Biochemistry Class Biologic Presentation Final Draft4

Advancement of Biopharmaceuticals:

The synthesis of Erythropoietin

ByMichael English And Asefa Dinagde

Erythropoietin is a signaling glycoprotein

• Glycoproteins are proteins (amino acid chains) that have covalently attached oligosaccharides side chains (linked carbohydrate sugars).

• Oligosaccharides are typically oxygen or nitrogen linked to amino acids in proteins.

Figure 1:

Linked Carbohydrate Sugars

Protein Amino Acid Chain

• Multiple carbohydrate sugars can be N-linked to asparagine or O-linked serine and threonine.

Wales Jimmy, Larry Sanger et al. “Glycoprotein.” Wikipedia. March, 2015. Wikimedia Foundation. 3 March 2015 < https://en.wikipedia.org/wiki/Glycoprotein >

https://en.wikipedia.org/wiki/Glycoprotein

Motif of glycoprotein• An asparagine amino acid in a protein can accept a carbohydrate side chain only if

it is part of a specific sequence.

Asn - X - Ser or Asn - X - Thr• The X can be any amino acid except proline.• This pattern allows, potential glycosylation sites to be detected based on a

protein's amino acid sequence.• However, not all potential sites are glycosylated.• Which sites are glycosylated depends on the cell type that expresses the

glycoprotein. Amino Acid Figure 2:

Glycosidic Nitrogen Bond Glycosidic Oxygen Bond

Carbohydrate Sugar

Berg Jeremy M., John L. Tymoczko and Lubert Stryer. Biochemistry 7th Ed. New York: W. H. Freeman and Company, 2011. Page 330.

Motif of glycoprotein• All N-linked oligosaccharides have in common a 5-saccharide core consisting of 3

mannose and 2 N-acetylglucosamine residues.• Other sugars are attached to this core to form a great variety of oligosaccharide

patterns found in glycoproteins.

Figure 3:

Common 5-Saccharide Core


Erythropoietin

• Erythropoietin is a hormone that controls erythropoiesis (red blood cell production) and is found in the plasma.

• Erythropoietin is produced naturally in the kidney and the liver.• Under hypoxic (oxygen starved) conditions, the kidney produces erythropoietin

to increase the production of red blood cells.• Patients who have anemia due to kidney dysfunction, do not produce enough

erythropoietin to maintain healthy red blood cell levels.• Anemia can also be a side effect of Aids and cancer treatments.• Erythropoietin’s amino acid backbone is highly conserved but it’s oligosaccharide

side chain locations vary.• This makes the in vitro synthesis of a homogenous erythropoietin that can be

administered clinically difficult.• Currently most Erythropoietin is manufactured in vivo using biologic technology.

Wang, Ping, et al. “Erythropoietin Derived by Chemical Synthesis.” Science. Dec. 2013. American Association for the Advancement of Science. 19 Jan 2015

What is a Biopharmaceutical?• Biopharmaceuticals are medical products that are manufactured in living

organisms.• Biologic drugs are manufactured by cells that have been ‘programmed’ by

recombination.• Recombinant cells are made by using restriction enzymes to cut specific genes

out of a cell.• These genes are then inserted into a host cell that expresses this gene.• Host cells then act as factories and produce valuable proteins that can be

extracted and purified.• These proteins can then be administered to patients to treat diseases.• Biologics are currently being used to treat a wide variety of diseases that

includes cancer, autoimmunity and diabetes.• Biologics are typically high molecular weight proteins that are difficult to

manufacture synthetically.

C. Hsieh-Wilson, Linda and Matthew E. Griffin. “Improving Biologic Drugs via Total Chemical Synthesis.” Science. Dec. 2013. American Association for the Advancement of Science. 19 Jan 2015.

Biologic Erythropoietin• The gene for human Erythropoietin was spliced from human liver cells.• Restriction enzymes are then used to place this gene into cells who express this

gene.• These cells translate Erythropoietin and secrete it into their culture medium.• Cells tend to produce heterogeneous proteins due to post transcription enzyme

activity and existing chaperone proteins. • This problem is worse when glycoproteins are manufactured.• Glycoproteins have oligosaccharide chains that are assembled after translation.• N-linked oligosaccharide chains are added in the endoplasmic reticulum and in

the Golgi apparatus of a cell.• O-linked oligosaccharides are also added in the Golgi apparatus of the cell, but

there is no consensus on how specific glycosylation sites are determined.• This results in biologic Erythropoietin with heterogeneous oligosaccharide side

chains.


