Pharma Exam Revision

Main Types of Biological Products + Examples

Proteins

• Antibodies

• Enzymes

• Collagen

Metabolites

• Antibiotics

• Ethanol

• Vitamins

Cells

• Stem Cells

• Yeast Extract

• Baking/Brewing Yeast

Protein Structures

Primary Structure: Sequence of amino acids in the chain (covalent bonding)

Secondary Structure: Local structure of the peptide backbone (hydrogen bonding) e.g. α-helices & β-pleated sheets

Tertiary Structure: Overall 3D Shape of a protein (side chain intramolecular interactions)

Quaternary Structure: Arrangement of folded polypeptide chains

Protein Production Steps

1. Transcription - DNA is transcribed to an RNA copy of the gene by RNA polymerase

2. Translation - RNA is transcribed into a polypeptide by ribosomes

Metabolites

Chemical compounds that are intermediates or end-products of cellular metabolism

Primary Metabolites

Essential metabolites involved in normal cell growth and reproduction

e.g. Ethanol, Citric acid, Amino acids

Secondary Metabolites

Non-essential metabolites that have specialised roles in ecological interactions

e.g. Antibiotics, Antifungals, Chemotherapy drugs

Biological Sources of Metabolites Examples

Bacteria

• Ethanol

• Citric Acid

• Amino Acids

Plants

• Terpenoids

• Alkaloids

• Flavonoids

Key Industrial Microbes + Biological Products they produce

Bacteria

• Escherichia coli – widely used for protein production in research and industry

• Lactic acid bacteria – can grow at relatively low pH so widely used in food & beverage production

 

Fungi

• Saccharomyces cerevisiae (Baker’s yeast) – used industrially for ethanol production and insulin

• Aspergillus niger (Black mould) – widely used filamentous fungi producing citric acid and enzymes

Key differences between bacteria, yeast, fungi

Metabolic & Oxygen Classification

Autotrophs: use inorganic carbon (CO₂) to produce organic molecuules

• Photoautotrophs: use energy from sunlight

• Chemoautotrophs: use chemical energy from the oxidation of inorganic compounds

Heterotrophs: use organic carbon sources

• Photoheterotrophs: use sunlight for energy and organic carbon for carbon

• Chemoheterotrophs: use organic carbon molecules for both energy and carbon

  • Aerobes: use aerobic respiration for ATP production

  • Anaerobes: use anaerobic fermentation for ATP production

    • Obligate anaerobes die in the presence of oxygen

    • Facultative anaerobes use aerobic respiration if oxygen is present, or fementation if not

Nutritional Requirements of Microbes

• Macronutrients: microbes need these elements in relatively high amounts

  • N, C, H, O, P, S (components of carbohydrates, lipids, proteins and nucleic acids)

• Micronutrients: microbes also need various metals in trace amounts

  • Mn, Zn, Co, Mo, Ni, Cu

• Growth Factors: 

  • Amino acids

  • Purines & pyrimidines

  • Vitamins

Types of Microbial Growth Media: Difference + Pros/Cons

Defined (synthetic media): concentrations of all chemical compounds are known

• Pro: allows for consistent control of nutrients in industrial application

• Con: need to know nutritional requirements of the microbe

Complex media: rich nutrient sources with no precisely known compositions

• Pro: supports rapid growth and cultures wide range of microbes

• Con: no precise control

Bioreactor Key Features

SMMALT

• Sampling: aseptic sampling port

• Measurement: sensors for temperature, pH, dissolved oxygen, foaming

• Mixing: motorised impeller

• Aeration: sparger, gas inlets and outlets

• Liquid addition: feed pumps and inlets for nutrient, acid/base and anti-foam additions

• Temperature control: heating/cooling coils or jackets

Microbial Growth Phases

1. Lag phase: cells adjust to environmental conditions

2. Exponential growth phase: cells replicating at constant growth rate

3. Deceleration phase: growth rate slows due to nutrient depletion or build-up of toxic waste products

4. Stationary phase: cells enter a non-growing state, no net cell growth occurs but secondary metabolites are produced

5. Death phase: cells die due to nutrient exhaustion or toxicity

How to monitor cell growth rate in reactor

• Turbidity: via spectrophotometer

• Capacitance: via cell membrane polarisation

• Substrate depletion: via enzymatic probe

• Respiration rate: via rate of oxygen uptake

Differences Between Cell Culture Modes

Feeding Control Strategies for Industrial Fed-Batch Culture

• Feedforward Control: assuming constant specific growth rate using equation

• Feedback Control: based on measurement data which has a stronger correlation to the condition of the bioreactor

Key Features of a Plasmid

• Promoter: recruits transcription machinery

• Restriction enzyme sites: for gene insertion

• Selectable markers: to distinguish native and transcofrmed cells

• Affinity tag sequences: for protein purification

• Origin of replication: controls replication of plasmid inside cells

Steps to Produce Protein from a Foreign Source

1. Extract target gene & replicate via PCR

2. Insert gene into plasmid

• Cut plasmid & PCR product with restriction enzyme (matching sticky ends)

• Join fragments using DNA ligase to create recombinant DNA

• Alternatively, do Gibson assembly via overlapping regions between DNA

3. Transformation

• Introduce plasmid into microbe via chemical transformation (CaCl₂ + Heat Shock) or electroporation

4. Selection

• Plate cells on a medium containing antibiotic, only those with plasmid with antibiotic resistance gene survive

