Iran J Sci Technol Trans Sci (2022) 46:373–383

https://doi.org/10.1007/s40995-022-01269-7 (0123456789().,-volV)(0123456789().
,- volV)

RESEARCH PAPER

Industrial Scale Production of Recombinant Human Insulin using

Escherichia coli BL-21
Satish Babu Kaki1 • A. Naga Prasad2 • Anjani Devi Chintagunta1 • Vijaya Ramu Dirisala1 •

N. S. Sampath Kumar1 • S. J. K. Naidu3 • B. Ramesh3

Received: 9 February 2021 / Accepted: 9 February 2022 / Published online: 15 March 2022
Ó The Author(s), under exclusive licence to Shiraz University 2022

Abstract
Insulin is a well-characterized peptide produced by recombinant DNA technology that is widely used in maintaining blood
glucose level in the diabetic patients. In this study, we have used vector pET-9a to express the human insulin in E. coli BL-
21 DE3. The recombinant E. coli cells were cultured by fed-batch fermentation (20.0 L) process which resulted in a dry cell
mass of 20 g/L. Pro-Insulin was expressed as inclusion bodies (IBs) and it was 20% of the total protein measured by
densitometry. The IBs were then solubilized and refolded to get the native biological structure. The proinsulin form has
been converted into active insulin by enzymatic digestion using trypsin and carboxypeptidase-B. The active insulin was
purified using ion-exchange and reverse phase HPLC. The final purified insulin has achieved a purity of 98% with Des-
Insulin impurity B 0.5%, when analyzed in analytical HPLC as per the United States Pharmacopiea (USP) method. This
optimized process of recombinant human insulin may be used as a model for insulin analogues such as glargine, aspart,
lispro etc.

Keywords Diabetes
Enzymatic digestion
Insulin
Inclusion bodies
Pro-insulin

Abbreviations 1 Introduction
HPLC High Performance Liquid Chromatography
IBs Inclusion Bodies Diabetes mellitus is a metabolic disorder that pares the
E. coli Escherichia coli body competence to exploit or process blood glucose levels
USP United States Pharmacopeia (Sampath Kumar et al 2021; Shaheena et al. 2019). In
DNA Deoxyribonucleic acid mammals, insulin is a short peptide hormone produced by
WHO World health organization the beta cells of pancreatic islets that regulates the diges-
tion of sugars and fats to maintain blood glucose levels by
elevating the uptake of glucose from the blood to muscles
and adipose tissue (Sonksen and Sonksen 2000). Beta cells
are sensitive to glucose levels in the blood; in case of high
glucose levels it releases insulin and stops its discharge
after reaching to the normal level, and disturbance in this
homeostasis is considered as diabetes. Depending upon the
availability of insulin, diabetes is classified into 3 types
viz., type-1, type-2 and gestational diabetes (Mellitus
& N. S. Sampath Kumar 2005). Type-1 diabetes or Juvenile diabetes, is the chronic
drnssk_bt@vignan.ac.in; nssk84@gmail.com condition resulting due to autoimmune destruction of beta
cells of pancreas. On the other hand, type-2 diabetes occurs
1
Department of Biotechnology, Vignan’s Foundation for due to resistance to insulin action, defect in insulin secre-
Science, Technology and Research,
Vadlamudi, Guntur 522213, Andhra Pradesh, India
tion and excess production of hepatic glucose. According
2
to the ‘National Institute of Diabetes and Digestive and
Wockhardt Research Center,
Aurangabad 431210, Maharashtra, India
Kidney Disease (NIDDK)’ type-2 is the most widely rec-
3
ognized kind of diabetes. Gestational diabetes occurs in a
Vcare Biolabs Pvt. Ltd., Hyderabad, Telangana 500090, India

