Cell components are separated from the two desired insulin chains. Gel filtration and ion-exchange chromatography methods are used to remove impurities64.

The isolated purified protein has an insulin chain fused with β-galactosidase, as it has been linked with the gene incorporated into the plasmid and translated along with it. Cyanogen bromide is used to separate the insulin chains from β-galactosidase, as it splits the protein at the methionine residue which begins the β-galactosidase protein65.

The insulin chains (A and B) are treated with sodium dithionite and sodium sulfite to form the disulfide bonds that bridge the chains. This whole process is called reduction-reoxidation, and it is induced by β-mercaptoethanol and air oxidation to synthesize human insulin58.

RP-HPLC is performed to remove the remaining impurities or reagents. The purified, active human insulin can then be packed and sold by the industry66.

Recombinant human insulin is usually commercially produced in E. coli or S. cerevisiae. With the E. coli expression system, the inclusion bodies of insulin precursors (IPs) are produced, which are then solubilized and refolded to create active insulin. The yeast expression system yields the IPs in culture-supernatant, therefore it is often selected for commercial production. Several other insulin expression systems are being studied37. Some yeast strains are being manipulated to explore insulin production. Additionally, many mammalian cells and plants are under investigation for their suitability for large-scale production67. To fulfill the world’s requirement for insulin of 16,000 kg per year, novel and efficient expression systems must be developed37, 68.

The E. coli expression system was the first expression system used to produce human insulin in 1978, with the use of recombination technology in the two-chain method. This expression system is preferred for the large-scale production of R-insulin, as E. coli grows rapidly on cheap media, is easy to handle, and can be genetically manipulated69. However, several aspects limit its usefulness, including loss of plasmids, development of antibiotic properties, lack of post-translational modifications (PTMs), inclusion bodies’ accumulation within the cells, improper folding, proteolytic digestion, and poor secretion that makes protein recovery difficult70. Further research and the development of new technologies have tried to solve these problems. Some of the approaches are mentioned here.

Recently a new PCR-based approach was used for the cloning of human insulin, eliminating several steps such as affinity tags, tedious protein renaturation, inclusion body recovery steps, and the enzymatic cleavage of the C-peptide of insulin, making this method novel and more efficient45.

In 2019, Zieliński et al. reported the construction of the novel pIBAINS expression vector and the establishment of 20 novel E. coli strains, which provide greater efficiency of recombinant insulin production per liter of medium46.

Yeast is another commercially-used expression system for heterogeneous protein production with recombinant technology because of its PTMs. Such PTMs include acylation, phosphorylation, and N- and O-linked glycosylation of proteins74. Yeast can easily be scaled up in bioreactors and its use is cost-effective, which makes it a potential candidate as an expression system for the production of recombinant insulin. However, a factor of concern when using yeast as an expression system is that proteins synthesized in yeast have high-mannose N-glycosylation. This shortens the half-life of recombinant proteins in vivo and activates an immune response in humans because proteins with this modification are considered foreign antigens. Thus, yeast has to be humanized before use by modifying its N-glycosylation pathways to create less immunogenic products for humans75.

S. cerevisiae has been used commercially as a yeast expression system for insulin since the 1980s by creating proinsulin, which links the A and B chains via a short synthetic C-peptide37, 64. The α-factor signal sequence helps in the synthesis of the chains and increases proinsulin expression to 80 mg/ml76. Proinsulin is converted to active insulin by a trypsin-mediated transpeptidation reaction with a threonine ester. Other insulin analogs synthesized using yeast include:

