Lecture 4 Delivery of large and small molecules

Question 1

Describe the central dogma of molecular biology.

Answer 1

It is a theory stating that genetic information flows in one direction, from DNA to RNA to protein.

Question 2

What is recombinant DNA?

Answer 2

It is spliced DNA from different sources that have been cleaved by restriction enzymes and joined by ligases.

Question 3

Define transcription.

Answer 3

It is the process where a DNA segment is read and transcribed into a single-stranded sequence of RNA.

Question 4

Explain translation.

Answer 4

It is the process where the RNA sequence is translated into a sequence of amino acids to form a protein.

Question 5

What is a recombinant organism?

Answer 5

It is an organism whose DNA has been modified by joining together segments of DNA using genetic modification techniques.

Question 6

How is a recombinant DNA vector used?

Answer 6

It is a DNA molecule used to carry a specific DNA segment into a host cell as part of cloning or recombinant DNA techniques.

Question 7

Describe the sources of biopharmaceuticals.

Answer 7

They can be extracted from prokaryotes, yeast, fungi, mammalian cells, transgenic animals, insect cells, and plant cells.

Question 8

What are recombinant proteins?

Answer 8

They are proteins synthesized intracellularly by an introduced gene, with their amino acid sequence encoded by a cloned gene.

Question 9

Explain replication.

Answer 9

It is the process where a double-stranded nucleic acid is duplicated to produce identical copies.

Question 10

Describe the process of recombinant DNA technology.

Answer 10

Obtaining DNA fragment with gene of interest, cutting it with a restriction enzyme, ligating it into a plasmid, inserting the recombinant plasmid into a host cell, and expressing the gene/protein of interest.

Question 11

What is the role of DNA ligase in recombinant DNA technology?

Answer 11

DNA ligase catalyzes the joining of the DNA fragment containing the gene of interest and the plasmid to produce a recombinant plasmid.

Question 12

How are recombinant proteins purified from bacteria in biotechnology?

Answer 12

Recombinant proteins are purified by isolating them from the bacteria and then purifying to homogeneity using high resolution chromatography.

Question 13

Define the advantages of using prokaryotic cells like E. coli in recombinant DNA technology.

Answer 13

Advantages include simple physiology, short generation times, and high yield of protein expression.

Question 14

What are the disadvantages of using eukaryotic cells, such as mammalian cells, in recombinant DNA technology?

Answer 14

Disadvantages include slower growth compared to prokaryotic cells and the need for purification of tagged proteins.

Question 15

Describe the process of purifying recombinant proteins to homogeneity.

Answer 15

The process involves removing all contaminants that could cause infections and immunological reactions, typically carried out using high resolution chromatography.

Question 16

Explain the structure of the insulin molecule, including the number of amino acids in each chain and how they are linked.

Answer 16

Insulin consists of two chains: A chain with 21 amino acids and B chain with 30 amino acids. These chains are linked by two disulfide bonds.

Question 17

Describe the historical development of insulin isolation, including the contributions of Banting and Best. How did the methods evolve over time?

Answer 17

Banting and Best purified insulin from dog pancreas in 1921. Later, methods were developed to isolate insulin from cattle and pigs, with differences noted in porcine and bovine insulin compared to human insulin.

Question 18

Explain the significance of Eli Lilly’s production of insulin using E. coli in 1982. What was unique about this achievement?

Answer 18

Eli Lilly’s production of insulin using E. coli in 1982 marked the first genetically engineered pharmaceutical product, Humulin. This achievement revolutionized insulin production and reduced allergic reactions from animal- derived insulin.

Question 19

Describe the process of insulin production using recombinant technology with Method 1.

Answer 19

A and B chains are produced separately in separate bioreactors, then joined using lysosomes and cyanogen bromide, followed by purification through chromatography.

Question 20

How is insulin production carried out in the Proinsulin process?

Answer 20

After fermentation, insulin is isolated and additives like Zn2+ added to delay absorption in body

Question 21

Define Analog insulin and explain its significance in insulin production.

Answer 21

Analog insulin involves amino acid sequence changes, allowing for short acting insulins to act more rapidly and longer acting insulin longer duration of action.

Question 22

What are the factors that can affect insulin absorption?

Answer 22

Small molecules vs. Biologics, Types of Biopharmaceutics, Proteins, Nucleic Acids, Cell-based therapies.

Question 23

Describe in vitro stability barriers for large molecules.

Answer 23

In vitro stability barriers for large molecules involve susceptibility to chemical, environmental, and manufacturing conditions, leading to issues like self-association, formation of multimers, aggregation, and precipitation.

Question 24

What are some challenges posed by in vivo stability barriers for large molecules?

Answer 24

In vivo stability barriers include proteolysis, where proteins are broken down into smaller polypeptides or amino acids by proteases, and nucleases break down nucleic acids. Factors like low pH or high temperature can also cause non-enzymatic proteolysis.

Question 25

Define proteolysis in the context of large molecules.

Answer 25

Proteolysis refers to the breakdown of proteins into smaller polypeptides or amino acids, which can occur in vivo through enzymatic catalysis by proteases.

Question 26

How do absorption barriers impact the delivery of large molecules?

Answer 26

Absorption barriers can hinder the effective delivery of large molecules by impeding their absorption into the bloodstream or target tissues, affecting their bioavailability and therapeutic efficacy.

Question 27

Explain the concept of distribution and excretion barriers for large molecules.

Answer 27

Distribution and excretion barriers refer to challenges faced by large molecules in terms of reaching their target tissues or organs due to factors like limited distribution within the body and obstacles in the excretion process, impacting their overall pharmacokinetics and effectiveness.