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374
few women during gestational period wherein the body
becomes insensitive to insulin. In all these cases, the
patients need insulin therapy externally day-by-day to
endure their life.
Human insulin is made up of 51 amino acids in two
chains A and B which are connected by two disulfide
bridges with a molecular weight of 5,808 Daltons (Weiss
et al. 2015). Insulin structure differs from species to spe-
cies, but human insulin has shown close resemblance with
porcine insulin and earlier used to treat diabetes (Weiss
et al. 2015). These days, biosynthetic human insulin is
manufactured by recombinant DNA technology, which has
also prompted the accessibility of insulin analogs, like
glargine (Son et al. 2009). Presently, industries are utilizing
E. coli and yeast (Saccharomyces cerevisiae and Pichia
pastoris) systems for production of recombinant insulin.
Employment of yeast system is less profitable compared to
E. coli as it has high efficiency because of the formation of
inclusion bodies (IBs). Besides, E. coli can be easily han-
dled and manipulated and incurs less incubation cost.
Globally, by 2030, approximately 79 million people will be
in need of insulin for treatment of diabetes thus, the
industries need to create colossal amounts of insulin to
meet the demand (Shaw et al. 2010).
In view of futuristic demand for insulin, a simple, easy
to scale-up and cost effective process has been developed
in the present study. E. coli BL21 DE3 was cultured
through fed batch fermentation process to obtain inclusion
bodies which were lysed with high pressure homogenizer.
After lysis, proinsulin containing IBs were washed, solu-
bilized with 8 M urea and refolded. The refolded proinsulin
was concentrated by capturing on cation exchange chro-
matography followed by enzymatic digestion of proinsulin
to insulin. Subsequently, pure insulin was obtained by
loading on to cation exchange chromatography and reverse
Fig. 1 Schematic representation
of process involved in insulin
production
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Iran J Sci Technol Trans Sci (2022) 46:373–383
phase high performance liquid chromatography (RP
HPLC) (Fig. 1).
2 Materials and Methods
2.1 Materials
Restriction enzymes NdeI and BamHI were obtained from
Thermo Fisher Scientific. Forward and backward primers
are received as courtesy from Vcare Biolabs Pvt, Ltd.
Modified terrific broth, yeast extract, soya protein,
K2HPO4, KH2PO4, NaCl, EDTA, glucose, Triton X-100,
isopropyl alcohol, cystine, and citraconic anhydride are
procured from Merck. The proteolytic enzymes such as
trypsin and carboxy-peptidase-B were acquired from
Richcore Lifesciences.
2.2 Vector and Strain
The region coding for mature peptide of insulin precursor
(UniProtKB—P01308) was amplified with primer sequen-
ces containing NdeI and BamHI recognition sequences.
The amplified product was digested with NdeI and BamHI
and ligated into pET-9a expression vector that was already
digested with the same restriction enzymes. The ligated
product was transformed with E. coli BL 21 (DE3) to
obtain the transformants.
2.2.1 Expression of Proinsulin Peptide
Transformed E. coli were cultured at 37 °C for 6 h in a
flask containing modified -TB media along with kanamycin
(70 lg/ml) as a seed culture. These cells were inoculated
aseptically into 1 L of fermentation media containing yeast
Iran J Sci Technol Trans Sci (2022) 46:373–383
extract - 5 g/L, soya protein-10 g/L, K2HPO4-6 g/L,
KH2PO4-3 g/L, NaCl-5 g/L and glucose-5 g/L) in a cir-
culated air through fermentor (Shanghai Baoxing Bio-
Engineering Equipment CO., Ltd, China). Fermenter was
agitated for 20–24 h under various parameters like pH:
6.86, dissolved oxygen: 100–0%, wind stream: 0.5 vvm,
OHP: 0.2–0.3 bar. Fed-batch fermentation is the most
preferred mode of operation in industries because of its
controlled addition of substrate which allows increased
yield and productivity of the desired product. During fer-
mentation, cells were fed with elevated concentration of
glucose as supplement to maintain the set parameters. The
glucose feeding was connected to a pump with acid. It was
automatically added by the controller whenever the pH
exceeded the set point. The growth of the cells in the fer-
menter is measured using UV spectrophotometer (UV1800,
Shimadzu, Japan).
2.2.2 Isolation of Inclusion Bodies
Harvested broth was centrifuged (Hanil Scientific Inc.,
Korea) at 10,000 g for 45 min to amass the cell pellet. The
