CN113797317B - Composition, and preparation method and application thereof - Google Patents

Composition, and preparation method and application thereof Download PDF

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Publication number
CN113797317B
CN113797317B CN202111251289.0A CN202111251289A CN113797317B CN 113797317 B CN113797317 B CN 113797317B CN 202111251289 A CN202111251289 A CN 202111251289A CN 113797317 B CN113797317 B CN 113797317B
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composition
interferon
ascorbic acid
interferon alpha
preparation
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CN113797317A (en
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杨经安
苏婷婷
鄢成伟
秦锁富
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Kexing Biopharmaceutical Co ltd
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Kexing Biopharmaceutical Co ltd
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Abstract

The invention relates to a composition for preserving an interferon stock solution, a preparation method and application thereof, and belongs to the field of biological medicine. The composition comprises an interferon, L-ascorbic acid, a buffer salt and optionally a surfactant. The composition has better stability, prolonged storage life and is beneficial to industrialized production.

Description

Composition, and preparation method and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a composition, a preparation method and application thereof.
Background
Human Interferon (IFN) is a cytokine protein drug produced by activated fibroblasts and certain epithelial cells, and has antiviral infection, antitumor and strong immunoregulatory effects. Most of the interferons have the disadvantages of short half-life and strong immunogenicity in application.
The use of propylene glycol, amino acids (glycine, alanine) or human serum albumin as a protective agent has been described in the literature to improve the stability of interferon stock solutions during processing and storage, but the applicant has found during the course of implementation that the protective agents for high-concentration interferon stock solutions employed in the prior art have the following problems: propylene glycol is haemolytic, there is a risk of injection, the cost of amino acids (glycine, alanine) or human serum albumin is high, new impurities may be introduced, or detection may be affected.
Thus, there remains a need for a safe, environmentally friendly, low cost, stable interferon composition.
Disclosure of Invention
In order to solve the problems, the invention provides the following technical scheme.
In a first aspect, the present invention provides a composition of an interferon stock.
A composition comprising an interferon, L-ascorbic acid and a buffer salt. The invention adopts L-ascorbic acid, is beneficial to improving the chemical stability of interferon, avoids the reduction of the specific activity of the interferon and the increase of related proteins, can obviously prolong the freezing time of the composition, brings convenience to industrial production management and reduces the outdated loss of stock solution.
The composition may also include a surfactant. The surfactant is beneficial to reducing activity loss caused by adsorption and aggregation of a gas-liquid interface when the interferon is frozen.
The interferon may comprise recombinant interferon or interferon prepared by other methods.
The interferon may comprise an interferon selected from a human or an animal. In some embodiments, the interferon comprises at least one of interferon alpha, interferon beta, interferon omega, interferon kappa, interferon lambda, interferon epsilon, or interferon gamma. In some embodiments, the interferon comprises at least one selected from human interferon alpha, human interferon beta, or human interferon gamma. In some embodiments, the interferon comprises a member selected from interferon alpha 1, interferon alpha 2, interferon alpha 3, interferon alpha a, interferon alpha B, or interferon alpha C. In some embodiments, the interferon comprises a polypeptide selected from human interferon alpha 1a, human interferon alpha 1b, human interferon alpha 2a, and human interferon alpha 2b. In some embodiments, the interferon comprises a human interferon alpha 1a selected from the group consisting of a pegylated or fusion protein modified human interferon alpha 1b, a pegylated or fusion protein modified human interferon alpha 2a, and a pegylated or fusion protein modified human interferon alpha 2b. In some embodiments, the interferon is a kana-resistant human interferon. In some embodiments, the interferon is a kana-resistant human interferon alpha 2b.
The purity of the L-ascorbic acid can be more than or equal to 90 percent. In some embodiments, the purity of the L-ascorbic acid is greater than or equal to 95%. In some embodiments, the purity of the L-ascorbic acid is greater than or equal to 98%. The higher the purity of the L-ascorbic acid, the more advantageous is the improvement of the specific activity of the interferon in the composition.
The composition may further comprise water.
The buffer salt may include at least one selected from phosphate, citrate, or acetate.
The phosphate may include at least one selected from potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate, or sodium hydrogen phosphate.
