Therapeutic proteins: developments, progress, challenges, and future perspectives

Abstract

Proteins are considered magic molecules due to their enormous applications in the health sector. Over the past few decades, therapeutic proteins have emerged as a promising treatment option for various diseases, particularly cancer, cardiovascular disease, diabetes, and others. The formulation of protein-based therapies is a major area of research, however, a few factors still hinder the large-scale production of these therapeutic products, such as stability, heterogenicity, immunogenicity, high cost of production, etc. This review provides comprehensive information on various sources and production of therapeutic proteins. The review also summarizes the challenges currently faced by scientists while developing protein-based therapeutics, along with possible solutions. It can be concluded that these proteins can be used in combination with small molecular drugs to give synergistic benefits in the future.

Introduction

Protein-based treatments remain a top priority for many pharmaceutical businesses in the modern era of drug discovery and development (R&D). Recombinant biologics have been widely employed as the medicine of choice for treating a variety of diseases, in large part due to their specificity in identifying and modifying disease-related pathways (Walsh 2018). Human insulin was the first recombinant therapeutic protein, and its discovery in 1970 has increased interest in and expectations for the creation of new protein-based medicines. The 1st therapeutic protein other than antibodies, Insulin, was purified from the animal pancreas (bovine and porcine pancreas) and administered to patients suffering from diabetes mellitus type I (DM‑I) and type II (DM-II) in 1922. It was the first paradigm shift. DM-I is a disease whose primary cause is the absence of the protein hormone insulin, which provides signals to the cells so that the cells can perform functions related to glucose metabolism. However, its use was limited due to its unavailability, cost, and immunogenicity. Then, it took 60 years for the second paradigm to produce the first recombinant therapeutic protein, i.e., humulin (human insulin), with the application of genetic engineering, which was approved by the US FDA in 1982. Later, a murine mAB Muromonab-CD3 (OKT3) was the first therapeutic protein approved for clinical use in 1986, but it was withdrawn from the market in 2010.

Protein-based therapeutics are the fastest-growing medicines in the pharmacological industry (Lagassé et al. 2017). Most protein therapeutics are recombinants, and more than 170 are used in medicine worldwide. In contrast, many of them are in clinical trials used for multiple applications such as diagnosis, prevention therapy of cancers, immune disorders, infections, and disease management. They have become an essential part of the pharmaceutical industry, and the pharma industries are focused on research using these proteins to identify novel effective therapeutics (Kintzing et al. 2016). These therapeutics have been known to represent 1/3rd of the entities launched as new medicines under biology. More than 6 million people have lost their lives due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making the outbreak the deadliest global health crisis since the influenza pandemic of 1918 (Aleem et al. 2023). Human recombinant soluble ACE2 protein is also one of the proposed COVID-19 treatments. This Recombinant human-soluble ACE2 protein prevents the SARS-CoV-2 from binding to ACE2 (Zhou et al. 2020; Monteil et al. 2020; Tai et al. 2020). Additionally, peptide-based therapies were developed and evaluated to target COVID-19, focusing on the N-protein and cytolytic T-cell response (Amawi et al. 2020). Neutralizing antibodies were also evaluated for treating COVID-19 infection using similar mechanisms, which involve inhibiting virus particle binding with ACE2 receptors (Du et al. 2021).

According to La Merie's financial reports, the global value of therapeutic biologics increased annually, reaching an estimated $800 billion between 2014 and 2018 (Walsh 2018). The global market for therapeutic proteins has been proliferating in recent years, with estimates suggesting a compound annual growth rate of 8.5% between 2020 and 2027 (Research and Markets, 2021). In 2020, the market size was valued at USD 168.5 billion, with North America and Europe accounting for the largest share of the market. The therapeutic protein market in India is also growing, driven by factors such as a large patient population, rising disposable incomes, and the increasing prevalence of chronic diseases. According to a report by Grand View Research (2021), the Indian market for therapeutic proteins was valued at USD 1.34 billion in 2020 and is expected to grow at a compound annual growth rate of 8.8% between 2021 and 2028. Therapeutic proteins represent a significant and growing market globally and in India, with potential for further growth as new drugs are developed and personalized medicine becomes more widespread. Drug discovery and development have shifted their focus to therapeutic proteins (with improved human safety profiles), but they still face some obstacles. Immune responses and other side effects have been linked to various protein-based therapies.

The field of peptide drug discovery has demonstrated significant dynamism in recent times such as the development of new peptide-focused industries and the emergence of big-pharma business (Albericio and Kruger 2012; Henninot et al. 2018; Al Shaer et al. 2020; De La Torre and Albericio 2020). Therapeutic peptides are a distinct class of pharmaceutical drugs made of well-arranged sequences of amino acids, most of which have molecular weights between 500 and 5000Da (Henninot et al. 2018). Peptides are known to be extremely effective and selective while also being quite safe and well-tolerated. With over 7000 naturally occurring peptides known, many have important functions in human physiology, acting as growth factors, hormones, neurotransmitters, ion channel ligands, or anti-infectives, among other things (Giordano et al. 2014; Robinson et al. 2014; Padhi et al. 2014; Buchwald et al. 2014; Fosgerau and Hoffmann 2015). Peptides are highly effective and selective signaling molecules that attach to particular cell surface receptors, including ion channels or G protein-coupled receptors (GPCRs), to initiate intracellular actions. The production complexity of peptide therapeutics is generally lower than that of protein-based biopharmaceuticals. Consequently, the production costs of peptide therapeutics are also less than those of small molecules (Fosgerau and Hoffmann 2015). Naturally occurring peptides (e.g., cyclosporin, oxytocin and insulin (Marx 2005; Tsomaia 2015)) due to their shortcoming such as poor physical and chemical stability as well as short half-life period in plasma circulation also a big challenge. In recent times such shortcomings are resolved using models such as “traditional design” (e.g., SWOT analysis). In addition to traditional peptide designs, a variety of peptide technologies have emerged such as peptide drug conjugates, multifunctional and cell penetrating peptides along with alternate routes of drug administration, signifying the potential and future possibilities in the peptide domain (Fosgerau and Hoffmann 2015).

Peptides mediate numerous vital biological activities, including signal transduction, heart rate regulation, food intake, and growth. They are distinguishable from proteins by their smaller size (50 amino acids or less). Peptides have a strong and selective ability to bind large protein targets, which means they are less likely to cause toxicity and off-target adverse effects than small molecule medications (Craik et al. 2013; Tsomaia 2015; Wang et al. 2022). In contrast to small molecules that might have adverse effects by generating toxic metabolites that build up in various organs, peptides break down into amino acids, hence reducing the potential for toxicity (Manna et al. 2018; Wang et al. 2022). Peptides are more stable at room temperature, have a cheaper manufacturing cost, and have a greater capacity to penetrate tissues because of their smaller size than recombinantly generated antibodies and designed protein medicines (Patel et al. 2019; Lee et al. 2019; Barman et al. 2023). Furthermore, a peptide sequence can be edited to include non-natural building blocks and other chemical scaffolds, resulting in a palette of modified peptides with a variety of functionalities and chemical diversity (Oeller et al. 2023). With the dawn of the twenty-first century, peptide drug development entered a new age marked by a major acceleration in structural biology, recombinant biologics and new synthetic and new analytical technologies (Wang et al. 2022). Currently, metabolic illnesses and oncology are the two main disease categories that are driving the therapeutic usage of peptide medicines. There are now around 170 peptides undergoing clinical trials, and numerous others are in preclinical development. Table 1 shows some of the FDA approved (2015–2020) therapeutic peptide drugs in clinical use and for management of various diseases.

Table 1.

List of some FDA approved (2015–2020) therapeutic peptide drugs in clinical use and for management of various diseases

S. no	Therapeutic peptide	Target name	Indications	Year of approval	References
1	Setmelanotide	Melanocortin-4 receptor	For chronic weight management of obesity	2020	Kamermans et al. (2019), Haws et al. (2020), Wang et al. (2022)
2	Edotreotide gallium Ga-68	Somatostatin receptors	Used to identify neuroendocrine tumors that are positive for the somatostatin receptor	2019	Moyade and Vinjamuri (2019, Evangelista et al. (2020)
3	Lutetium Lu 177 dotatate	Somatostatin receptors	In treatment of gastroenteropancreatic neuroendocrine tumors	2018	Das et al. (2019), Kaewput and Vinjamuri (2022)
4	Etelcalcetide	CaSR	In treatment of secondary hyperparathyroidism	2016	Friedl and Zitt (2018)
5	Angiotensis II	AT1 Receptor	In treatment of sepsis and septic shock	2017	Fernandes et al. (2021)
6	Insulin degludec Tresiba®	-	In treatment of diabetes	2015	Kesavadev et al. (2022)
7	Bremelanotide	MC receptors	In treatment of hypoactive sexual desire disorder	2019	Rao and Andrade (2020)
8	Plecanatide	Guanylate cyclase C	In treatment of idiopathic constipation	2017	Sharma et al. (2019)
9	Macimoreline Macrilen®	Ghrelin receptor (7TM receptor)	In treatment of Growth Hormone deficiency	2017	Garcia et al. (2021); Yuen et al. (2023)
10	Semaglutide	GLP-1 receptor	In treatment of Type-2 diabetes mellitus	2017	Pearson et al. (2019)

In the current review, we offer detailed insights into the classification of therapeutic proteins and various fundamental factors that may play an essential role in the production of therapeutic proteins and may influence their production, formulation, or delivery as drugs. Moreover, we have also addressed the challenges associated with the production of therapeutic proteins.

