CN111329996A

CN111329996A - Composition of recombinant human growth hormone and preparation method thereof

Info

Publication number: CN111329996A
Application number: CN202010137883.6A
Authority: CN
Inventors: 刘章; 张姗; 汤火龙
Original assignee: Shanghai United Cell Biotechnology Co ltd
Current assignee: Shanghai United Cell Biotechnology Co ltd
Priority date: 2020-03-03
Filing date: 2020-03-03
Publication date: 2020-06-26
Anticipated expiration: 2040-03-03
Also published as: CN111329996B

Abstract

The invention discloses a recombinant human growth hormone composition and a preparation method thereof. The composition of the present invention comprises recombinant human growth hormone, histidine, mannitol, poloxamer, phenol and disaccharide. The recombinant human growth hormone composition provided by the invention obviously reduces deamidation reaction, oxidation and high polymer aggregation.

Description

Composition of recombinant human growth hormone and preparation method thereof

Technical Field

The invention relates to a recombinant human growth hormone composition and a preparation method thereof. In particular, the present invention relates to a composition of recombinant human growth hormone comprising recombinant human growth hormone, histidine, mannitol, poloxamer, phenol and disaccharide, a preparation method and uses thereof.

Background

Human growth hormone (hGH) is a protein hormone produced and secreted by the anterior pituitary somatotrops. Human growth hormone plays a key role in the physical growth of children and in the metabolism of adults through metabolic actions on proteins, carbohydrates and fats.

Human growth hormone is a single chain polypeptide consisting of 191 amino acids (Bewly et al, 1972) with two disulfide bonds. One between Cys-53 and Cys-165, forming a macrocycle within the molecule. The other between Cys-182 and Cys-189, forming a small loop near the C-terminus.

In solution, hGH exists predominantly as monomers, with small fractions of dimers or higher molecular weight oligomers. Under certain conditions, hGH may be induced to form large amounts of dimers, trimers or higher oligomers. The formation of human growth hormone aggregates leads to increased immunogenicity and thus to safety problems in vivo.

Under appropriate conditions, asparagine and glutamine residues in proteins are susceptible to deamidation reactions. It has been reported in the literature that this reaction of hGH results in the conversion of Asn-152 to aspartic acid and Gln-137 to glutamic acid (Lewis et al, 1981). It is known that, under certain storage conditions, biosynthetic hGH may undergo degradation, leading to deamidation of another asparagine (Asn-149). Asn-152, Gln-137 and Asn-149 are the main sites for deamidation. Although studies have shown that the deamidated products of hGH are not toxic and do not alter their receptor binding ability and biological activity, deamidation is still undesirable and needs to be controlled as a pharmaceutical (Cleland et al, 1993; Jenkins et al, 2008).

Methionine residues in proteins are susceptible to oxidation to form sulfur oxides. Sulfoxidation of Met-14 and Met-125 in hGH can occur (Becker et al, 1988). Met-14 and Met-125 are the main oxidation sites in hGH. The sulfoxide structure generated by oxidation of Met-14 and Met-125 reduces the stability of hGH compared to native hGH.

Liquid formulations of hGH tend to be unstable and are susceptible to chemical reactions such as: deamidation reactions or oxidation, while also forming dimers or aggregates of high molecular weight due to physical instability (Becker et al, 1988; Becker et al, 1989; Cleland et al, 1993; Ablinger et al, 2012).

The recombinant human growth hormone (rhGH) has the same amino acid content, sequence and spatial conformation as the endogenous human growth hormone, and has the same biological activity. rhGH is approved for the treatment of growth hormone deficiency in children and adults, turner's syndrome, cryptorchism-dwarfism-obesity-hypo-mental syndrome, childhood nephropathy, and the like.

Due to the instability of recombinant human growth hormone in solution, rhGH drug formulations are typically freeze-dried and stored in freeze-dried form at 2 to 8 ℃, which must be re-dissolved prior to use. In order to improve the convenience of doctors and patients and to improve the stability of rhGH, many studies have been made in the art on liquid formulations of rhGH.

International patent publication No. WO94/03198 discloses a stable liquid formulation comprising human growth hormone, a buffer, a non-ionic surfactant and a neutral salt, mannitol or a preservative. The buffer may be histidine, and citric acid may also be preferred. This application discloses a preferred formulation comprising citric acid and sodium chloride as buffers and polysorbate 20 for stabilization. This combination prevents protein polymerization, but also produces significant amounts of deamidated growth hormone.

