20/01/2024

Structure & Manufacturing

of Insulin and Erythropoietin
SAI HS . BODDU
PROFESSOR OF PHARMACE UTI CS
A JMAN UNIVE RS ITY

Contents
By the end of this topic, students should be able to:
▪Identify the structure of insulin
▪Define the manufacturing steps of insulin manufacturing
▪Identify the structure of erythropoietin
▪Define the manufacturing steps of erythropoietin manufacturing

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Introduction
▪Purified insulin was first isolated from pig or cow pancreas for the treatment of type I diabetes
in the early 1920s by F. Banting & J. Macleod.
▪In 1923, awarded the Nobel Prize for their work.
▪Before insulin was discovered in 1921, patients with diabetes did not have long lives.
▪The most successful therapy was to place diabetic patients on stringent carbohydrate-restricted
diets.
▪This could provide patients with a few more years of life, but could not entirely cure them.
▪Patients have died of hunger as a result of strict diets consisting of only 450 calories per day

Introduction
▪Genes encoding human insulin and growth hormone were cloned and expressed in E. coli in
1978 and 1979 respectively.
▪First licensed drug produced using recombinant DNA technology was human insulin, which was
developed by Genentech and licensed as well as marketed by Eli Lilly in 1982.
▪The incidence of diabetes is increasing at an alarming rate and it has been speculated that the
number of diabetic patients worldwide would increase to approximately 300 million by the year
2025
▪Consequently, the requirement for insulin will increase manifold (approximately more than
16000 kg/ year).

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Structure of insulin
▪Fredrick Sanger (1955) gave the first complete description of
insulin structure (in 1958 awarded the Nobel Prize for his
work).
▪Chemically, human insulin is small, simple protein composed
of 51 amino acids sequences and has a molecular weight of
5808 g/mol.
▪Insulin consists of two polypeptide chains, chain A & chain B 3 sulfa
which are linked together by two disulphide bonds between linkage
two cysteine residues, one at position 7 of both chain A & B present in
and other at position 20 of chain A & at position 19 of chain B. human
▪Chain A is composed of 21 amino acids, while chain B is
insulin
composed of 30 amino acids

Structure of insulin
▪Human insulin differs from pigs’ insulin by one amino acid, where threonine found at position 30
of chain B in humans is replaced by alanine in pigs insulin.
▪While cow insulin differs by three amino acids, where threonine found at position 30 of chain B
in human is replaced by alanine in cows insulin, while threonine found at position 8 of chain A in
human is replaced by alanine and isoleucine at position 10 of chain A in human is replaced by
valine in cow insulin.

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active insulin
Insulin production using biotechnology
▪Several recombinant protein based drugs, produced by various expression systems are approved
by FDA.
▪Among prokaryotes, Escherichia coli has always been preferred for production of recombinant
proteins as it offered several advantages including high growth rate, simple media requirement,
easy to handle, high yield, and very cost effective.
▪Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris are
very commonly used for production of recombinant proteins
▪Genetech’s method of production used chemically synthesized cDNA encoding for insulin A and
B chains separately. Thus, the two chains were purified and co-incubated under preferred two chain method
conditions to accelerate generation growth of intact disulfide bond formation
▪Alternatively, Eli Lilly used a single chemically synthesized cDNA encoding for human proinsulin
with subsequent purification and excision of the C-peptide, yielding an active insulin product

know the steps of the method

General Manufacturing Method

1. Human insulin is extracted from pancreas cells and an insulin-producing gene is isolated.
Human insulin gene consists of 1425 base pairs which is located on chromosome 1
2. A plasmid DNA is extracted from a bacterium and cut with restriction enzyme, forming
plasmid vector. ECoR1 = example of restriction enzyme cuts
palindromic sequence "6 pair"
3. Insert human insulin-producing gene into the bacterial plasmid vector to form the
recombinant DNA of human insulin-producing gene.
4. Introduce this recombinant DNA into a bacterial cell to form the recombinant bacterium.
5. The recombinant bacteria multiply in a fermentation tank and produce human insulin.
6. Insulin is extracted, purified and bottled. It is then ready to be injected into diabetic
patients.

https://academic.oup.com/edrv/article/42/3/374/6042201

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General Insulin Manufacturing Method

General Insulin Manufacturing Method

know the steps in order

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General Insulin Manufacturing Method

https://www.youtube.com/watch?v=y4AMgE6B5jI

Insulin Manufacturing (Two chain method)

▪A chain and the B chain of insulin are produced
separately and then fused.
▪Here, these 2 polypeptides are cultured in
bacteria in two different fermenters and then
purified. B-gal is stabilizing the A chain

▪Purified A and B chains are then incubated

under oxidizing conditions to form the disulfide
bonds that are present in human insulin
CNBr = cyanide bromine used to separate B-gal from the A chain
ampicillin resistance = Amp
tetracycline resistance = Tet
primer = P
starting from the primer the recombinant plasmid is read
β-Galactosidase: lactose to galactose and glucose