Classification of therapeutic proteins:

Therapeutic proteins have been categorized into four groups based on their function and therapeutic applications (Leader et al. 2008; Naz et al. 2022). Group I and II are FDA-approved, while Group III and IV were investigated in vivo or in vitro. Group I includes therapeutic proteins having enzymatic or regulatory activity. It is further sub-divided into three sub-groups: 1a, Ib, and Ic. Group II includes therapeutic proteins with unique targeting activity. It is divided into two sub-groups: IIa and IIb. Group III consists of those proteins that act as prophylactic and therapeutic vaccines. Hepatitis B surface Ag (HBsAg) for Hepatitis B infection and anti-rhesus (rh) IgG vaccine, used in routine postpartum prevention of Rh(D) immunization in Rh(D) negative women (Crosnier et al. 1981; MacKenzie et al. 2004) are the examples of Group III therapeutic proteins. Group IV consists of proteins used in disease diagnostics, such as infectious disease detection and cancer detection as an imaging agent (Sodee et al. 2000; Campos-Neto et al. 2001). Table 2 summarizes the types of therapeutic proteins, their sub-groups, their function, and examples.

Table 2.

Classification of therapeutic proteins, along with functions and examples

Group	Sub-group	Functions	Examples	References
Group-I	Ia	Replace a protein that is deficient or abnormal Treat metabolic disorders or endocrine dysfunctions such as IL-1 Ra for type 2 diabetes mellitus and disorders like haemophilia A	Factor VIII (Hemophilia A)	Roth et al. (2001), Akash et al. (2012b, a)
	Ib	Stimulate various haematological and body responses	INF-α (Hepatitis C) Erythropoietin (chronic anaemia)	Corwin et al. (2002), van Zonneveld et al. (2004)
	Ic	Modify the pathophysiology of the disease	Botulinum toxin type A and B (dystonia) Lepirudin (heparin-induced thrombocytopenia)	Oates et al. (1991), Eriksson et al. (1997)
Group-II	IIa	—Either activate a signalling pathway or impair their ability to function	• Cetuximab-Monoclonal antibody that binds to EGFR to treat colorectal, neck and head cancer • Anakinra (IL-1Ra)	Saltz et al. (2004)
	IIb	—Can deliver proteins specifically and on-target	• Ibritumomab tiuxetan, denileukin diftitox for treating non-Hodgkin’s lymphoma and persistent cutaneous T-Cell lymphoma	Witzig et al. (2002), Ohya and Matsuda (2005)
Group-III	IIIa	Protecting against a harmful foreign agent	• Hepatitis B surface Ag (HBsAg) for Hepatitis B infection • Anti-rhesus (rh) IgG vaccine, used in routine postpartum prevention of Rh (D) immunization in Rh(D) negative women	Crosnier et al. (1981), MacKenzie et al. (2004)
	IIIb	Treating an autonomous disease
	IIIc	Treating cancer
Group-IV		Group IV proteins are not used to treat disease, but they are mentioned here because they are crucial in the decision-making process before treating and managing many diseases. These proteins are purified, and recombinant proteins are used for medical diagnostics (both in vivo and in vitro)	• Recombinant purified protein derivatives (DPPD) for diagnosis of tuberculosis exposure • Capromab pendetide for Prostate cancer detection • Apcitide for Imaging of acute venous thrombosis	Duchin et al. (1997), Coler et al. (2000), Sodee et al. (2000), Campos-Neto et al. (2001)

Sources of therapeutic proteins

Producing therapeutic proteins is one of the fastest-growing areas of molecular medicine, and it plays an important role in preventing and curing several disorders. Various sources of therapeutic proteins have been discussed below:

Microbes as a source of therapeutic proteins

Since nature is an attractive source of therapeutic products, huge chemical diversity is found in millions of species of plants, animals, marine organisms, and especially microorganisms. These microbes have been used for years for the large-scale production of various therapeutic products. They play an essential role as host expression systems (prokaryotic and eukaryotic) in having potential as natural sources of drugs for the treatment and prevention of several diseases such as Crohn’s disease, diarrhea, diabetes, cancer, atopic dermatitis, ulcerative colitis, etc. (Kota et al. 2018). Depending on their ability to produce the desired chemical, prokaryotic and eukaryotic microorganisms have been utilized as hosts for producing therapeutic proteins. Prokaryotes generally express smaller proteins and eukaryotes express larger ones due to their different cellular structures. Prokaryotic systems such as E. coli are used for the cost-effective, easiest, and fastest expression of therapeutic proteins. In contrast, insect systems, mammalian cells and fungi are better for the proteins requiring glycosylation (Schillberg et al. 2019). Yeasts can give higher yields of native glycosylated proteins (~ 50 kDa) at lower costs (Demain and Vaishnav 2009). Mammalian cell lines are most frequently used to assemble recombinant mammalian proteins. Genetically modified animals can also be exploited to produce therapeutic proteins, which they may secrete in their blood, urine, or milk (Jazayeri et al. 2018; Hunter et al. 2019).

Bacteria

E. coli is the earliest and most preferred microorganism employed for the expression of heterologous proteins, accounting for around 30% of the overall production of recombinant proteins in the past (Terpe 2006; Ferrer-Miralles et al. 2009). Due to improved genetic tools and a better understanding of its transcriptional and translational machinery, E. coli has been used as an expression host for large-scale production of proteins, particularly non-glycosylated proteins (Terpe 2006; Baeshen et al. 2015; Naz et al. 2022). The lac, tac, trc promoters of E. coli can be utilized to enhance protein productivity using simple and inexpensive media ingredients (Maksum et al. 2020). Solubilization of bacterial therapeutic proteins can be improved using denaturants and reducing agents to eliminate S–S bonds (Baeshen et al. 2015). Gram-positive Bacilli bacteria can also be employed for heterologous/homologous enzyme expression. Recombinant proteins are expressed in their native form using Bacillus sp. Furthermore, it has improved growth characteristics and a robust metabolism without producing exo- or endotoxins, resulting in the cost-effectiveness of a therapeutic peptide. Protein secretion into the medium eliminates the need for cell disruption and chemical processing and assists in downstream processing. Although the protein outputs from Bacillus are quite high, the production of proteases may degrade the recombinant protein (Pero and Sloma 1993). Neutral protease (metalloprotease) and Subtilisin (serine protease) account for approximately 96–98% of extracellular protease activity. So far, six to eight extracellular proteases have been identified. Murashima et al. (2002) (Murashima et al. 2002) had developed a strain lacking these eight extracellular proteases using genetic engineering techniques. Bacillus licheniformis, an exo-protease-deficient host strain, is utilized specifically for heterologous protein expression (Cai et al. 2017). Bacillus brevis is beneficial for manufacturing therapeutic proteins because it produces proteinase inhibitors (Sagiya et al. 1994; Udaka and Yamagata 2020). However, bacteria frequently cannot synthesize a recombinant human protein of the wild type due to its simple expression machinery. Bacterial cells are not developed with post-translational modifications (PTM), a feature of higher organisms, and glycosylation cannot be fully accomplished in bacterial cells. However, the low cost and simplicity of culturing bacteria are two advantages of bacterial cells over others, making them the preferred choice for lab and industrial-scale production (Kamionka 2011). But limitations of bacterial expression systems, such as lack of PTM and glycosylation, eukaryotic expression systems, such as yeast cells, fungal cells, and algal cells, were developed for therapeutic protein production (Fahad et al. 2015).

Yeasts

Yeasts are microscopic unicellular eukaryotic cells which play an important role being a good expression system for the production of therapeutic proteins because of their rapid growth rate, ease for genetic manipulation, less expensive growth medium requirements, availability of entire genome sequences of yeast and ability to provide PTMs (Nielsen 2013; Fletcher et al. 2016; Kim and Kim 2017; Vieira Gomes et al. 2018; Baghban et al. 2019; Huertas and Michán 2019). Pichia pastoris (non-conventional yeast) and Saccharomyces cerevisiae (traditional baker’s yeast) is the most used expression host systems (Love et al. 2018); however, Hansenula polymorpha and Yarrowia lipolytica have also been in use. They can readily accommodate genetic modifications, grow in simple media, and incorporate post-transformational modifications. Yeasts are effective at producing insulin, glargine, and other monoclonal antibodies (mAbs) (Mengdai et al. 2020). S. cerevisiae has several benefits over bacteria as a cloning host, including the ability to carry glycosylation and secrete heterologous proteins into the medium by incorporating signal sequences. However, glycosylation using S. cerevisiae is prohibited for mammalian proteins because O-linked oligosaccharides contain only mannose (Gellissen et al. 1992). This yeast may also induce immunological issues, as well as a decrease in activity and receptor binding at glycosylated N-linked regions. Urate oxidase, granulocyte–macrophage colony-stimulating factor (GM-CSF), insulin, glucagon, platelet-derived growth factor, hepatitis B surface antigen, and hirudin are all therapeutic compounds produced by S. cerevisiae (Gellissen et al. 1992). Recombinant yeast, mould, mammalian cells, or insects can glycosylate human chorionic gonadotropin (HCG) or erythropoietin (Saul and Sudbery 1985). Additional carbohydrates related to the oxygen moiety of threonine or serine are found in fungal therapeutic proteins (Nunberg et al. 1984). P. pastoris has been genetically modified and is used to produce antimicrobial peptides (AMP) (Shrivastava et al. 2023). This synthetic protein has been successfully isolated and proved useful against E. coli-based infections (Cao et al. 2018).

Fungi

Yeast has certain limitations as they tend to hyperglycosylated secreted proteins, reducing its half-life in vivo, and affecting its efficacy. Also, the expression levels and plasmid stability were found to be low, limiting their use, for example- in S. cerevisiae. Therefore, filamentous fungi with a low degree of hypermannoglycosylation can be used during glycosylation. High levels of post-translationally modified therapeutic proteins can be achieved using filamentous fungi such as Aspergillus niger (Ward et al. 2004). Trichoderma reesei is another important host for the synthesis of enzymes such as cellulases and hemicellulases with high yield and production efficacy. Engineered protease deletion strains have been used to synthesize several therapeutic proteins such as bovine chymosin, Ab Fab fragments, interferon α2b and IgG Abs (Belén et al. 2020). Protein expression platforms based on the glyceraldehyde-3-phosphate promoter have been developed in A. unguis. High levels of secreted protein were observed using heterologous signal peptides. This expression system was used for the production of Human interferon β (Madhavan et al. 2017). Matthews et al. (2017) also report the production of human lactoferrin, mucor rennin, and proteinases in A. oryzae (Matthews et al. 2017). Alkaline protease and hirudin can be effectively synthesized by Acremonium chrysogenum (Zhang and Lan 2018). High amounts of therapeutic proteins have been produced using Chrysosporium which makes very little protease (Legastelois et al. 2017).

