(6)Large Scale Production of Proteins from Recombinant Microorganisms

Introduction

• Requirements for commercial product production using G.E. microorganisms:

  • Molecular Biologists

  • Biochemical engineers

• Scale-up considerations:

  • Parameters: pH, temperature, mixing rate, oxygen demand

  • Optimal conditions change with volume increases

  • Reactor design: sterility, online monitoring of parameters

• Stepwise process:

  • Medium formulation and sterilization

  • Equipment sterilization

  • Stock cultures (5-10ml) → Shake flask (200-1000ml) → Seed fermenter (10-100L) → Production fermenters (>100L)

  • Cell separation from culture medium

Principles of Microbial Growth

• Types of cultures:

  • Batch: no fresh medium added

  • Fed-batch: nutrients added incrementally; no removal of medium until the end

  • Continuous: fresh medium added, and spent medium removed continuously

Batch Fermentation

• Changes during the process:

  • Medium composition

  • Microbe concentration

  • Internal chemical composition of microbes

  • Amount of target proteins produced

• Six phases of growth:

  • Lag: adaptation period; depends on medium composition

  • Acceleration

  • Log: constant growth rate

  • Deceleration

  • Stationary

  • Death

• Mathematical representation of cell growth:

  • \frac{dX}{dt} = μX (biomass concentration)

  • \frac{dN}{dt} = μN (cell number)

• Specific growth rate (μ):

  • μ = \frac{μ{max}S}{KS + S}

• Generation time (t):

  • Time for cell number or biomass to double.

• Practical implications:

  • When S >> Ks, μ = μ_{max}

  • Ks = half velocity constant

  • At the end of the log phase, substrate depletion leads to the deceleration phase.

  • Stationary phase reached after depletion of critical substance or production of inhibiting products.

Fed-Batch Fermentation

• Substrate added in increments

• Prolonged log and stationary phases

• Avoid stationary phase to prevent proteolytic enzyme production

• Changes in pH, organic acids, and carbon dioxide are substrate consumption indicators.

• Higher yield (25%-1000% more) compared to batch processes

• Applications in mammalian and insect cell cultures for therapeutic proteins

Continuous Fermentation

• Constant cell number and volume in bioreactor

• Steady state condition: \frac{dX}{dt} = 0

• Continuous medium addition and removal

• Dilution rate (D):

  • D = \frac{F}{V}, where F is volumetric flow rate and V is constant liquid volume

  • D = μ (specific growth rate)

• Advantages:

  • Smaller bioreactors

  • Smaller equipment for downstream processing

  • No downtime

  • Uniform cell physiological state

• Drawbacks:

  • Long process duration (500-1000hrs)

  • Maintaining sterile conditions

  • Medium quality assurance

Maximizing Fermentation Efficiency

• Monitor parameters: oxygen, pH, temperature, mixing.

• Dissolved oxygen concentration:

  • Q{max} = \frac{X μ{max}}{Y_{O2}}

  • Continuous supply is necessary.

• pH:

  • Optimum range: 5.5-8.5.

  • Affected by metabolite production; requires acid or base addition.

• Temperature:

  • Below optimum: low growth and productivity.

  • Above optimum: premature protein expression.

• Agitation:

  • Essential for nutrient supply, waste removal, and effective mixing.

  • Excessive agitation: hydromechanical stress or temperature increase.

Other Considerations

• Country-specific rules for using G.E. microbes.

• Recombinant microbes must be nonviable before disposal.