pellet was suspended in washing buffer (20 mM Tris,
1 mM EDTA, pH 7.5) at 100 g/L concentration and cen-
trifuged at 10,000 g, 4 °C for 30 min to collect washed
cells. Washed cells were re-suspended in lysis buffer
(20 mM Tris, 1 mM EDTA, pH 7.5) and lysed by high
pressure homogenizer (GEA Niro soavi, Italy). The
obtained lysate was centrifuged at 10,000 g at 8 °C for
45 min to congregate the inclusion bodies (IBs). IBs con-
taining proinsulin were consecutively washed with TE
buffer, TE buffer containing 1% Triton X-100, 10% iso-
propyl alcohol at 50 g/L deliberation. The IBs were col-
lected by centrifugation at 10,000 g at 8 °C for 45 min.
2.2.3 Solubilization, Refolding and Concentration of IBs
The IB pellet was solubilized in the buffer (8 M Urea,
20 mM Cysteine HCl, 2 mM EDTA and 20 mM Tris pH
9.5) for 6 h at ambient temperature. The IB solution was 10
times diluted with refolding buffer (1 mM Cystine, 2 mM
EDTA and 20 mM Tris, pH 9.5) and incubated at 2–8 °C
for 36 h under continuous stirring. After incubation, pH
was adjusted to 3.5 using 2 M citric acid and filtered (1 l
filter). The filtrate containing refolded proinsulin was
captured by cation-exchange chromatography connected to
Akta Pilot (GE Healthcare Biosciences, USA) on a pre
equilibrated column. The bounded proteins were separated
with elution buffer containing100 mM Tris pH 9.5.
375
2.2.4 In vitro Conversion of Proinsulin to Insulin
Citraconic anhydride was supplemented drop wise to the
refolded proinsulin (2 g/g of protein) and pH of the solu-
tion was maintained throughout the procedure at 8.5 by
adding 6 M NaOH. To this solution, hydrogen peroxide
(10 mM) was added and incubated at room temperature for
2 h. Subsequently, trypsin and carboxy-peptidase-B have
been supplemented at a concentration of 1:2000 ratio (w/w)
and subjected to incubation at room temperature for 6 h
under stirring condition. For decitraconylation, the pH of
the solution was adjusted to 2.0 using 0.1 M HCl and
incubated for 6 h at room temperature.
2.2.5 Purification of Insulin by Cation Exchange
Chromatography
After decitraconylation, the solution containing insulin
(active form), proinsulin, C-peptide, and other impurities
was loaded onto SP-Sepharose FF resin packed in XK 50
column connected to Akta Purifier (GE, Healthcare Bio-
sciences) for purification. The bound insulin was eluted
with 150 mM sodium sulfate containing 25% acetonitrile
and 20 mM citric acid (pH 3.5). Each fraction was moni-
tored at 280 nm and the fractions containing active insulin
were analyzed by loading onto C18 column connected to
analytical RP-HPLC (Agilent, USA).
2.2.6 Polishing of Insulin by Preparative RP-HPLC
Fractions containing purified insulin were loaded onto an
YMC DAC 50 mm (Japan) preparative HPLC column (50
9 250 mm) packed with YMC–Triart C8 resin (particle
size 10 lm) connected to Akta Purifier. Chromatography
was executed by using buffer (200 mM sodium sulfate, pH
2.3) with acetonitrile combination. Solvent A is buffer with
10% acetonitrile and solvent B is buffer with 50% ace-
tonitrile at a flow rate of 75 cm/h. The bound protein was
eluted by applying linear gradient of 35.0–38.0% B in 6
column volumes and fractions were monitored at 280 nm.
2.2.7 Precipitation and Lyophilization of Insulin
The active HPLC fractions with a purity C 98.0% (pri-
marily based at the USP product-associated materials
impurities method) were pooled and precipitated by 1.0%
zinc chloride (w/w) at pH 6.0, 2–8 °C for overnight. Sub-
sequently, centrifugation was carried out at 5000 g for
20 min and the pellet was collected. The moisture content
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in the zinc pellet was removed using lyophilizer (Biocool,
China) at 20 Pascal’s vacuum for 24 h. After the comple-
tion, vacuum was released gradually and powder was
stocked at - 30 °C.
2.2.8 Quantification and Quality Assessment of Insulin
Insulin was quantified by analytical HPLC (Agilent, USA)
connected to 5 C18AR 300 analytical column
(250 9 4.6 mm, particle size 5 lm; Cosmosil, Japan)
using mobile phase containing acetonitrile (ACN), water
and trifluroacetic acid (TFA). Mobile phase A was pre-
pared with 10% ACN ? 0.1% TFA in water and mobile
phase B was prepared with 80% ACN ? 0.1% TFA in
water. The flow rate is maintained at 1 ml/min by applying
the liner gradient of 10–90% of mobile phase B in a span of
20 min. The test samples were injected in the range of 20
to 100 lL based upon the protein concentration and mon-
