Production of Recombinant Pharmaceutical Proteins

Production of Recombinant
Pharmaceutical Proteins 4
Abstract
The proteins produced in the body control and mediate the metabolic
processes and help in its routine functioning. Any kind of impairment in
protein production, such as production of mutated protein, or misfolded
protein, leads to disruption of the pathway controlled by that protein. This
may manifest in the form of the disease. However, these diseases can be
treated, by supplying the protein from outside or exogenously. The supply
of active exogenous protein requires its production on large scale to fulfill
the growing demand. The process is complex, requiring higher protein
expression, purification, and processing. Each product needs unique
settings or standardizations for large-scale production and purification. As
only large-scale production can fulfill the growing demand, thus it needs
to be cost-effective. The tools of genetic engineering are utilized to pro-
duce the proteins of human origin in bacteria, fungi, insect, or mammalian
host. Usage of recombinant DNA technology for large-scale production of
proteins requires ample amount of time, labor, and resources, but it also
offers many opportunities for economic growth. After reading this chapter,
readers would be able to understand the basics about production of recom-
binant proteins in various hosts along with the advantages and limitations
of each host system and properties and production of some of the important
pharmaceutical compounds and growth factors.
4.1 Introduction these diseases can be treated, by supplying the

protein from outside or exogenously. The supply
The proteins produced in the body control and of active exogenous protein requires its produc-
mediate the metabolic processes and help in its tion on large scale to fulfill the growing demand.
routine functioning. Any kind of impairment in The process is complex, requiring higher protein
protein production, such as production of mutated expression, purification, and processing. Each
protein or misfolded protein, leads to disruption product needs unique settings or standardizations
of the pathway controlled by that protein. This for large-scale production and purification. As
may manifest in the form of the disease. However, only large-scale production can fulfill the growing
© Springer Science+Business Media Singapore 2017 77

V. Gupta et al., Basic and Applied Aspects of Biotechnology, DOI 10.1007/978-981-10-0875-7_4
78 4 Production of Recombinant Pharmaceutical Proteins
demand, thus it needs to be cost-effective. The have three RNA polymerases: polymerase I tran-
tools of genetic engineering are utilized to produce scribes ribosomal RNA genes (18S,5.8S and
the proteins of human origin in bacteria, fungi, 28SrRNA), polymerase II transcribes all protein-
insect, or mammalian host (Fig. 4.1). Usage of coding genes (mRNA and small RNA), and poly-
recombinant DNA technology for large-scale merase III transcribes the genes for 5SrRNA and
production of proteins requires ample amount of tRNA. In prokaryotic cells (E. coli), RNA poly-
time, labor, and resources, but it also offers many merase consists of five subunits—two identical α
opportunities for economic growth. After reading subunits and one subunit each of β, β′, and σ sub-
this chapter, readers would be able to understand unit. The σ subunit dissociates after polymerization
the basics about production of recombinant pro- ensues. Thus, the term “holoenzyme” is used for
teins in various hosts along with the advantages complete enzyme and “core enzyme” is used with-
and limitations of each host system and proper- out σ subunit. The process of transcription is initi-
ties and production of some of the important ated by binding of RNA polymerase to DNA
pharmaceutical compounds and growth factors. molecule at a very specific site called “promoters.”
The promoters are critical for the start of the tran-
scription. The promoter sites are nearly 40 bp long
4.2 Expression of Foreign Gene and are mostly located before the first base, which is
copied into RNA (Fig. 4.2a). This first base is called
In all living cells, the expression of gene occurs as start point of transcription and is denoted by +1.
where genetic information contained in DNA is
passed on to RNA in the process of transcription
and from RNA to protein in the process of transla- 4.2.1 Promoters
tion. Thus, the body synthesizes RNA and then pro-
teins according to the instructions from the Promoters for RNApol II allow differential
DNA. DNA-dependent RNA polymerase or RNA expression of genes and determine the rate at
polymerase carries the transcription. Eukaryotes which the genes are transcribed. There are some
Fig. 4.1 The gene of interest Gene of interest

is cloned in suitable vector.
For expression of the cloned Selective gene Essential transcriptional
gene, the gene is with all the DHFR (nucleoside metabolism) regulatory elements
essential regulatory elements
required for transcription of Glutamine synthase (glutamine
the gene. The gene is attached synthesis)
to the selective gene, which
helps in the selection of the
clones with gene of interest. Selection
The cells are screened for the
synthesis of desirable product
and then processed for Maintained in single cell
large-scale production in the
bioreactor with optimum
condition for high yields Screening for production
of recombinant protein
One/few cells/cell line is

choosen for production and
grown on large scale in a
bioreactor
4.2 Expression of Foreign Gene 79
a
E.Coli promoter
TTGACAT TATAAT Transcription Structural gene

-35box -10 box start site
Transcription
Eukaryotic promoter
start site
Exon 1 Exon2
Various GGGCGG CCAAT TATAAAT Intron
59 UTR 39 UTR
other GC box CAAT box -30 box
signals ~100 ~70 to -90
b Eukaryotic transcription and translation
Exon1 Exon2 Exon3 Exon4 heterogeneous RNA

Intron Intron Intron
Splicing for removal of introns
and joining of exons
Translation
mRNA
c Codon bias
Human gene codon for proline
CCA CCA CCA CCA
E.coli gene codon for proline
CCG CCG CCG
Fig. 4.2 The figure shows the problems encountered by intronless version of gene is used in E. coli. (c) shows
E. coli due to sequence of foreign gene, when it is cloned codon bias in bacterial and human system. As one amino
and expressed in it. (a) It shows the promoter for E. coli acid is encoded by more than one codon, for example,
and eukaryotes. As there are differences in the promoter proline, where preferential codons in humans are CCA
of E. coli and eukaryotes, thus eukaryotic promoter might whereas E. coli prefers CCG, if the gene containing the
not work in E. coli, and the gene is placed under the con- preferential codons for humans is used in E. coli, then it
trol of E. coli promoter. (b) In the eukaryotes the splicing might result in inefficient translation. Thus, these problems
machinery removes introns from the target gene. Whereas need to be taken care of while using E. coli as host
the E. coli does not have any such system, therefore, the
promoters that cause the inserted genes to be approximately −70–90 and −100, respectively.
expressed all the time; in all parts of the system, The location of promoter is always on the same
they are known as “constitutive” promoters. DNA molecule which they regulate. They are
Others allow expression only at certain stages/ referred as cis-acting elements. The spacing of
certain tissue/organ of individuals and at certain various elements is more important and much is
time points. Gene expression is under temporal dependent on locus-specific activators, either at
and spatial regulation. core promoter or at distant sites. Various other
In prokaryotes the position before start site at signals as enhancers are also involved which are
−10 and −35 can interact with σ subunit of holo- far apart from the target gene. They exert stimula-
enzyme of RNA polymerase. In eukaryotes the tory effects on promoter activity and can be
sequence (analogous to −10—consensus upstream, downstream or in the middle of the
sequence of prokaryotes), TATAAAA, is present gene. Promoters of housekeeping genes or genes
at −30 position in the promoter region. with complex patterns of expression have CpG
Eukaryotic promoter is shown (Fig. 4.2a) with islands rather than TATA box. The gene to be
TATA box forming the core promoter at −30 transcribed has 5ʹ-untranslated region (5ʹ UTR),
position (from −30 to −100), upstream of tran- exons (coding region) separated by introns (non-
scription start site. CAAT box and GC box are at coding region). The end has 3ʹ-untranslated
region (3ʹ UTR). In the cloning for gene expres- • Appropriate modification with higher specificity,
sion, usually intronless version of the gene is increased half-life, and improved functionality.
used. In eukaryotes, the transcription is further • Allow to create critical changes for better
enhanced by enhancers, which may be 2,000– specificity and activity.
3,000 bases away from the promoter region but
are able to affect the rate of transcription.
4.2.3 High Protein Expression
in the Host
4.2.2 General Considerations
for Protein Production Expression and production of eukaryotic protein
require cloning of its cDNA in an expression vec-
The simplest host for the work of recombinant tor and subsequent transfer of the vector in suit-
DNA technology is prokaryotic bacterial system. able host. DNA is modified, cloned, and expressed
In the early 1980s, the first recombinant FDA- in other host for the production of the protein;
approved pharmaceutical, the human insulin thus, optimum production conditions are required
(Humulin-US/Humuline-EU), was obtained from in each host. However, there are yield variations
genetically engineered Escherichia coli (E. coli) in different expression systems but high-level
for treatment of diabetes. expression of the protein may be achieved by
Due to increasing demand, many strains of considering the following points:
microbial species are being designed with
increased throughput and better recovery of the • The recombinant gene should be with all the
therapeutic protein from large-scale culture. necessary elements for effective transcription
The recombinant proteins approved by FDA are initiation.
obtained either from Escherichia coli or other • Use is made of strong viral or cellular pro-
prokaryotes; from Saccharomyces cerevisiae or moter/enhancer for efficient driving of tran-
other fungal species; from insect cells, mamma- scription. The usage of viral T7 promoter in
lian cells, or human cells; or from transgenic bacteria can result in higher yield of recombi-
plants or animals. Cloning and production of nant proteins. Transgene may be expressed
protein in a particular host system are dependent under the control of either polyhedrin pro-
upon a property of host to clone and express the moter of baculovirus, E1 promoter of adeno-
desirable size of protein-encoding genes; produc- virus, or p7.5 promoter of Vaccinia virus.
tion of correctly modified, folded, and functional These are suitable for wide range of cell types.
protein; high yield of the protein; and low-cost Mammalian cells may use promoter and
requirements. The choice of host systems requires enhancer of SV40 or the long-terminal-repeat
best system, which can fulfill the requirements promoter and enhancer of the Rous sarcoma
[17]. virus or early promoter of the human cyto-
The advantages of producing proteins using megalovirus (refer to Chap. 2 for promoters
recombinant DNA technology are: and expression vectors).
• Polyadenylation signals are helpful in eukary-
• As human gene may be cloned and expressed, otic genes. These terminators are required for
it minimizes the risk of immune reaction and defined 3′ end to the mRNA which extends by
the specific activity of the protein is high. addition of poly A tail ultimately increasing
• The therapeutic protein can be produced effi- the stability of RNA and facilitating its export
ciently, maintaining its cost-effectiveness. to the cytoplasm.
• It minimizes the risk of transmission of • Removal of 3′ and 5′ UTRs (untranslated
unknown pathogens present in animal and repeats) may influence gene expression. They
human sources. may interfere with initiation of translation and
4.3 Microbial System for Production of Therapeutic Protein 81
their secondary structure prevents efficient 4.3 Microbial System