The citrate may include at least one selected from citric acid, potassium citrate, or sodium citrate salt.
The acetate may include at least one selected from acetic acid, ammonium acetate, sodium acetate, or potassium acetate.
The surfactant may include at least one selected from tween 80, tween 20, poloxamer P188. In some embodiments, the surfactant is tween 80.
The L-ascorbic acid may be present in an amount of 1mmol/L to 5mmol/L based on the total volume of the composition. In some embodiments, the L-ascorbic acid is present in an amount of 1.5mmol/L to 5mmol/L based on the total volume of the composition. In some embodiments, the L-ascorbic acid is present in an amount of 3mmol/L to 5mmol/L based on the total volume of the composition. In some embodiments, the L-ascorbic acid is present in an amount of 4mmol/L to 5mmol/L based on the total volume of the composition.
The buffer salt may be present in a concentration of 5mmol/L to 50mmol/L based on the total volume of the composition. In some embodiments, the buffer salt is present at a concentration of 5mmol/L to 40mmol/L based on the total volume of the composition. In some embodiments, the buffer salt is present at a concentration of 5mmol/L to 30mmol/L based on the total volume of the composition. In some embodiments, the buffer salt is present at a concentration of 5mmol/L to 20mmol/L based on the total volume of the composition. In some embodiments, the buffer salt is present at a concentration of 5mmol/L to 15mmol/L based on the total volume of the composition. In some embodiments, the buffer salt is present at a concentration of 8mmol/L to 12mmol/L based on the total volume of the composition.
The pH of the composition may be from 4.0 to 7.0. In some embodiments, the pH of the composition is from 5.0 to 7.0. In some embodiments, the pH of the composition is from 6.0 to 7.0.
The surfactant may be present in an amount of 0.001wt% to 0.05wt% based on the total mass of the composition. In some embodiments, the surfactant is present in an amount of 0.01wt% to 0.05wt% based on the total mass of the composition. In some embodiments, the surfactant is present in an amount of 0.02wt% to 0.05wt% based on the total mass of the composition.
The interferon may be present in an amount of 0.1mg/mL to 3.0mg/mL based on the total volume of the composition. In some embodiments, the interferon is present in an amount of 1.0mg/mL to 3.0mg/mL based on the total volume of the composition. In some embodiments, the interferon is present in an amount of 0.2mg/mL to 2.5mg/mL based on the total volume of the composition. In some embodiments, the interferon is present in an amount of 0.5mg/mL to 2mg/mL based on the total volume of the composition. In some embodiments, the interferon is present in an amount of 0.5mg/mL to 1.5mg/mL based on the total volume of the composition.
The composition may not comprise propylene glycol, amino acids (glycine, alanine) or human serum albumin as protective agents.
In a second aspect, the present invention provides a method of preparing the composition of the first aspect.
A method of preparing the composition of the first aspect, comprising: dissolving buffer salt, L-ascorbic acid and optional surfactant in water, mixing with interferon, sterilizing, and filtering.
In some embodiments, a method of preparing the composition of the first aspect comprises: dissolving buffer salt, L-ascorbic acid and optional surfactant in water, mixing with interferon, sterilizing, filtering, packaging, and filling nitrogen into the headspace.
In a third aspect, the present invention provides the use of a composition according to the first aspect.
Use of a composition according to the first aspect for the preparation of a pharmaceutical formulation.
The formulation may be a liquid formulation.
The liquid preparation is injection, aerosol inhalation, spray or capsule.
The injection is solution type injection, suspension type injection or emulsion type injection.
In a fourth aspect, the present invention provides the use of a composition according to the first aspect.
Use of a composition according to the first aspect for the preparation of a medicament for immunomodulation, or for the treatment and/or prophylaxis of viral infectious diseases and tumours.
In some embodiments, the use of a composition according to the first aspect for the manufacture of a medicament for the treatment and/or prophylaxis of acute and chronic viral hepatitis, condyloma acuminatum, chronic cervicitis, cervical erosion, vaginitis, diseases caused by endopapilloma virus, diseases caused by herpes simplex virus, hairy cell leukemia, chronic granulocytic leukemia, lymphoma, aids-related kaposi's sarcoma, or malignant melanoma.