High Density Cell Cultures

• High cell densities for high productivity

• Optimized growth medium is crucial

• Oxygen can become a limiting factor; increase air/oxygen supply and agitation

Increasing Plasmid Stability

• Plasmid loss is a significant problem

• Solution: delete essential gene from chromosomal DNA and place on plasmid

Bioreactors

• Types:

  • Stirred tank reactor (STR)

  • Bubble column reactors

  • Airlift reactors

Stirred Tank Reactor (STR)

• Internal mechanical agitation

• Advantages:

  • Flexible operating conditions

  • Commercially available

  • Efficient gas transfer

• Air added through a sparger

• Impellers disperse gas and enhance residence time

• Made of glass or stainless steel

• Cooling jackets/coils remove heat

• Steam sterilization required

• Fewer ports preferred

• Antifoaming agents or mechanical foam breakers control foaming

Advantages of Other Bioreactors over STR

• Agitation via air injection rather than stirrers

• Lower shear stress, which is uniformly distributed

Bubble Column Reactors

• Air introduced under high pressure for agitation

• Disadvantage: uneven gas distribution and foaming

Airlift Bioreactors

• Internal or external loop

• Mixing achieved by gas introduction

Typical Large Scale Fermentation Systems

• Primary metabolites: cells grown till late log phase

• Secondary metabolites: cells grown till deceleration or stationary phase

• Cloned genes under strong promoter

• Optimal protein production process:

  • Grow cells to certain density under optimum conditions

  • Induce transcription

• Two connected bioreactors can optimize growth and induction separately.

Two Stage Fermentation Examples

• Tandem airlift reactors:

  • E.coli NM989 with T4DNA ligase under pL promoter and temperature-sensitive cI repressor.

  • Growth stage at 30°C; induction stage in a second loop.

• Stirred Tank Reactor:

  • AGßgal fusion protein production.

  • Cells grown at 30°C, then temperature increased to 40°C to induce expression.

Batch Vs. Fed-Batch Fermentation

• Fed-batch can produce both high cell density and high target protein levels.

Harvesting Microbial Cells

• Separate cells from culture medium using high-speed centrifugation or membrane microfiltration.

• Membrane Filtration:

  • Cells accumulate on the membrane surface

  • Overcome reduced flow rate by increasing pressure or using cross-flow filtration

• Cross-flow Filtration:

  • Cell suspension sweeps the membrane clean.

Downstream Processing

• If product is in medium: concentrate medium (ultrafiltration) and purify protein.

• If product is intracellular: disrupt cells before purification.

Disrupting Microbial Cells

• Methods: chemical, physical, and biological.

• Should break cell walls without denaturing product

• Chemical Methods:

  • Alkali, organic solvents, or detergents.

  • Detergents solubilize cell membranes.

• Biological Method:

  • Enzymatic lysis (e.g., lysozyme, ß-1-3-glucanase).

• Physical Methods:

  • Osmotic shock, freeze-thawing, sonication, wet milling, high pressure homogenization, impingement

• Wet Milling:

  • Cell suspension pumped into a chamber with glass beads

  • Shear forces disrupt cells

• High Pressure Homogenization:

  • Cells pumped into a valve assembly under high pressure

  • Rapid pressure decrease causes lysis

• Impingement:

  • Cells hit a stationary phase at high velocity

Down Stream Processing

• Remove cell debris after lysis.

• Purify protein from lysate.

• Enrich target protein or concentrate and fractionate by cross flow ultrafiltration, dialysis.

Protein Solubilization

• Aqueous two-phase liquid extraction for proteins in soluble and insoluble forms.

Large Scale Production of Plasmid DNA

• Consider host cell, growth, metabolism, plasmid size, and cell lysis.

Comparison of Batch, Fed-Batch, and Continuous Fermentation

1. Batch Fermentation:

   • A closed system where all nutrients are added at the beginning, and no additional nutrients are added during fermentation. Conditions change over time affecting the cultural organisms.

   • Lag Phase: Normal.

   • Acceleration Phase: Normal.

   • Log Phase: Proceeds normally until a nutrient becomes limiting or inhibitory metabolite accumulates.

   • Deceleration Phase: Slowing of growth as nutrients deplete and waste accumulates.

   • Stationary Phase: Growth rate equals death rate; nutrient depletion and waste accumulation are significant.

   • Death Phase: Decline in viable cell count due to toxic conditions.

2. Fed-Batch Fermentation:

   • Nutrients are added incrementally without removal of culture volume. This avoids substrate inhibition and extends the log phase. Conditions are better controlled than Batch.

   • Lag Phase: Normal.