itored at 214 nm.
Quality of purified insulin was analyzed using (USP
product related materials and impurities approach) analyt-
ical HPLC connected to Vertisep HCS, C18 analytical
column (250 9 4.6 mm, particle size 5 lm; Vertical
Chromatography, Thailand). Mobile phase was prepared by
mixing 0.2 M sodium sulfate (pH 2.3) with phosphoric acid
in acetonitrile. Buffer A and buffer B were prepared with
acetonitrile and salt solution in 18:82 and 50:50 ratio
respectively. Isocratic gradient elution was adjusted to
obtain insulin peak between 15 and 25 min with the A-21
desamido insulin prior to loading the purified insulin
(Sonksen and Sonksen 2000).
3 Results and Discussion
3.1 Confirmation of pET-9a Vector
and Expression of Proinsulin Peptide
The presence of inserts in the transformants was confirmed
by digestion with NdeI and BamHI. Followed by it, plas-
mid DNA was subjected to sequencing using T7 forward
and reverses primers which confirmed that the cloned insert
was in frame. The E. coli BL-21 DE3 is a laboratory strain
derived from BL21, which is extensively used for recom-
binant protein expression. The B lineage lack lon and
ompT proteases, which made the strain as unique protein
expression host. Besides, these bacteria contain prophage
DE3 derived from bacteriophage k that carries gene for T7
RNA polymerase controlled by lacUV5 promoter (Jeong
et al. 2015).
Recombinant proinsulin peptide was effectively
expressed in fed batch fermentation of the recombinant
strain. The cell growth was measured at 600 nm and optical
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Iran J Sci Technol Trans Sci (2022) 46:373–383
density was observed to be 40–45 after 24 h of incubation
(Fig. 2). From 1.0 L of culture broth, the yield obtained
was 16 -18 g (dry wt.). Zieliński et al. (2019) constructed a
new vector, pIBAINS and expressed it in E. coli 20. The
cell growth at 600 nm was measured to be 54.4 and yield
obtained was 90 g (dry wt.). Similarly, Govender et al.
(2020) worked with E. coli BL21 (DE3) comprising
pCMV6-XL5 vector and found that the yield of human
proinsulin expressed in presence of isopropyl b-D thio-
galactoside was 04–0.7 g/L. Whereas, Polez et al. (2016)
reported a method for human insulin production using the
vector IP pPIC9K in Pichia pastoris where the yield
obtained was 1.5 to 3.84 g/L (dry wt.). From the mentioned
reports, it can be understood that the yield obtained in the
present work is significantly acceptable.
In order to analyze the expression of the proinsulin
peptides, the E. coli cells were collected at different time
intervals (18 h, 20 h, 22 h and 24 h) of fermentation pro-
cess. These cells were lysed and inclusion bodies were
solubilized for the discharge of proinsulin. The proinsulin
released from cells collected at different time intervals
were run on SDS-PAGE. Figure 3, confirmed that the
expression was exceptional from 18 h onwards. The
molecular weight of expressed proinsulin peptide was
found to be around 10 kDa. Moreover, the expressed pro-
tein quantity is around 20% of the total protein.
3.2 Separation, Washing and Solubilization
of Inclusion Bodies
The cells separated from the harvested broth were lysed by
suspending in lysis buffer (20 mM Tris, 1 mM EDTA pH
7.5) following by passing through high pressure homoge-
nizer. In several studies, use of lysozyme, which cleaves
the backbone of peptidoglycan present in the bacterial cell
wall, was reported (Hwang et al. 2016; Vilcacundo et al.
2018). But, in the present study mechanical means of cell
lysis was used to make the process cost effective at
industrial scale. The lysate containing inclusion bodies
were separated by centrifugation and washed with three
different buffers viz., TE buffer, TE buffer with 1% Triton
X-100 and 10% IPA to cast off the cellular particles and
other unwanted proteins. Triton X-100 was used to elimi-
nate lipoproteins and phospholipids; IPA is known for
removing of hydrophobic impure proteins. A series of
washing steps were carried out to reduce the impurity load
in the lysate and the same was evident from the Fig. 4.
Upon observing the lanes 1, 3, 5 and 7, there is a significant
decrease in number of bands representing impurities after
every wash. In general, washing is carried out either by TE
buffer or by combination of TE buffer and Triton X-100
which does not remove impurities significantly leading to
extensive steps of purification (Baeshen et al. 2014;
Iran J Sci Technol Trans Sci (2022) 46:373–383
