Proteins and Interferons - snq

Biotech Drugs - Proteins and Interferons

Course and Lecturer Information

  • Course Code: BIT

  • Course Title: Biotech Drugs - Proteins and Interferons

  • Year: 2nd Year

  • Lecturer: Dr. Kulwinder Kaur

Lecture Learning Outcomes

The following learning outcomes are expected from this lecture:

  • Describe the basis of protein product

  • List the methods of biotech medicine production

  • Describe the characteristics of an “ideal” protein product

  • Explain the process of upstream processing

  • Describe the significance of contamination in parenteral products

  • Explain the use of bacterial and mammalian expression systems in the production of therapeutic proteins

  • Describe the use of transgenic plants and animals in the production of therapeutic proteins

  • Explain the generation and use of the Master Cell Bank

Overview of Biotech Drugs Production

  • Drug production in biotechnology is dependent on the type of drug being produced.

Hybridoma Technology

  • Definition: Monoclonal antibodies which are proteins that act similarly to antibodies that naturally protect the body against unwanted substances.

Recombinant DNA and Expression Systems

  • Cytokines: Small proteins important for controlling growth and activity of immune system cells and blood cells.

  • Interferons: Man-made versions of proteins naturally produced by the body.

  • Interleukins: Proteins expressed by leukocytes and other body cells.

  • Anti-TNFs (Fusion Proteins): Suppress immune activity by blocking TNF (Tumor Necrosis Factor) secreted by inflammatory cells.

Protein Production Methods

Recombinant DNA

  • Process: Involves joining two or more DNA molecules to create a hybrid.

Systems Used:

  1. E. Coli

  2. Animal Cell Culture Systems

  3. Yeasts

  4. Fungi

Transgenic Organisms

  • Transgenic Animals: Animals whose genome has been altered to include DNA from another species.

  • Transgenic Plants: Plants whose DNA is modified using genetic engineering techniques, for example, Bt cotton.

Recombinant Protein Expression Systems

  • The bulk of biopharmaceuticals is produced through genetic engineering using recombinant systems.

Distribution of Production:

  • Mammalian Cells: 60% (e.g., Chinese hamster ovary cells (CHO), NSO murine myeloma cell expression systems)

  • Microbial Expression Systems: 30% (e.g., E. coli)

  • Yeast Systems: 10% (e.g., Saccharomyces cerevisiae, Pichia pastoris)

Characteristics of the DNA-RNA-Protein Flow

  • Transcription Process:

    • The information in DNA is passed to messenger RNA (mRNA) in the nucleus.

  • Translation Process:

    • mRNA interacts with ribosomes in cytoplasm, each codon (sequence of three nucleotides) coding for an amino acid.

    • Transfer RNA (tRNA) assembles proteins one amino acid at a time until reaching a stop codon.

  • Central Dogma of Molecular Biology: This concept illustrates the flow of genetic information from DNA to RNA to proteins.

Insulin Production Example

  • Recombinant DNA Technology:

    • Inserting a human gene into a bacterial plasmid to create a recombinant bacterium that can produce human insulin.

    • The process involves fermentation in a tank to produce insulin, which is then purified.

Production of Pharmaceutical Proteins

Process Overview

  1. Development of Recombinant Expression System:

    • Creation of Master Cell Bank (MCB) and Working Cell Bank (WCB)

  2. Upstream Processes:

    • Cell culture

    • Protein expression

    • Harvest and recovery

  3. Downstream Processes:

    • Purification

    • Concentration

    • Formulation

Ideal Protein Product Characteristics

  • Active Protein Produced:

    • Correctly folded, stable, and functionally active

    • Glycosylated: involves the addition of sugar moieties to specific amino acids.

    • High expression levels, maximization of yield, and cost of production considerations.

Commercial Products

  • Mammalian Cell Products: e.g., Enbrel™ (Etanercept), Elaprase™ (Idursulfase)

  • Bacterial Expression: e.g., Optisulin™ (Insulin)

  • Yeast Products: e.g., Twinrix™ (Vaccine), Regranex™ (PDGF)

  • Transgenic Animal Products: e.g., ATryn™ (Human Antithrombin – obtained from goat milk)

Upstream Processing

  1. Cell culture

  2. Protein expression

  3. Harvest and recovery

Expression Systems Explained

E. Coli as an Expression System

  • Advantages:

    • Well-characterized genetics

    • Rapid growth

    • High cell density

    • Inexpensive media

    • Availability of numerous vectors

  • Disadvantages:

    • Proteins accumulate intracellularly, complicating extraction

    • Inability to perform complex post-translational modifications

    • Presence of lipopolysaccharides

    • Inclusion body formation promoting aggregation

Mammalian Cell Expression Systems

  • Common Host Cells: CHO cells, Human embryonic kidney (HEK) cells, mouse myeloma cells.

  • Advantages:

    • Capable of post-translational modifications (e.g., glycosylation which aids in protein folding and stability).

  • Disadvantages:

    • Slower growth relative to E. coli

    • Complex nutritional needs

    • Increased production costs due to fragility of animal cells

Master Cell Bank Production

  • Definition: A Master Cell Bank is created from a cell line extensively characterized for various factors including growth, productivity, and purity.

  • Purpose: Represents a renewable source of cells for biopharmaceutical manufacturing ensuring consistent production.

Upstream Processing Detailed Steps

  • Process involves cell culture, protein expression, and harvesting, followed by downstream purification methods.

Contamination Considerations

Viral Contamination Impact

  • Viral particles can integrate into genome and potentially transmit disease.

  • FDA Guidelines:Recommend three viral removal steps during production:

    1. Heat treatment at elevated temperatures (35°C to 60°C).

    2. Filtration (using 0.1 to 0.2 µm filters).

    3. UV irradiation treatments.

Types of Contaminants

  • Microorganisms: Can lead to severe infections.

  • Viral Particles: Risk of severe viral infections.

  • Pyrogenic Substances: Result in fever response and can be lethal.

  • Contaminating Proteins: Can cause immunological reactions.

Final Processing Steps

  1. Initial recovery and purification from cellular debris.

  2. Concentration and further purification techniques like column chromatography.

  3. Characterization of protein to determine purity.

Large Scale Cell Culture Challenges

  • Key Issues:

    • Maximizing protein expression

    • Maintaining product quality through all phases of production

    • Minimizing contamination through careful monitoring.

Transgenic Production

Overview

  • Involves introducing specific genes into animals or plants to produce therapeutics in larger quantities.

  • Process: Microinjection of DNA constructs into fertilized eggs and subsequently implanting them to produce offspring capable of producing desirable proteins, such as antithrombin in goats.

Plant Expression Systems

  • Involves inserting DNA sequences into plants to produce proteins.

  • Methods of DNA Introduction: Infection with Agrobacterium or direct injection into plant cells.

Advantages and Disadvantages of Plant Systems

Advantages:

  • Scalability, cost-effectiveness, freedom from human pathogens.
    Disadvantages:

  • Low expression levels, challenges in post-translational modification.

Summary of Protein Production Techniques

  • A comprehensive overview of different methods used to ensure the production of ideal protein products across diverse biological systems and the processes involved in overcoming contamination risks and optimizing yield and quality.