International patent publication No. WO93/19776 discloses formulations of growth hormone-containing protein solutions that include citric acid as a buffer and are more stable than formulations that include phosphate buffers. The formulation comprises amino acids such as glycine and alanine and/or mannitol or other sugar alcohols and/or glycerol and/or other carbohydrates and optionally a preservative such as phenylethyl alcohol.

International patent publication No. WO2005/105148 discloses liquid growth hormone formulations comprising citric acid or phosphate as a buffer, and polyethylene-polypropylene glycol and an alkali metal salt or a pseudo alkaline earth metal salt.

US patent publication No. US6448225 discloses a liquid formulation of rhGH comprising human growth hormone, mannitol, a buffer and a non-ionic surfactant, of which citrate buffer is preferred.

International patent publication No. WO97/39768 discloses a stable pharmaceutical formulation containing growth hormone, which formulation comprises an amino acid selected from aspartic acid, isoleucine, valine, leucine or histidine, or a peptide comprising at least one basic amino acid and at least one acidic amino acid, and a non-ionic detergent, such as a polysorbate or poloxamer.

Korean patent publication No. KR1019980052483 discloses a pharmaceutically stable liquid formulation of human growth hormone, in which a buffer solution consisting of sodium acetate and glutamic acid is preferred.

The example disclosed in chinese patent publication No. CN20110267989.9 is preferably citrate buffer system, poloxamer, glycine, mannitol to prepare stable human growth hormone injection.

NovoNordisk A/S currently marketed Norditroping SimpleXx injection, which contains recombinant human growth hormone, histidine buffer, non-ionic surfactant, sodium chloride and preservative.

In patent WO1993012812a1, the use of sucrose to improve bulking agents for frozen human growth hormone is disclosed, but the stability problems of human growth hormone solutions are not discussed either.

The study by Sumitra et al found that trehalose did not protect the rhGH mer and that different types of cyclodextrins and sugars such as glucose, maltose and trehalose did not protect the rhGH aggregation in 4.5M GuHCl solution and there was no significant difference between these sugars.

The research of Mark et al found that trehalose may increase partial protein oxidation, and sucrose increases the oxidation rate of MET of FVII protein (see Mark et al, 2010), and it was also reported that mannitol and the like, although scavenging oxygen free radicals, did not mention reduction of protein oxidation level.

The study by Maya et al found that sucrose and trehalose slowed deamidation of lyophilized hGH. However, there is no mention that both are capable of reducing deamidation of hGH liquid formulations.

As described above, although many attempts have been made to prepare liquid formulations of growth hormone, there is still a need for more efficient methods capable of reducing deamidation, oxidation and aggregation of macromolecules of rhGH.

Disclosure of Invention

In view of the above, the present invention provides a recombinant human growth hormone composition for reducing deamidation, oxidation and polymer aggregation of rhGH, and a method for preparing the same.

In one aspect, the present invention provides a composition of recombinant human growth hormone, comprising recombinant human growth hormone, histidine, mannitol, poloxamer, phenol and a disaccharide.

Specifically, the composition of the present invention may include 1.0mg/ml to 4.0mg/ml of recombinant human growth hormone, 8.0mM of histidine, 9.1mg/ml to 45.5mg/ml of mannitol, 3.0mg/ml of poloxamer, 3.0mg/ml of phenol, and 17.1mg/ml to 85.5mg/ml of disaccharide.

The composition of recombinant human growth hormone according to the present invention has prolonged stability, in particular, reduced deamidation reaction, reduced oxidation and reduced polymer aggregation.

Wherein the disaccharide is sucrose or trehalose.

Wherein the content ratio of the disaccharide to the mannitol is 1.93:1 to 9.40:1, preferably 4.7: 1.

The recombinant human growth hormone composition provided by the invention obviously reduces deamidation reaction, oxidation and high polymer aggregation.

In another aspect, the present invention provides a method for preparing the recombinant human growth hormone composition of the present invention, the method comprising the steps of: preparing poloxamer solution, histidine-histidine hydrochloride buffer solution, sucrose solution, trehalose solution and phenol solution by using water for injection respectively;

and fully and uniformly mixing the prepared poloxamer solution, histidine-histidine hydrochloride buffer solution, sucrose solution and/or trehalose solution, phenol solution, water for injection, mannitol and recombinant human growth hormone to obtain a mixed solution, and adjusting the pH value to be 6.0-6.3, wherein the premise is that for the poloxamer solution and the phenol solution, after one solution is fully mixed with other components, the other solution is added.