translation. for Production
• Kozak’s consensus 5′-CCRCCAUGG-3′ with of Therapeutic Protein
purine at −3 position and guanine at +4 posi-
tion affects transgene expression. Though various host systems are available for
• Inclusion of one intron sequence, which is production of recombinant proteins, microbial
located between the promoter and cDNA coding hosts offer several advantages over other sys-
sequence, gives better yield; thus, most expres- tems, as production is fast, cheap, and
sion vectors include at least one intron sequence. economic:
Intron presence is of importance when transgene
needs to be expressed in mammalian cells. 1. The molecular biology and physiology is well
• For eliminating the slow rates of translation characterized and documented.
due to codon biasing, the gene may be con- 2. Easy to maintain and manipulate.
verted to a high-expressing gene by changing 3. Utilizes inexpensive nutrition sources.
the t-RNA codons to the most abundant ones. 4. Rapid growth and biomass accumulation to
• The integration site has a major effect on the achieve high cell densities.
rate of the transcription of the recombinant 5. Scale-up is easy and convenient.
gene (position effect). 6. Their expression machinery can be with vari-
• Some selective genes like dihydrofolate reduc- ety of strong inducible promoters.
tase (DHFR) or glutamine synthetase (GS) gene
are incorporated. The genes are involved in
nucleotide biosynthesis and glutamine synthe- Inducible Promoters
tase, respectively; the selection occurs when the The inducible promoter may require an
appropriate metabolite is missing preventing the inducer, or the depletion or addition of a
growth of non-transformed cells. specific nutrient, or pH change or changes
• For increasing the yield of the recombinant in physicochemical factors in order to initi-
protein, the protein is often fused with endog- ate the process of gene expression. The
enous protein sequence (see Chap. 5 for inducible systems suffer from the disad-
selectable markers, reporters). vantage that chemical inducers may be
• To aid purification, the protein-encoding DNA expensive and toxic and would require
contains coding DNA for specific protein or elimination during downstream processing
peptide that can be a target for affinity when the product is intended for human
chromatography. usage. Thus, usage of thermoregulated sys-
• For affinity purification, two systems are read- tems has been used for production of
ily employed, (1) glutathione S-transferase recombinant pharmaceutical proteins as
(GST)–glutathione affinity and (2) polyhisti- expression is dependent upon strong heat-
dine–nickel ion affinity. GST has high affinity regulated promoter minimizing the risks of
for glutathione and protein with side chains of any addition of chemical agent.
histidine has high affinity for nickel ions.
• Stable transfection gives high yield of protein.
After recombinant DNA is transfected into
animal cells, it either can be integrated (stable As with the cellular structure of bacteria, it
expression) into the host genome or main- can rapidly adapt to culturing conditions with
tained in episomal form (transient expres- very short replication time (20 min). The media
sion). In stable expression systems, the foreign requirement of bacterial cell is simple and con-
gene is passed on to the next descendants, and sists of simple carbon and nitrogen source.
the expression is maintained generation after Thus, the overall inputs in bacteria are 90 %
generation. lower than for mammalian cells. Some of the
approved bacteria-derived (by either the approach may be co-expression of rare

European Union or FDA, USA) therapeutics t-RNAs in E. coli (strains of E. coli, BL21
include hormones (human insulin and insulin codon plus, and Rosetta were designed for
analogs, calcitonin, human growth hormone, this purpose). For addition of amino acids
glucagons, parathyroid hormone, somatropin, during the process of translation, as there are
and insulin-like growth factor 1), interferons more than one codon for several amino acids,
(alfa-1, alfa-2a, alfa-2b, and gamma-1b), inter- thus, codon biasing occurs which is the pref-
leukins 11 and 2, light and heavy chains raised erence of a particular codon of amino acid in a
against vascular endothelial growth factor-A, particular species (Fig. 4.2c).
tumor necrosis factor alpha, cholera B subunit 5. Complexity: Eukaryotic cells have the advan-
protein, granulocyte colony-stimulating factor, tage of producing fully functional and prop-
and plasminogen activator [3]. erly folded proteins. The antibodies with four
The microbe of first choice for production of subunits may be secreted by eukaryotic cells
recombinant protein is enterobacterium E. coli. in fully functional form. On the other hand, it
The system offers quick and easy modifications, is very difficult to obtain multidomain protein
ease of growing in manageable environmental from E. coli. Even if protein is obtained, the
conditions and short life cycle. The bacterial renaturation and folding in laboratory condi-
cell can tolerate and adapt to changes in the tion may either be very expensive or protein
environment rapidly, thus scale-up is easier. may lack its activity.
However, the system suffers from some of the 6. Lack of posttranslational modifications
disadvantages [11,16]. (PTMs) is the problem, which cannot be
solved, and is mandatory for activity of many
1. Human or mammalian genes cloned in bacteria therapeutic proteins. The glycosylation is the
cannot undergo splicing due to lack of splicing most common modifications, and others are
machinery, thus intron-less version of the gene phosphorylation and formation of disulfide
is cloned for optimum results (Fig. 4.2b). bond, which are essentially required for the
2. The signals involved in transcription of genes full functional capability of many human pro-
may vary; thus, the gene of interest is usually teins. PTMs play an important role in proper
fused with bacterial gene under the control of protein folding, processing, stability, tissue
its promoter, and the protein is obtained as a targeting, activity, immune reactivity, and
fusion product, which can be later cleaved, half-life of the protein. Lack of these results in
purified, and used. insoluble, unstable, or inactive product.
3. Lac promoter is one of the most popular However, the N-linked glycosylation sys-
bacterial promoters. However, for high-level tem of Campylobacter jejuni has been suc-
expression, T7 promoter is also preferred cessfully transferred to E. coli, thus opening a
(present in pET vectors). It can drive the target possibility for the production of glycosylated
protein expression to nearly 50 % of total cell protein in it. Certain mutant E. coli are being
protein. Gene of interest can be placed under developed to promote disulfide bond forma-
the control of regulated promoter of phage. tion (AD494, Origami, Rosetta-gami) with
4. For translation, abundance of t-RNA is related reduced protease activity (BLZ1).
to the frequency of appearance of different 7. Overproduction of recombinant protein in
codons (codon biasing). Therefore, codons bacteria might result in the loss of solubility
rare for E. coli may cause amino acid misin- and deposition of many protein species as pro-
corporation or premature termination affect- tein aggregates or inclusion bodies. Alteration
ing the yield of the therapeutic protein. This in growth conditions might render the product
can be solved by either site-directed replace- in insoluble form. Many eukaryotic proteins
ment of rare codons to the codons (for the are found trapped in inclusion bodies with
same amino acid) preferred by E. coli. Another resistance to further processing. Success has
4.3 Microbial System for Production of Therapeutic Protein 83
been obtained in purification of insulin and

betaferon from inclusion bodies. The retrieval and two N-acetylglucosamine residues.
of proteins using denaturating condition with The core may attach to different oligosac-
subsequent refolding and renaturation might charides to form different glycoproteins.
not always be easy and prove to be extremely
Man Man
expensive.
8. With the E. coli host, it is very difficult to Man
obtain protein larger than 60 kDa in soluble
form. GlcNAc
Due to certain limitations for production of pro-
teins in E. coli, other host systems are being GlcNAc
discussed for production of proteins. --Asn—
Pentasaccharide core
Glycosylation Glycosylation is one of the important

