Tutorial 3: Recombinant Proteins Flashcards

0
Q

What are the advantages of recombinant proteins?

A

Transcription and translation of an exact human gene can lead to a higher specific activity of the protein and a decreased chance of immunological rejection (just keep domain that has activity).
Recombinant proteins often produced more efficiently and inexpensively, and in potentially limitless quantity (limited availability of source tissue).
reduction of exposure to animal or human diseases (eg viruses).
Allows modification of a protein or selection of a particular gene variant to improve function, activity or specificity, or even create new function (increase half life, penetration and stability).

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1
Q

Describe the production of protein therapeutics.

A

A small number of protein therapeutics are purified from a native source (eg pig pancreatic enzymes, pooled human plasma).
Most protein therapeutics are now produced by recombinant DNA technology, and purified from a wide range of organisms.
The system of choice is dictated by the cost of production, or the protein modifications required for biological activity. for example, bacteria do not perform glycosylation reactions, and each biological system produces a different type or pattern of glycosylation.

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2
Q

Describe the innitial isolation of insulin, and the production of insulin using recombinant DNA technology.

A

In 1922, insulin was purified from bovine/porcine pancreas and injected into diabetic patients. A number of problems hindered the widespread use of this protein:
- availability of animal pancreas for purification
- cost of insulin purification from animal pancreas
- immunological reaction of some patients to animal insulin
These problems were addressed by isolation the human insulin gene and engineering E. coli to express human insulin by using recombinant DNA technology.
- by growing vast quantities of these bacteria, large-scale prodction of human insulin was achieved.
-resulting insulin was abundant, innexpensive, of low immunogenicity and free from other animal pancreatic substances
- recombinant insulin was approved by the US FDA in 1982
- First commercially available recombinant protein therapeutic, and has been the major therapy for T1D

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3
Q

How can protein drug therapy be improved?

A

Consider route of administration: IV and oral routes do not allow a drug to cross the BBB.
Dose: increase frequency or amount of drug in dose
Find a way to increase the absorption and distribution of the drug
Find a way to decrease the metabolism and excretion of the drug
Increase plasma half life and protein stability (eg encapsulation to prevent enzymatic degradation)
Increase specificity of binding to receptor, improve targeting, decrease side effects.
Increase binding efficacy to receptor to amplify effect of drug.

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4
Q

describe things affecting the half life and stability of a protein.

A

The half life of a protein is affected by inherent stability and rate of clearance.
Physical instability may relate to a protiein’s absorption, aggregation or denaturation.
Chemical instability may be the result of susceptibility to hydrolysis, oxidation or reduction.
Factors affecting the clearance of a protein include its size (small peptides more easily cleared by the kidney), action of proteases in blood or inside cells, nature of the N-terminal residue/hydrophillic properties.

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5
Q

describe protein degradation (PEST hypothesis, N-end rule and the ubiquitin proteosome pathway).

A

PEST hypothesis: polypeptide and protein sequences rich in porline (P), glutamate (E), serine (S) and threonine (T) are more rapidly degraded than others.
N-end Rule: The identity of the N-terminal residue of a protein determines its in vivo half life.
The ubiquitin-proteosome pathway targets proteins for degradation through their destabilising N-terminal residues (controls many physiological processes)

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6
Q

How can an improved EPO drug be developed?

A

Protein glycosylation patterns can have a dramatic effect on the activity, half life and immunogenicity of recombinant protein.
EPO is a sialo-glycoprotein. Sialic acid residues on EPO are responsible for maintaining in vivo biological activity. When EPO is dsialylated in vivo, it binds to receptors in the liver, thus being cleared from the plasma.
Thus, there is a direct correlation between the amount of sialic acid-containing carbohydrates, plasma half life and in vivo biological activity of EPO.
The half life of native EPO can be increased by increasing the glycosylation of protein. This discovery led to the production of several dozen analogues of rHuEPO with AA substitutions which were screened to create one or more new carbohydrate addition sites –> more potential sialic acid residues on the molecule.

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7
Q

What are the steps involved in creating darbepoetin-alpha?

A
  1. site-directed mutagenesis –> change in the nucleic acid sequence encoding one or more AAs of a human EPO cDNA clone.
  2. the clone encoding each new candidatie analogue was then transfected into mamalian cells and the expressed protein analysed.
  3. testing: only a few of the several dozen analogues tested were fully glycosylated, had the proper tertiary structure, and retained biological activity.
    the EPO analogues each had one extra N-linked carbohydrate chain or 2 extra N-linked carbohydrate chains.
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8
Q

What are the differences between rHuEPO and darbepoetin?

A

rHuEPO has up to 14 sialic acid residues, while darbepoetin has up to 22.
rHuEPO has the same AA sequence as human EPO
AA sequence of darbepoetin-alpha differs from that of native human EPO at 5 positions (5 AA substitutions).
Darbepoetin has a higher carbohydrate content than rHuEPO.
Darb has a higher molecular weight and a greater negative charge than rHuEPO due to its increased sialic acid content.
Darbepoetin has a 3-fold larger circulating half life than native EPO or rHuEPO.

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9
Q

Compare the efficacy of darbepoetin.

A

rHuEPO has an IV half life of 4-9 hours, and a subcutaneous half life of more than 24 hours.
Darbepoetin has less affinity for EPO receptor than native EPO, however this is compensated for by its increased potency.
Due to pharmacokinetic differences, the relative potency of the 2 molecules varies as a function of dosing frequency in CKD patients.
- Darbepoetin is 3.6x more potent than rHuEPO in increasing haematocrit when each is administered twice weekly.
- When admin frequency is reduced to once weekly, darbepoetin is 13-14x more potent than rHuEPO.
A new 3rd gen EPO called CERA has been produced.

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