Drawings

Fig. 1 shows rhGH monomer content over time in a composition of recombinant human growth hormone according to an embodiment of the invention;

FIG. 2 shows rhGH multimer content over time in a composition of recombinant human growth hormone according to an embodiment of the invention;

fig. 3A to 3F show the particle size distribution of rhGH after 15 days of nortzia and compositions 1 to 5 of recombinant human growth hormone according to an embodiment of the present invention, respectively;

fig. 4 shows the content of deamidation products of rhGH in the composition of recombinant human growth hormone according to an embodiment of the present invention over time;

fig. 5 shows the content of total related impurities in the composition of recombinant human growth hormone according to an embodiment of the present invention over time.

Detailed Description

The present inventors have conducted extensive studies on the stability of recombinant human growth hormone in order to slow the growth of related protein impurities in the recombinant human growth hormone injection and reduce the instability of recombinant human growth hormone in solution. Specifically, the present inventors studied the effect of buffers, disaccharides (including sucrose and/or trehalose), mannitol, phenol, poloxamer, and the like on the stability of recombinant human growth hormone.

In order to prolong the stability of the human recombinant growth hormone and slow down the increase of related protein impurities in the stability experiment of the growth hormone injection, the inventor carries out experimental Design (DOE) on a prescription of the recombinant human growth hormone injection, and the specific design scheme is shown in Table 1:

TABLE 1 DOE design of growth hormone injection

The growth hormone injection DOE experiment preparation steps are as follows:

solution preparation stock solution

1) Thawing: the stock solution was thawed overnight in a refrigerator at 2-8 ℃.

2) Liquid changing: the protein was subjected to buffer exchange using a G25 column, 20mM citrate buffer and 1.34mg/ml histidine buffer.

3) And (3) filtering: the G25 peak protein solution was subjected to a single clarification filtration using a 0.5 μm MCE disposable syringe filter and a second clarification filtration using a 0.22 μm PVDF syringe filter, respectively. And sampling after primary filtration of the protein solution and secondary filtration of the protein solution, and sending to a detection HPLC (high performance liquid chromatography) for content determination.

Preparation of semi-finished product

The prescription is divided into 2 parts, one part is protein liquid and buffer salt and is 150ml, the other part is auxiliary liquid and is 150ml, firstly, the pH value required by the auxiliary liquid is tested by a pre-experiment, and the pH value of a semi-finished product obtained by mixing the two parts of solutions is between 6.0 and 6.3.

The solutions were prepared according to the respective formulations in table 1, and it was noted that, for poloxamer and phenol, the mother solutions were prepared separately and then mixed with the other components in table 1, in other words, one of poloxamer and phenol was mixed well with the other component, and then the other of poloxamer and phenol was added, otherwise irreversible turbidity would be caused.

Filtration filling

Firstly, washing a filter by using auxiliary material solution of each batch number, then filtering a semi-finished product, and receiving materials by using a dry-baked triangular flask. Filling the semi-finished product into a bottle with a plug by a peristaltic pump, and finally rolling the cap and placing the bottle in a refrigerator at 2-8 ℃ for later use. The filtration pipeline is washed by corresponding auxiliary material solution before filling the semi-finished product.

The results of the DOE experiments are shown in table 2 below:

TABLE 2 DOE results of growth hormone injection formulation DOE experiments on protein-related impurities at 15 deg.C

Through the results of table 2 in the above DOE experiment, the inventors unexpectedly found that:

compared to formulations containing mannitol and no mannitol (

samples

3, 4, 5 and 13), formulations containing sucrose and no mannitol (

samples

1, 7, 10 and 15) showed a significant decrease in the level of the relevant protein (p <0.05) upon acceleration at 15 ℃ for 2.5 months, and a significant decrease in the slope of the increase in the relevant protein throughout the acceleration (p < 0.05). This suggests that sucrose has a significant inhibitory effect on the growth of the relevant protein.