The recombinant hepatitis B vaccine produced in the non-conventional yeast H. polymorpha has been commercialized (Seo et al. 2008). The H. polymorpha-produced hepatitis B virus antigen (HbsAg) was found to be assembled into yeast-derived lipid membranes. Previous studies have indicated that this lipoprotein particle structure is essential for the antigenicity of the HBsAg (Rutgers et al. 1988). The methanol induction condition, which is routinely used for high-level production of recombinant proteins in the methylotrophic yeast H. polymorpha, is favorable condition for lipid membrane formation and thus advantageous to produce the recombinant hepatitis B vaccine with more desirable antigenicity. Another methylotrophic yeast P. pastoris was recently approved as a host for biopharmaceutical production. The first recombinant biopharmaceutical, a Kallikrein inhibitor (Kalbitor), developed in P. pastoris was approved by the FDA in 2009, and several others are undergoing evaluation in clinical trials (Walsh 2010). Table 3 summarizes the examples of various microbes, such as bacteria, yeasts, and fungi, with therapeutic proteins and their clinical uses.

Table 3.

Summary of some microbial therapeutic proteins and their clinical uses

Microbes	Examples	Therapeutic Protein	Clinical Use	References
Bacteria	E. coli	Interleukin-1 receptor antagonist	Auto-immunity disorder	Kamionka (2011), Adan Gökbulut and Arslanoğlu (2013), Ahmad et al. (2014a), Wang et al. (2014)
		L-asparaginase	Lymphoma, mast cell tumor
		Insulin	Diabetes mellitus
		Streptokinase	Acute myocardial infarction
		Human growth factor	Hypopituitary dwarfism
		INF-α	Leukaemia, hepatitis-B, cancers
		INF-β	Sclerosis
		INF-γ	Chronic glaucomatous disease
		IL-2	Renal cell carcinoma
	Erwinia sp.,	L-asparaginase	Lymphoma, mast cell tumor	Pieters et al. (2011)
	Pseudomonas sp., P. putida	Methionine gamma- lyase, L-glutaminase	Infectious disease, cancer, Leukemia	Sato and Nozaki (2009)
	Bacillus sp.,	L-glutaminase	Leukemia	Potla Durthi et al. (2019, Gomaa (2022)
	Aeromonas veronii	L-glutaminase	Lymphocytic leukemia	Jesuraj et al. (2017)
	Bacteroides thetaiotaomicron	α-galactosidase	Immune response modification	Chaudet et al. (2012)
Yeast	Saccharomyces cerevisiae	Hepatitis B vaccines	H. influenzae type B and hepatitis B infection in infants	Martemyanov et al. (2001), Ahmed et al. (2013)
		Lepirudin	Heparin-induced thrombocytopenia type II	Kim et al. (2014)
		Desirudin	Venous thrombosis	Eriksson et al. (1997)
		Insulin	Diabetes mellitus	Adan Gökbulut and Arslanoğlu (2013)
		HPV vaccine	Cervical cancer caused by human papillomavirus (HPV)	Wang et al. (2020)
		Rasburicase	Hyperuricemia	Cammalleri and Malaguarnera (2007)
		PDGF	Lower extremity diabetic neuropathic ulcer	Kim et al. (2014)
		HGH	Dwarfism, pituitary turner syndrome	Kim et al. (2014)
		GM-CSF	Cancer, bone marrow transplant	Kim et al. (2014)
		Glucagon	Hypoglycaemia	Kim et al. (2014)
		GLP-1	Type-2 diabetes	Kim et al. (2014)
		Insulin aspart and Insulin detemir	Diabetes mellitus	Furman (2017)
	Pichia pastoris	Ecallantide	Hereditary angioedema	Craig et al. (2015)
		Insulin	Type 2 diabetes	Polez et al. (2016)
		Human serum albumin	Blood volume expansion	Zhu et al. (2021)
		Hepatitis vaccine	Hepatitis B	Rahimi et al. (2019)
		INF-α 2B	Hepatitis C and cancer	Katla et al. (2019)
		Ocriplasmin	Vitreomacular adhesion (VMA)	Baghban et al. (2020)
		Anti-IL- 6R Ab	Rheumatoid arthritis	Van Roy et al. (2015)
		Anti-RSV Ab	Respiratory syncytial virus (RSV) infection	Kim et al. (2014)
		HB-EGF	Treatment of interstitial cystitis/bladder pain syndrome (IC/BPS)	Kim et al. (2014)
		Collagen	Medical research reagent/ dermal filler	Xiang et al. (2022)
	Hansenula polymorpha	HBV vaccine	Hepatitis B	Caetano et al. (2017)
	Yarrowia lipolytica	Pancrelipase	Exocrine pancreatic insufficiency	Turki et al. (2010)
Fungi	Lentinula edodes	α-galactosidase	Treatment of fabry disease, blood group conversion and removal of α- type immunogenic epitopes in xenotransplantation	Xu et al. (2019), Bhatia et al. (2020)
	Schizophyllum commune	Hydrophobin,	Potential for modification of hydrophobic nanomaterials and solubilization of lipophilic drugs	Bayry et al. (2012)
	Trametes sp.	Laccase	Natamycin functionalization	Tong et al. (2007), Ji et al. (2022)
	Taxomyces andrenae, F. oxysporum and A. niger	Paclitaxel (Taxol)	Cancer	Jozala et al. (2016)
	A. foetidus	β- galactosidase	Lactose intolerance, GM1-gangliosidosis	Weesner et al. (2022)
	Monascus ruber, A.terreus	Lovastatin (statin)	High blood cholesterol and reduce the risk of cardiovascular disease	Aravindan et al. (2008)
	M. hiemalis, A. niger, A. flavus,A. nidulans, A. terreus	L-asparaginase	Acute lymphoblastic leukemia and non-Hodgkin lymphoma	El-Gendy et al. (2021)
	Claviceps purpurea	Ergot alkaloids	Parkinson’s disease, cluster headaches and migraine	Meade et al. (2022)
	Hericium erinaceus	Hericenones and erinacines (cyathane derivatives)	Alzheimer’s and Parkinson’s disease	Meade et al. (2022)
	Aspergillus and Penicillium species	Proteases/proteinases/peptidases	Cardiovascular disease, emerging agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis	Shankar et al. (2021)
	Penicillium nalgiovense	Amphotericin B (AMP B)	Antifungal, treatment of invasive fungal infections mucormycosis, aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, and cryptococcosis	Svahn et al. (2015)
	P. griseofulvum	Griseofulvin	Antifungal, treatment of dermatophytoses	Aris et al. (2022)

Microalgal source

Microalgae represent a promising platform for developing recombinant proteins with therapeutic value due to their safety and capacity to produce extracellular vesicles (Banerjee and Ward 2022). Microalgae can be cultured in enclosed systems. Microalgae, like plants, have solid cell walls that protect them from severe gastrointestinal conditions. Microalgae have built-in benefits such as a fast growth rate, low and inexpensive input requirements such as light source, water source, carbon source, supplementary nutrients, and photoautotrophic nature. Transformation and production of recombinant therapeutic proteins in microalgae require minimal time (Kaye et al. 2015; Hempel et al. 2017). Microalgae can produce a variety of bioactive compounds, such as carotenoids, polysaccharides, vitamins, and lipids. In addition, they have a wide range of potential applications in the biomedical, pharmaceutical, cosmetics, environmental, and animal feeding industry (Rizwan et al. 2018; Fu et al. 2019). There are three transformable sites in microalgae, i.e., chloroplast, nucleus, mitochondria, and out of these, nucleus and chloroplast sites are nowadays attracting the attention of scientific community for the development of recombinant proteins (Dehghani et al. 2020). Transgene expression in the model microalga via chloroplasts Chlamydomonas reinhardtii can produce protein concentrations > 5% of total soluble protein higher than plant systems (Manuell et al. 2007; Specht and Mayfield 2014; Barbosa et al. 2023). Due to their safety and solitary chloroplast, which expresses proteins with great accumulation, microalgae (particularly C. reinhardtii) are appropriate options to be employed as vaccine carriers (Khavari et al. 2021). In a related study, Demurtas et al. (2013) engineered C. reinhardtii chloroplast to express the E7GGG protein needed for therapeutic vaccinations via inserting the HPV16 E7 protein gene into the (C. reinhardtii) chloroplast's genome (Demurtas et al. 2013). Nowadays, the majority of monoclonal antibodies produced by cell lines derived from Chinese hamster ovary are pretty expensive and carry a risk of pathogen contamination. Therefore, microalgae are regarded as effective alternative host cells because of their advantages (Khavari et al. 2021).

These microalgae can appropriately handle the post-translational changes of human recombinant proteins than bacterial cells. In a study, Hempel et al. (2017) developed a monoclonal IgG antibody against the Marburg virus's nucleoprotein, which is a significant cause of hemorrhagic fever in western Africa (Hempel et al. 2017). Microalgal antiviral substances have an impact on viral infections at various stages. For instance, the first step is hampered by sulfated polysaccharides (Fu et al. 2016). In a study on this subject, Hayashi et al. (2019) were able to extract a monogalactosyl diacylglycerol (MGDG) from Coccomyxa sp. that led to physical alterations in HPV that caused it to encapsulate. The virus was unable to connect to the host cell, showing that MGDG has antiviral properties (Hayashi et al. 2019). A sulfated polysaccharide called fucoidan, which is extracted from various microalgae including Fucus vesiculosus, Sargassum henslowianam, Cladosiphon fucoidan, and Coccophora longsdorfii, inhibits angiogenesis and metastasis by lowering down kinase activity and activating caspase-3/7 in human colon cancer, melanoma, breast cancer, etc. (Deniz et al. 2017). Miceli et al. (2019) reported selective apoptosis of colon cancer cell line (HCT-116) and haematological cancer cell line (U-937) using monoacylglycerides extracted from Skeletonema marinoi through the activation of caspase-3/7. Still, no apoptosis was induced in normal cells (Miceli et al. 2019). Astaxanthin from microalgae, Haematococcus pluvialis can reduce the cytokine storm, averting acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) in COVID-19 patients (Talukdar et al. 2020).

