Expression Systems Lecture Review

Flashcards covering biopharmaceuticals, selection criteria for expression systems, specific systems (E.coli, mammalian, insect, yeast, molecular farming), and their advantages/disadvantages.

What are biopharmaceuticals?

Biopharmaceuticals are produced by genetic engineering using various recombinant expression systems.

What are recombinant proteins?

Recombinant proteins are those that are expressed by an organism that does not normally have the genetic material to produce that protein.

What is the general process for producing a recombinant protein?

The process involves inserting a gene for a protein into a target ‘expression system’ to make it produce the protein that we want.

What are the key criteria for selecting an expression system?

Protein complexity and need for post-translational modifications (PTMs), yield and scalability, cost and infrastructure, and regulatory and speed considerations.

What are the characteristics of yeast or bacteria as expression systems for simple proteins?

They offer simpler fermentation, are scalable, and have a lower cost of goods, but may result in post-translational modifications that are not human-like.

What are the characteristics of mammalian cell lines as expression systems for complex proteins?

They involve complex culture, are less scalable, have a high cost of goods, but produce human-like Post-Translational Modifications (PTMs).

What type of organism is E.coli?

E.coli is a Gram negative, rod-shaped, facultative anaerobic bacterium.

What was the first recombinant protein produced in E.coli cells?

Human insulin.

What are some advantages of using E.coli as an expression system?

Ease of culture & genome modifications, rapid expression, high yields, ease of scale-up, and it is inexpensive.

What are some disadvantages of using E.coli as an expression system?

Proteins produced with endotoxins, acetate formation resulting in cell toxicity, proteins are intracellular, and proteins often produced as inactive inclusion bodies requiring refolding.

What are endotoxins?

Endotoxins are pyrogenic lipopolysaccharides, which are major constituents of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria.

Name some common methods for endotoxin removal.

Ultrafiltration, anion-exchange chromatography, histamine-immobilised affinity resins, and the Limulus amebocyte lysate (LAL) assay.

What is the Limulus amebocyte lysate (LAL) assay?

An in-vitro assay developed in the 1960s using an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, based on its blood clotting reaction to endotoxin.

Why is the LAL assay preferred for endotoxin detection?

It is the most sensitive, reliable, and specific detection method for pyrogen producing Gram-negative bacterial endotoxins, and it is faster, more economical, and more sensitive than the rabbit pyrogen test.

What are some general characteristics of mammalian cell lines used in expression systems?

Expensive culture system, susceptible to infection (e.g., viruses), and can produce high titres of recombinant proteins.

What are two common mammalian cell lines used for biopharmaceutical production?

Chinese Hamster Ovary (CHO) cells and Baby Hamster Kidney (BHK) fibroblast cells.

What are the advantages of using CHO cells in particular?

They can secrete proteins with complex post-translational modifications very similar to those produced by human cells, proteins are secreted extracellularly, and they are extremely tolerant to changes in oxygen concentrations, pH, temperature, and pressure.

What are the disadvantages of using mammalian cell lines for protein expression?

High production cost, expensive media and culture conditions, low protein yield, fragility and susceptibility to shear, and susceptibility to viral infections.

What is a key characteristic of insect cell lines regarding protein expression?

They are eukaryotic and efficiently express complex recombinant proteins similar to mammalian cell lines.

What are some examples of insect cell lines?

Spodoptera frugiperda (sf9 & sf21).

What are the advantages of using insect cell lines for protein expression?

They allow post-translational modifications, can produce large complex proteins, offer high protein yield, can be used for large-scale production, and facilitate stable integration of the Gene of Interest (GoI).

What are the disadvantages of using insect cell lines for protein expression?

Prone to lysis, lack some glycosylation pathways necessary for human proteins, more expensive than bacterial or yeast cell lines, have less extensive regulatory records, and naturally occurring proteases can degrade expressed proteins.

What are two common yeast species used in expression systems?

Saccharomyces cerevisiae and Pichia pastoris.

What is an example of an antibody produced in Pichia pastoris approved for clinical use?

Eptinezumab (Vyepti), approved in 2020 for migraine prevention.

What are the advantages of using Saccharomyces cerevisiae as an expression system?

Rapid growth (very high cell mass densities) & scalable, easy to manipulate, simple & inexpensive media requirements & culture conditions, and allows for post-translational modifications of recombinant proteins.

What are the disadvantages of using Saccharomyces cerevisiae as an expression system?

Difficult to disrupt cells due to thick and hard cell walls, and hyperglycosylation of proteins, which can affect protein function and immunogenicity.

What is 'Molecular farming' and what is the most used expression host?

Molecular farming is the use of plants to produce recombinant proteins, with Nicotiana benthamiana (tobacco plant) being the most used expression host.

What key benefit does molecular farming with Nicotiana benthamiana offer?

Protein yields can reach gram levels of product per kilogram of leaves within 5–7 days post-DNA delivery, which is a key benefit over transgenic procedures and mammalian cell-based expression systems.

What advantages does molecular farming provide?

It is rapid & affordable (only needs water & an energy source), offers optimized growth conditions, is free from pathogen & bacterial toxin contamination, and allows for PTMs.

What are the disadvantages of molecular farming?

Limited glycosylation capacity and expression levels can vary.

Why are CHO cells often used for antibody production (e.g., monoclonal antibodies)?

They enable the production of antibodies with human-like glycosylation, which is crucial for therapeutic efficacy.

Why is E.coli suitable for insulin production?

It efficiently produces recombinant human insulin, ideal for simpler protein expression needs.

What type of biopharmaceutical product has been successfully manufactured using yeast systems?

Hepatitis B vaccine, demonstrating effectiveness in producing vaccine antigens safely and reliably.