Advantageous effects
Compared with the prior art, the invention has at least one of the following beneficial technical effects:
(1) The invention is beneficial to improving the stability of the interferon composition by adding the L-ascorbic acid, avoiding the reduction of the specific activity of the interferon and the increase of related proteins under long-term conditions (-25+/-5 ℃) and acceleration conditions (5+/-3 ℃), and remarkably prolonging the effective period.
(2) The higher the purity of the L-ascorbic acid added, the more advantageous is the improvement of the specific activity of the interferon in the composition.
(3) The invention is beneficial to reducing the activity loss caused by adsorption and aggregation of the gas-liquid interface when the interferon is frozen by adding the surfactant, and can avoid the reduction of the specific activity of the interferon under long-term conditions (-25+/-5 ℃) and acceleration conditions (5+/-3 ℃).
(4) The L-ascorbic acid and the surfactant have synergistic technical effects, and the combination of the L-ascorbic acid and the surfactant can greatly improve the stability of the specific activity of the kana resistant interferon.
(5) The invention does not need to add propylene glycol, amino acid or human serum albumin as a protective agent, is beneficial to avoiding the irritation of the substances to the human body or the intolerance of the substances to the human body, is beneficial to avoiding the degradation of the substances in the placing process to bring new impurities, and is beneficial to improving the safety of medication; in addition, the invention does not need propylene glycol with hemolysis, glycine, alanine or human serum albumin with high storage difficulty, preparation difficulty and cost, and the invention has the advantages of less required materials, low cost, safety, green and environment-friendly property and is beneficial to industrialized production.
(6) The preparation method provided by the invention is simple to operate and is beneficial to industrial production.
Definition of terms:
in the invention, "room temperature" means the ambient temperature, which can be 20-30 ℃; in some embodiments, 22 ℃ to 28 ℃; in some embodiments, 24 ℃ to 26 ℃; in some embodiments, 25 ℃.
The term "interferon" may be a recombinant interferon or an interferon prepared by other methods, such as an extracted natural interferon, e.g., "human interferon alpha 2b" includes recombinant human interferon alpha 2b or human interferon alpha 2b prepared by other methods (e.g., extracted natural human interferon alpha 2 b).
In the context of the present invention, all numbers disclosed herein are approximations, whether or not the word "about" or "about" is used. Based on the numbers disclosed, there is a possibility that the values of each number may differ by less than + -10% or a reasonable difference as recognized by those skilled in the art, such as + -1%, + -2%, + -3%, + -4%, or + -5%.
The terms "optional," "optional," or "optionally" mean that the subsequently described event or circumstance may, but need not, occur. For example, "optional surfactant" means that the surfactant may or may not be present.
The term "weight percent" or "percent by weight" or "wt%" is defined as the weight of an individual component in a composition divided by the total weight of all components of the composition and then multiplied by 100.
The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.
As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or moderating the disease state, and alleviating or improving prognosis.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
In this application, the "composition" may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.
Detailed Description
In order to better understand the technical solution of the present invention, some non-limiting examples are further disclosed below to further describe the present invention in detail.
The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.
Comparative example 1: preparation of Interferon alpha 2b stock solution and stability investigation thereof
Construction of two different resistance engineering bacteria of ampicillin (ampicillin Lin Kangxing) and kana (kanamycin resistance) by E.coli to express interferon alpha 2b (respectively named as ampicillin resistant human interferon alpha 2b and kana resistant human interferon alpha 2 b) in a fermentation mannerThe engineering bacteria adopts the same culture medium and fermentation conditions, under the similar purification process and parameters, the final step of purifying and collecting the interferon alpha 2b stock solution contains the interferon with the concentration of 1mg/l-3mg/ml and the specific activity of 1.05X10 8 IU/mg-1.5×10 8 IU/mg. The two stock solutions contained: 1mg/L-3mg/ml interferon degree alpha 2b, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer salt 10mmol/L. The two stock solutions were subjected to long term (-25.+ -. 5 ℃) and accelerated (5.+ -. 3 ℃) investigation and the final investigation results are shown in tables 1, 2, 3 and 4.
