Human growth hormone (hGH), a 191 amino acid single-chain polypeptide produced by somatotropic cells within the anterior pituitary gland, is the most important growth-promoting hormone in the human body, whose main function is to promote the growth of soft tissues and bones, and to regulate the metabolism of the body and growth and development.
Growth hormone deficiency (GHD) is a rare disorder characterized by insufficient secretion of the growth hormone somatropin by the pituitary gland, occurring in approximately 1 in 4,000 to 10,000 children. If left untreated, the child will experience persistent growth attenuation, a very short height in adulthood, and puberty may be delayed. Children with GHD also face physical and mental health challenges.
Currently, in vitro growth hormone supplementation is the only treatment modality for children with growth hormone deficiency.
The History of Human Growth Hormone
Since the beginning of human pituitary growth hormone in 1958 and its synthesis today using genetic engineering techniques, growth hormone has undergone five generations of development.
First Generation Growth Hormone (1950s-1970s)
First-generation growth hormone refers to human pituitary-derived growth hormone, which is the earliest clinically applied growth hormone and was first successfully extracted from human cadaveric pituitary glands by Raben in 1958, and subsequently began to be used in the treatment of dwarfism in children. However, the first generation of growth hormone had the disadvantages of restricted source, scarce production, low relative purity, and susceptibility to donor virus contamination, and was withdrawn from the market in 1985 after being banned by the U.S. FDA.
Second Generation Growth Hormone (early 1980s)
The second generation of hGH is Met-rhGH, a recombinant hGH containing 192 amino acids, developed by Genentech using inclusion body technology in Escherichia coli (E. Coli). Compared with natural hGH, the second generation of hGH has an additional methionine residue at the N-terminus, also known as somatrem. It is clinically tested to be identical to the physiological activity of extracted human growth hormone.
However, the second-generation growth hormone has the disadvantages of a complicated extraction and replication process, easy contamination, high antibody production rate and low purity, leading to its elimination.
Third Generation Growth Hormone (mid-1980s)
The third-generation growth hormone is a recombinant human growth hormone containing 191 amino acids synthesized by gene expression technology in common E. coli, but it is not suitable for long-term use due to structural differences with human pituitary growth hormone.
Fourth Generation Growth Hormone (late 1980s)
The fourth-generation growth hormone is synthesized by recombinant DNA technology in mammalian cells, and its structure is closer to that of natural growth hormone. However, due to the high requirements of cell culture, slow propagation speed, low yield, and the danger of adenovirus contamination, the fourth-generation growth hormone is currently used by very few manufacturers.
Fifth Generation Growth Hormone (1990s)
In 1995, Genentech produced the world's first growth hormone aqueous injection, which was synthesized by E. coli secretory gene expression technology, its amino acid content, sequence and protein structure are identical with human pituitary growth hormone. The biological activity, potency, purity and absorption rate of the fifth-generation growth hormone are incredibly high, and its specific activity can be as high as 3.0 IU/ mg of protein, with an antibody production rate of ≤1%. This ensures the safety, efficacy and stability of the product while minimizing the cost of treatment. Due to its higher biological activity and lower antibody production rate, it has gradually become the mainstream of the market and is still in use today.
Growth Hormone Dosage Form
Currently, short-acting formulations of growth hormone can be categorized into two dosage forms: powder injection and aqueous injection. There is no significant difference between the therapeutic effect of powder injection and aqueous injection, and the safety is similar, but from the perspective of drug stability and convenience of use, the two dosage forms have their own advantages. Powder injection is more stable after lyophilization process, but the re-solubilization process reduces the biological activity and makes it inconvenient to use.
In practical treatment, regardless of powder or aqueous injections, short-acting preparations require frequent injections, which brings inconvenience and pain to patients' lives, leading to poor treatment compliance and decreased therapeutic efficacy due to missed injections in patients with GHD.
Therefore, it is imperative to design appropriate long-acting growth hormone (LAGH) formulations that not only improve the safety of growth hormone, but also reduce the frequency of injections, minimize pain, and effectively improve patient compliance as well as reduce immunogenicity, which further improves the benefit of clinical treatment for patients.
Development of Long-acting Growth Hormone (LAGA)
Technologies used to increase half-life include depot formulations, PEGylated formulations, pro-drug formulations, non-covalent albumin binding growth hormone and growth hormone fusion proteins. Currently, there are only five approved long-acting growth hormone drugs on the market, including Declage™ (somatropin biopartners, LG Life Sciences, approved in South Korea), Jintrolong® (GenSci, approved in China), Sogroya® (Somapacitan, Novo Nordisk), Skytrofa® (Lonapegsomatropin, Ascendis Pharma) and Ngenla (Somatrogon, Pfizer/OPKO).
1. Microsphere technologies
The preparation of LAGH using microsphere technology was one of the earliest attempts to make a formulation of unmodified growth hormone adhered to biodegradable microspheres, resulting in a sustained release of growth hormone over a period of 1 month. The main disadvantage of this technique is that due to the limited loading of rhGH within the microspheres, a large volume of microspheres needs to be prepared to load a sufficient dose, but too large a volume of microspheres poses an increased risk of adverse events, in addition to the incidence of burst of GH release, delayed release after burst, the risk of protein aggregation, and the difficulty of preparing microspheres in larger sizes.