Chemical Synthesis of Erythropoietin

• Studies suggest that specific oligosaccharide locations improve the effectiveness of Erythropoietin.

• In 2002, Ping Wang et. al. determined that an effective “wild type” erythropoietin has 4 conserved glycosylation sites.

• 3 sites are N-linked and 1 site is O-linked. EPO Protein Amino Acid Chain

Figure 4:

1 of 4 EPO

oligosaccharides

Wang, Ping, et al. “Erythropoietin Derived by Chemical Synthesis.” Science. Dec. 2013. American Association for the Advancement of Science. 19 Jan 2015.


• Erythropoietin can be synthesized in vitro by using 4 crucial techniques:

a) Native chemical ligation: This technique combines two smaller unprotected protein fragments into a larger protein. One protein must contain a thiolate group on a N-terminal cysteine residue. The other protein must C-terminal thioester. A trans-thio-esterification and a S,N-acyl shift combines both proteins.

b) O-mercaptoaryl ester rearrangement mediated ligation: An ester re-arranges it’s sulfur and oxygen groups so it can be used to combine two protein fragments together.

c) One-flask aspartylation: Attaches an oligosaccharide to an amino acid by a Glycosidic Nitrogen Bond .

d) Metal free desulfurization: Uses light and a free radical to remove sulfur from cysteine to convert the amino acid to alanine.

Wang, Ping, et al. “At Last: Erythropoietin as a Single Glycoform.” NCBI. Medicine. Sep. 2012.Bethesda: United States National Library of Medicine. 1 March 2015.

< http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500780/ >

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500780/


Figure5: Four Critical EPO Synthesis Techniques

Wang, Ping, et al. “At Last: Erythropoietin as a Single Glycoform.” NCBI. Medicine. Sep. 2012.Bethesda: United States National Library of Medicine. 1 March 2015.

< http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500780/ >

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500780/

Role of Erythropoiesis • Erythropoiesis is from the Greek word ‘erythro’ meaning ‘red’ and from ‘poiesis’

meaning ‘to make’.• Red blood cell production is stimulated by a decrease in O2 levels.• O2 levels are detected by the kidneys.• When low O2 levels are detected, peritubular capillary endothelial cells in the

kidneys secrete the hormone erythropoietin to stimulate the production of red blood cells within red bone marrow.

Figure: 6

Wales Jimmy, Larry Sanger et al. “Erythropoiesis.” Wikipedia. Sep., 2014. Wikimedia Foundation. 3 March 2015

< https://en.wikipedia.org/wiki/Glycoprotein >


Regulation of erythropoiesis• A functioning erythropoiesis feedback loop allows for the number of red blood cells

to maintain stable levels.• When the body is in a diseased state, blood cells that are lost are replaced so

sufficient oxygen levels are maintained.• Erythropoietin levels in circulation increase as the levels of red blood cells decrease.

Figure 7:

Wales Jimmy, Larry Sanger et al. “Erythropoiesis.” Wikipedia. Sep., 2014. Wikimedia Foundation. 3 March 2015 < https://en.wikipedia.org/wiki/Glycoprotein >


Regulation of erythropoiesis• Erythropoietin acts on stem cells in bone marrow.• It stimulates stem cells to fully mature into red blood cells.

Figure 8:



Blood doping• Blood doping is the practice of boosting the number of red blood cells in order to

increase athletic performance.• This enhances bloods ability to deliver oxygen to the lungs and improves the

athlete's aerobic capacity.• Some endurance athletes have used recombinant human EPO to

increase their red blood cell count.

Figure 9:

Wales Jimmy, Larry Sanger et al. “Erythropoiesis.” Wikipedia. Sep., 2014. Wikimedia Foundation. 3 March 2015

< https://en.wikipedia.org/wiki/Glycoprotein >



Blood doping• Recombinant erythropoietin can increase an athlete's maximal oxygen uptake a

significant amount.• EPO blood doping is difficult to detect since other types of blood doping resulted

in a gradual decrease in blood cell levels after a few days.• EPO blood doping can keep red blood cell levels up for weeks.



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Biochemistry Class Biologic Presentation Final Draft4