Fig. 2 Growth profile of E.coli
BL21 at different time intervals
under following conditions:
temperature - 37 °C, pH: 6.86,
wind stream: 0.5 vvm and OHP:
0.2–0.3 bar
Fig. 3 SDS-PAGE analysis of expressed proinsulin peptide at
different time intervals (Lane-1: Broad range protein ladder, Lane-
2: Negative control, Lane-3: prolinsulin at 18 h culture, Lane-4:
prolinsulin at 20 h, Lane-5: prolinsulin at 22 h, Lane-6: prolinsulin at
24 h)
Fig. 4 SDS-PAGE analysis of Inclusion Bodies wash (Lane-1 and 2:
Wash-1 supernatant and pellet, Lane-3 and 4: wash-2 supernatant and
pellet, Lane-5 and 6: wash-3 supernatant and pellet, Lane-7 and 8:
wash -4 supernatant and pellet)
Zieliński et al. 2019). But in the present study, washing was
done consecutively at three stages, which directly reduces
the impurity load on the purification systems.
After washing and separation of inclusion bodies, IBs
were treated with solubilisation buffer for release of
377
proinsulin. The novelty of the present study lies in the use
of 8 M Urea, 20 mM Cysteine HCl, 2 mM EDTA and
20 mM Tris pH 9.5 for solubilizing the inclusion bodies.
The high pH buffer improves the refolding yield by
retaining the secondary structure in stabilized form. Several
researchers have reported the use of combination of urea
and detergents such as Lauroyl-L-glutamate; b-mercap-
toethanol, n-propanol and low concentration of urea
(Chura-Chambi et al. 2013; Singh et al. 2015). Besides use
of organic solvents like alcohol has also been reported to
solubilize the inclusion bodies, but they interact with the
protein and effects their secondary and tertiary structure
(Perham et al. 2006).
The proinsulin is refolded using refolding buffer and the
refolding yield is around 80 mg/L after 36 h of incubation.
After filtration, the filtrate was passed through SP-
Sepharose chromatography to concentrate the proinsulin.
At this stage, the proinsulin recovery was &80% and the
same was confirmed by injected into analytical RT-HPLC
(Fig. 5). A major peak at retention time 15.389 indicates
the pro-insulin peptide. By employing direct method of
refolding, total volume of refolding solution and processing
steps for human insulin production can be decreased and
production yield can be increased. Hence, this process may
be more viable for insulin production at a large scale (Kim
et al. 2015). Moreover, from the recovery percentage, it is
evident that the proinsulin was obtained in intact form. It is
confirmed that the treatment of proinsulin with
chaotropic/denaturing salt have contributed for proper
maintenance of disulfide bonds (Sarker et al. 2019).
3.3 Conversion of Proinsulin to Insulin
by Enzymatic Digestion with Citraconylation
The concentrated proinsulin was specifically tailored with
trypsin and carboxypeptidase-B proteolytic enzymes for
the conversion of proinsulin into insulin. Trypsin is serine
protease that cleaves peptide chains at carboxyl group of
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Fig. 5 Confirmation of Pro-Insulin at 15.389 min by analytical RP-HPLC chromatogram connected to SP-Sepharose FF resin packed in XK 50
column
amino acids lysine and arginine. Carboxypeptidase-B
hydrolyses the fundamental amino acids of lysine and
arginine from the C-terminal end of polypeptides (Son
et al. 2009). Both the enzymes have been added at a con-
centration of 1:2000 ratio and kept under continuous stir-
ring at an adequate temperature for 6 h. From 4th h
onwards conversion samples had been analyzed for reac-
tion progress. Peak RT shift and increase in peak area were
determined at every hour and found maximum at 9 h
(Fig. 6). Conversion reaction was stopped by changing the
pH to 2.5 with 6 M HCl. The major peak at RT 15.94 in
RP-HPLC chromatogram indicates insulin. Along with
insulin, the enzymatic digestion results in the formation of
other insulin derivatives, the purification of which is a
major hurdle. By subjecting the proinsulin to citraconyla-
tion prior to enzymatic conversion, unexpected cleavage of
the human proinsulin due to enzymatic action can be
blocked, due to which there will be significant reduction in
the formation of des-threonine insulin (Wu et al. 2018).
Hence, by employing citraconylation step, the yield of the
active insulin can be improved (Son et al. 2009).
3.4 Purification and Polishing of Insulin
The sample after enzymatic digestion was injected onto the
SP-Sepharose chromatography to purify the active insulin
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Iran J Sci Technol Trans Sci (2022) 46:373–383
