Lecture 1

Question 1

What are biologics?

Answer 1

A biologic is any pharamceutical drug product that may be produced through biotechnology in a living system, such as a microorganism, plant cell or animal cell

Question 2

What are the differences between biologics and small molecule drugs?

Answer 2

1. Small molecular drugs have a smaller molecular weight (<1000Da) compared to biologics
2. Small molecular drugs have a simple and well defined structure thats always known, whereas biologics have a more complex structure that may not always be known
3. The production of Small molecular drugs are chemical processes based on a series of controlled and predictable reactions, whereas biologics are derived from living sources, so there is a complex synthesis process
4. The production of Small molecular drugs yields a homogenous product whereas the production of biologics may yield heterogenous products.
5. The process of producing Small molecular drugs is standardized, the final structure is easily verified and the contaminants are quantifiable whereas biologics are not easily characterised and refined to a high degree of purification, so there is batch to batch variation.

Question 3

What are the advantages of using biologics?

Answer 3

1. Unlike small molecular drugs, biologics are more specific because of their larger size ad greater extent of interaction with the target protein –> hence they behave more predictably
2. Since biologics are bigger and more specific, there is a lesser chance of off target binding, so lower toxcitiy
3. Unlike small molecular drugs, macromolecules are broken down more predictably by hydrolysis, so its easy to see if its metabolites are toxic

Question 4

What are the limitations of using biologics?

Answer 4

1. Biologics are more heat sensitive and have a limited shelf life compared to small molecular drugs.
2. Biologics are more easily degraded by enzymes.
3. Biologics may be potentially immunogenic
4. Biologics are too big to be absorbed through the gastrointestinal tract, so it has to be administered through invasive methods which makes them less accessible
5. It cant cross through the cell membrane, so it either has to be modified to be able to enter, or its function is only limited to the cell surface

Question 5

What are the different biopharmaceutical modalities?

Answer 5

1. recombinant proteins
2. Peptides
3. Antibodies
4. Nucleic acids
5. Engineered cells

(4 and 5 are emerging modalities btw)

Question 6

What are biosimilars?

Answer 6

It’s a type of biologic that is almost an identical, alternative version of the innovator biologic, which is manufactured after the innovator biologic’s patent expires

Question 7

Can a biosimilar biologic be entirely identical to the innovator biologic?

Answer 7

1. For biologics of low MW, it could be identical because its development process is more straightforward
2. For antibodies, they are larger proteins with post translational modifications so its impossible to engineer a 100% identical biosimilar

Question 8

What are the steps in gene cloning?

Answer 8

1. PCR to amplify gene of interest
2. Digestion of vector and gene of interest with restriction enzymes
3. Ligation
4. Transformation into host cells
5. Culture to proliferate cells
6. Selection of required rDNA

Question 9

What are the different temperatures used in the different steps of PCR?

Answer 9

1. Denaturing - 95degC
2. Annealing - 50-60degC
3. Elongation - 72degC

Question 10

What are the limitations of PCR?

Answer 10

1. The sequences of primer annealing sites must be known
2. There is a limit of the length of DNA that can be amplified using PCR
3. DNA replication may not be 100% accurate and mutations may be introduced into the DNA which then gets amplified

Question 11

What is the purpose of Sanger sequencing?

Answer 11

To determine the nucleotide sequence of DNA

Question 12

What is the process of Sanger sequencing?

Answer 12

1. Gene of interest is cloned into vectors, and then the double-stranded DNA in the vector is converted to single stranded DNA by denaturation with alkali or boiling.
2. Thermal cycle sequencing is carried out by DNA polymerase using one primer. Involves enzymatic DNA polymerase synthesis of a second strand of DNA, complementary to existing template.
3. Fluorescent dideoxynucleotides are added at low concentrations, and get randomly incorporated into the new strand. They lack the hydroxyl group so the chain gets terminated. This process creates different lengths of PCR products labelled with terminal fluorescent dNTP.
4. This process is repeated separately with each of the 4 dideoxy bases (4 concurrent strand synthesis reactions). Thus, 4 separate reactions result in 4 families of terminated strands.
5. The double stranded DNA can be separated by heating, and the fragments are separated by electrophoresis.
6. Fluorescence (of each dideoxy base) detected and read by the detector.

Question 13

What are some pharmaceutical applications of Sanger sequencing?

Answer 13

1. Sequencing of genetic mutations associated with diseases allow us to identify causal genes and design drug targets.
2. It allows us to create personalized medication by analysing the patients genome to design more specific drugs and predict drug gene interactions to prevent adverse drug reactions