The high cell-density, methylotrophic yeast P. pastoris is also used commercially for insulin production80. A special feature of this yeast expression system is the presence of the methanol-inducible alcohol oxidase-1 promoter (AOX-1), which increases the cell density under simple cultivation strategies to help in the large-scale production of recombinant proteins. Unlike S. cerevisiae, P. pastoris does not hyperglycosylate its secreted heterogeneous proteins, proving advantageous for use in the creation of human therapeutics81. Both yeast expression systems do carry out high-mannose N-glycosylation, but in P. pastoris, the oligosaccharide chain consists of 8-14 mannose residues/chain, which is far shorter than the 50-150 mannose residues/chain added by S. cerevisiae. Heterogeneous protein expression attained by P. pastoris is approximately 30% of its total cell protein, which is much higher than S. cerevisiae82. Such characteristics make P. pastoris an attractive expression system for use in the commercial production of recombinant human insulin and its analogs. The highest yield of insulin precursor, 1.5 g of IP/L of culture broth, was recorded from the P. pastoris expression system83.

Of recent interest in the field is the production of recombinant or heterogeneous proteins from transgenic plants, which have the advantage of low cost and high protein quality84. Plants do not have human pathogens and because they are eukaryotic, their PTM machinery is more similar to humans than that previously described in yeast85. The mechanism involved in producing human insulin in transgenic plants is illustrated in Figure 5.

With recent experimentation, the human insulin gene has been successfully expressed in oilseeds of the Arabidopsis thaliana plant37. This plant has a short generation time of almost 6 weeks and can easily grow under laboratory conditions with limited sunlight. The genome of A. thaliana is completely known, making it easier to perform further research on recombinant insulin production. Each plant is capable of generating approximately 10,000 to 30,000 seeds86. The insulin is expressed in subcellular organelles of plant-like oil bodies which, along with a high amount of protein expression, makes its recovery easier. Inside the oilseeds, the oil bodies are encapsulated by a hydrophobic tri-acylglycerol phospholipid membrane, and their outer wall is made up of oleosins87. These oil bodies can be easily separated from other seed components by liquid-liquid phase separation methods, reducing the need for chromatographic steps to purify the insulin. Experimental results showed that the insulin accumulation in transgenic seeds is up to 0.31% of the total seed proteins88. Trypsin digestion is used to cleave the oleosin from the oil bodies, which also purifies and matures the active insulin via simple purification methods. The use of seeds as an expression system is also advantageous because the insulin can be stockpiled until it is required.

The tobacco plant exhibits higher seed germination and survival rates, while the lettuce plant is widely consumed across the globe and is a very attractive expression system89, 90. The chloroplasts of these plants are used to synthesize proinsulin. The insulin chains (A and B) and the C-peptide become fused with subunits of cholera toxin-B91, 92. The reported data show that old leaves of tobacco plants contain almost 47% of proinsulin out of their total leaf proteins91. In old leaves of lettuce, up to 53% of total leaf proteins were recorded as proinsulin91. The extracted insulin is found to be very stable and has up to 98% purity when cleaved by Furin protease treatment to release the insulin peptides. Oral delivery of plant cells also shows similar results as commercially available insulin. The recorded yield of proinsulin by these plants is 3 mg/g of leaves, which indicates that one acre of such plants could produce 20 million daily doses of insulin every year37, 72.

Strawberry (Fragaria ananassa Duch.) is one of the world's most significant and popular fruits and contains several vitamins, minerals, anthocyanins, and essential amino acids that contribute to human health93, 94. However, the edible nature of the strawberry makes it a useful plant for insulin production, as it can be used as a vehicle for oral administration. Also, strawberries contain levulose, which is quickly absorbed and decomposed in the body and does not affect people with diabetes95. Therefore, the strawberry should be studied to determine if oral insulin administration is a viable option for people with diabetes. According to Mendel's rules of inheritance, if a gene is delivered into the nucleus of a plant cell, at least one-fourth of the plant will be non-transgenic in the following generation96. However, the presence of runners in strawberry plants, which are clones of the mother plant, allows each generation to have a mother plant95. Because of these qualities, the strawberry plant is a suitable expression system for human proinsulin gene transformation and recombinant protein production.