Covalent attachment of carbohydrate group posttranslational modifications, which
to the protein to form glycoprotein is called occurs inside the lumen of the endoplasmic
glycosylation. In glycoproteins, proteins con- reticulum (ER) and in Golgi complex. The
stitute a major fraction. These play important ER and Golgi complex are important in
roles in various physiological processes and protein targeting and transport. N-linked
are components of cell membranes. glycosylation starts in the endoplasmic
The carbohydrates commonly attached to reticulum and continues in the Golgi com-
proteins may be fucose (Fuc), galactose plex after the polypeptide is synthesized on
(Gal), N-acetylgalactosamine (GalNAc), glu- ribosomes. However, O-linked glycosyl-
cose (Glc), N-acetylglucosamine (GlcNAc), ation exclusively occurs in the Golgi
mannose (Man), and sialic acid (Sia). complex.
These sugar moieties may associate In the process, oligosaccharide to be
through amide nitrogen atom of side chain attached to the protein associates with a
of asparagine (Asn) termed as N-linked specialized lipid present in ER, dolichol
glycosylation or to the oxygen atom in the phosphate, which consists of about 20 iso-
side chain of serine (Ser) or threonine (Thr) prene (C5) units. Through phosphate of
termed as O-linked glycosylation. dolichol phosphate, oligosaccharide is
Not all the asparagine (Asn) present in transferred to specific asparagine residue
polypeptide can accept the carbohydrate of polypeptide chain on ribosomes. The
moiety. The residues with the sequence enzyme responsible for glycosylating pro-
Asn-X-Ser or Asn-X-Thr, where X is any tein and activated oligosaccharide are
amino acid except proline, are targets for located on the lumen side of ER.
glycosylation. Not only the residual Then these are transported to the Golgi
sequence but other aspects of the structure complex, where the carbohydrate units are
of the protein and cell type determine the altered and finalized. Golgi complex is
glycosylation site. responsible for O-linked sugar attachment
All the N-linked sugar residues have a and modification of N-linked sugar. Then
common core of pentasaccharides. These the proteins are targeted and transported to
pentasaccharide consists of three mannose their destination.
(continued)
-Asn- -Asn-
-Asn- Pichia pastoris Sacharomyces cerevisiae
-Asn- (Fungi)
MAMMALIAN (Fungi)
HUMAN
Glc/Man
Fig. 4.3 Many of the human proteins are glycosylated cerevisiae) for production of recombinant proteins results
(O-linked or N-linked glycosylation). Glycosylation is one in hyper-glycosylation. The figure shows glycosylation pat-
of the important posttranslational modifications. The E. coli tern in human, mammalian, Pichia, and Saccharomyces
is unable to perform glycosylation. Fungi are simplest systems. The hyper-glycosylations or abnormal glycosyl-
eukaryotic systems which can perform glycosylation. The ations can make the protein highly immunogenic making it
use of fungal host (Pichia pastoris and Saccharomyces unsuitable for therapeutic purposes
4.4 Production of Recombinant Protein Folding and Molecular Chaperons

Protein in Fungal Hosts Chaperones are family of highly conserved
different proteins. The important functions
Due to the problems encountered in E. coli for of chaperones are:
production of larger proteins or modified proteins,
the next cost-effective, fast, high-density, and easy • Prevention of aggregation and misfolding
to handle system is of fungi. Saccharomyces cere- of newly synthesized polypeptide chain.
visiae (yeast) was the system of choice when it was • They prevent irreversible aggregation of
difficult to obtain therapeutic protein in soluble nonnative conformation and maintain the
form and with appropriate posttranslational modifi- protein on the productive folding pathway.
cations in bacterial host. In yeast, mutants are avail- • They prevent nonproductive interac-
able which can give high yield. The approved tions with other components of the cell.
products obtained from yeast are hormones, vac- • They help and guide the direct assembly
cines, recombinant granulocyte macrophage col- of multisubunit protein complexes and
ony-stimulating factors (GM-CSF), albumin, larger proteins.
hirudin, and platelet-derived growth factor (PDGF). • The chaperones involved in folding recog-
The advantages of S. cerevisiae are the follow- nize nonnative substrate proteins mainly
ing: (1) it secretes recombinant protein in the cul- via their exposed hydrophobic residues.
ture, (2) protein is properly folded, and (3) it
performs most posttranslational modifications. The major classes of molecular chaper-
With the yeast system, high amounts of recombi- ones are:
nant protein are obtained, and yeast is also capa-
ble of performing posttranslational modifications Heat shock proteins are present in a variety
as O-linked glycosylation, phosphorylation, acet- of systems and prevent damage to the
ylation, and acylation but differs drastically in proteins under high heat.
patterns of N-linked glycosylation (Fig. 4.3).
(continued)
4.5 Production of Recombinant Protein in Insect Cell 85
HSP60 GroEL
• It is tetradecameric mitochondrial • It is a bacterial chaperone.
chaperonin. • It binds with partially folded and mis-
• It is implicated in protein import and folded proteins.
macromolecular assembly. • For its functionality GroEL requires its
• Required for folding of precursor poly- cofactor GroES.
peptides in ATP-dependent manner.
• Prevents aggregation and mediates
refolding of protein after heat shock. Differences in N-glycosylation in yeast are
with high or hypermannose which is highly
HSP70 immunogenic. Unmodified proteins are suitable
• They are central components of the cel- for production in yeast.
lular network of folding catalysts and Other members of fungi are Pichia pastoris,
molecular chaperones. Pichia methanolica, Candida boidinii, and Pichia
• They assist in different types of processes angusta, which are facultative methylotrophic
of protein folding in the cell by transient yeast having great potential. Pichia pastoris is
association of their substrate binding favored as high cell densities can be obtained;
domain with short hydrophobic peptide. protein is secreted in high concentration (1 g/l),
• They bind and release their substrate by less hypermannosylation as compared to yeast
switching to low-affinity ATP-bound state and thus less immunogenic.
and the high-affinity ADP-bound state. However, the disadvantage is that it requires
• They form complex network of folding methanol to induce gene expression as transgene
machines [15]. is under the control of the promoter of alcohol
oxidase 1 (AOX1) gene. Methanol may be flam-
HSP90 mable and is toxic to cells and humans if not
• It is highly abundant chaperone. thoroughly removed. Because of hypermannose-
• It plays an important role in many cel- type glycosylation, the fungi are also unsuitable
lular processes, for example, cell cycle for production of many recombinant proteins.
control, cell survival, and hormone and
other signaling pathways.
• It is a key player in maintaining cellular 4.5 Production of Recombinant
homeostasis during stress. Protein in Insect Cell
• Has ATPase activity, whose binding and
hydrolysis affects conformational Insect cells: Insect cell can be infected with
dynamics of the protein. baculoviruses which are double-stranded cir-
• It has become a major therapeutic target cular DNA viruses with arthropods as host.
for cancer, and its role is being explored Baculovirus-mediated gene expression in insects
in neurodegenerative disorders and is a method of choice and is cost-effective, giving
infectious diseases [10]. the much higher yield of recombinant protein
compared to other systems. It is possible to pro-
CCT: Chaperonin Containing TCP1 duce large protein resulting in production of cor-
• This is eukaryotic chaperonin tailless rectly processed and biologically active protein.
complex polypeptide 1 (TCP1) ring A baculovirus Autographa californica nuclear
complex (TRiC). polyhedrosis virus (AcMNPV) is used as a clon-
• It facilitates the proper folding of many ing vector for insect cell lines. In this viral poly-
cellular proteins. hedron protein is used, which is required in its
normal habitat and exhibits high rate of transcrip-
(continued)
tion, but is not needed in cell culture. Thus, the 4.6 Production of Recombinant
coding sequence of the gene is replaced with for- Protein in Mammalian Cell
eign DNA. The gene is transcribed under the con-
trol of powerful polyhedron promoter with high Mammalian Cells: Production of complex pro-
yields (~30 % of total cell protein). The observed teins requires extensive processing and posttrans-
yield may be variable due to the course of virus lational modifications. Mammalian cells have the
infection and viral titer. advantage of performing PTMs (Fig. 4.3) cor-
The production of recombinant protein in rectly; they secrete recombinant protein into the
insect cell is time consuming (as compared to medium in their natural form, thus skipping the
bacterial system) as cell growth is slow and the critical steps of renaturation and refolding which
cost of medium is high. Every time fresh cells are sometimes leads to inactive proteins. Therefore,
required, viral infection is lethal for cells. It also major therapeutic proteins (60–70 %) are pro-
has limitations in performing posttranslational duced in mammalian cells primarily Chinese
modifications as it performs non-syalated hamster ovary (CHO) cells and baby hamster
N-linked glycosylation. All the other optimiza- kidney cells (BHK). CHO cells are relatively
tions need to be perfect as yield depends upon the easy to manipulate and their properties favor
virus titer and time taken from infection to large-scale production in them [24]. The proteins
expression. Insect cells are preferred when active produced are safe to use in humans with no
protein is difficult to obtain in E. coli system. adverse reactions because of similar glycosyl-
Genetic engineering has been used to select ation pattern. Chinese hamster ovary (CHO) and
MIMIC™ (Invitrogen) and SfSWT-3, which are baby hamster kidney (BHK) cells are the promi-
transgenic cell lines expressing all necessary nent producers of recombinant proteins (Fig. 4.4).
enzymes to obtain humanized, complex N-linked
glycosylation pattern. The system has been Roller bottle
extensively used for structural studies as correctly
folded eukaryotic proteins may be obtained in
secreted form simplifying purification protocols.
Some of the approved biopharmaceuticals from Medium
CHO cells
infected insect cell line Hi Five are Cervarix
(recombinant papillomavirus C-terminal truncated
Slowly rotated with regular
major capsid protein L1 types 16 and 18, used as wetting and oxygen supply
cancer vaccine).
Glycosylation is a problem which is encoun-
tered when insect cells are used for production of Cells adhere making confluent culture
recombinant human glycosylated proteins. Lots of
genetic engineering is required to produce human-
Product harvesting
like glycosylation in insect cell. Thus, the pre-
ferred system for therapeutic human protein
production is mammalian system (Chinese ham- Scaled up by number
ster ovary cell line). Due to time and difficultly in of roller bottles in parallel
maintaining insect cells, the mammalian cells
were explored for production of recombinant pro- Yield upto 50-200mg/l
tein. Mammalian cells, because of their properties
of protein folding, assembly, and posttranslational Fig. 4.4 The figure shows the culturing of mammalian
cells using roller bottles. These cells are maintained in
modifications, have become the preferred system
number of roller bottles. For adherent cells, the microcar-
for protein production and are now accounting for rier beads are used. The cells adhere to beads and the
major recombinant protein production. beads are maintained in suspension culture
4.6 Production of Recombinant Protein in Mammalian Cell 87
In mammalian cells, genes can be expressed Plasma-free manufacturing involves the elimi-
either transiently or stably. Obtaining stably nation of plasma derivatives in every step (like
transformed cell lines requires the usage of some cell line development, upstream processing,
selectable marker. Another major advantage of downstream processing, and final formulations)
these cells is they can be grown in suspension, in of the process with appropriate postproduction
serum-free (SF), protein-free, and chemically checks. The manufacturers shifted from the use
defined media. The product is safe without the of serum to serum-free cell culture media with
risk of prions of bovine spongiform encephalopa- animal product-free media and ultimately to
thy (BSE) from bovine serum albumin and infec- protein-free, completely synthetic chemically
tions of variant Creutzfeldt–Jakob disease (vCJD) defined media. The media consists of protein
from the human serum. hydrolysates derived from yeast, soy, and wheat
Presently due to virus and prions in the donor with amino acids, peptides, carbohydrates, vitamins,
plasma or blood samples, the manufacturers are and essential elements, which are ultrafiltered to
opting for plasma-/serum-free growth medium remove any unwanted contaminants [8].
for culturing the cell lines. Cells require some of
these components (albumin, transferring) for
their growth. Nowadays recombinant human
albumin is available like human insulin (pro- Case Study
duced in E. coli and yeast) and yeast-derived The recombinant therapeutic product used
animal-free recombinant human transferrin is for clinical applications was produced from
available. These support plasma-free mammalian mammalian cells. Mammalian cells were
cell culture. Else, CHO cells may be engineered maintained in serum-/blood-/plasma-based
to produce its own transferring or insulin-like medium; therefore, the presence of any
growth factor. The therapeutic protein obtained infectious agent in the product might be
by mammalian cell is treated and tested for the deleterious if not properly removed.
presence of any pathogenic agents or viruses as Infections may range from HIV, coronavi-
contaminant. The therapeutic products obtained rus (severe acute respiratory syndrome
during processing are treated with various agents (SARS), non-lipid-enveloped (NLE)
to remove inactive virus, but the presence of viruses as circoviruses (Torque tenovirus
asymptomatic virus poses a serious risk. Common (TTV) and Torque tenominivirus (TTMV)),
virus inactivation technique is using solvent/ HBV, HCV, HTLV (human T-cell lympho-
detergent, which is effective against lipid- tropic virus), to West Nile virus. Prions that
enveloped viruses such as human immunodefi- are self-replicating infectious proteins may
ciency virus (HIV), hepatitis B and C virus (HBV also be present which may lead to variant
and HCV), human T-cell lymphotropic virus Creutzfeldt–Jakob disease (vCJD).
(HTLV), and West Nile virus (WNV). Non-lipid- Pathogen transmission was a major con-
enveloped viruses such as parvoviruses, enterovi- cern in the manufacture of blood-derived
ruses, and circoviruses are resistant to inactivation coagulation factor. In the early 1980s, the
via solvent detergent treatment. Human herpesvi- factor replacement products derived from
rus 8, responsible for Kaposi’s sarcoma, is shown plasma, which were used to treat hemophilia,
to be transmitted through blood and blood prod- were found to be contaminated with HIV,
uct. Following outbreaks, strict requirements HBV, and HCV viruses. In the year 1984, up
were imposed on manufacturers of biologics and to 78 % of US-based hemophilic patients
medical devices. Such concerns prompted manu- were infected with HIV and 74–90 % were
facturers to switch to sugar-based final formula- infected with HCV. Parvoviruses, B19
tions and develop recombinant plasma-free (B19V) and PARV4, were present as con-
albumin produced in yeast for usage in biophar-
maceutical manufacturing. (continued)
4.8 Transgenics for Protein