Comparing the formulations containing both sucrose and mannitol (samples 6 and 16) to the formulations containing the same level of sucrose but no mannitol (samples 15 and 7), the formulations with mannitol (samples 6 and 16) had a lower slope for the increase of the protein of interest at 15 ℃ for 2.5 months of acceleration and the increase of the protein of interest throughout the acceleration. This indicates that the combination of sucrose and mannitol has a more significant inhibitory effect on the growth of the protein of interest than sucrose alone.

Formulations containing both mannitol and sucrose (

samples

2, 6, 8, 9, 11, 12, 14 and 16) had a significant reduction in the relevant protein levels (p <0.01) at 15 ℃ accelerated for 2.5 months compared to formulations containing mannitol but no sucrose (

samples

3, 4, 5 and 13). The inhibition effect of the combination of sucrose and mannitol at different ratios on the increase of the protein of interest was different, wherein the water level of the protein of interest was less than 10% at the acceleration of 2.5 months at 15 ℃ and the slope of the increase of the protein of interest was less than 3 throughout the acceleration, for the formulations (samples 6, 8, 11, 14 and 16) in which the combination of sucrose and mannitol ranged from 1.92:1 to 9.40: 1. In particular, the inhibitory effect of sample 16 was the best for the inhibitory effect of the growth of the relevant protein. This indicates that different sucrose and mannitol content ratios have different effects on inhibiting the growth of the relevant protein.

On this basis, the present inventors have provided a composition containing recombinant human growth hormone which is stable over a prolonged period of time through an exploratory study of disaccharides, including sucrose and trehalose, and the ratio of the combination of disaccharide and mannitol.

1. Experimental materials and sources thereof

The recombinant human growth hormone stock solution used in the invention is from Shanghai combined Sell bioengineering limited company with the batch number of GBB 1201904; the preparation process of the recombinant human growth hormone stock solution used in the invention comprises the following steps: carrying out shake flask fermentation, tank fermentation, fusion protein capture, enzyme digestion and liquid exchange, fine purification (hydrophobic chromatography, ion exchange chromatography and gel filtration chromatography), and sterilizing and filtering on escherichia coli to obtain a stock solution, wherein the content of the recombinant human growth hormone in the stock solution is 17.7 mg/ml.

Sucrose was purchased from Pfanstiebl, inc; trehalose was purchased from japanese forest origin; mannitol was purchased from Tianrui, Zhejiang; poloxamers are purchased from BSAF; phenol was purchased from Merck; histidine-histidine hydrochloride buffer was purchased from shanghai synephrine.

2. The invention provides a composition of recombinant human growth hormone

The present inventors provide compositions of recombinant human growth hormone having stability over an extended period of time. The recombinant human growth hormone used in the invention has the same amino acid content, sequence and spatial conformation as endogenous human growth hormone, and has the same biological activity. The content of recombinant human growth hormone in the composition is 1.0mg/ml to 4.0mg/ml, preferably 3.3 mg/ml.

Disaccharides are included in the compositions of the present invention, which have the effect of preventing or slowing the deamidation reaction of recombinant human growth hormone. The disaccharide includes sucrose, Sargassum, etc. Preferably, the disaccharide is sucrose. The content of disaccharide in the composition is 17.1mg/ml to 85.5mg/ml, preferably 85 mg/ml.

The composition of the present invention further includes a buffer solution, which includes a histidine-histidine hydrochloride buffer solution, a histidine buffer solution, a citric acid buffer solution, and the like. The pH of the buffer is 6.0 to 6.3, preferably 6.1 to 6.2. The content of histidine buffer in the composition was 8.0 mM.

The composition of the present invention further comprises mannitol, wherein the mannitol is contained in an amount of 9.1mg/ml to 45.5mg/ml, preferably 18 mg/ml.

The composition of the present invention further comprises phenol, wherein the content of phenol is 3.0 mg/ml.

The composition further comprises poloxamer, wherein the content of the poloxamer is 3.0 mg/ml.

The ratio of sucrose to mannitol in the composition of the invention is 1.93:1 to 9.40:1, preferably 4.72: 1.

3. Preparation of the composition

Poloxamer solution, histidine-histidine hydrochloride buffer solution, sucrose solution, trehalose solution and phenol solution are prepared by using water for injection respectively.

Sequentially adding the prepared poloxamer solution, histidine-histidine hydrochloride buffer solution, sucrose solution and/or trehalose solution, phenol solution, water for injection and mannitol into the recombinant human growth hormone, uniformly mixing to obtain a mixed solution, and adjusting the pH value to 6.0-6.3.

