Winter exam questions

Question 1

What is the best expression host to use and why?

Answer 1

Mammalian cells are preferable as they have a higher PTM modification compared to bacteria. Although they are slow and expensive

Question 2

Talk about upstream and downstream

Answer 2

Upstream: Cells are modified to express the gene of interest. They’re grown in large quantities. They manufacture the protein of interest and secrete it into the media

Downstream: 1: media is filtered to reduce bulk volume and retain product. 2: Purification steps. 3: Bioanalytical analysis. 4:Antiviral. 5: Sterile fill and vialing

Question 3

Which form of amino acids is found in proteins?

Answer 3

L-form of zwitterionic molecules

Question 4

Define enzymes

Answer 4

Highly selective catalysts that convert substrates into products. In the biopharma industry, Enzymes
accelerates the reaction by lowering the activation energy of a reaction
a good example is urease
They don’t take part in the reaction

Question 5

Define cellular respiration

Answer 5

The generation of ATP in all living things. Lipid oxidation is where the most ATP is produced. Red cells do not respirate

Question 6

Talk about cox 1 and cox 2

Answer 6

Asprin inhibits by binding to enzyme Cox 2. This prevents production of prostiglandins. Cox 2 normally binds to archeadonic acid and converts to prostiglandins which cause an inflammatory immune response (pain) . An off target side effect is that cox 1 is inhibited which produces cytoprotective prostoglandins to prevent gastric mucosa. This results in ulcers and indegestion

Question 7

What is the active site?

Answer 7

The part of the protein in which the substrate binds to. its the region in the enzyme that comprises of amino acid residues. 5-8 amino acids makes up the active site. The protein needs a 3D shape so the Amino acid can bind to the substrate
Characteristics:
Amino acids might be separated
The protein must be properly folded for the amino acid to bind
Substrate binds via hydrogen bonds
Enzymes can have more than one substrate

Question 8

What are the 4 types of specificity in enzymes and describe what they are

Answer 8

Bond specificity: When an enzyme only reacts with a specific chemical bond
Group specificity: When an enzyme will only react to substrates with similar functional groups
Optical/stereo specificity: When the enzyme is not specific to substrate but also optical configuration
Dual specificity: Can convert two substrates at once, may act on one substrate by two different reaction types

Question 9

What is an Apoenzyme?

Answer 9

Apoenzyme is an enzymatically inactive protein part of an enzyme, which requires a cofactor for its activity

Question 10

Differences between cofactors and co-enzymes

Answer 10

Cofactors are non-proteins which assist enzymes in performing
catalytic actions.
Cofactors are metals i.e. cations
Co-enzymes are more organic, i.e. vitamins e.g. ATP
Some enzymes require 1 or both

Question 11

Give three examples of cofactors and the enzyme/protein they code for

Answer 11

Zinc = Carbonic anhydrase
Zinc = Alcohol dehydrogenase
Potassium and magnesium = Pyruvate phosphokinase

Question 12

Name a cofactor that is permanently attached

Answer 12

Haem in Haemoglobin

Question 13

What are the 6 classes of enzymes based on reaction catalysed and what is the reaction carried out

Answer 13

Oxidoreductase: Transfer of reactions, i.e. alcohol dehydrogenase
Transferases: Transfer of C,N or P groups, i.e. Hexokinase
Hydrolases: Bonds cleavage by adding water i.e. Trypsin
Lyases: Cleavage of the same elements to make a double bond i.e. Pyruvate decarboxylase
Isomerases: Forms optical or geometric isomers i.e. Maleate isomerase
Ligases: Hydrolysis of high energy phosphates to form new bonds i.e. pyruvate carboxylase

Question 14

What is the most common structural characterisation of enzymes involved in glycolysis

Answer 14

Oligomeric enzymes

Question 15

What does “ “ refer to in EC classification
A:
B:
C:
D:

Answer 15

A: type of reaction
B: Subclass indicating type of substrate
C: Sub-sub classes, precise bond/reaction catalysed
D:Individual reaction

Question 16

What is the differences between continuous assay and discontinuous assays

Answer 16

Continuous assay are measured in real time with continuous monitoring this means that the reaction proceeds without stopping which results in gathering immediate data and detailed kinetic information.
Discontinuous assays collects data from samples taken at specific intervals and the reaction is stopped at set points. This results in limited kinetic data

Question 17

What is a reversible inhibitor?

