterilization in the pharmaceutical Industry
- Sterilization cycle development & lethal rate calculation (Fo concept)
- Functions and process cycles of heat sterilizers - Qualification of dry heat sterilizer & autoclave
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- Familiarization and operation of industrial double door heat sterilizers
- Generation of P&ID and valve matrix for all cycles (Standard, HPHV and liquid)
- IQ: verification of designed system vs actual system
- OQ: Bowie dick test / Vacuum leak test / thermal mapping of a sterilizer
- PQ: Heat penetration studies for standard, porous and liquid loads with temperature probes/ biological/ chemical indicator & correlation between thermal death time and Fo
- Evaluation of endotoxins inactivation after dry heat sterilization with CSE and LAL test
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| Intro to Bioprocess Equipment Engineering (15 days) |
- Design cell culture bioreactors
- Design microbial fermentors
- cGMP compliant valves
- cGMP compliant components
- Design of CIP systems
- Design of process vessels
- Design of tangential flow filtration system
- Design of dead end filtration system
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- Preparation of P & ID using ISA based symbols for all process equipments
- Preparation of bill of material for all process equipments
- Preparation of valve matrix for process sequences, CIP and SIP for some process equipments
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| Intro to Regulatory Affairs (15 days) |
- Validation master plan
- Quality risk management
- Facility validation approach
- Qualification of utility Systems
- Qualification of process equipments
- Process validation
- Method validation
- Good documentation practices
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- Writing of VMP
- Approach to risk assessment
- Writing of validation and qualification Protocol
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| Project Work |
Duration: 1 to 4 months |
| ANIMAL CELL CULTURE |
Project 1: Scale-up of suspension mode BHK-21 cells in a 5 L stirred tank bioreactor
Project 2: Scale-up of adherent BHK-21 cells using microcarrier technology |
The activities for both the experiments would include:
- Thawing of cells from cell bank and viability assessment
- Set up of monolayer and suspension cultures
- Staining of cells and examination of cell biology - Mono & Polychromatic
- Cell enumeration
- Media preparation and filtration
- Effect of serum supplementation on cell proliferation
- Growth curve establishment and analysis
- Sub-culturing and maintenance of adherent cultures
- Seeding suspension culture 100ml
- Scale up of adherent cultures using microcarriers
- Scale up of suspension cultures in a 5 L bioreactor
- Set up of 5 L lab fermentor and autoclaving
- Calibration of pH / DO Probes
- Inoculation of lab fermentor
- Monitoring & maintenance of process condition (Agitation / Temperature / pH / DO)
- Sampling and analysis
- Harvest and cleaning of lab fermentor
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| DOWNSTREAM PROCESSING |
Project 1: Purification of recombinant proteins from E.coli, Yeast, and animal cell culture fermentation
Project 2: Purification of non recombinant proteins from fermentation
Project 3: Purification of therapeutically important carbohydrates or polysaccharides from fermentation broth |
The activities for all the experiments would include:
- Clarification by centrifugation / Microfiltration
- Target product quantification
- Concentration of samples by tangential flow filtration system
- Diafiltration for removal of impurities and salts by TFF
- Purity analysis of target product
- Removal of impurities by chromatography
- Ion exchange chromatography -- anion & cation exchange chromatography
- Gelfiltration chromatography
- Hydrophobic chromatography
- Affinity chromatography
- Pure product quantification and yield calculations buffer exchange of purified samples
- Sterile filtration and packing
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| MICROBIAL FERMENTATION |
Project 1: Optimization of biomass production of Escherichia coli at shake flask and lab scale fermentor
Project 2: Optimization of biomass production of Pichia pastoris at shake flask and lab scale fermentor |
The activities for both the experiments would include:
- Media preparation for Shake flasks and 5 L bench top fermentor
- Preparation of material and 5 L bench top fermentor for sterilization by autoclave
- Inoculum preparation
- Aseptic inoculation of shake flasks and 5L bench top fermentor
- DO and pH probes calibration
- Fermentation parameters control
- Sampling and sample analysis
- Fermentation process optimization at shake flask level
- Media and Media components
- pH ( volume to volume )
- Inoculum percentage at 5L bench top fermentor level
- Aeration
- Agitation
- Nutrient shot dosing
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