4. Stability evaluation of the compositions

For the composition of human growth hormone of the present invention, the following evaluation was performed.

A. Measurement of rhGH Polymer content

Measurement was performed using size exclusion-high phase liquid chromatography (SEC-HPLC), where mobile phase: 0.063mol/L phosphate buffer (pH7.0) (97%): isopropanol (3%); column temperature: sample chamber temperature at 25 ℃: 5 ℃; detection wavelength: 214 nm; flow rate: 0.6 ml/min; a chromatographic column: TSKgelG2000SW_xl7.8*300，5μm。

Appropriate amounts of a sample to be tested and a control (China institute for food and drug testing, Cat: 140635) were taken, diluted to 1mg/ml with 0.025M phosphate buffer (pH7.0), run for 30 minutes, and two portions were prepared in parallel for measurement.

B. Dynamic Light Scattering (DLS) detection

The particle size distribution and the monomeric protein content were measured by dynamic light scattering. Taking a proper amount of reference substance and sample to be tested, adding into black 96-well plate, setting machine parameters (model: DAWNHELEOS II), obtaining 20 times each time, repeating for 3 times, and taking average value.

C. Measurement of deamidation product

Measuring the deamidation product by using a capillary electrophoresis apparatus, respectively adding water into the solution to be tested and the reference solution to dilute to 1mg/ml, and separating under the following separation conditions:

capillary tube: uncoated-fused silica capillary tube with inner diameter of 50 μm;

the length of the capillary tube is 100cm, and the effective length is 90 cm;

temperature of the clamping groove: 30 ℃;

temperature of the sample chamber: 8 ℃;

detection wavelength: 200 nm;

sample introduction amount: 0.7psi, 5 seconds;

separation time: for 80 minutes, the voltage was 22 KV.

D. Measurement of Total related impurities

The measurement was performed by high phase liquid chromatography, in which mobile phase a: 0.05M Tris-hydrochloric acid buffer (pH7.5) (71%); mobile phase B: n-propanol (29%); column temperature: 45 ℃; detection wavelength: 220 nm; a chromatographic column: sepax Bio-C44.6 x 250mm,

(PN：110045-4625)。

taking a proper amount of a reference substance and a sample to be tested, diluting the reference substance and the sample to be tested to 2mg/ml by using the mobile phase A, running for 50 minutes, and calculating the content of related impurities according to an area normalization method.

According to the results of the stability evaluation, it was found that the rhGH monomer content in the sucrose and trehalose-containing composition was higher than that in the disaccharide-free composition and Naoze (a commercially available recombinant human growth hormone injection solution consisting of mannitol, histidine, poloxamer 188, phenol and water for injection) after being left at 40 ℃ for 15 days. The content of rhGH multimer in the sucrose-and trehalose-containing composition was lower than that in the disaccharide-free composition and that in Norze, and the rate of increase of rhGH multimer was also slower in the sucrose-and trehalose-containing composition. In terms of particle size distribution, it can be seen that the majority of the compositions without disaccharide and the noni had formed polymer after 15 days, while 30% to 50% of rhGH was still present in monomeric form in the compositions with sucrose and trehalose. The content of deamidation product in the sucrose and trehalose-containing composition is lower than the content of deamidation product in the disaccharide-free composition and the noni; the total level of related impurities in the sucrose and trehalose-containing composition is lower than the total level of related impurities in the disaccharide-free composition and the noni.

Therefore, the human growth hormone composition provided by the invention reduces deamidation reaction, oxidation and high molecular polymerization of the recombinant human growth hormone, thereby prolonging the stability of the recombinant human growth hormone.

The compositions provided herein are useful for treating a human growth hormone deficiency-related disorder selected from the group consisting of childhood and adult growth hormone deficiency, turner's syndrome, cryptorchism-dwarfism-obesity-hypo-mental syndrome, childhood nephropathy, and the like.

The method and concept of the present invention are further illustrated by the following specific examples, but the scope of the invention is not limited by the examples.

Examples

Example 1

Preparing 2L of 16mM histidine-histidine hydrochloride buffer mother solution by using water for injection; 200ml of 500mg/ml sucrose solution is prepared; weighing 100g of trehalose, and preparing a trehalose solution with a constant volume of 500mg/ml by using a 200ml volumetric flask; dissolving 6g of poloxamer in 200ml of water for injection to prepare a 30mg/ml poloxamer solution; 6g of phenol was dissolved in 200ml of water for injection to prepare a 30mg/ml phenol solution.