In a recent study, Tavizi et al. infected strawberry explants (leaves, petiole, and buds) with A. rhizogenes and A. tumefaciens that contained insulin-producing genes. The hairy roots that grew as a result of the treatment with A. rhizogenes were subcultured in a bioreactor for several days, while the explants infected with A. tumefaciens were micropropagated. The transmission and expression of the insulin genes in the hairy roots and regenerated plants were analyzed through PCR, RT-PCR, and ELISA. Additionally, the proinsulin was purified from the transgenic plants and hairy roots and injected into diabetic rats. 60 ng of the purified insulin was able to significantly reduce the blood sugar levels in these rats95.

Stem cells are specialized cells that eventually develop into the tissues and organs of the body. Throughout its life, the body relies on stem cells to repair damaged tissues and cells that are lost regularly, as they can self-renew and differentiate. Embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) have all been developed into insulin-producing cells (Figure 6)2, 97.

ESCs can develop into endoderm, mesoderm, and ectoderm cells and are extracted from blastocysts. When ESCs are transplanted into diabetic mice, they can develop into insulin-producing cells, which can release insulin in response to glucose stimulation and restore proper blood glucose levels98, 99. Human ESCs can also be differentiated into endocrine cells, however, this carries the risk of promoting tumor formation100.

Like ESCs, iPSCs have an indefinite capacity for self-renewal and the ability to differentiate into a wide range of cell types. Undifferentiated iPSCs can be maintained as cell lines, which holds tremendous potential for disease modeling and the development of autologous cell therapies101. iPSCs generated from mouse skin fibroblasts were able to develop into β-like cells, comparable to the natural, endogenous insulin-secreting cells, and helped diabetic mice regulate hyperglycemia102. Human ESCs and iPSCs have been developed into mature pancreatic cells capable of secreting insulin and C-peptide103. Insulin-producing cells were also grown in vitro from iPSCs using small chemicals and growth factors104.

MSCs can be extracted from adipose tissue, mobilized peripheral blood, fetal liver, periodontal ligament, umbilical cord blood, placenta, fetal lung, dental pulp, umbilical cord, synovial membrane, endometrium, compact bone, trabecular and dental pulp, synovial membrane, endometrium, periodontal ligament, and trabecular and compact bone105. MSCs can differentiate into mesodermal, endodermal, and even ectodermal cells when cultured under the right circumstances106. They can secrete growth factors and immunoprotective cytokines during transplantation107. MSCs have also been injected directly into the pancreas as niche-providing cells, which helped relieve diabetic symptoms in animal models by improving metabolic regulation, counteracting autoimmunity, boosting islet engraftment and survival, and acting as a source of growth factors and cytokines108. MSC injections enhance pancreatic function while also alleviating symptoms such as diabetic foot, nephropathy, and neuropathy. Although MSCs appear to have a significant impact, it is more likely to be a niche effect than genuine regeneration109.

Human bone marrow (BM)-derived MSCs can be maintained in vitro for up to 44 weeks without losing their morphological, phenotypical, functional, or karyotype features110. MSCs from the BM suppress the immunological response of T cells towards freshly generated β cells111. As a result, stem cell therapy may be the most effective treatment for patients with type 1 diabetes. Stem cells migrate to the injured area, develop, and initiate structural and functional repair, which aids in the treatment of diabetes and the normalization of insulin levels. Mouse bone marrow cells developed into functional β cells in an in vivo experiment112. In another investigation, iPSCs from BM-MSC rats were shown to cure chronic hyperglycemia in diabetic rats.

Adipose-derived stromal cells (ADSCs), also known as adipose tissues isolated from human lipoaspirates, can be obtained in high quantities and have differentiation capabilities comparable to BM-MSCs. When AD-MSCs were grown in a fibroblast growth factor-containing medium, they produced markers such as Isl1 mRNA, which are required for the formation of pancreatic islet cells113. Several studies have shown that AD-MSCs from the mouse epididymis can develop into cells that express PDX1, Ngn3, NeuroD, Pax4, Glut2, and produce insulin and C-peptide114. After 38 days of co-culture with islet cells, AD-MSCs were shown to be differentiated into iPSCs115. Compared to islet transplants alone or co-transplantation of islets and differentiated BM-MSCs, combining differentiated AD-MSCs and islet cells resulted in better diabetes recovery116.

