biotech and bioengineering lec 1 part 2

Unit Operations and Flow Sheets

Unit operation is a term for describing a particular task.

Example: heat sterilizer for industrial fermentation media, where media is pumped through a heat coil to heat, hold at sterilizing temperature, and then cool before the next stage.

Unit operations are numbered in flow sheets for identification.
Unit operations generally have one or more inputs and one or more outputs.
Mass and energy balances can be performed on unit operations.
Energy can be conserved by using the exiting hot stream to preheat the incoming material.
Energy requirements can be characterized via a heat balance equation such as:
- energy required $=$ mass flow rate $(kg^{-1})$ x Heat Capacity $(Cp)$ x Temp. Change $(Celsius)$

$Q= m*Cp (T1-T2)$ —> to find energy required

where Q represents the total energy needed for the process, m is the mass flow rate of the material, Cp is the specific heat capacity, and T1 and T2 represent the initial and final temperatures respectively.

The goal of a sterilization unit operation is to lower or eliminate contaminants in a process stream.

Batch, Continuous, and Fed-Batch Processes

Unit operations can be operated as batch, continuous, or fed-batch processes.

Batch Process:
- Media and culture are added to a sterile container, sealed with a vent, and left to ferment for a set period without process control.
- Example: kombucha making where tea, sugar, flavors, and a kombucha culture (mother) are combined and left to ferment.
- Volume is typically constant throughout the fermentation phase.
Continuous System:
- After inoculation and initial growth, new substrate is continuously fed into the process while material is continuously removed.
- Aims to maintain a constant volume in the reactor (Inflow rate $=$ Outflow rate).
- Ideally operates at steady-state for long periods.
Fed-Batch:
- Starts as a batch culture.
- At some point, new substrate is fed into the reactor without any withdrawal of material.
- Volume increases over time during the feeding phase.
- Useful for controlling substrate concentration and avoiding inhibition.

Changes within Unit Operations

Unit operations can involve changes in composition, temperature (e.g., heat exchanger), or phase of materials (e.g., liquid to gas in flash distillation).

Bioprocess Flow Sheet Examples

Process flow sheets illustrate the flow of materials and the unit operations involved in a bioprocess.

Recombinant Human Insulin Production

Complex process using genetically modified E. coli to produce recombinant human insulin.
Materials start at the top left, and the product exits at the bottom right of the flow sheet.
Each block represents a unit operation or individual process step.
Lines represent material flows.
Insulin, a relatively small protein with an A chain of 21 amino acids and a B chain of 30 amino acids, linked by two disulfide bonds.
Requires correct disulfide bond formation and folding in downstream processing.

Process steps include:

Bioreactor:
- Genetically modified E. coli generate cells containing recombinant insulin as insoluble inclusion bodies.
- This is the primary fermentation process.
Cell harvesting by centrifuge:
- Separates target cells from the liquid culture broth.
Cell disruption to release intracellular protein:
- Breaks open cell walls to access the inclusion bodies.
Inclusion body (IB) recovery via another centrifugation step:
- Isolates the dense inclusion bodies from cell debris.
Solubilization of inclusion bodies using chaotropic agents like guanidine:
- Converts insoluble protein aggregates into soluble forms.
Diafiltration: Buffer exchange system.
Filtration.
Chemical cleavage of the single chain into two chains.
Protection of cysteine sulfhydryl groups.
Sulfatolysis.
Buffer exchange.
Multiple chromatography steps for purification.
Refolding step.
Enzymatic conversion to cleave off the carrier peptide.
HPLC (High-Performance Liquid Chromatography): purification step often used for analytical purposes, but can be scaled up for production.
Concentration.
Crystallization.
Centrifugation to obtain the final product.
- Multiple chromatography and buffer exchange steps are involved in purifying recombinant insulin.
- Chromatography steps and HPLC are specifically for purification of recombinant insulin.

Invertase Production

Simpler flow sheet producing the enzyme Invertase from Saccharomyces cerevisiae (baking yeast).

Steps include:

Cell disruption to break open cells and recover Invertase.
Acid titration to lower the pH.
Heating and holding step.
Cooling step.
Buffer change to return pH from acidic to neutral.
Centrifugation to separate solids (broken yeast cells) from liquid.
Cross-flow ultrafiltration to concentrate the Invertase.

The process exploits Invertase's stability at lower pH and elevated temperature, which helps to denature other proteins.
The simplicity and low cost of the process are essential due to its use in food applications.
Invertase hydrolyzes sucrose (table sugar) into glucose and fructose, creating a sweeter mixture and allowing less sugar to be used.
Because the yeast is generally regarded as safe (GRAS), the Invertase can be used directly in food or beverage applications.

Tissue Plasminogen Activator (tPA) Production

Comparison of downstream flow sheets for producing tPA, a therapeutic enzyme, using Chinese hamster ovary (CHO) cells vs. E. coli fermentation.

Mammalian (CHO) cell process:

Recombinant proteins are secreted into the liquid media in their correctly folded state, which simplifies downstream processing.
Downstream processing involves removing cells and using chromatography and filtration to recover the protein product.
Protein is correctly folded and glycosylated directly by the host cells.