taminant in plasma-derived factor Production
VIII. Therefore, the production urgently
required regulatory measures. 4.8.1 Transgenic Animals
Then came first recombinant factor
VIII, Advate (Baxter), in the USA in 2003. The transgenic animals are successfully used for
Advate was produced in CHO cells grown production of recombinant proteins (for details
in serum-free and protein-free medium refer to Chap. 5). Protein production poses great
with ultrafiltered soybean peptides with risk in terms of safety as transmission of infectious
subsequent purification by immunoaffinity agents, allergic responses, immune reactivity,
chromatography. The usage of Advate and autoimmune responses might occur.
helped in eliminating the risk of transmit- ATryn was the only approved (approved in
ting emerging blood pathogens. 2006 by European medical agency and in 2009
Prions pose a serious risk, as they are highly by FDA) recombinant biopharmaceutical using
resistant to physical/chemical inactivation. transgenic animals. It contains human antithrom-
Early stage of prion infection is almost bin (432AA) with 15 % glycosylated moieties
impossible to detect in plasma donor. and is secreted into milk of transgenic goats.
Iatrogenic transmission of prions has Rhucin intended for acute attacks of angio-
occurred in patients who received human- edema in patients with congenital C1 inhibitor
derived pituitary hormones as human activity deficiency, obtained from transgenic rab-
growth hormone (hGH) and gonadotro- bit, was denied approval. Transgenic plants are
pins. CJD was transmitted to over 160 being explored as recombinant protein producers
recipients of cadaveric pituitary hGH for research and diagnostic uses.
before its withdrawal. Cadaveric pituitary-
derived gonadotropins for infertility were
associated with iatrogenic transmission of 4.8.2 Transgenic Plants
CJD. Later on cadaveric pituitary hGH and
gonadotropins have been replaced with Obtaining therapeutic proteins from their natural
recombinant GH (produced in microbial source poses threat for spread of diseases.
system) and recombinant gonadotropins Therefore, alternative systems for the production
(produced in CHO cell lines). of therapeutic agents have their own benefits.
Molecular farming in plants has been widely
explored for production of recombinant pharma-
ceutical proteins. Their advantages are low cost,
high mass production, scale-up, lack of human
4.7 Using Human Cells pathogens, and addition of eukaryotic PTMs. The
for Protein Production first recombinant protein obtained in 1986 from
tobacco plants was human growth hormone.
Human cell lines: Human cell line-derived However, sometimes plant-specific PTMs might
Dynepoerithropoietin (erythropoietin with result in adverse immune reactivity.
increased shelf life), Elaprase-irudonate-2- Production of recombinant heterologous pro-
sulfatase (lysosomal enzyme), and teins in plants is simple and is used for production
Replagal-alfa-galactosidase A (lysosomal hydro- of non-naturally occurring proteins as single chain
lase) have been approved by the European Union Fv fragments (ScFvs). High yield of recombinant
(EU) or Food and Drug Administration (FDA, protein is the main goal of production system in
USA). As these products are fully glycosylated transgenic plants. Therefore appropriate expression
when expressed in human cell lines and used as vectors and constructs are designed to achieve high
therapeutics in human beings. yields of the engineered gene products [7, 12].
4.9 Challenges of Production of Therapeutic Proteins 89
Protein Production in Plant System • Subcellular targeting is also very impor-

The transgene in expression construct is tant factor which affects the process of
chimeric structure as it is surrounded by folding, assembly, and posttranslational
various active regulatory elements. modification and can be efficiently
Polyadenylation sites play an important achieved by inclusion of an N-terminal
role for the high level of expression of signal peptide.
transgene. Cauliflower mosaic virus • Position of transgene integration.
(CaMV) 35S promoter works well with • Structure of transgene locus.
dicots. It is a strong constitutive promoter • Gene copy number.
that is made more active by duplicating the • Presence of truncated or rearranged
enhancer region. However, in monocots transgene copies.
maize ubiquitin-1 promoter is the preferred • Affinity tags as His or the FLAG epit-
promoter. The presence of an intron in the opes can be used to ease the process of
5′-untranslated region (5′-UTR) enhances purification; however, these modifica-
transcription in monocots. tions not only affect the primary struc-
For obtaining high yield of the protein, ture but also the properties of the
several factors may be appropriately protein.
considered:
Figure 4.5 shows the general vector
• The incorporation of polyadenylation pCAMBIA (it is small in size (7–12 kb)
sites may be from CaMV 35S transcripts maintained in high copy number with
or the Agrobacterium tumefaciens nos pVS1 replicon that imparts chlorampheni-
gene or the pea ssu gene. col or kanamycin resistance and high sta-
• The yield can be controlled by placing bility in Agrobacterium) that is used for
the gene under the control of the pro- transgene expression in the plants. The
moter which is active in a particular tis- modified vector has shown success in insu-
sue or developmental stage or particular lin production.
environment (e.g., rice glutelin, pea
legumin).
• Usage of inducible promoter (e.g., Nowadays plant system is efficiently engineered
tomato hydroxyl-3-methylglutaryl CoA to produce human growth hormone, human
reductase-2 (HMGR2)) which has serum albumin, erythropoietin, α-interferon,
mechanical gene activation system antibodies and ScFvs, toxins, subunit vaccines,
developed by Cramer (Crop Tech Corp., and insulin [20].
Virginia, USA). Transcription starts
when harvested tobacco leaves are
sheared during processing. 4.9 Challenges of Production
• Codon bias in the host plant may be of Therapeutic Proteins
overcome by engineering of transgene
at positions, which might lead to trunca- With increasing demand from the consumer, the
tion and/or misincorporation, or slowing companies are trying to increase the productivity
the process. [17]. Few challenges with large-scale production
of proteins are:
(continued)
CaMV 35S promoter