MSCs from umbilical cord blood and unrestricted somatic stem cells from hUCB can develop into iPSCs with the same cell markers and characteristics as MAPCs (multipotent adult progenitor cells)117. After incubation in a medium supplied with no particular cytokines or growth factors besides fetal calf serum, UCB cells express genes necessary for differentiation into pancreatic endocrine tissue, including Isl1, PDX1, Pax4, and Ngn3113. In vitro and in vivo, insulin and the C-peptide were released by iPSCs generated from UCB-MSCs in response to a glucose challenge. After intravenous injection of hUCB-MSCs into 25 NOD type 1 diabetic mice with insulitis, glycemic profiles improved along with histological improvements in the islets118. UCB-MSCs are widely accessible, have minimal risk of immunological rejection, and show improved capabilities for growth and differentiation into iPSCs, making them a viable choice for the treatment of diabetes.

Humanized gene expression is better in eukaryotic systems than in prokaryotic systems. The human insulin gene, which is eukaryotic, cannot be immediately transferred to a prokaryotic expression system, because the gene contains introns that must be spliced before translation, followed by PTMs to create the active insulin product. Instead, cDNA of the desired gene is created and then injected into a prokaryotic cell. E.coli has historically been the favored prokaryotic expression system for large-scale recombinant protein production because of its ease of genetic modification, cost-effectiveness, and high growth and recombinant protein synthesis rates119, 120. However, this expression system comprises a large number of uncommon codons and has a high incidence of translational mistakes such as amino acid substitutions, premature translation termination, and frame-shift mutations. In the case of therapeutic proteins like insulin, such mistakes might degrade the quality, causing an immunogenic reaction in humans121. PTMs, loss of plasmids, antibiotic properties, and the downstream procedures to extract the required proteins from chaperones are some of the drawbacks of employing an E. coli expression method.

Humanized yeast expression systems, which use single-celled organisms with eukaryotic post-translational modification processes, can be employed to overcome the aforementioned constraints. On a large scale, yeast can be readily maintained using a basic medium. Among yeast species, S. cerevisiae plays a crucial role, particularly in the production of proinsulin analogs. With naturally occurring plasmids, it has been described to have strong related promoters. It only produces a few proteins. Regardless of its high copy number and the presence of LUE2, yeast episomal plasmids are most commonly utilized for manufacturing122. S. cerevisiae is used to produce Insulin Aspart, a fast-acting insulin derivative. P. pastoris, on the other hand, is a better choice for industrial-scale production due to its plasmid degradation and lesser extent of hyperglycosylation. In both bacterial and yeast expression methods, the total space-time yield is similar, although yeast, particularly P. pastoris, is more appropriate123.

As transgenic plants do not contain human diseases, they are thought to be a safer expression method for human insulin. A. thaliana seeds, tobacco and lettuce plant leaves, and strawberry root hairs are among the transgenic plants being studied for use in proinsulin production. Physiologically active insulin can be readily isolated from subcellular oil bodies of A. thaliana seeds using liquid-phase chromatography124. At a larger scale, enzymatic purification can be employed. The shockability and low cost of seeds as a bioreactor are the two most essential advantages. However, difficulties with protein stability remain a key concern when utilizing plant expression systems. To prevent problems like these, tobacco and lettuce plant leaves were chosen as proinsulin bioreactors125. In their global usage, tobacco provides protection against food-chain contamination, while lettuce does not126. However, the chloroplast-based expression mechanism utilized in both plants has chloroplast glucan interference, nonhuman glycosylation, and may require PTMs. As a result, before employing such expression methods, changes to the production method are necessary127. Strawberry is another intriguing plant for proinsulin synthesis, and its root hairs are being studied as a bioreactor. It is, so far, the safest option, as there is no gene escape128. Unfortunately, it can induce allergic responses in people when consumed orally.