   • Acceleration Phase: Normal.

   • Log Phase: Extended by feeding limited nutrients, avoiding inhibitory concentrations.

   • Deceleration Phase: Less pronounced due to controlled nutrient feed.

   • Stationary Phase: Extended as nutrients are managed to avoid complete depletion.

   • Death Phase: Occurs eventually due to waste accumulation or other limitations.

3. Continuous Fermentation:

   • Nutrients are continuously added, and products/waste are removed at the same rate, maintaining a constant volume. Conditions are kept constant, allowing for steady-state growth.

   • Lag Phase: Normal upon initial setup.

   • Acceleration Phase: Normal upon initial setup.

   • Log Phase: Maintained indefinitely at a steady state by controlling nutrient addition and waste removal.

   • Deceleration Phase: Minimal, as conditions are kept constant.

   • Stationary Phase: Not typically observed under ideal conditions.

   • Death Phase: Can occur if the system is disrupted or if inhibitory substances accumulate excessively.

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Benefits and Drawbacks of Continuous Fermentation

• Benefits:

  • High Productivity: Continuous product formation.

  • Consistent Product Quality: Steady-state operation ensures uniform product.

  • Automation: Well-suited for automated control, reducing labor costs.

• Drawbacks:

  • Risk of Contamination: Susceptible to infections, which can halt production.

  • Technical Complexity: Requires sophisticated equipment and controls.

  • Strain Instability: Prolonged culture can lead to genetic drift and reduced productivity.

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Parameters Affecting Fermentation Reactions

1. Dissolved Oxygen Concentration:

   • Effect: Aerobic organisms require sufficient dissolved oxygen for respiration and growth. Insufficient O_2 leads to reduced growth rates and altered metabolic pathways.

   • Control: Maintained by sparging (bubbling air or oxygen), agitation, and optimizing bioreactor design.

2. pH:

   • Effect: Affects enzyme activity, nutrient solubility, and cell membrane function. Optimal pH varies by organism. Deviations can inhibit growth or alter product formation.

   • Control: Monitored and adjusted by adding acids (e.g., H2SO4) or bases (e.g., NaOH).

3. Temperature:

   • Effect: Influences enzyme activity, membrane fluidity, and protein stability. Too high or too low temperatures can inhibit growth or cause cell death.

   • Control: Maintained using heating or cooling systems integrated with the bioreactor.

4. Cell Density:

   • Effect: High cell densities can lead to nutrient depletion, waste accumulation, and O_2 limitation. Can also affect broth viscosity and mixing efficiency.

   • Control: Managed through feed rates in fed-batch and continuous cultures; optimized inoculation densities.

5. Agitation:

   • Effect: Ensures homogenous mixing, nutrient distribution, and gas transfer (i.e., O2 supply and CO2 removal). Inadequate agitation leads to gradients and reduced productivity. Excessive agitation can cause shear stress, damaging cells.

   • Control: Adjusted by varying impeller speed and bioreactor design.

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Comparison of Continuous Fermentation Bioreactors

1. Stirred Tank Bioreactor:

   • Design: Cylindrical tank with an impeller for mixing. Baffles are often included to improve mixing efficiency. Air is sparged into the bottom of the tank.

   • Benefits: Well-mixed, good control over temperature and pH, suitable for a wide range of organisms and processes.

   • Drawbacks: High energy consumption for agitation, potential for shear stress, can be challenging to scale up while maintaining homogeneity.

2. Bubble Column Bioreactor:

   • Design: Vertical column with gas sparged from the bottom. Mixing is achieved by rising bubbles. No mechanical agitation.

   • Benefits: Simple design, low energy consumption, reduced shear stress.

   • Drawbacks: Poor mixing compared to stirred tanks, limited to low-viscosity fluids, difficult to control gas distribution, not suitable for organisms requiring high oxygen transfer rates.

3. Airlift Bioreactor:

   • Design: Similar to bubble columns but with a draft tube or baffle to promote circulation. Gas is sparged into one section (riser), and liquid circulates through the other section (downcomer).