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Insulin Manufacturing (Two chain method)

▪Gene Isolation: Complementary DNA (cDNA) molecules encoding chain A and chain B are
obtained from human insulin mRNA using reverse transcription. The cDNAs of both chains are
amplified by PCR.
▪Insertion into the Plasmid: Two plasmids are cut using restriction enzymes to insert the DNA
sequence for the A chain or the B chain separately.
▪Transfection: The entry of recombinant plasmids into bacterial cells is called transfection.
Different techniques can be used for the transformation of E. coli, such as treatment with CaCl 2
or electroporation techniques. After the plasmid’s entry, the cells become transformed bacteria.
▪Medium Preparation: Lysogeny broth (LB) is used as a culture medium for E. coli.

Insulin Manufacturing
(Two chain method)
Bioreactor Fermentation: A and B chains are synthesized as their respective E. coli strains
replicate in separate fermenters.
Crude Product Isolation: The bacterial cells are removed from the bioreactor tank and must be
lysed with one of several methods, such as enzyme digestion, sonication, or freezing and thawing
of cells. The use of lysosome enzymes is preferred for large-scale operations, as it digests the
bacterial outer layer to release the insulin into the surrounding media, so detergents can later be
added to remove the cell wall.
Purification: Cell components are separated from the two desired insulin chains. Gel filtration
and ion-exchange chromatography methods are used to remove impurities.
Insulin Chain Isolation and Chain Joining: Insulin chains (A and B) are treated with sodium
dithionite and sodium sulfite to form the disulfide bonds that bridge the chains. This whole
process is called reduction-reoxidation, and it is induced by β-mercaptoethanol and air oxidation
to synthesize human insulin.

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Manufacturing human proinsulin

▪Another approach involves the expression of a single chemically synthesized cDNA encoding for
human proinsulin in E. coli followed by purification and subsequent excision of C-peptide by
proteolytic digestion.
▪This approach was more efficient and convenient for large scale production of therapeutic
insulin as compared to the two chain combination approach and has been used commercially
since 1986 increase the stability of B chain
and A chain otherwise the
chains will degrade

Insulin Manufacturing
▪After synthesizing the human
insulin, high performance liquid know these two only
chromatography is used to
detect any impurities.
Fast acting
Long acting
slight change in the amino acids are done to
make the insulin fast acting or long acting or
normal action

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EPO =
Erythropoietin-Background
▪Anemia is a common complication of advanced chronic kidney disease (CKD).
▪Pathogenesis of anemia of CKD is multifactorial, the decreased production of erythropoietin
with declining renal mass is considered the primary etiologic factor.
▪Anemia is associated with fatigue, weakness, and dyspnea, as well as worsening quality of life
and performance status.
▪Exogenous replacement of EPO by rHuEPO, along with iron, quickly became a standard of care
for the treatment of anemia in patients with CKD after the isolation of introduction of the EPO
gene into CHO cells.
▪EPO stimulates the production of red blood cells in the bone marrow in
response to falling levels of oxygen in the tissues.

recombinant human erythropoietin = rHuEPO

bigger than insulin

Structure of Erythropoietin
▪Erythropoietin (EPO) is an amino acid glycoprotein
hormone synthesized and secreted by interstitial cells to
stimulate the production of red blood cells in the bone
marrow in response to falling levels of oxygen in the
tissues.
▪Molecular mass- 30.4 kDa. Its 165 amino acid residues
chain forms four antiparallel α-helices, two β-sheets and
two intra-chain disulfide bridges (Cys7-Cys161, Cys29-
Cys33).
▪Carbohydrate portion (40% of the molecule) comprises
three N-glycans (at Asn24, Asn38, and Asn83) and one O-
glycan (at Ser126).
▪N-glycans serve a variety of functions, including the
protection of Epo from proteases and the modulation of
its receptor binding affinity

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Erythropoietin-History
▪Human EPO was first isolated from the urine of anemic patients in 1977, and its gene was later
isolated in 1983.
▪One year later, two groups succeeded in cloning the EPO gene and expressing it in Chinese
hamster ovary (CHO) cells, enabling development of recombinant human erythropoietin
(rHuEPO) as a drug.
First rHuEPO, epoetin alfa, was manufactured by Amgen and sold as Epogen ® in the US for
dialysis patients (in 1989).

Genetic engineering of Erythropoietin

Briefly, the genetic engineering of Human recombinant erythropoietin (rHuEPO) is as follows:
1) identification of the gene that produced erythropoietin
2) isolation of the gene
3) introduction of the gene into CHO cells
4) production of rHuEPO in CHO cells
5) purification of rHuEPO in a stable and biologically active form.

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