BioTech (Upstream Processing): Proteins & Interferons

Question 1

Hybridoma Technology Outline

Answer 1

2 cells mixed together. Eg monoclonal antibodies (antibodies made in lab)

Question 2

Recombinant DNA Outline

Answer 2

2 types of DNA mixed together. Egs; Cytokines, Interferons, Interleuken and anti-TNF. Types of DNA: E. Coli, Animal cells, yeasts and fungal cells created by transgenic plants and transgenic animals

Question 3

Transcription Outline

Answer 3

DNA section (coding) -> complementary mRNA in nucleus

Question 4

Translation Outline

Answer 4

mRNA binds to ribosome in cytoplasm. tRNA drops complemenatry anticodons. mRNA to DNA

Question 5

What codes for protein

Answer 5

Codon

Question 6

How does an mRNA vaccine work

Answer 6

Bypasses transcription. Inserts mRNA directly into body for translation creating protein

Question 7

3 stages of Pharmaceutical Protein Manufacture

Answer 7

Recombinant expression system (master and working cell banks), upstream process and downstream process

Question 8

Further stages of pharmaceutical protein manufacture

Answer 8

Host cell transformation, gene of interest isolation, target protein ID, cloning expression vector, vector fermentation and protein expression, protein purification, protein formulation, manufacturing, packaging and storage

Question 9

Benefits of Alpha Helix protein Expression

Answer 9

Elastic

Question 10

Benefits of beta sheet protein expression

Answer 10

Compact

Question 11

Ideal Protein Product Characteristics

Answer 11

Correct folding, glycosylated, high expression, soluble, easy purification, maximised yield and low cost

Question 12

Example of Mammalian Cell Protein Pharmaceutical Product

Answer 12

Enbrel (Entaracept) and Elaparasale (Irdulusafe)

Question 13

Example of Bacterial Cell Protein Pharmaceutical Product

Answer 13

E. Coli and insulin

Question 14

Example of Yeast Cell Protein Pharmaceutical Product

Answer 14

Twinrix Vaccine

Question 15

Example of Transgenic Animal Protein Pharmaceutical Product

Answer 15

Human antithrombin in goat’s milk

Question 16

Upstream Processing Outline

Answer 16

Cell culture, protein expression and harvest & recovery

Question 17

Expression Vector Outline

Answer 17

Allows transcription and translation of a foreign gene in host. Eg; Plasmid

Question 18

Plasmid Outline

Answer 18

Circular DNA molecules separate from nucleus in bacterial cells

Question 19

Protein Production Process in Cell Expression Systems

Answer 19

Gene of interest transfected into expression system, purification, protein of interest and further processing into pharmaceutical formulation

Question 20

E Coli Expression System Advantages

Answer 20

Well characterised, genetics, fast growth, high cell density, inexpensive media and several available vectors and mutant hosts

Question 21

E Coli Expression System Disadvantages

Answer 21

Protein’s produced intracellularly (hard extraction), can’t make post-translational modifications, aggregation risk

Question 22

Mammalian Cell Advantages

Answer 22

Posttranslational modification (glycosylation, acylation and hydroxylation. Allows mature protein folding)

Question 23

Mammalian Cell Disadvantages

Answer 23

Fermenter conditions (pH, gas, nutrients), shear stress susceptible (no cell wall) and slow

Question 24

What does animal cell cultures produce

Answer 24

Recombinant biopharmaceutics, Vaccines, mAbs and Interferons

Question 25

Master Cell Bank Outline

Answer 25

Manufactured from 1 cel line: growth, productivity, product genes integrity, expression stability and microbial purity. Consistent and renewable source of cells for manufacturing. Critical raw material

Question 26

Large Scale Cell Challenges

Answer 26

Protein expression maximisation, product quality, contamination minimisation and control environmental conditions

Question 27

Critical Barriers to Large Cell Culture

Answer 27

Limitation oxygen supply, waste product accumulation, sophisticated process control, shear sensitivity

Question 28

Are suspended or adherent cell lines better for use

Answer 28

Suspension as cells have more space to proliferate and less competition

Question 29

Transgenic Animal Outline

Answer 29

Intro specific functional genes to produce large quantities of biopharmaceuticals. Gene modified, implanted into animal egg, mutation present in the offspring, protein produced in milk

Question 30

Plant Expression Treatment Outline

Answer 30

Agrobacterium infect plant cells ,plants produce heterologous proteins. Advantages: easy scale up. Disadvantages: Low expression levels

Question 31

Viral Contamination of Animal Cells

Answer 31

Virus integrates DNA into genome, transmitting disease. 3 methods of removal: Elevated temps (35-60 C) for several hours, multiple filtrations (0.1-0.2 mcm) and UV Irridation

Question 32

What happens once protein is produced in expression system

Answer 32

Protein recovery, rmoval of whole cell debris, primary purification, chromotography (purity determination) and protein characterisation

Question 33

Stages in Development

Answer 33

Cells medium adapation and establishment of master and working cell banks

Question 34

Upstream Processing Steps

Answer 34

Culture media fermentation in bioreactor, extracellular protein extraction (filtration)

Question 35

Downstream Processing

Answer 35

Multistep purification (eg ion exchange rsin) evaluation by chromtography and viral inactivation

Question 36

Final Formulation

Answer 36

Sterile filtration and aseptic vial filling