Nco I Reporter gene
Bgl I
Lac Z alpha Spe I Nhe I
Histidine tag
Pml I
Multiple cloning site Bst EII
Nos Poly-A
T-Border (right)
CaMV 35S promoter
Plant selection gene

General structure of pVS1 sta
CaMV 35S polyA pCAMBIA vectors
T-Border (left)
Bacterial selection gene

pVS1 rep
pBR322 ori
pBR322 bom
Fig. 4.5 The figure shows the structure of pCAMBIA reporter gene (GUS or GFP may be used). The vector can
vector (cambia.org) used for plant transformation. The be modified to express genes for insulin (tomato) or Hep-B
vector has CaMV35S promoter, multiple cloning site, and surface antigen (HBsAg) for recombinant therapeutics
1. Loss of expression: For high output, it is very biological activity, and circulation half-life.
important that gene of interest should give Getting correctly glycosylated and folded pro-
adequate protein production. However expres- tein is required for therapeutic usage. In pro-
sion may be lost due to many factors, like, if karyotes, with the discovery of N-glycosylation
there are structural changes in the recombinant system in Campylobacter jejuni, several other
gene or inactivation or disappearance of the systems of O-glycosylation were unraveled in
gene from host cell. Other factors influencing both pathogenic and symbiotic bacteria. The
yield may be increase in the copy number of production of recombinant proteins is com-
insert, maintenance of optimum temperature, monly done by using E. coli, yeast, or cell
and toxicity of the expressed protein to the lines derived from insects (SF9), mice (SP2/0),
host. Chromosomal integration of the foreign or CHO, but obtaining fully human PTMs is a
gene might overcome the problem of expres- challenging task. The commonly used mam-
sion stability, but in plasmid-based system, malian cell lines of rodent origin (such as
high copy number leads to increased yield. SP2/0, CHO, or BHK) are able to perform
2. Posttranslational processing: Protein folding humanlike glycosylation. However, some
requires foldases (accelerates protein folding) human components are missing (such as the
and chaperones (prevents protein formation of 2,6-linked sialylation), and a number of non-
nonnative insoluble folding intermediates). human components have been found to sig-
Glycosylation is complex PTM requiring con- nificantly increase (terminal galactose linked
secutive steps and enzymes. Glycosylation is to another galactose or terminal sialic acids)
important as it determines protein stability, which increases the high risk of immunogenic
solubility, antigenicity, folding, localization, reactions. For this reason, human cell lines
4.10 Some Important Biopharmaceuticals 91
providing a human glycosylation pattern have to have polyethylene glycol with prolonged
increased attention, and the efforts are being presence and reduction in enzymatic degrada-
made for the development of novel glyco- tion and renal clearance, thus extending its pres-
engineered cell lines for production of fully ence with lesser immunogenicity) [17].
glycosylated protein therapeutic.
3. Overexpression of therapeutic protein might
result in the formation of inclusion bodies in
prokaryotic system. Rapid intracellular pro- 4.10 Some Important
tein accumulation and expression of large pro- Biopharmaceuticals
teins increases the probability of aggregation.
Aggregation protects proteins from proteoly- 4.10.1 Tissue Plasminogen Activator
sis and can facilitate protein recovery. When (tPA)
the expressed protein is toxic to the host, the
presence of protein in the inclusion body tends Ischemic stroke and myocardial infarction are
to protect it. one of the leading causes of cardiovascular mor-
bidity and mortality in the world. Important
Precautions Recombinant protein production thrombolytic agents are urokinase (obtained from
requires some precaution resulting in a loss of urine), tissue plasminogen activator (tPA), and
yield and/or product: streptokinase (obtained from bacteria). Among
these, t-PA is largely used commercially.
1. Contamination: At any stage, contamination Plasminogen activators are serine proteases,
might occur from any source. It poses big which are responsible for conversion of inactive
challenge and may be adverse when contami- proenzyme plasminogen (Plg-a single chain gly-
nated material is put for human usage. coprotein) to serine protease called plasmin
2. Immune response: For the therapeutic agent, (Plm). The plasmin degrades the network of
the body can mount the immune reactions fibrin of the blood clots (Fig. 4.6a). There are two
which lead to deposition of immune com- immunologically unrelated groups of plasmino-
plexes in various tissues, and condition of ana- gen activators, the 55 kDa urokinase-type-PA
phylactic shock might occur (e.g., when some (u-PA) and 72 kDa tissue-type PA (t-PA) (EC
essential agent is lost since birth like factor 3.4.21.68). The t-PA is the physiological vascular
VIII, then the patient might raise antibody activator consisting of single polypeptide chain
response against the treatment). This also of 72 kDa consisting of 527 amino acids. It shows
occurs when antibodies are used as therapeutic strong activity in the presence of fibrin [23].
agents in the treatment of variety of cancers. The potential inhibitors of the thrombolytic
3. Protein aggregation: Any nonfunctional con- cascade are type I plasminogen activator inhibi-
dition might result in aggregation or loss of tor (PAI-1 or serpin E1) and PAI-2 (secreted by
activity of recombinant protein. placenta and present in significant amount during
4. Posttranslational modifications and folding: pregnancy). They act by competing with t-PA for
After purification, the proper modifications binding sites on fibrin thus preventing the fibrino-
and refolding are required for therapeutics. lytic cascade. PAI-1 complexes with t-PA for
5. Disulfide bond formation: It stabilizes protein binding to fibrin. Thus, truncated t-PA in which
structure; thus, strategies for specific extracellu- the residues responsible for interacting with
lar excretion pathway or overexpression of chap- PAI-1 (296–299) are replaced with four alanine
erones is required for optimum production. amino acids and three domains (finger, epidermal
6. Degradation: Sometimes proteins, which are growth factor (EGF), and kringle 1 domains)) are
active in host cells like proteases or protein- deleted, and chimeric tetrapeptide Gly-His-Arg-
modifying chemicals, might degrade the recom- Pro (GHRP) with high affinity to fibrin was
binant protein (e.g., PEG interferon is modified added. Reteplase is the deletion mutant with a
b Transfected CHO cell lines for

the gene of tissue-plasminogen Chinese hamster
activator ovary cell lines
Clones selected
on antibiotic
a
Plasminogen
Transfected
tissue-plasminogen CHO cell Antifoam
Activator (t-PA) lines
expressing pH probe Dissolved oxygen
Plasmin
truncated probe
Acts mutant t-PA
on in CHO-
Fibrin Impellers
DG44 in
serum free Medium
Fibrin medium in
degraded bioreactor
Stirred tank bioreactor
Blood clot removed
Purification
High yield of tissue