   • Benefits: Better mixing and gas transfer than bubble columns, lower shear stress than stirred tanks, energy-efficient.

   • Drawbacks: More complex design than bubble columns, performance depends heavily on bioreactor geometry, may not be suitable for highly viscous cultures.

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Tandem Bioreactors for Temperature-Sensitive Promoter

• Setup: Two or more bioreactors connected in series. The first bioreactor is maintained at a temperature that represses the temperature-sensitive promoter, allowing for cell growth without protein production. The culture is then transferred to the second bioreactor, where the temperature is shifted to induce protein expression.

• Benefits:

  • High Cell Density: Achieve high cell densities before inducing protein expression, maximizing total product yield.

  • Reduced Metabolic Burden: Prevent the metabolic burden of protein production during the growth phase, improving cell viability and growth rate.

  • Optimized Production: Separate optimization of growth and production phases allows for fine-tuning of conditions to maximize overall yield.

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Harvesting Microbial Organisms

1. Centrifugation:

   • Process: Uses centrifugal force to separate cells from the fermentation broth.

   • Benefits: Effective for high cell densities, relatively simple and scalable.

   • Drawbacks: Can be expensive for large volumes, may damage cells, does not remove soluble impurities.

2. Membrane Filtration:

   • Process: Uses a membrane to separate cells based on size. Typically uses microfiltration or ultrafiltration membranes.

   • Benefits: Effective for removing cells and large debris, can be used for clarification and sterilization.

   • Drawbacks: Membranes can foul and clog, may require pre-treatment, can be expensive for large volumes.

3. Cross-Flow Filtration (Tangential Flow Filtration):

   • Process: The feed stream flows tangentially across the membrane surface, reducing fouling. Cells are retained, while permeate (liquid) passes through.

   • Benefits: Reduces membrane fouling, can handle high cell densities, suitable for continuous operation.

   • Drawbacks: More complex than dead-end filtration, requires careful optimization of flow rates and pressures, can be expensive.

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Purification of Recombinant Product

1. Extracellular, Secreted, Soluble Product:

   • Process:

     1. Clarification: Remove cells and debris by centrifugation or filtration.

     2. Concentration: Concentrate the product using ultrafiltration or evaporation.

     3. Purification: Use affinity chromatography, ion exchange chromatography, or size exclusion chromatography.

2. Extracellular, Secreted, Insoluble Product:

   • Process:

     1. Separate Solid: Separate by centrifugation or filtration.

     2. Solubilize Product: Dissolve the insoluble product using denaturing agents (e.g., urea, guanidine hydrochloride).

     3. Refold Product: Remove the denaturant gradually to allow the protein to refold into its active conformation.

     4. Purify: Use chromatography techniques.

3. Intracellular, Soluble Product:

   • Process:

     1. Cell Disruption: Break open the cells to release the product.

     2. Clarification: Remove cell debris by centrifugation or filtration.

     3. Purify: Use chromatography techniques.

4. Intracellular, Insoluble Product (Inclusion Bodies):

   • Process:

     1. Cell Disruption: Break open the cells to release inclusion bodies.

     2. Separate Inclusion Bodies: Collect inclusion bodies by centrifugation.

     3. Solubilize Product: Dissolve the inclusion bodies using denaturing agents.

     4. Refold Product: Remove the denaturant gradually to allow the protein to refold.

     5. Purify: Use chromatography techniques.

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Methods for Disrupting Cells

1. Chemical Methods:

   • Alkali: Use of alkaline solutions (e.g., NaOH) to disrupt cell membranes.

     • Benefits: Simple, cost-effective.

     • Drawbacks: Can denature proteins, requires careful pH control.

   • Organic Solvents: Use of solvents (e.g., ethanol, acetone) to dissolve lipids in the cell membrane.

     • Benefits: Effective for some cells.

     • Drawbacks: Can denature proteins, may be hazardous.

   • Detergents: Use of detergents (e.g., SDS, Triton X-100) to solubilize cell membranes.

     • Benefits: Effective for a wide range of cells.