• Alternatively, do blue-white screening for visual identification

5. Expression

• Pick a colony and grow in a cell culture

• Control gene expression via constitutive (always on) or inducible (on/off) promoters

6. Harvest & Purify

• Lyse cells to release protein

• Use affinity tags and chromatography to isolate the target protein

PCR Reaction Components & Steps

Components

• Template DNA: contains target gene to be amplified

• Nucleotides: four DNA bases (ACGT)

• Primers: match start & end of DNA region to be amplified

• DNA polymerase: enzyme that copies DNA

Steps

1. Denaturation: double-stranded DNA heated, strands separates

2. Annealing: primers bind target sequences

3. Extension: DNA polymerase binds to annealed primer, nucleotides incorporated into new DNA strand

How ‘tags’ can assist in purification of proteins

Tags are peptides joined to the end of a recombinant protein. In affinity chromatography, tags attached to the protein are attracted to the affinity resin

Key Issues with Protein Production in Microbes & Solutions

• Inclusion bodies: aggregates of insoluble protein formed by many recombinantly expressed proteins in microbes

  • Avoid by optimising culture conditions, using weaker promoter, or lower copy number plasmid

• Low expression levels

  • Optimise culture conditions and media components

  • DNA sequence optimisiation

Algae Cell Growth at Various Levels of Light

Weak-Moderate light

• Carbon fixation proportional to light recieved

Intense Light

• Photoinhibition can limit growth

• Photodamage occurs when excessive light is maintained

No Light

• Aerobic respiration of cellular organic carbon used to power and maintain cellular activities

Algae products

Products

• Pigments (β-carotene, etc)

• Lipids (biofuels, etc)

• Proteins

• Carbohydrates

Algae Commercial Advantages

• Genetic diversity with wide range of physiological and biochemical characteristics

• Potential to be bioengineered for strain improvements

• High growth rates (compared to terrestrial plants)

Classifications of algae

• Macroalgae: large, multicellular; source of polysaccharides

• Microalgae: microscopic, unicellular; source of pigments, lipids, proteins, etc)

Algae Growth Systems Pros/Cons

• Open ponds: low cost, contamination risk, low control

• Photobioreactors: high control, small footprint, high cost

Photobioreactor Scale-Up Considerations

• Changes in illumination

• Gas transfer

• Temperature

• Turbulence

Algae Production Flow Charts

1. Discovery

• Identification of metabolite

• Species selection

2. Production

• Cultivation

3. Harvest

• Screening

• Thickening

• Dewatering

• Drying

Challenges in Engineering & Processing of Algae

• Low productivities

• High recovery cost

• Light penetration, O₂ accumulation, mixing, scaling up

Plant-derived Pharmaceuticals Examples

• Morphine (from opium poppy)

• Paclitaxel (anti-cancer drug - from Pacific yew tree but now in plant cell culture)

• Artemisinin (anti-malaria drug - from sweet wormwood but now from yeast)

Why use plant cell culture vs obtain from plants

• Independent of crop supply

• Controlled environment (no crop disease, weather)

• Improved yields (metabolites normally at low concentration)

• Easier genetic manipulation of cells vs plants

• Simplify metabolite extraction (less contaminants)

Disadvantages of plant cell culture vs microbial cells

• Slower growth rates

• Lower yields

• Higher medium costs

• Intracellular product accumulation

• Higher shear sensitivity

• Formation of cell aggregates

Plant vs microbial cell culture bioreactor & downstream differences

Bioreactor Design

• Plant cells larger, so more shear sensitive - needs gentle mixing such as airlift or bubble column

• Cell aggregation causes diffusion limitations and metabolic hetereogeneity , so gentle circulation is required to minimise gradients

• Intracellular product accumulation means that continuous immobilised systems are not suitable

Downstream Processing

• Secondary metabolites retained within vacuoles, requiring cell disruption to release products

• High viscosity and suspended aggregates complicate filtering, separation and extraction

• Lower yields and slower growth make process intensification and recovery efficiency more critical

Need for Growth Regulators in Plants vs Microbes

Plant cells need growth regulators (e.g. auxins and cytokinins) to mimic the hormonal signals that regulate growth and differentiation in whole plants

Enhancing Metabolite Production

• Removal of growth regulators or starving cells or nutrients

• Addition of product precursor molecule

• Elicitors to initiate defence or stress response

Plant Cell Culture Production Steps

1. Take plant tissue, wound it, grow on solid media with growth regulators

2. Extract callus (mass of undifferentiated plant cells)

3. Start suspension cell culture

4. Optionally genetically modify

5. Scale-up into bioreactor

Genetic Modification of Plant Cells

• Agrobacterium tumefaciens transformation

  • Bacterium creates tumors in plants, containing a plasmid transferring a segment of its DNA into plant cell genomes

• Particle bombardment (gene gun)

  • DNA-coated particles are propelled into plant cells which enter the nucleus and integrate into the plant genome

Pros/Cons of Mammalian Cells vs Microbial

Types of Post-Translation Modifications

Applications of Mammalian Cell Culture

• Viral vaccines

• Monoclonal antibodies

• Therapeutic glycoproteins (interferons, hormones, blood clotting factors)

• Tissue/organ replacement

• Medical research applications

Steps for the preparation and culture of animal cells

Differences between normal/transformed cells

Difference between stem/differentiated cells

Fermenter/growth set ups for adhesive and suspension cell cultures

Components within media and importance to cell growth and process development & optimisation