Bacterial (E. coli) process:

More steps are required because the protein is produced inside the cells as an insoluble inclusion body.
The process involves:
1. Cell disruption.
2. Unfolding and refolding of the protein.
3. Chromatography and filtration.
- Large losses can occur during the refolding step, which is often inefficient.

Cost Analysis

Although mammalian cell fermentation is initially more expensive than bacterial fermentation, a process economic analysis showed that the E. coli process was ultimately more costly.
- This was primarily due to the poor recovery of functional protein from inclusion bodies.
The E. coli process:
- Required larger capital investments.
- Incurred higher waste treatment costs due to the poor refolding yield.
The mammalian cell process:
- Benefits from the eukaryotic refolding machinery.
- Allows correct protein folding without a separate refolding step, simplifying downstream processing.

Key Differences Highlighted

Cell disruption and inclusion body solubilization are necessary specifically for the bacterial process.
Reacting with sulfhydryl groups is required in the bacterial process due to disulfide bond formation issues.
There is a low refolding yield in the bacterial process, leading to significant product loss.

Computer Modeling of Upstream Fermentation

Purpose: Computer modeling software simulates the mass and energy balances in the upstream fermentation process for insulin production.
Process Operation (Batch Mode):
- Sterile media is pumped into a sterile reactor.
- An inoculum is then added.
- Air mixed with ammonia is continuously pumped in while the fermentation proceeds.
- Air exiting the fermenter is continuously filtered.
Model Simplifications Include:
- Media is simplified as water, glucose, and salts.
- Air mixed with ammonia provides oxygen and nitrogen, respectively.
- A barrier filtration sterilizes the airstream to prevent the release of genetically modified organisms (GMOs).
Inputs Modeled As:
- Water
- Oxygen (from air)
- Salts
- Glucose
- Ammonia
- Inoculum (note: not explicitly shown in model diagrams).
Outputs Modeled As:
- Continuous air stream (containing carbon dioxide).
- Liquid stream (containing cells, recombinant protein in inclusion bodies, and acetate).
- biomass containing recombinant protein as inclusion bodies, co2, accetate (fermentation product)

Upstream and Downstream Operations

Upstream operations include:
- Mixes and sterilizers to prepare the media.
- Substrate conversions or bioreactors.
- Chemical reactions, such as transesterification, that convert biological products.
- Bioreactors can be cell-based or non-cell-based.
- Bioreactors can be operated in batch, continuous, or fed-batch modes.
- More complicated ways to operate are cell recycle systems and perfusion bioreactors.
- Common bioreactor designs include stirred tank reactors and column or tower fermenters.
- Enzyme reactors can be stirred tank reactors or plug flow designs.
Downstream operations focus on purification steps:
- Physical separations like filters or centrifuges.
- Fractionating at a molecular scale (ultrafiltration, nanofiltration).
- Precipitation steps (selective precipitation).
- Affinity methods (chromatography).
- Volatility-based steps (distillation, molecular sieving).
- Final purification with further chromatography and filtration.
- Product formulation to stabilize the product.

Stirred Tank Fermenter

Design: Common bioreactor design featuring:
- A mechanical agitator.
- Impellers.
Functions of Impellers:
- Provide bulk mixing.
- Enhance gas exchange, particularly oxygen transfer.
Temperature Control: Achieved with either:
- An internal coil.
- A jacket surrounding the vessel.
Ports: Included for various operations such as:
- Media input.
- Inoculum addition.
- Gas sparging.
- Harvesting the product.

Airlift Bioreactors

Mixing Mechanism: Utilize gas flow for mixing.
- Do not employ mechanical agitation.
Operation: Gases are sparged into the vessel, creating mixing and circulation within the reactor.

Batch and continuous operation of stirred reactor and mass balancing

(mass flow rate entering reactor) - (mass flow rate leaving the reactor) + (mass generated in the reactor) - (mass consumed in the reactor) = (mass change in the reactor

continous example.

feed coming in and volume coming out.
Volume doesn’t change but it is intrinsically different because fresh feed is coming in.

batch

nothing is entering and nothing is leaving

Perfusion and cell recycle

volume V
Total biomass Xt
Viable biomass Xv
substrate S
Product P

Mobilised and immobilised enzyme

A mobilized enzyme is an enzyme that is free and dissolved in a solution, capable of moving freely. In contrast

Immobilized enzyme is an enzyme that is fixed or confined to a specific location, often on a support material, allowing it to be reused repeatedly.

product recovery and purification

gas liquid

distillation, ethanol purification

liquid liquid

decnating centrifuge, biodesisel pruification

liquid solid

filtration
centrifugation

liquid solute

chromatography

homogenisers - intracellular products

break cells open

some bioproducts are located within cells or tissues

inclusion bodies
recombinant proteins
biopolymers
enzymes
palsmid DNA

cell lysis is required to recover these products

mechanical methods
chemical methods

Downstream operations

biomass separation from supernatant

centrifuge
filtration

cross flow filtration systems

used to facilitate solid liquid separation
separation liquid liquid exchanges
buffer exchange