Cytokines and Proteins - snq

Biotech Drugs - Cytokines and Proteins Class Notes

Course Information

• Course Code: BIT Biotech Drugs - Cytokines and Proteins

• Lecturer: Dr. Kulwinder Kaur

• Academic Level: 2nd Year BSc (ATT)

Lecture Learning Outcomes

• Explain the process of downstream processing

• Describe the steps to recovery and purification of the protein product

• Describe the steps to concentrate the protein product

• List techniques used in the characterization of the product

• Describe the role of cytokines and interferons in the immune system and in clinical applications

• Explain the method of manufacture of a TNF-alpha fusion protein

• Explain the method of manufacture of recombinant human interferon alpha

Overview of Processes

Upstream Process (Previous Lecture)

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

Downstream Processing

Key Components

1. Purification

2. Concentration

3. Formulation

Factors Influencing Purification

• Source Material

  • Origin of protein (microbial vs mammalian) and location (intracellular vs extracellular)

• Level of Expression

  • Low expression levels necessitate larger volumes of source material

• Physicochemical Properties of the Protein

• Purpose of Purification

  • Achieve purification to homogeneity (remove all contaminants) for therapeutic proteins

  • Consider economic and technical factors, balancing costs of equipment, consumables, labor, and effectiveness of methods

Three Phase Purification Strategy

1. Phase 1: Initial Recovery

   • Rapidly isolate target protein from bulk mixture (e.g. cell lysate or broth) while removing most contaminants

2. Phase 2: Intermediate Purification/Polishing

   • Remove major contaminants and bulk impurities, concentrate the protein while retaining activity

3. Phase 3: Final Polishing / High-Resolution Purification

   • Achieve high purity, removing trace contaminants, endotoxins, and aggregates

Initial Recovery

• Most engineered proteins are extracellular

• Recovery involves removing whole cells from fermentation media via filtration or centrifugation; the protein is extracted in dilute form

• Intracellular Proteins:

  • More complex to extract due to cell wall structures in microbial cells

  • Involves cell disruption and efficient handling with reduced volumes

Cell Disruption Techniques

• Mammalian Cells:

  • Techniques include physical disruption methods such as homogenization and microfluidization

• Microbial Cells:

  • Techniques include lysozyme digestion at lab scale, sonication, and chemical treatments (solvents, detergents) at large scale

  • Must avoid denaturing effects and ensure they can be removed in subsequent purification steps

• Agitation with Glass Beads

• High Shear Forces

Removal of Whole Cells and Debris

Techniques

• Centrifugation

• Filtration

  • Filters should not shed fibers into the product

Concentration of Protein

Methods

1. Precipitation

   • Target protein becomes insoluble and separates from contaminants

2. Ion Exchange Chromatography (IEX)

   • Separates proteins based on net surface charge

3. Ultrafiltration

   • Uses semipermeable membranes for separation based on molecular size

4. Vacuum Dialysis

   • Rapidly removes small molecules from protein solutions

5. Freeze Drying

   • Preserves proteins in stable, solid form by removing water

   • The first three methods are utilized in large-scale setups

Precipitation Details

• Neutral Salts Used:

  • Typically ammonium sulfate, known for high solubility and low cost

  • Process called "salting out" decreases solvation, leading to protein aggregation and precipitation

• Organic Solvents:

  • Involve selectively dissolving a compound of interest and then adding a non-solvent to cause precipitation

  • Must be conducted below 0°C to prevent denaturation

Ion Exchange Resins

• Consist of cation and anion exchange resins

  • Cation exchange binds positively charged proteins

  • Anion exchange binds negatively charged proteins

• Proteins are eluted by altering salt concentration or pH

• Effectiveness depends on net charge of proteins, pH of buffer, and ionic strength

• Used for initial concentration and some purification

• Added to fermentation broth and recovered via centrifugation

Column Chromatography

Principles

• Separation based on size, shape, overall charge, and binding characteristics

• Commonly employed methods include 3 to 5 high-resolution chromatography steps

• Recovery rates range from 25% to 95%

Types of Column Chromatography

1. Ion Exchange Chromatography (IEX)

   • Stationary Phase: Resin interacts with proteins based on charge

   • Mobile Phase: Buffer affects separation by altering pH and salt concentration

2. Size Exclusion Chromatography

   • Separation based on molecular size; larger proteins elute first

   • Uses porous beads, gentle on proteins

   • Does not modify protein structure

3. Affinity Chromatography

   • Separation based on specific binding between protein and ligand on resin

   • Effective but costly

Ultrafiltration

• Process relies on pressure-driven filtration through a semipermeable membrane

• Retains proteins & larger molecules while allowing small molecules (e.g. water, salts) to pass through

• Membranes range from 1 to 20 nm in pore size, suitable for retaining low molecular weight proteins

• Membrane materials include PVC, polycarbonate, cellulose acetate

• Results in high recovery rates and is scalable for industrial use

Protein Characterization

Importance

• Therapeutic proteins must undergo extensive characterization for bioactivity and purity

Characterization Techniques

1. Functional Activity:

   • Bioassay and ligand interaction assessment

2. Purity Evidence:

   • Techniques such as SDS-PAGE, 2D-Gel Electrophoresis, IEF, HPLC, Mass Spectrometry

3. Structural Studies:

   • Include determination of molecular mass and structural analysis

4. Size and Surface Charge Analysis:

   • Isoelectric focusing and chromatofocusing

5. Identity Confirmation:

   • N-terminal sequencing and peptide mapping

Cytokines and Interferons in the Immune System

Role and Types of Cytokines

• Cytokines are small signaling proteins that regulate growth, differentiation, and immune responses.

• Types include:

  • Interferons

  • Interleukins

  • Chemokines

  • Tumor Necrosis Factor (TNF)

Mechanism of Action

• Bind to specific receptors and activate intracellular signaling pathways

• Induce transcription of genes related to proliferation or immune responses

Advantages of Cytokine Therapy

• Targeted immune modulation and potential to restore immune function

Disadvantages

• Short half-life, systemic toxicity, production costs, and delivery limitations

Interferons

Characteristics and Types

• Produced in response to pathogens by host cells

• Types:

  • Type 1 IFN (IFN-α, IFN-β): Induces antiviral state, activates NK cells

  • Type 2 IFN (IFN-γ): Activates macrophages and enhances presentation of antigens

Production Methods for Interferons

1. Cell Culture:

   • Mammalian cells cultured for large-scale production, e.g., Namalwa cell line

2. Recombinant DNA Technology:

   • Involves gene cloning, transfection, protein expression, purification, and formulation

Cytokine-Based Therapies in Clinical Use

Etanercept (Enbrel®)

• A biologic TNF inhibitor for controlling arthritis and psoriasis

• A recombinant human soluble TNF receptor fusion protein that neutralizes TNF-α

• Manufacturing involves recombinant DNA technology using CHO cells, requiring careful processing to avoid misfolding

Summary

• Topics covered include downstream purification, cytokine, and interferon production, and their roles in pharmaceuticals for clinical treatments.