from other impurities. Bound insulin was eluted with the
aid of linear gradient elution in 5 column volumes from
0–200 mM sodium sulfate. As shown in Fig. 7a, two peaks
were observed in the chromatogram based on the UV
absorption at 280 nm. Insulin was expected to elute at 80%
gradient, despite of that, both the peaks were loaded on to
analytical HPLC and second peak has shown a prominent
peak at 15.92 RT confirming the elutant as insulin
(Fig. 7b). Elution has a purity of &85% with a recovery of
&70% through the quantification procedure.
To achieve further purity, the eluted portion was diluted
with water to diminish the acetonitrile concentration to
20% and loaded on to preparative HPLC. Upon raising the
linear gradient of solvent B from 36–38%, the peak
absorbance commenced to increase and reached over 100
mAU at 280 nm (Fig. 8a). Column outlet was collected as
50 mL portions until the peak absorption decreased to 50
mAU. Each and every portion was evaluated for purity and
quantity by loading on to analytical HPLC. As evident
from Fig. 8b, 98% of purity was achieved through second
round of purification and all such portions have been
pooled and allowed for precipitation by addition of 1% zinc
chloride at pH 6.0. Zinc induces hexamerization of insulin
precursor, stabilizes the molecule being specific to insulin-
like molecules and hence, preferred metal for precipitation
(Smith et al. 1984). Further, the precipitate was collected
Iran J Sci Technol Trans Sci (2022) 46:373–383
Fig. 6 RP-HPLC chromatogram of insulin after successful enzymatic conversion from pro-insulin. Major peak at RT 15.94 gradually increased
during enzymatic digestion and exhibited present height after 9 h of incubation
and lyophilized for removal of moisture content and stored
at - 20 °C.
3.5 Quantification and Quality of Insulin
The quality and quantity of lyophilized insulin was deter-
mined by the injecting the sample on to analytical HPLC
connected to 5C18 AR300 column. Further confirmation
was achieved by injecting in vertisep HCS C18 column
connected to analytical HPLC (Fig. 9a and b). Similar
approach was carried out for both standard insulin and
purified insulin.USP product related materials and impu-
rities approach. The purity of the insulin was found to be
97.73% which was in proximity with that of the standard
insulin (99.69%). The retention time for purified insulin
and the standard insulin were 25.67 and 27.26 respectively
which shows that the purified insulin is in line with that of
the standard.
Till date, in the literature, two major routes of recom-
binant insulin production have been reported (Baeshen
et al. 2014). One route is the use of E. coli for production of
insulin precursor which needs solubilization and refolding
steps (Kaki et al. 2021). Another route is the use of yeast
expression system where the insulin precursor is released
into culture medium, the recovery and purification of which
379
includes approximately fifteen steps (Mollerup et al. 2009).
At industrial scale, the purity and yield of the product
depends upon the number of steps involved in the down-
stream processing (Kumar et al. 2021). With increase in
number of purification steps, the purity of the product will
certainly increase but there will be substantial decrease in
the yield (Banerjee et al. 2017). At large scale, along with
purity, yield also plays a key role hence, one should for-
mulate the steps in such a way that maximum yield and
purity could be obtained in less purification steps with low
input cost (Chintagunta et al. 2017).
By implementing the process set forth in the present
work, from 20 L of culture broth, 2.1 g/L of recombinant
human insulin with 98% purity has been obtained in two
purification steps (Table 1). Govender et al. (2020) worked
on the biosynthesis and optimization of expression of
human insulin in E. coli and obtained the insulin yield of
520.92 (mg/L). Similarly, Zieliński et al (2019) obtained
2700 IU of upto 99% purity recombinant human insulin
from 1 L of the culture media in three step purification
process. Polez et al. (2016) worked with Pichia pastoris for
insulin production and employed three purification chro-
matography steps to obtain yield of 51% with 98% purity.
Upon comparing with the reported processes, in the present
process, significant yield of insulin was obtained within
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380 Iran J Sci Technol Trans Sci (2022) 46:373–383