ESCs are a popular target for researchers because they are produced from unused or unfertilized embryos at in vitro fertilization clinics, but there are few limitations in utilizing these cells. They should only be utilized for clinical studies with the prior consent of the donor. However, in the majority of cases, cells from the embryo are acquired by destroying the embryo, which raises ethical concerns regarding the origin of life and the right to annihilate the embryo129. Human ESCs can be differentiated into endocrine cells, but they may also promote tumor growth130.

The lack of ethical issues and minimal probability of teratoma development are two advantages of utilizing iPSCs131. The most common method for transforming somatic cells into iPSCs is to use viral transfection of transcription factors. The use of harmful genomes, which can cause mutations and affect normal iPSC function, differentiation ability, and tumorigenesis, is an important drawback of this technology131. Similarly, MSCs seldom differentiate spontaneously in the host tissue. Thus, their therapeutic utility relies on the capacity to regulate their in vivo differentiation into functional cells with high efficiency and purity132. Additionally, MSCs can develop into undesirable mesenchymal lineages, which might restrict their therapeutic effectiveness133. A comparative analysis of the different expression systems that have been discussed for insulin production is provided in Table 1.

Table 1

Expression Systems	Advantage	Year of FDA Approval	Limitations	Advancements	References
Prokaryotic Expression System
Escherichia coli	Versatility and cost-effectiveness for large scale production	US FDA in 1982	Plasmid loss, high probability of translation errors which can cause adverse immunological responses in human	Colony PCR strategies for similarity verification, strain modifications, codon optimizations and addition of chaperones	45, 59
Yeast Expression System
Saccharomyces cerevisiae	Eukaryotic model systemwhich enables the quality production and proper folding.	US FDA in 2001	Plasmid degradation and Hyper-glycosylation on large scale. Time consuming purification methods.	Modification in recombinant sequence of B chain cDNA to prolong the insulin effect duration.	121
Pichia pastoris	High cell density of Methylotrophic yeast with methanol-inducible alcohol oxidase-1 (AOX-1)promoter, do not hyper glycosylate insulin	US FDA in 2009	High mannose structures and glycosylation heterogeneity can cause batch-to-batch variation	Engineered strains for higher secretion levels, AOX1-based methanol-free protein manufacture, modified N-glycosylation machinery	122, 123
Transgenic Plants
	Large amount of seed generation in 6 weeks, easy purification of oil bodies, no human pathogen, shockable	Approval required	Protein stability issues Inappropriate containment strategies	Finding tissue-specific promoters to avoid toxicity	124
Tobacco	High number of leaves yield contains proinsulin, high purity protein with minimal PTMs requirement, less transgene containment.		Chloroplast glycoproteins interference	Transformed chloroplast, CTB-insulin fusion	125, 126
Lettuce	Long term stability in leaves, more leaf protein yield then tobacco plant		Lack of regulatory approval due to food-chain contamination, Improper glycosylation	Transformed chloroplast	126
Strawberry	Oral consumption, using hairy roots as bioreactor		lack of regulatory approval, unwanted allergic reactions	No gene escape	127, 128
Stem cells
Embryonic stem cells (ESC)	Can differentiate into any cell type	Approval required	Ethical issues, Chances of immune rejection, genetic instability, teratocarcinomas formation	Generation of pancreatic progenitor cells through ESCs	134, 135, 136, 137
Induced pluripotent stem cells (iPSCs)	No ethical issues, less immune rejection chances, variable sources to derive iPSCs, can differentiate into variety of cell types		Require reteroviruses to generate iPSCs, chances of cancer formation	Use of non-viral reprogramming factors for generation of iPSCs	138, 139, 140
Bone Marrow derived Mesenchymal stem cells (BM-MSCs)	Easy isolation, no ethical issues, can differentiate in variety of cell types		Minimal differentiation potential, differentiate into unwanted mesenchymal lineages	Use of nanotechnology and gene regulation enhanced the differentiation and proliferation ability of MSCs	132, 141
Adipose-derived Mesenchymal Stem cells (AD-MSCs)	No ethical issues, different isolation sources, high histocompatibility and low immune rejection		Low multipotency and proliferative ability, genetic instability, apoptosis occurs in newly produced cells		142, 143
Human Placenta derived Mesenchymal stem cells (HP-MSCs)	High proliferative and differentiation rate, Ease in extraction			Use of nanotechnology to promote drug delivery and detection of transplanted cells	144, 145, 146
Human Umbilical Cord derived Mesenchymal stem cells (HUC-MSCs)	High proliferative and differentiation rate, 10 times more stem cells than those collected from bone marrow, Anti-inflammatory properties, anti-fibrotic potential, low risk of immune rejection		Limited cells can be isolated for once from umbilical cord, less banks for storage of umbilical cords, require more time to graft		132, 147, 148