For microalgal production of recombinant therapeutic proteins, chloroplast transformation is a significantly utilized approach primarily for some of the inbuilt advantages such as the single promoter for expression of multiple genes, no gene silencing, no position-effect change, elevated stages of transgene expressions, etc. (Sproles et al. 2021). However, the major issue with the microalgal production of therapeutic proteins is low yield. Various genetics-based tools and approaches were made available to improve protein expression. Banerjee and Ward have summarized different ways and techniques for other microalgal species to enhance the yield of therapeutic proteins (Banerjee and Ward 2022). Agrobacterium-mediated transformation also proved to be a reliable method for transgenes to be transferred to the genome of many microalgal species. Using AMT process, active human interleukin-2 (hIL-2) was produced using C. reinhardtii, C. vulgaris, and D. Salina (Dehghani et al. 2020). A significant challenge for transgenic strain production of microalgae is randomized insertion of transgene leading towards heterogenicity. High-throughput techniques will be essential and needed to speed up the screening process and choose the finest clones. Microalgae are currently being grown in bioreactors for the expression of recombinant proteins. However, the consequences of the cultural conditions are appallingly understudied (Banerjee and Ward 2022). The scaling up of these cultures in bioreactors in a manner compliant with good manufacturing practices is one issue with the use of obligate phototrophic species of microalgae for the manufacture of recombinant proteins (Banerjee and Ward 2022). Table 4 summarizes some microalgal therapeutic proteins and their clinical applications.

Table 4.

Summary of some microalgal therapeutic proteins and their clinical uses

Sr. no	Host (microalgae)	Therapeutic proteins	Clinical use	References
1	P. tricornutum	CL4mAb with ER retention signal against hepatitis	Hepatitis B virus	Hempel et al. (2011), Vanier et al. (2015)
2	P. tricornutum	CL4mAb without ER retention signal against hepatitis	Hepatitis B virus	Hempel et al. (2011), Vanier et al. (2015)
3	P. tricornutum	IGA against Marburg virus	Marburg virus Disease	Hempel et al. (2017)
4	Chlorella sp.	Fish growth hormone	–	Kim et al. (2014)
5	Chlorella sp.	Trypsin-Modulating Oostatic factor	Mosquitoes decapeptide	Borovsky et al. (2016)
6	C. reinhardtii	Human erythropoietin	Anemia	Eichler-Stahlberg et al. (2009)
7	P. tricornutum	Hepatitis B surface Ag	Hepatitis B	Hempel et al. (2011)
8	Chlamydomonas sp.	HIV-1 P24 Ag	–	Barahimipour et al. (2016)
9	Chlorella sp.	IBDV VP2 protein	–	Reddy et al. (2017)
10	C. reinhardtii	IGA against herpes simplex virus	Herpes simplex virus	Mayfield et al. (2003)
11	C. reinhardtii	IgG1 against Anthrax	Anthrax	Tran et al. (2009)
12	C. reinhardtii	Immunotoxin against Lymphoma	Lymphoma	Tran et al. (2013)
13	C. reinhardtii	Human growth hormone	Turner syndrome	Wannathong et al. (2016)
14	C. reinhardtii	Angiotensin II fused to Hepatitis B virus capsid antigen (HbcAg)	Hepatitis B	Soria-Guerra et al. (2014)
15	C. reinhardtii, C. vulgaris, and D. salina	Human interleukin 2	Cancer	Dehghani et al. (2020)
16	P. tricornutum	RBD (Receptor binding domain) of SARS-CoV-2	COVID-19	Slattery et al. (2022)
17	C. reinhardtii	Human interferon alpha 2a	Cancer	Commault et al. (2020)
18	C. reinhardtii	RBD SARS-CoV-2	COVID-19	Berndt et al. (2021)
19	C. reinhardtii	Spike glycoprotein of SARS-CoV-2	COVID-19	Kiefer et al. (2022)
20	Schizochytrium sp.	BCB (multi-epitope protein)	Breast cancer	Hernández-Ramírez et al. (2020)
21	C. reinhardtii	Human VEGF-165 (human vascular endothelial growth factor)	Wound healing	Jarquín-Cordero et al. (2020)

Expression hosts for the production of therapeutic proteins

Recombinant therapeutic proteins represent a major and significant percentage of approved drugs. However, the effectiveness of these drugs may be compromised due to their short in vivo half-lives triggered by proteolysis and abrupt disposal. To get the desired therapeutic effect, large doses must be administered regularly, which entails a higher risk of toxicity, considerable patient discomfort and suffering, and a high cost (Du et al. 2019). PEGylation (Bailon and Won 2009) and the insertion of fusion tags such as Fh8, SUMO, and immunoglobulin (Ig) G Fc fragments (Levin et al. 2015) have been developed and used in the past to boost therapeutic protein in vivo activity and half-life (Kontermann 2011; Du et al. 2019). PEGylation of therapeutic proteins is a prevalent approach in the pharmaceutical business. However, studies suggest that the immunogenicity of PEG and the formation of anti-PEG antibodies, as well as the buildup of unused PEG in body tissues, are some of the harmful and devastating consequences (Verhoef et al. 2014; Chen et al. 2021). Therefore, there is a requirement of other protein modification techniques. PTM glycosylation (either N- or O-linked glycan) is naturally present in eukaryotic cells. These glycans improve protein stability, solubility, half-life, and biological functioning, among other things (Varki 2017). However, glycoengineering-based modifications and investments are required to improve therapeutic protein efficacy (Solá and Griebenow 2010). However, different expression methods can produce glycoproteins with a wide range of glycoform compositions. Some of the expression systems, viz., bacteria, yeast, insects, mammals, transgenic animals, and transgenic plants, are briefly discussed for therapeutic protein production.

Bacterial expression systems

Bacterial expression systems are one of the most critical industrial expression hosts due to their advantages of quick growth, well-characterized genetics, a well-established safety background, and high protein production. However, the restricted environment of bacterial cells, as well as the lack of PTM, are issues associated with bacterial expression hosts (Du et al. 2019). Nevertheless, cellular engineering approaches have enabled the expression of human proteins in bacterial cells. For example, the commercial strain of E. coli Shuffle from New England Biolabs offers an oxidizing cytoplasmic environment and promotes proper protein folding by increasing disulfide bond formation (Lobstein et al. 2012). Co-expression of E. coli with Saccharomyces cerevisiae's chaperone sulfhydryl oxidase and human protein disulfide isomerase (hPDI), as reported by (Nguyen et al. 2011), also improves protein folding. Attempts have been made to overcome the fact that E. coli does not natively manufacture N- or O-glycosylated proteins with mammalian-style glycans by demonstrating the feasibility of N- and O-linked glycosylation in E. coli (Pandhal and Wright 2010; Merritt et al. 2013). For instance, the expression of several glycosyltransferases in E. coli allowed for the synthesis and transfer of a tri-mannosyl chitobiose N-glycan to the designed target eukaryotic protein (Valderrama-Rincon et al. 2012).

Yeast expression systems

Yeast, as a unicellular eukaryotic microbial host cell, provides unique advantages for the production of biopharmaceutical proteins. The use of yeasts enables an ideal combination of steady development of the primary media in large-scale bioreactors, post-translational modifications, and ease of genetic manipulation (Mattanovich et al. 2012). Since the initial industrial manufacture of recombinant human insulin in S. cerevisiae in 1987, several pharmaceutical medicines have been marketed, many of which are highly successful (Walsh 2010). Non-saccharomyces yeast species are effective alternatives to S. cerevisiae as hosts for biotechnological applications owing to their superior secretion capability and faster growth on inexpensive carbon sources. Non-conventional yeasts have used conventional methods for developing toolboxes for genetically engineering host strains to increase protein production capacity, boost product quality, and simplify downstream processing (Mattanovich et al. 2012; Krainer et al. 2012; Saraya et al. 2014).

S. cerevisiae expression systems yield about 15% of protein-based biopharmaceuticals for human application (Wang et al. 2017). S. cerevisiae is superior to bacteria, other yeasts, and filamentous fungi in key physiological features related to the production of commercial ethanol. This physiological flexibility includes resistance to high osmotic pressure, high ethanol, and sugar concentrations, and a broad pH tolerance (Tesfaw and Assefa 2014; Baghban et al. 2019). Among eukaryotic hosts, S. cerevisiae is the most popular for investigating gene expression regulation, secretory pathways, and other associated issues (Baghban et al. 2019). Whenever heterologous glycoproteins are synthesized in S. cerevisiae, the resulting glycan structure is often hyper-mannosylated, which can reduce activity and increase immunogenicity. The synthesis of recombinant cellulases is improved and boosted when the Mnn2p and Mnn11p genes in the glycosylation modification pathway are disrupted, as revealed by Tang et al. (2016) (Tang et al. 2016). S. cerevisiae is a yeast cell that is utilized in the production of hepatitis B and human papillomavirus vaccines, both of which produce a protective immune response against wild-type viruses (Bill 2015). In S. cerevisiae, expressed proteins are frequently N and O-hyperglycosylated, which may impact protein immunogenicity (Rasala and Mayfield 2015). To address the issue of protein expression, methylotrophic yeasts such as Hansenulla polymorpha and Pichia pastoris (P. pastoris; syn. Komagataella phaffii) have been produced in recent years. P. pastoris has been the most popular because of its low cost and expression host system. This microorganism can create large amounts of recombinant proteins with glycosylation, which is similar to that of mammalian cells (Balamurugan et al. 2007). Overall, the advantages of protein production by the P. pastoris system include proper folding (in the endoplasmic reticulum, ER) and secretion of recombinant proteins to the cell's external environment (via Kex2 as signal peptidase) (Yang et al. 2013). Because some proteins produced by their original host are secreted out of the cell, P. pastoris is excellent for the creation of recombinant proteins because it has a secretion system (Ahmad et al. 2014b).