30ml of poloxamer solution, 94.1ml of histidine-histidine hydrochloride buffer, 51ml of sucrose mother liquor, 27ml of mannitol (200mg/ml), 12ml of water for injection, 30ml of phenol, and 55.9ml of recombinant human growth hormone stock solution (17.7mg/ml) were thoroughly mixed, and the pH was adjusted to 6.0 to 6.3, to obtain 300ml of liquid composition 1.

Example 2

Composition 2 of example 2 was prepared according to the same procedure as in example 1, except that trehalose was used instead of sucrose.

Comparative examples 1 to 3

Compositions 3 to 5 of comparative examples 1-3 were prepared according to the same procedures as in example 1, except for the specific compositions of compositions 3 to 5 listed in the following Table 3.

TABLE 3 compositions 1 to 5

The content ratio of sucrose and mannitol is not shown when the composition does not contain the sucrose or mannitol.

Evaluation examples

1. Measurement of rhGH monomer content

Compositions 1 to 5 were left at 40 ℃ for 15 days, protected from light, and sampled on day 0, day 5, day 10 and day 15, and the rhGH monomer content was measured according to the method in "B, Dynamic Light Scattering (DLS) detection" above, respectively. The results are shown in FIG. 1. In FIG. 1, the vertical axis represents the content of monomeric protein, and the horizontal axis represents the time of leaving.

As can be seen from figure 1, by day 15, the levels of rhGH monomer protein were significantly higher in the sucrose or trehalose containing compositions (examples 1 and 2 and comparative examples 2 and 3,

compositions

1, 2, 4, 5) than in the sucrose or trehalose free compositions (comparative example 1, composition 3) and norze. It can be seen that rhGH in sucrose or trehalose-containing compositions is more stable in monomeric form after 15 days of accelerated testing at 40 ℃ than the composition without sucrose or trehalose and rhGH in norze. Further, the compositions containing both sucrose or trehalose and mannitol (examples 1 and 2, compositions 1 and 2) had higher rhGH monomeric protein content than the compositions containing only sucrose or trehalose and no mannitol (comparative examples 2 and 4, compositions 4 and 5). It can be seen that the combination of sucrose or trehalose with mannitol further improved the stability of rhGH.

2. Measurement of rhGH Polymer content

Compositions 1 to 5 were left at 40 ℃ for 15 days, protected from light, and sampled on day 0, day 5, day 10 and day 15, and the rhGH multimer content was measured according to the "measurement of multimer content of A, rhGH" method, respectively. The results are shown in FIG. 2. In FIG. 2, the vertical axis represents the content of the multimeric protein, and the horizontal axis represents the time of the standing.

As can be seen from FIG. 2, by day 15, the rhGH multimer content was significantly lower in the sucrose and trehalose-containing compositions (examples 1 and 2, compositions 1 and 2) than in the sucrose and trehalose-free compositions (comparative examples 1 to 3, compositions 3 to 5) and Noreze. It can be seen that after 15 days of accelerated testing at 40 ℃, the compositions containing sucrose and trehalose had less rhGH polymers and were more stable than the compositions without sucrose or trehalose and norze. Further, the compositions containing both sucrose or trehalose and mannitol (examples 1 and 2, compositions 1 and 2) had lower content of the multimeric protein than the compositions containing only sucrose or trehalose and no mannitol (comparative examples 2 and 4, compositions 4 and 5). It can be seen that the combination of sucrose or trehalose with mannitol further improved the stability of rhGH.

3. Measurement of particle size distribution

Compositions 1 to 5 were placed at 40 ℃ for 15 days, protected from light and sampled on day 15, and the particle size distribution was measured according to the "B, Dynamic Light Scattering (DLS) detection" method described above, respectively. The results are shown in FIGS. 3A to 3F, in which the vertical axis represents the intensity of the particle size distribution and the horizontal axis represents the particle radius.

As can be seen from fig. 3A to 3F, the compositions without sucrose or trehalose (comparative example 1, composition 3) and norzel, after being left at 40 ℃ for 15 days, measured particle size distributions around 3.5nm (rhGH monomeric protein) were only 13.1% and 2.1% (see, fig. 3C and 3F). Whereas compositions containing sucrose or trehalose (examples 1 and 2 and comparative examples 2 and 3,

compositions

1, 2, 4 and 5) had a particle size distribution around 3.5nm measured after 15 days at 40 ℃ of about 30% to about 50% (see, fig. 3A, 3B, 3D and 3E). In other words, after 15 days at 40 ℃, the majority of the disaccharide free and the noni formed larger particle size polymers, while 30% to 50% of the rhGH was still present in the sucrose or trehalose containing compositions as monomers.