The two conventional methods of producing insulin by recombinant technology are the proinsulin method and the two-chain method. The proinsulin method was developed more recently and is more reliable because it involves fewer steps than the two-chain method, making it effective and cost-efficient. Bacterial and yeast expression systems are generally preferred for commercial production, but novel organisms and transgenic plants have recently been introduced. In E. coli and S. cerevisiae, the C-peptide of proinsulin is absent, which can be dealt with by certain genetic modifications in existing expression systems or by using new transgenic organisms like P. pastoris, A. thaliana, tobacco, strawberry, or lettuce. Additionally, transgenic plants can provide a higher level of protein expression. Even though human MSCs could be differentiated into functional pancreatic cells in vitro, the rate of trans-differentiation is limited, and the length of functional maintenance in vivo is challenging to assess. Considering the lack of defined protocols for the growth and production of insulin-secreting cells, clinical results are inconsistent. The use of novel approaches such as nanotechnology, signaling hormones, non-viral programming, or gene regulation of different types of stem cells can be utilized to develop insulin-producing cells that can serve as a useful treatment option for patients with diabetes. To achieve long-term stability, low-cost technologies should be promoted. Additionally, oral delivery of insulin is a safer method.

A. rhizogenes: Agrobacterium rhizogenes

A. thaliana: Arabidopsis thaliana

A. tumefaciens: Agrobacterium tumefaciens

AD-MSCs: Adipose-derived Mesenchymal Stem cells

AOX-1: Alcohol oxidase-1 promoter

BM-MSCs: Bone Marrow derived Mesenchymal stem cells

CNBr: Cyanogen bromide

CNS: Central nervous system

DTT: Dithiothreitol

E. coli: Escherichia coli

ESCs: Embryonic stem cells

GI: Gastrointestinal tract

GSH: Glutathione

HP-MSCs: Human Placenta derived Mesenchymal stem cells

HUC-MSCs: Human Umbilical Cord derived Mesenchymal stem cells

IP: Insulin precursors

iPSCs: Induced pluripotent stem cells

LB: Luria Broth

P. pastoris: Pichia pastoris

PSI: Pounds per square inch

PTMs: Post-translational modifications

RE: Restriction enzyme

Rpm: Revolutions per minute

S. cerevisiae: Saccharomyces cerevisiae

STR: Stirred tank reactor

T1DM patients: Type 1 Diabetes mellitus patients

The authors are thankful to the team of Big Bio for their support.

All the authors contributed equally in manuscript writing. J. Alyas, A. Rafiq, H. Amir and T. Sajid conceptualized study and wrote introduction of the manuscript. S. U. Khan, T. Sultana, A. Hameed and I. Ahmad, wrote about the insulin production and revised it critically. A. Ali and A. Kazmi designed and modified the figures, wrote manuscript and approved the final version of the manuscript for submission.

None.

Not applicable.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.