Insect cells as expression host

Insect cells have the benefit of executing PTMs that are more comparable to those in humans and are able to produce recombinant proteins with reasonable cost and yield (Dana 2018). Insect cells can be infected with baculoviruses, which are double-stranded circular DNA viruses with arthropods as host. Baculovirus-mediated gene expression in insects is a method of choice and is cost-effective, giving a much higher yield of recombinant protein compared to other systems. It is possible to produce large proteins, resulting in the production of correctly processed and biologically active proteins. Baculovirus-based vectors used in the insect cell expression system are primarily able to integrate the transgene into the insect cells' genomes (Osz-Papai et al. 2015; Kost and Kemp 2016). However, there are some problems associated with insect hosts, such as the creation of baculovirus vectors, which could take a long time and be difficult to create (Osz-Papai et al. 2015; Dana 2018). Humans do not possess the paucimannosidic glycans that are added by insect cells (Burnett and Burnett 2020). Insect cells also have limitations in performing post-translational modifications as it performs non-syalated N-linked glycosylation. All the other optimizations need to be perfect as yield depends upon the virus titer and the time taken from infection to expression. Insect cells are preferred when active protein is difficult to obtain in E. coli system. Glycosylation is another problem which is encountered when insect cells are used for the production of recombinant human glycosylated proteins (Gupta and Shukla 2016). Extensive genetic engineering is required to produce human-like glycosylation in insect cells (Clausen et al. 2022).

Mammalian cell expression system

Among all approved recombinant protein-based biopharmaceuticals, mammalian cells dominate the other recombinant protein-expression systems (Owczarek et al. 2019). Mammalian cells can express large and complex recombinant proteins. Mammalian cells have the advantage of performing PTMs correctly, and they secrete recombinant protein in their natural form into the medium (Gupta and Shukla 2016; Dana 2018). Although the glycosylation patterns of Chinese hamster ovary cells are generally regarded as safe (i.e., they don't include the hyper-immunogenic α-gal epitope), they do have limitations. For instance, they are inadequate production hosts for potent anti-inflammatory antibodies because they lack 2,6-sialyltransferase activity. The fact that CHO cells produce the Neu5Gc form of sialic acid despite being relatively moderately immunogenic raises concern (Ghaderi et al. 2010, 2012).

The majority (~ 84%) of the approved biopharmaceutical products on the market today are produced in CHO cell-based systems (57 products), with the remaining antibodies being expressed in NS0 cells (9 products) or Sp2/0 cells (2 products) (Walsh 2018). In mammalian cell lines, PTM is present, although their glycosylation pattern differs from the human type. Protein expression is also being investigated in the human cell lines- HEK293, HKB11, PER.C6, HeLa, and CAP (Bandaranayake and Almo 2014; Dyson 2016; Dumont et al. 2016; Hu et al. 2018; Gupta et al. 2019; Hunter et al. 2019). The expression of proteins modified in a way similar to humans is stimulated in a human cell line. HeLa cells were used to generate a recombinant protein effectively encoding the fully glycosylated form of connective tissue growth factor CCN2 (Nishida et al. 2017). However, large-scale commercial production of these cells is still in the research and development stages. Contamination with animal viruses is another issue with this method. Culture medium formulation for a cell line is challenging due to the need for several components, such as growth hormones, amino acids, reducing agents, and vitamins. There are several subtypes of CHO cells, including CHO-K1, CHO-S, CHO-DG44, and CHO-DXB11.

Reinhart et al. (2019) examined CHO-K1, CHO-S, and CHO-DG44 host cell-specific differences in mAb expression in batch, fed-batch, and semi-continuous perfusion cultures, revealing CHO cell line-specific preferences for mAb production. The host cell line and media also affected the quality attributes of mAb. It has also been demonstrated that cell engineering prevents the accumulation of ammonium and lactate and enhances cell proliferation (Kim and Lee 2007a). The cell line is optimized through codon optimization and other methods (Zhu 2012). In addition, glycoengineering is utilized to produce the desired glycoform of a protein to enhance its efficacy and create a high-quality product (Lalonde and Durocher 2017; Wang et al. 2017; Heffner et al. 2018). For mammalian expression systems, gene-editing technologies such as CRISPR/Cas9 have been effectively implemented to improve product quality. Li et al. (2019) conducted a study in which C1s protease was inactivated using CRISPR/Cas9 for the production of recombinant HIV envelope protein gp120 in CHO cells. Yang et al. (2019) identified CRISPR/Cas9-mediated site-specific integration as an efficient and dependable method for establishing stable recombinant HEK293 cell lines for biopharmaceutical production. Gene modification with CRISPR/Cas9 allowed HEK293 cells to bioprocess media without antibiotics (Román et al. 2019). CRISPR/Cas9 was used to generate Anxa2- and Ctsd-knockout CHO cell lines, which led to the elimination of the corresponding host cell protein (HCP) in cell lysates without compromising cell growth or viability, paving the way for the successful creation of recombinant proteins (Fukuda et al. 2019). CRISPR/Cas9-mediated deletion of microRNA-744 enhanced antibody titer in CHO production cell lines (Raab et al. 2019). Among gene-editing tools, CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE) technology will contribute most to the advancement of glycoprotein production in the near future.

Transgenic organisms as expression host

Transgenic animals

Transgenic animals have been used to express recombinant protein-based medicines, such as monoclonal antibodies (mAbs), vaccinations, hormones, enzymes, and growth factors. Animals with a transgene incorporated into their DNA that codes for a recombinant protein can pass that protein on to their progeny. Milk from transgenic mammals and eggs from transgenic chickens are two examples of where proteins are sourced nowadays (Moura et al. 2011; Maksimenko et al. 2013; Owczarek et al. 2019). The natural secretion of recombinant proteins provides correct post-translational modifications (PTMs) in this method. However, creating transgenic animals raises serious moral concerns. Protein preparations from transgenic animals may contain zoonotic diseases (Wang et al. 2013; Bertolini et al. 2016). Transgenic cattle have the potential to be used as bioreactors for the industrial production of protein in milk. However, there are currently inefficiencies in the methods used to achieve this goal (Monzani et al. 2016). To create animals that generate recombinant proteins in milk, (Shepelev et al. 2018) investigated targeted genome-editing tools and other methods for developing transgenic animals.

Transgenic plants

Recombinant biopharmaceutical production can be improved with the use of transgenic plants. Low cost, safety (low risk of contamination with animal pathogens), easy scale-up, stability, presence of metabolites, and ability to produce N-glycosylated proteins are some of the benefits of this system (Fahad et al. 2015; Yao et al. 2015; Xu et al. 2016; Lomonossoff and D’Aoust 2016; Park and Wi 2016; Buyel et al. 2017). In recent years, cancer immunotherapy drugs derived from plants have been developed (Chen et al. 2016; Hefferon 2017). The production and quality of plant-produced biopharmaceuticals can be improved by paying attention to a few key parameters, such as host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification strategies. Glycoengineering has been studied extensively to lessen the immunogenicity of plant-made recombinant proteins (Moustafa et al. 2016). Contamination of the final product by harmful plant metabolites and pesticides is a significant drawback of this approach. Controlling the level of transgene expression and the complexity of the purification process are additional challenges associated with this system. Most recombinant proteins are produced in plant systems, including cell culture, tissue-based systems, and transgenic plant building. Bacterial infection (agro-infection) or viral infection, as well as direct techniques like biolistic bombardment and the PEG-mediated procedure, are commonly used to introduce the transgene into plant cells. The ability to express the recombinant protein in the desired cell compartment or plant organ is a significant benefit of these expression systems. The glycosylation pattern of human therapeutic proteins generated in plants is generally more similar to plants than to humans. This problem is being tackled with the help of glycoengineering (Fischer et al. 2018; Owczarek et al. 2019). Both traditional and cutting-edge methods for improving the production of recombinant proteins, such as the use of gene-editing tools coupled with the insertion of target genes in euchromatin genome areas, can be used (Rozov and Deineko 2019).

Edible vaccines have been derived from transgenic plants such as rice, bananas, peas, potatoes, lettuce, and corn. Transgenic rice cell culture has used genetic engineering to produce proteins at levels as high as 247 mg/l (α1-antitrypsin) and 100 mg/l (antibodies) (Loh et al. 2017; Owczarek et al. 2019). Human recombinant β-glucocerebrosidase (taliglucerase α-approved by FDA in 2012) enzyme was produced on a large scale in carrot (Daucus carota) cell culture (ProCellEx™) for the treatment of Gaucher disease (Tekoah et al. 2015; Moustafa et al. 2016). The world's first plant-derived IgA mAb that detects the surface antigen I/II of the bacteria Streptococcus mutans has been licensed in Europe and is used to prevent tooth decay (Larrick et al. 2001; Loh et al. 2017). Biopharmaceuticals derived from plants are in various phases of clinical trials or implementation on the market (Yao et al. 2015; Park and Wi 2016; Dirisala et al. 2017; Owczarek et al. 2019). Examples include HAI-05 [Influenza Vaccine- for Influenza A virus H5N1, tobacco (Nicotiana tabacum) as the host plant, phase II], Insulin (SBS-1000) for diabetes, safflower (Carthamus tinctorius) as the host plant, phase III], ZMApp (monoclonal antibody cocktail) for Ebola virus, tobacco (N. benthamiana) as the host plant, phase II, human growth hormone for deficiency treatments, host plant-barley seed (Hordeum vulgare), status, commercialization] (Owczarek et al. 2019). The development of a new plant system based on carnivorous plants demonstrated the ability of biomimetic approaches to contribute to the production of novel recombinant proteins. Nevertheless, the modest protein yields disqualified these plants from an industrial platform (Miguel et al. 2019). The establishment of a new plant system based on carnivorous plants demonstrated the ability of biomimetic approaches to contribute to the production of novel recombinant proteins. Nevertheless, the modest protein yields disqualified these plants for an industrial platform (Miguel et al. 2019). Donini and Marusic (2019) (Donini and Marusic 2019) described recent developments in the production of mAbs using plant-based systems such as transgenic plants, tissue and cell cultures, and transient expression systems. Holtz et al. (2015) described a commercial biotherapeutics manufacturing facility for plant-based pharmaceuticals. Various methods for the plant-based production of recombinant proteins and recent advances in the development of plant-based therapeutics and biologics for the prevention and treatment of human diseases have also been described (Loh et al. 2017; Rozov et al. 2018; Schillberg et al. 2019; Tripathi and Shrivastava 2019).