Answer 17

A molecule that binds to an enzyme and temporarily blocks it which alters the 3D shape of the enzyme. It doesn’t permanently inactivate the enzyme and can be removed when the condition changes. There are 3 types, competitive, non competitive and uncompetitive

Question 18

What is meant by Q10?

Answer 18

Its the factor in which the rate of the enzyme increases with a 10 degree rise

Question 19

How are antibodies used in industry?

Answer 19

Therapies: Humeric
Biomarkers for disease: ELIZAS = Tyrosine
Research: GFP

Question 20

What is the differences between monoclonal and polyclonal **

Answer 20

Monoclonal: Identical protein molecules
Bind to only one epitope
Specific binding
Easier to manufacture
Production takes up to a year
Used directly in therapies i.e. IV (Due to IV’s going into the blood stream, it has to be done by a med professional which is inconvenient)

Polyclonal: A mixture of protein molecules (antibody)
They vary in the FAB region
They’re sensitive which is an advantage
Used in diagnostic assays i.e. ELISAS
They have some research applications

Question 21

What are the effector functions of antibodies?

Answer 21

They block ligand- receptor interactions (auto immune)
Cause cell lysis through activating complement dependent cytotoxicity (Fights cancer)
interacts with FC receptors on effector cells to engage antibody cell cytotoxicity (Cancer)
Signal for ingestion of a pathogen by a phagocyte (Cancer)

Question 22

Why is IgG used in commercial drug manufacturing?

Answer 22

Effector mechanism
Bioavailability ( stable in blood)
MAB purification (Affinity chromatography)
IgG1 is the major Ig in serum with fluid carrying vessels
IgG is the only class of Ig that crosses the placenta.
Binds macrophages and monocytes increases efficiency
Down stream purification is possible even in large volumes

Question 23

What are the 5 immunoglobin classes and functions?***

Answer 23

IgG: Monomer, capable of carrying out all effector functions from the serum and can travel around the body
IgM: Pentamer, the first Ig to be made by the fetus, is a good complement fixing Ig
IgA: Monomer, made in the plasma, found in tears and saliva doesn’t fix complement
IgD: Only exists as a monomer, found in low levels of serum
IgE: Monomer, least common serum as it binds to Fc receptors, involved in allergic reactions

Question 24

What are some advantages to immunoassays?

Answer 24

They are are highly sensitive, it detects low quantities of Ag
Highly specific, relies on specificity of AB component
High thorough put
Cost efficient

Question 25

What is protein A/G chromatography

Answer 25

Its used in Biopharma industry to make MAB. It has been largely confined to the use of protein A and protein G. Protein G is a cell wall protein from group c and G streptococci. It binds strongly to the FC region of Ig Protein A is a surface protein originally found in the cell wall of bacteria staphylococcus aerus

Question 26

What is affinity chromatography and the steps

Answer 26

Makes use of specific binding that occurs between molecules and is used extensively for the isolation of biological molecules Steps: Affinity purification: Ligand is immobilised to a solid support Specifically binds its partner under mild buffer conditions After binding to the partner, support is washed with buffer to remove unbound components Elution buffer is then added disrupting interaction between ligand and partner

Question 27

Explain and differentiate between the common assays ***

Answer 27

Direct immunoassay: A one step solution, needs to be added to detect the antigen Sandwich immunoassay: The analyte is bound between two antibody reagents Indirect immunoassay: A two step method. First a primary antibody is incubated with the antigen. Its then incubated with the secondary antibody (Which is tagged with a fluorophore) It recognises the primary antibody Indirect, non competitive sandwich: The primary antibody binds the antigen. The secondary antibody binds the primary antibody- labelling and signal amplification (bound to a coloured signal) Competitive: Unlabelled analyte in sample is measured by its ability to compete with a labelled antigen

Question 28

How is a monoclonal antibody made for mass production?

Answer 28

1) Make hybridoma 2) Select the clone 3) Determine Gene sequence 4) Humanise Antibody 5) Make GMO -> Mammalian cell 6) Send for recombinant drug production, upstream and downstream

Question 29

What are the steps for making a hybridoma?