plasminogen activator
Fig. 4.6 The figure shows the function and production of plasmin. (b) This figure shows the production of truncated
tissue plasminogen activator. (a) This figure shows the t-PA in CHO DG44 cell line. The mutated form of t-PA is
function of t-PA that it helps in degradation of blood clot resistant to inhibition by plasminogen activator inhibitor
by degrading fibrin by activation of plasminogen into and has better effectiveness in clinical usage
prolonged half-life, in which the finger, EGF, and tance to PAI-1 was expressed in a CHO DG44
kringle 1 domains of the full-length molecule are expression system. Therapeutic protein was pro-
all deleted; thus, it is not inhibited. duced in stably transfected CHO DG44 cell lines.
Deficiency of PAI leads to over fibrinolysis These cell lines were maintained in serum-free
and hemorrhagic diathesis (like deficiency of medium, with glutamine, hypoxanthine, and thy-
clotting factors). Tiplaxtinin is the inhibitor and mine in stirred tank bioreactor. The cells were
is used for remodeling of blood vessels [2]. grown at 37 °C, 140 rpm with 5 % CO2 and 85 %
humidity. The protein was then purified, with
4.10.1.1 Production higher yield (Fig. 4.6b).
Plasminogen activator has great clinical rele-
vance for the management of stroke and myocar-
dial infarctions. For production of t-PA, E. coli 4.10.2 Factor VIII
and yeast system did not work properly due to
lack of posttranslational modification and over Factor VIII is one of the important factors of all
glycosylation, respectively. blood clotting factors. Deficiency of factor VIII
A novel truncated form of t-PA with an causes bleeding disorder called hemophilia
improved fibrin affinity and an increased resis- A. The hemophilia may be mild to severe depend-
ing upon factor VIII concentration in the body. In about 10 mg/24 g of healthy and transformed
moderate and severe factor VIII deficiency, there cells. Production of recombinant insulin is shown
can be spontaneous bleeding episodes in the in (Fig. 4.7a, b).
joints. Hemophilia A affects 1 in 5,000–10,000
males. Replacement therapy is the treatment
option for hemophiliacs either with human 4.10.4 Human Growth Hormone
plasma-derived factor VIII (pdFVIII) or recombi- (HGH)
nant FVIII (rFVIII) [21].
Transfection of HEK 293 cell cultures in Growth hormone is produced by the anterior lobe
serum-free suspension is being tried for optimal of the pituitary gland and is released in multiple
yield. Recombinant factor VIII (rFVIII) is pro- pulses. The hGH is encoded by GHN gene clus-
duced by culturing mammalian cells as baby ter (an array of five closely related genes), which
hamster kidney (BHK) or Chinese hamster ovary is localized on chromosome 17. It belongs to
cells (CHO), using large-scale bioreactors. diverse gene family that has evolved by gene
Standardizations are done to maximize yields. duplication events and has lots of structural simi-
larity and some common functions. Of the vari-
ous forms, the predominant form of hGH is
4.10.3 Insulin 22 kDa protein of 191 amino acids with two
disulfide bonds.
Insulin is a peptide hormone consisting of 51 GH does not control the functions directly, but
amino acids. It is secreted by β cells of islets of acts on certain hormones or somatomedins for its
Langerhans of pancreas. The hormone is respon- activity, for example, insulin-like growth factor 1
sible for maintaining normal blood glucose level (IGF-1). It has a wide spectrum of roles to play as
in blood. Insulin is stored in the form of proinsu- promotion of long bone growth, promotion of
lin which contains two polypeptide chains, A normal sex organ development through puberty,
and B, and is connected with a third peptide C- regulation of metabolism, stimulation of tissue
chain), which before secretion is cleaved with growth and repair, anabolic/anti-catabolic effect
production of insulin and C-chain. The cleavage via improved nitrogen retention, modulation of
results in the removal of C-chain, and the A (21 bone mineral density and metabolism throughout
amino acids) and B chain (30 amino acids) are life, proliferation of some cell types of the
linked by disulfide linkage to form mature immune system, appetite stimulation, and break-
insulin. down of fat (lipolysis). In clinical conditions, the
In the beginning, efforts were made to isolate therapy of growth hormone is given in the
mRNA for pre- and proinsulin from rat islets of treatment of dwarfism, bone fractures, skin burns,
Langerhans of pancreas and to synthesize bleeding ulcers, and AIDS.
cDNA. Thereafter, it was inserted into a plasmid. Recombinant human growth hormone (rhGH)
The recombinant plasmids were transferred into the is 22 kDa consisting of long chain of amino acids.
E. coli cells, which secreted proinsulin [4, 5, 6, 19]. It is used in deficiency disorders of growth hor-
Scientists have chemically synthesized DNA mone. In children, it is used for growth abnor-
sequences for two chains, A and B, of insulin and mality as short stature and is used in chronic
separately inserted into two pBR322 plasmids by renal insufficiency. The therapy of growth hor-
the side of β-galactosidase gene. The recombi- mone is also approved for adult growth hormone
nant plasmids were separately transferred into E. deficiency. GH is one of the most widely used
coli cells which secreted fused β-galactosidase-A hormones with the estimated market of more than
chain and β-galactosidase-B chain separately. 1.7 billion USD. Long experience in its adminis-
These chains were isolated in pure form by tration has proven the therapy as safe and effec-
detaching from β-galactosidase with yields of tive in various conditions of growth abnormality.
a b
Insulin A chain Insulin B chain

with b-galactosidase with b-galactosidase
Proinsulin gene
Transformed E.coli cloned in vector
Transformation, Selection and expression

Selection and expression of recombinant fusion of recombinant protein
protein
Cyanogen bromide cleavage of fusion Authentic human proinsulin is

Protein in 70% formic acid at room temperaure split to yield insulin and C-chain
Biosynthetic human insulin
Renaturation and folding in appropriate conditions

A chain
(21 amino acids) Insulin A and B chains Genentech in agreement with Eli Lilly Inc. produced
with dislphide bridges HUMULIN or Biosynthetic Human Insulin (BHI)
B chain
(30 amino acids)
Fig. 4.7 The figure shows the production of insulin. (a) bacterial protein. The protein is renatured and provided
The A and B chains of insulin are cloned separately in the suitable conditions for folding. (b) This figure shows the
vector. The transformation is done and after selection, the production of insulin from proinsulin gene which yields
recombinant protein is obtained for both A- and B-chain insulin and C-peptide. This is preferred method for pro-
genes. Cleavage by using cyanogen bromide removes the duction of biosynthetic human insulin
Earlier pituitary-derived hGH was used but • rhGH increases IGF-1, osteocalcin, type I pro-
later on it was prohibited when found associated collagen pro-peptide (PICP), and bone den-
with Creutzfeldt–Jakob disease. Because of sity, when administered to children with GHD.
recombinant DNA technology, safe and abundant
recombinant hGH was produced in various heter- For optimal productivity, strong inducible
ologous systems. As the non-glycosylated human promoters are preferred as IPL, IPR, trc, and T7 in
growth hormone was biologically active, thus, E. coli. They are advantageous as they drive over-
the preferred system for its production is E. coli, production of recombinant proteins. Apart from E.
which allows its rapid and economical produc- coli, human somatotropin (hST) expression was
tion in large amounts [14]. tried in a biologically active, disulfide-bonded
Recombinant hGH (rhGH) is now used to form in tobacco chloroplasts. The hormone is used
treat: for the treatment of hypopituitary dwarfism in
children; additional indications are in treatment of
• GH-deficient (GHD) short-stature children. Turner syndrome, chronic renal failure, HIV wasting
• Acceleration of wound healing. syndrome, and possibly treatment of the elderly.
• Increase in insulin-like growth factor (IGF)-1 Growth hormone deficiency in human occurs both
levels. in children and adults [18, 22].
4.10.5 Interferons of erythropoietin, progenitor cells are stimulated