Production of therapeutic proteins and process optimization

We are all aware of the global health crisis caused by infectious illnesses. Therefore, there is an urgent need for the mass production of therapeutic proteins to combat these epidemics. Recombinant DNA technology has allowed the production of therapeutic proteins in heterologous expression systems (mammalian cells, bacteria, yeast, transgenic plants, and animals at laboratory and large scale) that can be used as therapeutics, vaccines, and diagnostic reagents. There are several advantages of using these recombinant proteins, such as low immunological rejection, low risk of infection, and reduced exposure to animal or human diseases (Leader et al. 2008). For the production of these recombinant proteins, some recent advances have also been made in the bioprocessing sector, such as the use of high-throughput devices (microtiter plates, bubble column or microplate-based mini-bioreactors, stirred mini-tank bioreactors), continuous upstream processing, quality by design, continuous chromatography to achieve a good quality product with higher yield (Tripathi and Shrivastava 2019). The strategies for microbial production of proteins are of great importance in developing application-based research and explaining the novel purification strategies used for industrial production. These strategies or processes play a vital role in effective therapeutic protein development at the industrial level. Some of the strategies adopted for the optimum production of therapeutic proteins have been discussed below:

Bioreactor conditions

Optimization of therapeutic proteins is primarily based on fed-batch cultures after initial development via a batch process, which provides enormous insight into the decoupling growth and production phase, improved cellular activity control, and balanced media uptake (Vandermies and Fickers 2019). Factors such as starvation, overfeeding, and high concentration and accumulation of media and nutrients for cells must be considered throughout production to maintain the constant growth of products. Trassaert et al. (2017) observed that reactor-scale continuous processes can be utilized as exploratory chemostats to determine the optimal promoter induction settings. At the industrial level, however, issues with continuous processes, such as contamination and product instability, must be addressed. By transitioning from shake-flask to bioreactor cultures, the oxygenation conditions and medium uniformity are enhanced (Gasmi et al. 2012). This transition from shake-flask to bioreactor increases productivity by 3 to 5 folds, and even more for complex non-enzymatic therapeutic proteins, such as a 416-fold increase in human interferon concentration after scaling up the culture from 250 ml to 5L batch reactor (Gasmi et al. 2012).

Complex media offers increased biomass and protein production yields. Still, non-defined composition and batch-to-batch variability affect production, which is why defined media are utilized for controlled processes such as the production of therapeutic proteins (Gasmi et al. 2012; Vandermies and Fickers 2019). The availability of oxygen is another crucial factor affecting production processes. The concentration of dissolved oxygen (DO) in culture media influences cell physiology. It provides information about the growth phase (exponential or stationary) and the health and condition of the cell population. While oxygen needs to diminish at the end of culture owing to decreased metabolic activity, it increases during the exponential growth phase, paralleling the expansion of substrate metabolism by a growing number of cells. In addition, abnormally elevated DO levels indicate cell starvation in media (Vandermies and Fickers 2019). Fixed agitation and aeration levels are the most uncomplicated operational designs for processes that do not require a large amount of oxygen (Darvishi et al. 2017). Manually or automatically, air flow and, more frequently, agitation are regulated for optimal oxygen transfer to the culture fluid (as measured by the oxygen transfer rate, OTR). Inadequate pH levels may affect the production of therapeutic proteins and be detrimental to the activity and stability of the proteins (Celińska et al. 2015, 2016). Although biomass load in the bioreactor receives less attention, this parameter influences the production process by affecting the growth kinetics of microorganisms. In fed-batch systems, adequate nutrient uptake results in an increase in cell density (Celińska et al. 2017; YaPing et al. 2017).

Cell line development and clone selection

During cell-line development, several factors were taken into consideration, such as the selection of host cells, expression vectors, transfection, and selection methods. High throughput devices such as ClonePix (Thermo) and FACS (BD and Beckman) are now being used for cell line development and screening. The selection of the expression system is based on its ability to ensure and meet high productivity along with quality criteria. Among the various expression systems in industries, E. coli (bacteria), S. cerevisiae, and P. pastoris are mainly used to produce therapeutic proteins and are in commercial use worldwide. The most preferable and widely used host for the production of therapeutic proteins is E. coli due to its low cost, rapid growth, good productivity, and well-known biochemistry (Baeshen et al. 2014; Gupta and Shukla 2017a). But there are some limitations also, such as lack of proper post-translational modifications (PTMs), formation of inclusion bodies (IB), production of endotoxins, etc. These limitations can be avoided by adding fusion tags such as Fh8, SUMO, TRX, His at N- or C- terminal, co-factors, co-expression of the protein with molecular/chemical chaperons, which enhances the solubility of proteins and help in affinity purification (Paraskevopoulou and Falcone 2018).

Further, lowering the temperature and glycoengineering E. coli cells also leads to better protein solubility and increases protein folding. However, some PTMs have been done in E. coli to produce therapeutic proteins. The addition of diverse carbon and nitrogen sources along with acetate metabolism knockout strains can redirect E. coli carbon-fluxes to different pathways and result in a fivefold increase in protein production (Chung et al. 2017; Huang et al. 2019; Klein et al. 2019; Lozano Terol et al. 2019).

Another expression system, i.e., S. cerevisiae can be rapidly grown in protein-free media, presence of PTMs, ability to secrete extracellular product. The gene encoding mannosyl transferase has been knocked out and ALG3/ALG11 double knockout was generated to prevent hypermannosylation of expressed protein (Parsaie Nasab et al. 2013). Then, the GlycoSwitch platform was developed in which hypermannosylation gene of yeast is removed and used to produce glycosylated proteins. However, the lower yield limits its commercial use. Pichia pastoris (Komagataella phaffi or K. pastoris) is methylotrophic yeast which as the ability to produce properly folded and functional proteins. They also provide reduced protein glycosylation and achieve high cell densities (Looser et al. 2015; Yang and Zhang 2018; Juturu and Wu 2018; Werten et al. 2019). However, proteolytic degradation or truncation of the product leading to low yield and loss of biological activity are the major problems associated with P. pastoris, which can be reduced using yeast peptone, casamino acids, protease inhibitors, lowering of pH and temperature is done. Nowadays, mammalian cell line such as CHO (Chinese hamster ovary cells) cell line dominate the other protein expression systems as they can express large and complex recombinant proteins. CHO cells having different lineages- CHO-K1, CHO-S, CHO-DG44 and CHO-DXB11 are mostly used as a host to produce recombinant therapeutic proteins including monoclonal antibodies (mAbs) and fusion proteins (Kelley 2009; Rita Costa et al. 2010; De Jesus and Wurm 2011; Fan et al. 2013; Dumont et al. 2016; Gupta and Shukla 2016; Zhu et al. 2017; Sharker and Rahman 2021). About 70–80% of all recombinant therapeutic proteins have been produced in CHO cells (De Jesus and Wurm 2011; Gupta and Shukla 2016).

Other factors such as cell growth pattern, stable production, cultivation in serum-free medium as a suspension culture, scalability in bioreactor, attributes of quality of product are also considered. The glycosylation pattern in mammalian cell lines is different from human-type glycosylation. The expression of proteins with human-like PTMs is enhanced by a human-cell line. Cell engineering helps to avoid accumulation of ammonium lactate and improves cell growth (Kim and Lee 2007b) and the cell line is optimized by codon optimization. A new modular cloning system has been developed for flexible de novo plasmid assembly, MoCloFlex which can be used to plan, build, and isolate a custom plasmid within 24 h, leading to low cost and less time consumption (Klein et al. 2019). CRISPR/Cas9 has also been used for the chromosomal integration of large DNA into E. coli successfully and able to integrate functional genes in diverse E. coli strains (Chung et al. 2017). There are some recent developments done in the field of metabolic engineering which includes the use of knock-in (KI) and knock-out (KO) gene-editing tools for successful clone and product development. Using CRISPR/Cas9, C1 protease was inactivated to produce recombinant HIV envelope protein group 120 in CHO cells (Li et al. 2020). CRISPR/Cas9 assisted native end-joining and editing offered a simple strategy for efficient genetic engineering in E. coli (Huang et al. 2019; Amiri et al. 2023). It is now possible to integrate large DNA in E. coli using CRISPR/Cas9 and can also be used for yeast engineering for site specific gene integration or knock-out of certain unwanted genes in an improved protein-production (Utomo et al. 2021; Ingle et al. 2023).

Manufacturing process development

Analysis of the medium spent in the fermentation of microbial system provides an insight into consumption and accumulation of constituents present in the culture supernatant. The nutrients such as carbon sources used in E. coli and yeast culture, can affect cell metabolism, protein production and quality significantly (Seyis and Aksoz 2005). The clone, medium and feed screening plays a crucial role at early stage for high productivity. With the advent of high-throughput cultivation systems such as miniature shaken vessel/ wells/ microtiter plates (MTPs), bubble column or microplate-based mini-bioreactors, and stirred mini-tank bioreactors, it has now become very easy to perform screening assays including optimization of processes which saves time and cost before proceeding to scale up the production of recombinant proteins (Fisher et al. 2019; Tripathi and Shrivastava 2019).