Fig. 7 Purification of insulin: a elution of insulin at 80% gradient via cation exchange chromatogram at 280 nm and b RP-HPLC chromatogram
confirming insulin at 15.92 RT with &85% purity

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Iran J Sci Technol Trans Sci (2022) 46:373–383 381

Fig. 8 Polishing of insulin: a Preparative HPLC at 280 nm and 38% of buffer B linear gradient showed maximum absorbance of 100mAU b RP-
HPLC chromatogram confirming insulin at 15.92 RT with &98% purity

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382 Iran J Sci Technol Trans Sci (2022) 46:373–383

Fig. 9 Chromatograms a USP standard and b purified insulin analytical HPLC connected to 5C18 AR300 column. The retention time for purified
insulin and the standard insulin were 25.67 and 27.26 min

Table 1 Insulin recovery and

Steps Protein (g/L) Recovery (%) Purity (%)
purity at various steps of the
purification Proinsulin refolding and concentration 0.094 80 n.d
Proinsulin to insulin conversion 0.31 45 57
Purification of insulin 4.37 70 85
Polishing of insulin 2.1 80 98
n.d not determined

two purification steps. Thus, it can be concluded that the
process put forward in the present study is viable and has
great application at industrial scale.
4 Conclusion
To augment the supply of human insulin, the pharmaceu-
tical biotechnology industries are striving hard to develop a
novel technique for production and purification of insulin.
Use of recombinant DNA technology has reduced the
dependency of industries on animal-derived pancreatic
tissue. The present work was framed in the same lines of
industrial requirement. Usage of urea in combination with
high pH for solubilization of inclusion bodies and
increasing the number of washing steps to reduce the load
on the purification systems are the amendments made in the
process which makes it distinct from the processes reported
till date. The recovery and purity of insulin obtained
through the study are 80% and 98% respectively. Through

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this work, an attempt was made to make the process of
insulin production and purification economical without any
compromise in the yield and purity of the product.
Acknowledgements The authors are grateful to Vcare Biolabs Pvt.
Ltd., Hyderabad for providing facilities to carry out the work. Mr.
K. Satish Babu is thankful to Management, VFSTR, India for pro-
viding opportunity to pursue his Ph.D.
Authors’ contributions KSB: Investigation; NPA: Conceptualization
and curation; ADC: Writing-original draft; VRD: Reviewing the
manuscript; NSSK: Writing-original draft and Reviewing the manu-
script; SJKN: Methodology; RB: Resource.
Funding No financial assistance was received either from govern-
ment or any private agency for the present work.
Declarations
Conflict of interest The authors declare that they have no conflicts of
interest with respect to the work described in this manuscript.
Human and animal rights statement This article does not contain any
studies with human or animal subjects.
Availability of data and material The data that support the findings of
this study are available on request from the corresponding author. The
data are not publicly available due to privacy or ethical restrictions.
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