in the bone marrow to form mature erythrocytes
Interferons are a group of proteins which are (red blood cells). Thus the patients with chronic
secreted in response to viral infections. Resistance kidney disease are unable to maintain adequate
imparted by INFs is short lived and does not last amount of erythropoietin for normal develop-
forever. They are family of naturally occurring ment of erythrocytes in blood, resulting in low
proteins that are made and secreted by all the numbers of red blood cells and subsequent anemia.
cells (INF-α and INF-β) and lymphocytes (INF- These patients either require blood transfusion or
γ). All these modulate the response of cells and erythropoietin from outside. As supply is limited
immune system to viruses, bacteria, and cancer. from the natural source that is kidney cells, thus a
Interferons are produced by either an estab- recombinant human erythropoietin EPOGEN®
lished cell line (lymphoblastoid) or fresh cells which is Amgen’s trade name for epoetin alfa is
isolated from blood. The production involves marketed for anemic condition involving erythro-
induction with virus and priming (incubation poietin. The human gene encoding erythropoietin
with some interferon) with interferon, resulting was cloned into the Chinese hamster ovary cell
in better yield. The affinity chromatography with line for production of the human protein. This
monoclonal antibodies packed in the column has cell line continues to be used today for the pro-
helped in purification of interferons. But before duction of EPOGEN®.
the clinical usage, the removal or inactivation of The half-life of erythropoietin can be increased
virus is very important. The interferon therapy is by incorporating the glycosylation of the protein
used for cancers and viral infections (INF-α), growth factor. Thus, Darbepoetin-α is an analog
multiple sclerosis (INF-β), and chronic granulo- which is engineered for two extra amino acids
matous disease (INF-γ). Multiferon is natural which are substrates for glycosylation. Thus, pro-
interferon-α, which consists of several subtypes. duction is done in CHO cell lines; the product has
In some of the cancers like Merkel cell carci- five N-linked sugar chains and has almost three
noma, type I interferons (multiferon, which is a times longer life than erythropoietin.
mix of various INF-α subtypes and INF-β) are
highly effective. In Chap. 11, interferons are
mentioned in protein therapeutics. Interferon is 4.10.7 Platelet-Derived Growth
marketed as Roferon-A, Infergen, Intron A, and Factor (PDGF)
so on.
PDGF regulates cell growth and division and
plays a significant role in blood vessel formation
4.10.6 Erythropoietin (angiogenesis), the growth of blood vessels from
already existing blood vessel in tissue, and may
Hematopoietic growth factors consist of cytokines act in autocrine and paracrine stimulation of
and protein hormones produced by the body cell growth in vivo. PDGF plays the role in devel-
which govern the production and maturation of opment, cell proliferation, cell migration, and
the various cells produced during the process of angiogenesis and has been linked to atherosclero-
hematopoiesis in the bone marrow from hemato- sis, fibrosis, and malignant diseases.
poietic stem cell. The precursor cells in the pres- PDGF has five different isoforms: PDGFA,
ence of a particular growth factor differentiate PDGFB, PDGFC, PDGFD, and AB heterodimer.
and become a specialized kind of cell as mono- PDGF-A and PDGF-B have 60 % similarity in
cyte, macrophage, lymphocytes, or red blood amino acid sequence, but experiments suggest
cell. different biological functions for the two chains
One of the important growth factor is erythro- and different locations of these under different
poietin which is a protein hormone produced by a transcriptional controls. PGDF-AA is released in
specific type of cells in the kidney. In the presence the medium, and PDGF-BB are insufficiently
secreted and remain attached to the plasma 4.10.9 Fibroblast Growth Factor
membrane, and of these PGDF-BB and (FGF)
PDGF-AB are strong mitogens and are probably
responsible for biological roles of PDGF. PDGF FGFs are commonly mitogens with multifunc-
receptor (PDGFR) is receptor tyrosine kinase tional proteins with a wide variety of regulatory,
(RTK) (alpha and beta type). Upon activation by morphological, and endocrine effects. There are 18
PDGF, these receptors dimerize leading to auto- mammalian FGFs (1–10 and 16–23) which affect
phosphorylation of several sites on their cytosolic growth and functions of a wide variety of mesen-
domain. PDGF being a mitogen promotes the chymal, endocrine, and nerve cells. The functions
proliferation of fibroblasts and smooth muscle of FGFs in developmental processes include meso-
cells in vitro. derm induction, anteroposterior patterning, limb
PDGF shows considerable heterogeneity with formation, neural tube induction, and brain devel-
sizes of 27–31 kDa; however, purified PDGF is opment and in mature tissue/systems angiogenesis,
cationic protein of 30 kDa. Recombinant human keratinocyte organization, and wound healing pro-
platelet-derived growth factor (rh-PDGF) was the cesses. FGF is very important during normal devel-
first recombinant protein to be approved by the opment of both vertebrates and invertebrates, and
US Food and Drug Administration for treatment any irregularities in their function lead to a range of
of chronic foot ulcers in diabetic patients developmental defects [1].
(Regranex, Ethicon Inc. Somerville, NJ). It has FGF1 and FGF2 show strong angiogenic prop-
the potential for use in bone regeneration and erties with the promotion of endothelial cell prolif-
increasing bone density in long bones and spine. eration and the physical organization of endothelial
PDGF is commercially produced by using E. coli cells into tubelike structures and the growth of new
and mammalian cells. blood vessels from the preexisting vasculature.
FGF7 and FGF10 (also known as keratinocyte
growth factors (KGF) and KGF2, respectively)
4.10.8 Epidermal Growth Factor stimulate the repair of injured skin and mucosal tis-
(EGF) sues by stimulating the proliferation, migration, and
differentiation of epithelial cells, and they have
Human EGF protein has 53AA and three intra- direct chemotactic effects on tissue remodeling.
cellular disulfide bonds and plays an important Most FGFs are secreted proteins that bind heparan
role in the regulation of cell growth and prolifera- sulfates and can therefore be caught up in the extra-
tion. It shows strong sequential and functional cellular matrix of tissues that contain heparan sul-
homology with human type- alpha transforming fate proteoglycans. This allows them to act locally
growth factor (hTGF alpha), which is a competi- in a paracrine fashion. However, the FGF19 sub-
tor for EGF receptor site. EGF acts by binding family (including FGF19, FGF21, and FGF23)
with high affinity to EGFR on cell surface and which binds less tightly to heparan sulfates can act
stimulates the intrinsic protein tyrosine kinase in an endocrine fashion on far away tissues, such as
activity. EGF has many biological activities. the intestine, liver, kidney, adipose, and bone. For
Initial observations were centered around their example, FGF19 is produced by intestinal cells but
proliferative effects on fibroblasts, keratinocytes, acts on FGFR4-expressing liver cells to downregu-
and epithelial cells. EGF modulates luteinizing late key genes in the bile acid synthase pathway;
hormone and thyroid hormone. EGF is produced FGF23 is produced by the bone but acts on FGFR1-
commercially by engineered E. coli. The other expressing kidney cells to regulate the synthesis of
systems are also being explored for optimum vitamin D and in turn affect calcium homeostasis.
EGF production [9]. FGF may be synthesized using E. coli as host
system.
4.10.9.1 Therapeutic Potential Nerve growth factor (NGF) is a small secreted