The most representative drug is Genentech's PLGA extended-release microspheres, NutropinDepot, which was approved by the FDA in 1999. However, due to difficulties in mass production and GH burst release, as well as problems with infection and protein degradation found in subsequent studies, NutropinDepot was discontinued and withdrawn from the market in 2004.
In addition, LG Life Sciences introduced a microsphere LAGH formulation, Declage™, which uses hyaluronic acid as the packaging material and is made by mixing growth hormone into sodium hyaluronate microspheres suspended in medium-chain triglycerides prior to injection, which enables once-weekly dosing. It is available for use in children in South Korea.
Polyethylene glycol (PEG) is a hydrophilic polymer with low immunogenicity. PEGylation of proteins and peptides can increase their effective volume and solubility. PEGylated rhGH injection has a significantly lower rate of renal filtration and prolonged half-life, which effectively prolongs the retention time of the drug in the body.
In addition, the PEG macromolecule in the PEGylated rhGH increases the spatial resistance between the growth hormone molecule and the receptor and reduces its binding force with the growth hormone receptor, which makes the peak concentration of the actual effective exertion of biological effects significantly lower than that of the short-acting growth hormone.
Jintrolong® (PEG-rhGH), developed by GenSc, bears a 40-kDa branched PEG at its N-terminus and was approved for use to treat pediatric hGH deficiency by the National Medical Products Administration (NMPA) of China in 2014.
Figures 1. Jintrolong Mechanism of Action
Lonapegsomatropin-tcgd (Skytrofa), developed by Ascendis Pharma, is a long-acting prodrug consisting of rhGH (somatropin), inert methoxy PEG carriers (TransCon carriers), and a linker (TransCon linker) for binding hGH and lonapegsomatropin-tcgd.
The carrier has a shielding effect that minimizes renal excretion and receptor-mediated clearance of lonapegsomatropin. After subcutaneous administration, lonapegsomatropin releases fully active somatropin via autocleavage of the TransCon Linker. Skytrofa was approved for marketing by the FDA in August 2021
Figures 2. The TransCon linker in SKYTROFA releases active somatropin in a predictable manner 
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3. Growth Hormone Fusion Protein
Although PEGylated growth hormone preparations can avoid glomerular filtration, they can still bind to the hGH receptor in the kidney and be metabolized by proteases after cytosolization. The application of fusion protein drug technology not only retains the biological activity of the original protein, but also extends its molecular weight after fusion, which is not easy to be filtered by the glomerulus, and has the effect of prolonging the half-life of the drug.
The fusion protein growth hormone is formed by combining a protein or protein fragment (e.g., albumin, immunoglobulin fragment, human chorionic gonadotropin C-terminal polypeptide, etc.) with growth hormone by gene fusion technology at the amino-terminus of growth hormone.
Somatrogon (Ngenla), jointly developed by Pfizer and OPKO, is a classic fusion protein growth hormone. It received regulatory approvals in Europe, Japan, Canada and Australia in 2022 and was launched in the U.S. on June 28, 2023.
Somatrogon is a new molecular entity that utilizes OPKO's proprietary C-terminal peptide (CTP) long-acting technology. It consists of the amino acid sequence of hGH fused to 3 copies of the C-terminal peptide from the β-chain of human chorionic gonadotropin, resulting in an extended half-life. At 12 months of treatment, the annual rate of height gain was 10.12 cm/year in the Ngenla group and 9.78 cm/year in the active control group.
Figure 3. Somatrogon mechanism of action
4. Albumin Binding Growth Hormone
One way to extend the half-life of a drug is to increase its binding to common serum proteins, such as albumin. Albumin is a single poly-peptide chain having 585 amino acid residues, characterized by low tryptophan and high cysteine content. Also, albumin is a common macromolecular carrier, which is beneficial in helping various endogenous compounds with limited solubility, to prolong their circulation in the body.
Sogroya (Somapacitan) is a long-acting recombinant human GH derivative with a single substitution in the peptide backbone (leucine [L] at position 101 replaced by cysteine [C]) and an albumin-binding moiety. The albumin-binding moiety (side chain) consists of a C16 fatty acid moiety and a hydrophilic spacer chemically conjugated to position 101 of the protein. The non-covalent, reversible binding to endogenous albumin delays the elimination of somapacitan, thereby prolonging its in vivo half-life (t½ and duration of action).
Sogroya is the first FDA-approved formulation of LAGH and is administered to adult patients by subcutaneous injection only once a week.
Figure 4. Somapacitan bound to albumin 
rGH has been shown to be an effective treatment for GHD, which can serve the therapeutic purpose of restoring longitudinal growth and improving quality of life. Compared with short-acting daily growth hormone, long-acting weekly growth hormone can significantly reduce the frequency of injections and effectively improve the treatment compliance and quality of life of pediatric patients.
A variety of LAGH formulations are currently on the market or under development, and these formulations have allowed the frequency of rhGH injections to be reduced from daily to weekly, or even monthly. Differences in the process of LAGH formulation may affect the binding of LAGH to target tissues, leading to differences in efficacy and safety. There are many practical issues that need to be addressed regarding the replacement of daily rhGH by LAGH preparations for the treatment of patients with GHD, such as methods of dose adjustment and insulin-like growth factor I monitoring.
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