     • Drawbacks: Can interfere with downstream purification, may denature proteins.

2. Biological Methods:

   • Enzymatic: Use of enzymes (e.g., lysozyme, glucanases) to degrade cell walls.

     • Benefits: Specific, gentle.

     • Drawbacks: Expensive, may not be effective for all cells.

3. Physical Methods (Non-Mechanical):

   • Freeze-Thaw: Repeated freezing and thawing to disrupt cell structure.

     • Benefits: Simple, suitable for small volumes.

     • Drawbacks: Time-consuming, may not be effective for all cells.

   • Osmotic Shock: Exposing cells to drastic changes in osmotic pressure.

     • Benefits: Simple, can be effective for releasing periplasmic proteins.

     • Drawbacks: Limited applicability, can damage cells.

4. Physical Methods (Mechanical):

   • Wet Milling (Bead Milling): Use of beads to grind cells.

     • Benefits: Effective for disrupting tough cell walls, scalable.

     • Drawbacks: Can generate heat, may damage proteins.

   • High-Pressure Homogenization: Forcing cells through a narrow space at high pressure.

     • Benefits: Effective for a wide range of cells, scalable.

     • Drawbacks: Can generate heat, may require multiple passes.

   • Impingement: Cells are forced to collide at high velocity.

     • Benefits: Efficient disruption, scalable.

     • Drawbacks: Can be expensive, requires specialized equipment.

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Ultrafiltration/Dialysis in Downstream Purification

1. One-Step Ultrafiltration/Dialysis:

   • Process: Use of a single ultrafiltration membrane with a specific molecular weight cutoff to concentrate the product and remove small impurities (e.g., salts, buffer components) through dialysis.

   • Benefits: Simple, rapid, can achieve significant concentration and buffer exchange.

   • Drawbacks: Limited selectivity, may not remove all impurities.

2. Two-Step Ultrafiltration/Dialysis:

   • Process:

     1. First Ultrafiltration: Concentrate the product and remove large impurities using a membrane with a high molecular weight cutoff.

     2. Second Ultrafiltration/Dialysis: Use a membrane with a lower molecular weight cutoff to further purify the product and remove smaller impurities through dialysis/diafiltration.

   • Benefits: Higher selectivity, can remove a wider range of impurities, better control over buffer composition.

   • Drawbacks: More complex, may require optimization of membrane cutoffs and operating

1. Batch Fermentation

   • A closed system where all nutrients are added at the beginning.

   • No additional nutrients are added during fermentation.

2. Fed-Batch Fermentation

   • Nutrients are added incrementally.

   • No removal of culture volume.

3. Continuous Fermentation

   • Nutrients are continuously added.

   • Products/waste are removed at the same rate.

   • Maintains a constant volume.

4. Lag Phase

   • Adaptation period.

   • Depends on medium composition.

5. Acceleration Phase

   • Increase in growth rate after the lag phase

6. Log Phase

   • Constant growth rate.

7. Deceleration Phase

   • Growth rate starts to slow down.

8. Stationary Phase

   • Growth rate equals death rate.

   • Nutrient depletion and waste accumulation are significant.

9. Death Phase

   • Decline in viable cell count due to toxic conditions.

10. Stirred Tank Reactor (STR)

    • Internal mechanical agitation.

    • Flexible operating conditions.

    • Commercially available.

    • Efficient gas transfer.

11. Bubble Column Reactor

    • Air introduced under high pressure for agitation.

    • Disadvantage: uneven gas distribution and foaming.

12. Airlift Reactor

    • Internal or external loop.

    • Mixing achieved by gas introduction.

13. Wet Milling

    • Cell suspension pumped into a chamber with glass beads.

    • Shear forces disrupt cells.

1. Impingement

   • Cells are forced to collide at high velocity.

   • Benefits: Efficient disruption, scalable.

   • Drawbacks: Can be expensive, requires specialized equipment.

2. High Pressure Homogenization

   • Forcing cells through a narrow space at high pressure.

   • Benefits: Effective for a wide range of cells, scalable.

   • Drawbacks: Can generate heat, may