Answer 29

1) A sample of B cells are extracted from the mouse spleen and is added to a culture of myeloma cells (To make cells immortal) 2) Cells are then fused together using PEG or electroporation 3) Results in the formation of hybridomas

Question 30

What is the selection of antibodies producing immortal cells

Answer 30

The non fused myeloma cells don't express the new antibody against the antigen whereas the non B cells do The hybridomas are grown in HAT medium to select for fused cells. Fused cells will have the immortality of the myeloma cells and the antibody production of the B cell lines

Question 31

Describe Competitive inhibitors*

Answer 31

Both the inhibitor and substrate compete for the active site. Increasing the substrate concentration will decrease the chance of inhibitor binding to the enzyme. It will require a higher concentration of substrate to achieve this so the KM will also be higher. The Vmax is unchanged and the Km is increased

Question 32

Describe Non- Competitive inhibitors*

Answer 32

This inhibitor reacts with either the enzyme- substrate complex or the enzyme and forms the rate of reaction to form the enzyme product complex The Vmax is decreased and the Km is unchanged

Question 33

Describe Un-Competitive inhibitors*

Answer 33

A rare class of inhibition. This inhibition binds to the enzyme and enhances the binding of the substrate but the resultant substrate only forms the product slowly This results in both the Km and Vmax decreasing

Question 34

What is glycosylation and CQA?*

Answer 34

Glycosylation is a biochemical process in which a carbohydrate is covalently attached to a protein or lipid. This modification typically ends in the endoplasmic reticulum and Golgi apparatus of cells and plays a crucial role in the structure stability and function of proteins CQA is the physical, chemical or biological property of a drug that must be controlled with predefined limits to ensure product quality and safety

Question 35

Why is IgG the main molecule used*

Answer 35

IgG is found in the blood which aids in efficiency. It also contains defences in the blood. It is relatively stable and it contains defences in the blood and it contains bioavailability. The blood carries the drug to the target. Theyre responsible for the majority of the effector responses. When the antibody gets there, it binds and inactivates the pathogen by telling the other molecules like mast cells to attach. This is the effector response. It can act as an antagonist by binding and blocking the pathogen. It binds to the growth receptor

Question 36

What is HAT medium

Answer 36

HAT medium stands for Hypoxanthine-aminopterin-thymidine medium. It is an election , medium for mammalian cell cultures which relies on the combination of aminopterin which is a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase with hypoxanthine and thymidine which are immediates in DNA syntheses

Question 37

Explain Gel filtration in chromatography

Answer 37

Pores present in the beads of the gel matrix. Size of the pores in the resin determine the exclusion of a GF column Proteins that are larger than all the pores in the gel matrix are said to be excluded and eluted in the void volume Proteins with sizes within the fraction range will elute according to their size. Larger proteins elute first and smaller proteins will elute last

Question 38

What is a LFD?

Answer 38

A lateral flow device/ Lateral flow test is a form of assay that detects the presence of a target without the need for specialised equipment. They're used in covid tests for detecting the virus spike protein or the HGC hormone in pregnancy tests

Question 39

How do LFD's work?

Answer 39

They run a sample along the membrane surface that use reactive molecules that show a visual positive or negative result . The membranes are based on a series of capillary beds that transport the fluid efficiently. The conjugate pad holds the reagents for a chemical reaction between the antigen and its antibody which has been immobilised. As the target molecules move on they are marked and moved to the test for detection, this test line often signals as a colour. LFT's can be a competitive assay or a sandwich assay.

Question 40

Effect of pH and temperatures on enzymes

Answer 40

The optimum temp for enzymes is 30-40. Any higher of a temperature denatures the enzymes due to the degradation of linkages in the polypeptide chain . Whereas low temperatures inactivates the enzyme due to the reduction in speed of molecular movement. The most favourable pH value is the point in which the enzyme is most active. Extremely high or extremely low pH values result in the complete loss of activity for most enzymes

Question 41

What is glycosylation

Answer 41

Glycosylation is a common PTM for IgG antibodies produced in mammalian cells. IgG1 molecules contain a single N linked glycans at ASN297 in each of the 2 heavy chains. During the synthesis of the N-glycans, multiple sugar minorities can be added to form different glycoforms. Glycosylation plays an important role in CDC through modulating the binding to the FC receptor. Its important as many therapeutic proteins are PTM by the addition of N linked glycans. Glycosylation is considered to be a CQA of biotherapies by regulatory authorities