Table 1: stability examination result of ampicillin-resistant human interferon a2b stock solution under long-term condition (-25+ -5 ℃)
Table 2: results of examination of stability of Canada resistant human interferon alpha 2b stock solution under long-term conditions (-25.+ -. 5 ℃ C.)
Table 3: stability test results of acceleration condition (5+ -3 ℃) of ampicillin-resistant human interferon alpha 2b stock solution
Table 4: results of examination of stability of kana-resistant human interferon alpha 2b stock solution under acceleration conditions (5.+ -. 3 ℃ C.)
Conclusion: the two stock solutions of ampicillin and kana have no obvious difference in the relative protein, specific activity and the like in 0 month. However, the stability studies of the two stock solutions under the same conditions show a significant difference. Specifically, the kana-resistant stock solution is inferior to the ampicillin-resistant stock solution in terms of activity decrease and the growth degree of related proteins when the stock solution is examined for a long time. The difference of the resistance genes causes the cell wall structure morphology of the thalli and the purification process to bring corresponding change influence, and finally causes the stability difference of the stock solution.
Example 1: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 5.
Table 5: interferon alpha 2b composition formulation (based on total volume or total mass of the composition)
The preparation method comprises the following steps: preparing a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate according to the concentration shown in Table 5, dissolving L-ascorbic acid to a target concentration, performing ultrafiltration exchange of the collection solution purified in the last step of the kana-resistant human interferon a2b by using the buffer solution containing the L-ascorbic acid (the preparation method is the same as that of comparative example 1), performing filtration sterilization by using a 0.2 mu m filter membrane, subpackaging, and filling nitrogen into a headspace part to obtain the interferon alpha 2b composition.
Example 2: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 6.
Table 6: interferon alpha 2b composition formulation (based on total volume or total mass of the composition)
The preparation method comprises the following steps: preparing a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate according to the concentration shown in Table 6, dissolving L-ascorbic acid to a target concentration, performing ultrafiltration exchange of the collection solution purified in the last step of the kana-resistant human interferon alpha 2b by using the buffer solution containing the L-ascorbic acid (the preparation method is the same as that of comparative example 1), performing filtration sterilization by using a 0.2 mu m filter membrane, subpackaging, and filling nitrogen into a headspace part to obtain the interferon alpha 2b composition.
Example 3: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 7.
Table 7: interferon alpha 2b composition formulation (based on total volume or total mass of the composition)
The preparation method comprises the following steps: preparing a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate according to the concentration shown in Table 7, dissolving L-ascorbic acid and Tween 80 to a target concentration, carrying out ultrafiltration exchange of the collection solution purified in the last step of the kana-resistant human interferon alpha 2b by using the buffer solution containing the L-ascorbic acid and the Tween 80 (the preparation method is the same as that of comparative example 1), finally filtering and sterilizing by using a 0.2 mu m filter membrane, subpackaging, and filling nitrogen into a headspace part to obtain the interferon alpha 2b composition.
Example 4: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 8.
Table 8: interferon alpha 2b composition formulation (based on total volume or total mass of the composition)
The preparation method comprises the following steps: preparing a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate according to the concentration shown in Table 8, dissolving L-ascorbic acid and Tween 80 to a target concentration, carrying out ultrafiltration exchange of the collection solution purified in the last step of the kana-resistant human interferon alpha 2b by using the buffer solution containing the L-ascorbic acid and the Tween 80 (the preparation method is the same as that of comparative example 1), finally filtering and sterilizing by using a 0.2 mu m filter membrane, subpackaging, and filling nitrogen into a headspace part to obtain the interferon alpha 2b composition.
Example 5: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 9.
Table 9: interferon alpha 2b composition formulation (based on total volume or total mass of the composition)
The preparation method comprises the following steps: preparing buffer solution of citric acid and sodium citrate according to the concentration shown in Table 9, dissolving L-ascorbic acid and Tween 80 to target concentration, carrying out ultrafiltration liquid exchange on the collection liquid purified in the last step of the kana-resistant human interferon alpha 2b by using the buffer solution containing the L-ascorbic acid and the Tween 80 (the preparation method is the same as that of comparative example 1), finally filtering and sterilizing by using a 0.2 mu m filter membrane, sub-packaging, and filling nitrogen into a part of the headspace to obtain the interferon alpha 2b composition.