The development process starts with the identification of protein expressing cells, which are first used for small scale and bioreactor culturing to analyze and check the cell growth and protein production levels. Optimization of batch and fed-batch production processes have also been established and improved to a large extent (Schmuck et al. 2021). BioLector (microbioreactor) is being used to perform high-throughput fermentation altogether with system (online) monitoring of the process parameters such as biomass, pH, dissolved oxygen (DO), florescence, etc. It works with non-invasive optical sensors and controls the shaking speed, temperature as well as humidity (Fink et al. 2021). In a study, 32 production clones were characterized in BioLector with production yield of 0–7.4 mg Fab/g of cell dry mass (Fink et al. 2021). In a different study, after comparing 51 cultivations, it was reported that the fully automated HTP cultivation speeds up the identification of the optimal expression systems and process conditions which leads to automated early-stage bioprocess development (Sawatzki et al. 2018) (Fig. 1).

Fig. 1.

Summary of the process involved in production of therapeutic proteins. The process comprised of cloning and expression, selection of hybrids, single cell cloning, optimization and growth evaluation. Further depending on the nature of protein, supernatant or cell pellets can be collected for extraction process. After purification, new formulations can be developed and packaged

Quality by design (QbD) or design of experiments approach (DOE)

This approach has also been studied and applied for upstream processes in which quality of product is integrated into the manufacturing process. According to one study, QbD resulted in a fivefold increase in target protein titer when compared to the basal medium and demonstrated its efficacy. In this process, a large amount of data was provided in a short span of time (Kumar et al. 2019a). For the process development and analytical evaluation of recombinant proteins, including mAbs, the QbD approach has been used (Pathak et al. 2014; Yu et al. 2014; Narayanan et al. 2019).

To reduce the time and money spent on experiments during upstream process development, the Design of Experiments (DoE) method has been applied to different recombinant protein production process parameters (Papaneophytou and Kontopidis 2012; Hanke and Ottens 2014; Shekhawat et al. 2019). Various design-of-experiments (DoE) methods, including full factorial design, Taguchi orthogonal arrays, fractional factorial design, and the response surface methodology, are used to optimize media to maintain a balance between the different components of the media, thereby increasing protein yield (Shekhawat et al. 2019; Kumar et al. 2019b). In one study, to produce recombinant antibody fragments in the periplasm of E. coli, the signal peptide was chosen using DoE, and the best growing conditions were developed (Kasli et al. 2019). In another study, a specified medium to increase the synthesis of human interferon gamma was developed using RSM (Unni et al. 2019).

Process analytical technology (PAT)

This process is used to ensure that the final product meets the desired specifications by designing, analyzing, and manufacturing control via periodic and/or continuous measurement and check on the critical quality and performance attributes (Shaikh et al. 2018). The reactor design, cell harvesting, process control and analytics are also a part of optimization process and are optimized individually and the main goal is to attain high productivity with defined quality (Gronemeyer et al. 2014; Reyes et al. 2022). To implement PAT for the advancement of continuous cultures, innovation in sensor technology is required, as well as improvements in its configuration and resilience (Fisher et al. 2019).

Downstream processing

Downstream process mainly focusses on purity parameter, i.e., process and product impurity removal as well as yield and productivity (Owczarek et al. 2019). We can use a wide range of techniques, approaches, and technologies for an efficient and cost-effective downstream process, such as building a platform process, QbD and DOE-based high-throughput methods, a single-use system, integrating modelling, and replicating mini-plant/pilot plant facilities. Conventionally, a mAb or any other therapeutic protein is purified using chromatographic techniques (Zydney 2016). The downstream process involves 2 major steps: Filtration and Chromatography. Filtration includes harvest clarification for biomass removal, virus filtration to remove the viruses and tangential flow filtration for protein concentration and polishing step. Primary cell clarification involves centrifugation, TFF-MF (Tangential flow microfiltration), and depth filtrations. TFF-MF is highly efficient process and helps in the removal of whole cell mass and its fragments based on size-exclusion and utilizes microfiltration membrane with pore size of up to 0.65 µm. Single use centrifugation device was developed by KSep, which was fully automated and can recover over 97% of product/ cell biomass, which can be used for harvest clarification of recombinant proteins and vaccines (Gupta and Shukla 2017b). After the harvest clarification, a chromatographic resin is used to capture the expressed protein from upstream bioreactor. There are various chromatographic techniques such as affinity, ion exchange, hydrophobic interactions and size inclusion or gel filtration which are used to achieve a biologically active highly purified product (Wilken and Nikolov 2012).

Affinity chromatography can be used for the purification of tagged proteins, bispecific Abs, cellular products, DNA-based biologics, viral vectors, and viruses using Hexahistidine (His), maltose-binding protein (MBP), glutathione S-transferase (GST) tags (Zhao et al. 2019; Łącki and Riske 2020). Protein A chromatography can be used for mAb purification, but there is certain limitation associated such as leaching of Protein A with non-specific binding of host cell protein (HCP), elevated cost of resins and short lifetime, protein A ligand modification and alternative formats such as monolith membrane and microspheres (Ramos-de-la-Peña et al. 2019). Ion-exchange chromatography can be used as a cost-effective method for purification of recombinant proteins. Impurities such as media components, endotoxins, remaining HCP and DNA, product variants are removed using Cation and anion exchange chromatography (CEX and AEX). The efficacy of a weak anion exchanger was reported on the isolation of rHBsAg VLPs (Virus-like particles) from aggregated structures and found to yield 94–97.5% content of rHBsAg VLPS within the acceptable quality level (Kimia et al. 2019). From yeast crude extract, it resulted in high purity (> 95%). It was also reported that using CEX in overloaded mode, it was possible to remove viruses during the manufacture of therapeutic proteins (Masuda et al. 2019). Hydrophobic interaction (HIC) chromatography depending on hydrophobicity of molecules can be used for purification of recombinant proteins in both small and large-scale purification of hormones and industrial enzymes (Bhuvanesh et al. 2010; Saraswat et al. 2013). In a recent study, the influenza A and B viruses were successfully purified using HIC and gave efficacy of about 96% virus recoveries and about 1.3% residual DNA using 96-well plate method (Weigel et al. 2019). Insulin like growth factor receptor (Levin et al. 2016) from E. coli was purified using Size exclusion/Gel filtration chromatography. In a study, a yellow fever vaccine was prepared using bioreactors, AEX membrane adsorber, a multimodal resin and beta-propiolactone inactivation. The overall recovery rate was 52.7%(Pato et al. 2019).

Expenditure

With the development of single-use bioreactor devices that are well-automated, it has become possible to regulate the environment actively. Compared to traditional stainless-steel systems, it needs less upfront investment, has lower operating costs, and provides greater adaptability. These are offered from approx. 50L–25000L size (Jacquemart et al. 2016; Mf et al. 2021). A study on integrated continuous processing reported that about 30% cost savings can be achieved using disposable technologies w.r.t SS batch process, examples- wave, orbital shaken (OS), stirred tank and pneumatically mixed bioreactors (Schmidt 2022). There is low risk of contamination also with these systems. Single-use bioreactor (microcarrier-based) can be used in case of viral vectors and vaccines (Gallo-Ramírez et al. 2015; Ton et al. 2023).

Genetic engineering of therapeutic proteins

Proteins regulate and mediate the body's normal, everyday functions and metabolic processes. Disruption of the normal protein and the usual pathway of protein leads to significant disorders, and any error during protein production or processing (such as mutation or misfolding) leads to disruption of the normal proteins. However, Gupta et al. (2017) report that such diseases may be cured through the administration of mutant protein. Such proteins must be manufactured in factories to ensure an external supply. The production process and the synthesis of human origin protein in bacterial, fungal, insect, or mammalian cell hosts can be assisted by a variety of genetic engineering methods (Gupta and Shukla 2017a). Methods such as host cell engineering, expression construct optimization, and media generation have all been employed to increase the yield of treatments (Tihanyi and Nyitray 2020). In host cell engineering, gene expression is modified using prior knowledge of linked biological pathways. Several techniques have been developed to modify genes, such as gene overexpression, gene knock-in and knock-out, and the clustered regularly interspaced short palindromic repeats—CRISPER associated protein 9 (CRISPER-Cas9)-mediated knock-in approach (Dana 2018; Kim et al. 2020). Site-specific gene knock-out and on-site heterologous gene overexpression in C. reinhardtii using CRISPR-Cas9 was investigated in a study (Kim et al. 2020). Optimizing expression constructs often makes use of targeted genome integration approaches. Transposons systems have been proven to increase integration efficiency (Rajendra et al. 2017; Balasubramanian et al. 2018), and recombinant CHO cells with a high production ability of > 7 g/L were discovered after only 2–3 weeks of integration. Gene integration mediated by transposons is still in its infancy and needs extensive testing before it can be put to practical use (Tihanyi and Nyitray 2020). Ubiquitous chromatin-opening elements (UCOEs) have been identified as a potential tool for preventing gene silencing and maintaining high production levels (Neville et al. 2017). Site-independent, stable, and high-level expression of the transgenes is also possible with the help of Bacterial Artificial Chromosomes (BACs). Some genetic engineering technologies that can aid in the exogenous synthesis of therapeutic proteins include zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), and CRISPR-CAS9 (Kim and Kim 2014; Lee et al. 2015; Sakuma et al. 2015). Emerging methods for host cell genomics and targeted expression of genes include the detection of transcriptional hot zones (Gaidukov et al. 2018). The development of omics technologies facilitated the study of cellular and molecular features. Genomic, transcriptomic, proteomic, and other data sets shed new light on these and other domains. Multiplex secretome analysis was used to investigate the impact of host cell proteins on the generation of recombinant therapeutic proteins in a study by Kol et al. (2020) (Kol et al. 2020). Therapeutic protein and host expression research have benefited greatly from advances in genetic engineering and omics technology (Tihanyi and Nyitray 2020). All of these, however, need the use of complex and expensive high-tech tools, materials, and information technology.