4. Measurement of deamidation product

Compositions 1 to 5 were left at 40 ℃ for 15 days, protected from light, and sampled on day 0, day 5, day 10 and day 15, and the measurement of the deamidation product was performed according to the "C, measurement of deamidation product" method described above, respectively. The results are shown in FIG. 4. In FIG. 4, the vertical axis represents the percentage of deamidation and the horizontal axis represents the time of standing.

As can be seen from fig. 4, in the compositions to which sucrose or trehalose was added, such as

compositions

1, 2, 4 and 5, although the content of deamidation product gradually increased over time, the rate of increase was significantly less than that of the composition to which no sucrose or trehalose was added, such as composition 3, and the knoeveness formula. It can be seen that sucrose or trehalose significantly reduced deamidation of rhGH.

5. Measurement of Total related impurities

Compositions 1 to 5 were placed at 40 ℃ for 15 days, protected from light, and sampled on day 0, day 5, day 10 and day 15, and the total relevant impurity content was measured according to the "D, measurement of total relevant impurity content" method described above, respectively. The results are shown in FIG. 5. In fig. 5, the vertical axis represents the content of the relevant impurities, and the horizontal axis represents the standing time.

As can be seen from fig. 5, in the compositions to which sucrose or trehalose was added, such as

compositions

1, 2, 4 and 5, the rate of increase was significantly less than that of the compositions to which no sucrose or trehalose was added, such as composition 3, and the knoeveness formula, although the total related impurity content gradually increased over time, after being left at 40 ℃ for 15 days, in terms of the total related impurity content, including deamidation products, oxidation products and protein partial degradation products.

It can be found from the above evaluation examples that the composition of the present invention has significantly higher rhGH monomer protein content, lower multimer content, more particle size distribution of the corresponding rhGH monomer, reduced deamidation and less related impurities after being left at 40 ℃ for 15 days. Thus, the rhGH compositions of the present invention have greater stability. The recombinant human growth hormone composition of the invention has stability in a prolonged period of time and has industrial application value.

Reference to the literature

Tangshu, et al, J.Med.Industrials 45:1071-1077 (2014);

ablinger et al, int.J.pharm.427:209-216 (2012);

becker et al, J.biol.chem, 262: 785-;

becker et al, Biotechnol. appl.biochem.10:326 (1988);

bewly et al, int.J.peptide and Protein Res.4:281-287 (1972);

cholewinski et al, pharm. actaHelv.71: 405-;

cleland et al, Crit. Rev. ther. drug Carrier Syst.10:307-377 (1993);

jenkins et al, mol.Biotechnol.39:113-118 (2008);

lewis et al, J.Bio.chem.256:11645 (1981);

salnikova et al, int.J.Pharm.358:108-113 (2008);

sumitra et al, Pharmaceutical Research, Vol.21, No.12 (2004);

mark et al, Pharmaceutical Research, Vol.27, No.4 (2010);

maya et al, International Journal of pharmaceuticals 358, 108-.

Claims

1. A composition of recombinant human growth hormone comprises recombinant human growth hormone, histidine, mannitol, poloxamer, phenol and disaccharide.

2. The composition according to claim 1, wherein the mannitol is at a concentration of 9.1mg/ml to 45.5mg/ml based on the total volume of the composition.

3. The composition according to claim 1, wherein the concentration of the disaccharide is from 17.1mg/ml to 85.5mg/ml based on the total volume of the composition.

4. The composition of claim 1, wherein the disaccharide is sucrose or trehalose.

5. The composition according to claim 1, wherein the disaccharide and the mannitol are present in a ratio of 1.93:1 to 9.40:1, preferably 4.72:1, by weight.

6. A method of making the composition of any one of claims 1 to 5, the method comprising the steps of:

respectively preparing poloxamer solution, histidine-histidine hydrochloride buffer solution, sucrose solution, trehalose solution and phenol solution;

7. The method of claim 6, wherein the recombinant human growth hormone is a recombinant human growth hormone stock solution.