of FGFRs protein which induces the differentiation and
FGF is involved in stimulating collateral vascu- survival of particular target neurons (nerve
larization and recovery from ischemia as well as cells). Little is known of the biological action of
enhancing wound healing, nerve regeneration, neurotrophin apart from NGF. The nucleotide
and repair of cartilage and has been alternately sequence of cDNA predicts that NGF is synthe-
referred to as “pluripotent” (capable of develop- sized as pre-pro-NGF. Upon removal of hydro-
ing into more than one cell type or tissue) growth phobic signal, either 34 kDa or 27 kDa pro-NGF
factors and as “promiscuous” (biochemistry and is generated depending on the size of the tran-
pharmacology concept of how a variety of mole- script. However, processing of the precursors in
cules can bind to and elicit a response from single different tissues is not well understood. They are
receptor) growth factors due to its multiple essential for normal development, growth, and
actions on multiple cell types. The FGFs and differentiation of the sympathetic and sensory
small-molecule FGF receptor kinase inhibitor are neurons and are also essential to maintain the
used in the treatment of cancer and cardiovascu- normal function of these cells in adults. Thus it
lar disease and have potential in the treatment of is important for maturation and survival of neu-
metabolic syndrome and hypophosphatemic rons and prevents degeneration of adult neurons.
diseases: Apart from its important role in the nervous sys-
tem, it has been shown to possess protective
• Receptor tyrosine kinase inhibitor (Sunitinib) action of human pressure ulcer, corneal ulcer,
is approved for indications in renal cell carci- and glaucoma. Reduced sensation may be
noma and gastrointestinal stromal tumors. observed in leprosy, wound healing, nerve injury,
• Small FGFR inhibitors, SU5402, PD173074, and diabetes. NGF may help to regulate the
and nordihydroguaiaretic acid, are effective in sensory fiber sensitivity and function directly or
multiple myeloma cell lines. indirectly by stimulating other effectors.
• PD173074 can induce cell cycle arrest in Administration of recombinant NGF may
endometrial cancer cells with mutated FGFR2. improve sensation and pain [13].
• Antibody against FGFR3 has been shown to Cholinergic neurons of the basal forebrain
effectively cause apoptosis in mouse models show receptors for NGF; specific mRNAs for
of multiple myeloma and bladder cancer. various NGFs have been identified in different
areas of the brain. Cholinergic neuron loss is a
Thus FGFR inhibition can be very effective in cardinal feature of Alzheimer’s disease. Nerve
the treatment of cancer. growth factor (NGF) stimulates cholinergic
function, improves memory, and prevents cho-
linergic degeneration in animal models of injury,
4.10.10 Nerve Growth Factor (NGF) amyloid overexpression, and aging. NGF acts on
intracellular calcium through tyrosine kinase
The nerve growth factor (NGF) is an important receptor mechanism. Nerve growth factor
member of the family of neurotrophins. Five pro- enhances early regeneration of severed exons
tein nerve growth factors of the neurotrophin and is also important in maintaining the bio-
family are important. They regulate the develop- chemical and morphological phenotype of
ment of the nervous system and play an impor- mature basal forebrain cholinergic neurons
tant role in maintaining the structure, plasticity, (BFCNLs) after lesions or injury of the central
and repair of the adult nervous system. All the nervous system (CNS). Thus, NGF may provide
neurotrophins are basic proteins of about 120AA, therapeutic option for preventing death of cholin-
share 50 % sequence homology, and are highly ergic neurons and other clinical conditions and is
conserved in mammalian species. produced using E. coli.
4.10.11 Transforming Growth activity and prolonged stay in the body, for exam-
Factor Alpha (TGF-α) ple, engineering of monoclonal antibodies to
have toxin or radioisotope or generation of bi-
TGF-α exerts its function in an autocrine and specific antibody. Still the technology is strug-
endocrine fashion for various cell types of ecto- gling hard to make the diseases completely a text
dermal origin, including most epithelial cells. It of books and having a society free of diseases and
is normally a transmembrane protein and func- the pathogens.
tions in cell communication through its ability to
activate a receptor tyrosine kinase. Ectodomain
of TGF-α is cleaved in a highly regulated man- 4.12 Chapter End Summary
ner, releasing soluble TGF-α which activates
paracrine signaling. The receptor is EGF/TGF • The production of proteins for therapeutic
alpha receptor; therefore, the focus is on under- purpose is very important not only because of
standing the important roles of TGF-α and EGF their specific action with minimum side effects
receptor signaling in carcinoma development. but also due to their unique form and
functions.
• Nowadays therapeutics (pancreatic enzymes
4.10.12 Transforming Growth from hog and pig pancreas or a-1-proteinase
Factor Beta (TGF-β) inhibitor from pooled human plasma) are not
extracted from their native sources (from
TGF-β is a large group of related proteins. Some of humans, animals) due to risk of transmission
its family members include bone morphogenetic of pathogens. Other problems faced for
protein (BMPs), growth, and differentiation factors extraction from native sources were the scarce
(GDP). It affects tissue remodeling, wound repair, availability of animal tissue for production,
hematopoiesis, morphogenesis, embryonic devel- high cost of purification with less yield, and
opment, adult stem cell differentiation, immune immunological reactions in the recipients.
regulation (is switch factor for IgA), and inflamma- • Majority of the biological therapeutic agents
tion. It exists as multiple forms as TGF-β1, TGF-β2, are produced by using various advance tools
and TGF-β3. It acts through transmembrane serine/ and technologies as cloning, selection, purifi-
threonine receptor kinase leading to the activation cation, and stability monitoring. It is also very
of Smads. It acts as tumor suppressor during cancer important to monitor risks and side effects of
initiation but promoter during tumor progression. It therapeutics (safety analysis) obtained from a
has a role in the control of embryonic development, wide range of cells.
cellular differentiation, hormone secretion, and • The various biological systems are available
immune function. Its role as mesenchymal differen- for production of recombinant protein as
tiation factor, with focus on the muscle, fat, and bacteria, fungal system (yeast), insect cell,
bone cell, might provide insights into its deregula- mammalian cell, human cell, and transgenic
tion in skeletal and developmental diseases and is plants and animals.
the area of active research. It is produced using • The system of choice is dependent upon the
CHO cell lines. total cost of production and obtaining fully
functional and appropriately modified (PTMs)
(glycosylation, phosphorylation, or properly
4.11 Future Prospects folded) protein. All these are essential for the
biological activity of the protein.
There is huge potential for future therapies using • The bacterial system is the simplest with short
proteins as therapeutic agents. The recombinant cell division times, easy maintenance, easy to
proteins are not only beneficial, but the research- modify, and cost-effective production. The
ers can further engineer them to improve their bacteria have limitation in production of
4.12 Chapter End Summary 99
large-sized protein and are unable to per- (b) Can easily perform splicing of foreign
form posttranslational modification as glyco- DNA
sylation. The glycosylation is very important (c) Produces processed and properly modi-
as it affects the activity, half-life, and immu- fied protein
nogenicity of the recombinant protein. (d) All of the above
• Due to limitations, the other host systems were 4. The limitation of E. coli which poses a prob-
used for production which could give better lem for recombinant protein production is:
product with minimal immune reactions and (a) Codon biasing
are cost-effective. Fungal and insect systems (b) Splicing
are utilized for protein production but some- (c) Posttranslational modification
times result in inappropriate glycosylation. (d) None of these
Improper glycosylation may result in nonfunc- 5. The fungal being eukaryote offers tremen-
tional and highly immunogenic protein. dous advantages in recombinant protein pro-
Mammalian cells are the most preferred sys- duction but it does:
tem for production of these proteins. CHO sys- (a) Proper protein folding
tem is the system of choice for therapeutic (b) Posttranslational modification
protein production. It accounts for nearly 70 % (c) Hypermannosylation
production of all products available in the (d) Easy scale-up
market. 6. Insect cells may be infected with which viral
• The protein products are having important vector for production of recombinant protein?
therapeutic role. They are approved for mar- (a) Retrovirus
keting and use by various government (b) Coronavirus
agencies like the Food and Drug (c) Baculovirus
Administration (FDA) and European Union (d) Parvovirus
(EU). The approved recombinant biotechnol- 7. Most commonly used mammalian cell line is:
ogy medicines include replacement products, (a) BHK
monoclonal antibodies, interferons, vaccines, (b) CHO
hormones, modified natural products, and (c) HeLa
many others. Many of them are in usage and (d) All of these
helping to alleviate the symptoms or cause of 8. Why is there a need of serum- or plasma-free
the diseases. medium in the production of therapeutic
protein?
(a) As their cost is high, thus the cost of
Multiple Choice Questions product is high
(b) Risk of transmission of unknown patho-
1. The function of proteins in the body is: gens leading to diseases
(a) Initiation of transcription (c) Variation in each lot results in different
(b) Mediators of metabolic processes yield
(c) Acts as enzymes (d) None of these
(d) All of the above 9. The first recombinant protein released in the
2. The purpose of recombinant DNA technology is: market was:
(a) Genes can be cloned in E. coli (a) Activase
(b) The product is cost-effective (b) Humulin
(c) Mammalian cells may be used for (c) Trastuzumab
production (d) All of these
(d) None of the above 10. Growth factor inhibitors are finding place in
3. The advantages of using E. coli as host for therapeutics because:
production of recombinant protein are: (a) Growth factors are harmful
(a) Easy scale-up and simple media (b) Growth factor deactivation leads to
requirement healthy body
(c) Growth factors are required for progres- Roncucci R, Schmelck PH (eds) Therapeutic agents
produced by genetic engineering “Quo Vadis”.
sion of tumors
Toulouse-Labege, Symposium Sanofi Group, Paris
(d) All of the above MEDSI, pp 137–46
11. Darbepoetin is used for the treatment of: 5. Galloway JA (1988) Chemistry and clinical use of
(a) Short stature insulin. In: Galloway JA, Potwin JH, Shuman CR
(eds) Diabetes Mellitus, 9th edn. Lilly, Indianapolis,
(b) Anemia
pp 106–137
(c) Blood clotting 6. Galloway JA, Hooper SA, Spradlin CT, Howey DC,
(d) None of these Frank BH, Bowsher RR, Anderson JH (1992)
Biosynthetic human proinsulin: review of chemistry,
in vitro and in vivo receptor binding, animal and
Answers
human pharmacology studies, and clinical trial expe-
1. (d); 2. (b); 3. (a); 4. (c); 5. (c); 6. (c); 7. (b); rience. Diabetes Care 15:666–692
8. (b); 9. (b); 10. (c); 11. (b) 7. Goldstein DA, Thomas JA (2004) Biopharmaceuticals
derived from genetically modified plants. Q J Med
97:705–716
8. Grillberger L, Kreil TR, Nasr S, Reiter M (2009)
Review Questions Emerging trends in plasma-free manufacturing of
recombinant protein therapeutics expressed in mam-
Q1. What are the advantages and disadvantages malian cells. Biotechnol J 4:186–201
9. Guglietta A, Sullivan PB (1995) Clinical applications
of using E. coli as host for production of
of epidermal growth factor. Eur J Gastroenterol
recombinant proteins? Hepatol 7:945–950
Q2. What is the role of codon biasing in expres- 10. Jackson SE (2013) Hsp90: structure and function. Top
sion of foreign gene? Curr Chem 328:155–240
11. Kamionka M (2011) Engineering of therapeutic pro-
Q3. What are the posttranslational modifications?
teins production in Escherichia coli. Curr Pharm
Q4. Write a note on fungal host for production of Biotechnol 12:268–274
proteins. 12. Ma JK, Drake PMW, Christou P (2003) The produc-
Q5. When are insect cells preferred for the tion of recombinant pharmaceutical proteins in plants.
Nat Rev Genet 4:794–805
recombinant protein production?
13. Manni L, Rocco ML, Bianchi P, Soligo M, Guaragna
Q6. Write a note on (a) insulin, (b) factor VIII, (c) M, Barbaro SP, Aloe L (2013) Nerve growth factor:
GH, and (d) tissue plasminogen activator. basic studies and possible therapeutic applications.
Q7. Write the trade names of insulin, tPA, and Growth Factors 31:115–122
14. Martínez JAA, Saldaña HAB (2012) Genetic engineer-
GH.
ing and biotechnology of growth hormones, genetic
engineering – basics, new applications and responsi-
bilities, Prof. Hugo A. Barrera-Saldaña (Ed.), ISBN:
978-953-307-790-1, InTech, Available from: http://
www.intechopen.com/books/genetic-engineering-
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sion of novel PAI-1-resistant t-PA in CHO DG44 pharmaceuticals. Microb Cell Fact 8:17
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tors. J Microbiol Biotechnol 21:1299–1305 Production of recombinant proteins. In: Balbas P,
3. Ferrer MN, Domingo EJ, Corchero JL, Vazquez E, Lorence A (eds) From methods in molecular biology.
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References 101
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human tissue-type plasminogen activator gene: cor-

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Production of Recombinant

4.1 Introduction these diseases can be treated, by supplying the

© Springer Science+Business Media Singapore 2017 77

Fig. 4.1 The gene of interest Gene of interest

One/few cells/cell line is

TTGACAT TATAAT Transcription Structural gene

b Eukaryotic transcription and translation

Exon1 Exon2 Exon3 Exon4 heterogeneous RNA

their secondary structure prevents efficient 4.3 Microbial System

approved bacteria-derived (by either the approach may be co-expression of rare

been obtained in purification of insulin and

Glycosylation Glycosylation is one of the important

4.4 Production of Recombinant Protein Folding and Molecular Chaperons

4.8 Transgenics for Protein

Protein Production in Plant System • Subcellular targeting is also very impor-

CaMV 35S promoter

Plant selection gene

Bacterial selection gene

b Transfected CHO cell lines for

High yield of tissue

Insulin A chain Insulin B chain

Transformation, Selection and expression

Cyanogen bromide cleavage of fusion Authentic human proinsulin is

Renaturation and folding in appropriate conditions

4.10.5 Interferons of erythropoietin, progenitor cells are stimulated

4.10.9.1 Therapeutic Potential Nerve growth factor (NGF) is a small secreted

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