Challenges associated with therapeutic protein production

Therapeutic proteins are a rapidly growing class of biologics that have revolutionized the treatment of various diseases, including cancer, autoimmune disorders, and infectious diseases. However, the production of these complex molecules presents numerous challenges that need to be addressed to ensure their safety, efficacy, and affordability. In this review, we will discuss some of the challenges associated with therapeutic protein production and possible solutions to overcome them. One of the greatest challenges in the use of therapeutic proteins was their fast degradation due to short half-lives. And because of their quick renal clearance and obvious enzymatic activity during systemic circulation, these therapeutic protein molecules have a short lifetime in blood. To compensate for this, a higher dose is usually required to maintain a desired concentration for a desired period in blood serum. So, a few technologies have been proposed and tested for the extension of half-life of therapeutic proteins such as amino acid manipulation, bio-conjugation/genetic fusion, post-translational modification by attaching the peptide to natural/synthetic polymers (PEGylation, polysialylation, HESylation) and carrier-mediated delivery (Tripathi and Shrivastava 2019). Another challenge of therapeutic protein production is the heterogeneity of the protein molecules. Unlike small molecules, which are chemically synthesized, therapeutic proteins are biologically produced in living cells, leading to variations in post-translational modifications, folding, and aggregation (Zhuang et al. 2023). This heterogeneity can affect the protein's stability, pharmacokinetics, and immunogenicity, leading to variability in efficacy and safety (Stebbings et al. 2009; Shih 2012; Jiskoot et al. 2012). To address this challenge, several approaches have been developed, including the use of advanced cell lines, optimized culture conditions, and novel purification techniques (Walsh 2018). Another significant challenge in therapeutic protein production is the high cost of production, which limits their accessibility to patients. The cost is driven by several factors, including the complexity of the manufacturing process, regulatory requirements, and intellectual property issues (Qarawi et al. 2019). To reduce the cost of production, various strategies have been explored, including process intensification, cell line engineering, and process optimization using advanced analytics and automation. Moreover, the production of therapeutic proteins also poses challenges related to scalability and manufacturing capacity (Chennamsetty et al. 2009; Hamid Akash et al. 2015). Many biologics are produced using mammalian cells, which require large-scale cell culture facilities and specialized equipment, making it challenging to produce enough proteins to meet the growing demand. Alternative expression systems, such as microbial cells and plant cells, have been explored to overcome this challenge (Buyel 2018).

Another challenge associated with protein therapeutics is immunogenicity. A modification in the protein's structure, which might happen as a result of post-translational changes such as product's administration, storage, or manufacturing process, sets off the immune response (Dingman and Balu-Iyer 2019). While a vaccine induced immune response is desired clinical response while immune response due to therapeutic proteins is undesirable. Today, a wide range of preclinical methods, including computational (HLA-binding algorithms) (Wang et al. 2008; De Groot et al. 2008; Chen et al. 2023), in vitro (Peptide/HLA-binding assay) (Justesen et al. 2009; Hamuro et al. 2017; Groell et al. 2018), ex vivo (LH/MS-based MHC-associated peptide proteomics, MHC-II tetramer-guided epitope mapping, protein-specific T-cell amplification, Human blood derived cell-based assay) (Bozzacco et al. 2011; Dudek et al. 2016) and animal models (HLA transgenic mice) (Jiskoot et al. 2016; Sauna et al. 2018), are employed to evaluate the immunogenicity risk of therapeutic proteins. The underlying assumption of most of these techniques is that the immunological reactions that are triggered by therapeutic proteins are dependent on T cells. A significant amount of research has demonstrated that the development of meaningful anti-drug antibodies (ADA) responses depends on T helper cells (Th cells: CD4 + T cells) responding to protein epitopes. Antigen-presenting cells (APCs) surface-expressed human leukocyte antigen (HLA) molecules (major histocompatibility complex class II, MHC-II) deliver these epitopes to Th cells (Matucci et al. 2019; Jawa et al. 2020; Ducret et al. 2022). Hence, it becomes prerequisite to identify potential HLA- restricted immunogenic epitopes because these are necessary conditions for eliciting an immune response to therapeutic proteins. Mitigating unwanted immune responses associated with therapeutic protein-based drug administration is crucial for disease management and cure. Some other approaches to reduce the induced immunogenicity include interfering lymphocytes activation and induction of regulatory B and T cells (Salazar-Fontana et al. 2017). Table 5 shows some of the immunogenicity-mitigating approaches used with therapeutic proteins.

Table 5.

Some immunogenicity-mitigating approaches with therapeutic proteins

S. no	Approaches	Details	Examples	References
	Deimmunization of therapeutic protein (PEGylation, fusion to polypeptides, reductive methylation, glycosylation and polysialylation)	Eliminating non-self-amino acid sequences to prevent triggering unintended immunological reactions Antibody optimization by the reduction of non-germline amino acid sequences is one of the more recent mAb deimmunization techniques that have been more geared explicitly toward possibly immunogenic portions of the primary amino acid sequence	Immunotoxins (Diphtheria toxin, Pseudomonas exotoxin A and ricin toxin)	Pastan et al. (2006), Cizeau et al. (2009), Sauna et al. (2018), Lin and Dinner (2019), Zinsli et al. (2021)
2	Immune Tolerance Induction (ITI)	ITI regimens should be taken into consideration when a patient's immune system renders a life-saving medication ineffective and no other treatment alternatives, such as bypass therapy, are available	Immune responses to enzyme replacement therapy (ERT) in Pompe disease Tolerogenic nanoparticles (tNPs) such as synthetic virus particles (SVPs) e.g., poly(lactidecoglycolide) (PGLA) encapsulated with immunomodulator rapamycin	Kishimoto et al. (2016), McCarthy et al. (2017), Mazor et al. (2018), King et al. (2018), Nakar and Shapiro (2019), Desai et al. (2020)
3	Targeting and Manipulating Dendritic cells	To control ADA responses. The term "professional APCs" refers to dendritic cells (DCs). Initiating an immune response to a foreign antigen, serve as the first step	Murine DCs treated with vitamin D3 can induce tolerogenic DCs in vivo	Penna et al. (2007), Caminschi et al. (2012), Macri et al. (2016), Salazar-Fontana et al. (2017)
4	Cell depletion through Immunosuppression	Negative selection leads to the reduction of auto-reactive cells, which is one of the innate mechanisms of central tolerance. Similar deletion processes are used in germinal centers to eliminate peripheral B cells that express auto-reactive antibodies	Cyclophosphamide	Witmer and Young (2013), Tie et al. (2022)

Protein engineering can improve protein expression and folding by modifying the protein sequence to enhance solubility, stability, and folding efficiency. This can be achieved by introducing amino acid substitutions, deletions, or additions (Zhu et al. 2017). For example, a study reported that the introduction of a disulfide bond in the Fc domain of a monoclonal antibody improved its folding and stability (Zeng et al. 2018). Cell line engineering can improve protein expression and folding by modifying the host cell to enhance protein secretion, reduce protein degradation, and improve PTMs. This can be achieved by modifying the cell's protein quality control machinery, such as chaperones and proteases, or by introducing genes for PTM enzymes (Luo and Lee 2013). For example, a study reported that the overexpression of an endoplasmic reticulum chaperone improved the folding and secretion of a therapeutic protein (McLaughlin and Vandenbroeck 2011; Luo and Lee 2013). Process optimization is another way by which we can improve protein yield and quality by optimizing the culture conditions, expression systems, and purification methods. This can be achieved by optimizing the pH, temperature, oxygen, and nutrient conditions during protein expression and purification (Shukla and Thömmes 2010; Choi and Geletu 2018) (Fig. 2).

Fig. 2.

Summary showing challenges and possible solutions for production of therapeutic proteins. Challenges with therapeutic protein production shown in left side, and right side showed the strategies using to combat those challenges

Conclusion and future perspectives

To treat critical human infectious diseases, macromolecular protein therapeutics have been proven to be effective and helped in evolving the medicinal area. Due to the advent of recombinant DNA technology (RDT), a huge number of therapeutic proteins have made their way to pharmaceutical market plus their demand is rising day by day. The continuous improvement in the bioprocessing sector has led to the production of quality products. Using the prokaryotic and eukaryotic host expression systems, modern molecular biology is at front foot to produce therapeutic proteins which are of importance in various cell therapies. The future potential of such therapies is huge as ‘n’ number of proteins are produced by microorganisms. These proteins can also be used in combination with small molecular drugs to give synergistic benefits. For example, treatment of EGFR-positive colon cancer.

To summarize, it can be stated that the advancement in biotechnology has contributed a lot in reverberating the field. However, there are still some aspects that need to be worked upon. Such as in case of integrated therapeutic bioprocessing, sterility is a matter of concern, so a sterile barrier is required between upstream and downstream. Feedback control systems also need to be developed as some parameters of USP can affect DSP operation. Even after adding some formulations to protein systems, clinical safety and immunogenicity have been an issue which can lead to protein instability. Gene annotation is also a limitation even after the genome sequences of some microbes such as filamentous fungi are available. Therefore, it should enable genome-wide screening in fungi so as to identify the candidate genes and can surely provide sustainable solutions for multiple diseases worldwide. In conclusion, the production of therapeutic proteins presents numerous challenges that need to be addressed to ensure their safety, efficacy, and affordability. While many solutions have been proposed, there is still a need for continued innovation and collaboration among academia, industry, and regulatory agencies to overcome these challenges and make these life-saving therapies accessible to all.

Declarations

Conflict of interest

The authors declare no conflict of interest.