Example 6: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 10.
Table 10: interferon alpha 2b composition prescription
The preparation method comprises the following steps: preparing a buffer solution of acetic acid and sodium acetate according to the concentration shown in Table 10, dissolving L-ascorbic acid and Tween 80 to a target concentration, performing ultrafiltration exchange of the collection solution purified in the last step of the kana-resistant human interferon alpha 2b by using the buffer solution containing the L-ascorbic acid and the Tween 80 (the preparation method is the same as that of comparative example 1), performing filtration sterilization by using a 0.2 mu m filter membrane, sub-packaging, and filling part of the headspace with nitrogen gas to obtain the interferon alpha 2b composition.
Example 7: preparation of Interferon alpha 2b compositions
Prescription: as shown in table 11.
Table 11: interferon alpha 2b composition prescription
The preparation method comprises the following steps: preparing a buffer solution of disodium hydrogen phosphate and sodium dihydrogen phosphate according to the concentration shown in table 11, dissolving Tween 80 to a target concentration, performing ultrafiltration exchange on the collected solution purified in the last step of the kana-resistant human interferon alpha 2b by using the buffer solution containing Tween 80 (the preparation method is the same as that of comparative example 1), performing filtration sterilization by using a 0.2 mu m filter membrane, sub-packaging, and filling nitrogen into a part of the headspace to obtain the interferon alpha 2b composition.
Example 8: stability detection
The interferon alpha 2b compositions obtained in examples 1 to 7 were examined under accelerated conditions (5.+ -. 3 ℃ C.) and the final examination results are shown in Table 12.
Table 12: stability test results under acceleration conditions (5.+ -. 3 ℃ C.)
Analysis of results: from the results in Table 11 and tables 1 to 4, it can be seen that:
(1) From the results of example 1, example 2 and tables 1 to 4, it is understood that the present invention is advantageous to improve the specific activity of the Canada resistant interferon under accelerated conditions and the stability of the relevant proteins by adding an appropriate amount of L-ascorbic acid, and in particular, the specific activity of the Canada resistant interferon under accelerated conditions and the stability of the relevant proteins can be improved significantly by adding an appropriate amount of high-purity L-ascorbic acid, and the higher the purity of L-ascorbic acid is, the better the specific activity of the Canada resistant interferon under accelerated conditions and the stability of the relevant proteins are.
(2) From the results of example 2 and example 3, it was found that the addition of the surfactant, on the basis of L-ascorbic acid, can improve the stability of the specific activity of the interferon against kana and can improve the initial specific activity 0 days after the preparation.
(3) From the results of example 3, example 4 and example 6, it is understood that an appropriate increase in the content of L-ascorbic acid is advantageous in improving the stability of the kana-resistant interferon.
(4) From the results of example 2, example 4 and example 5, it is understood that if example 5 is not adjusted to pH 4.5 but to pH 7.0, the change in specific activity of the accelerating condition-dependent interferon should be theoretically between that of example 2 and example 4, but that of example 5, pH is adjusted to pH 4.5, and that of example 2 and example 4, the change in specific activity of the accelerating condition-dependent interferon is much better than that of example 5, indicating that the preferred pH range of the present invention is 6.0-7.0.
(5) The L-ascorbic acid and the surfactant have synergistic technical effects, and the combination of the L-ascorbic acid and the surfactant can greatly improve the stability of the specific activity of the kana resistant interferon.
In combination, the L-ascorbic acid has outstanding effect on the stabilization of interferon, can comprehensively solve the influence caused by the upstream process, prolongs the storage period of stock solution, and saves the optimization time cost of the upstream process. In addition, through researches, the stability of the interferon stock solution is further improved by collocating the components of the stock solution composition, such as adding the surfactant, and the preservation time of the stock solution is remarkably prolonged.
While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and combinations of the methods and applications described herein can be made and applied within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.

Claims (2)

1. A composition comprising human interferon alpha 2b, L-ascorbic acid, phosphate buffer salt, and tween 80;
the purity of the L-ascorbic acid is more than or equal to 98%;
the L-ascorbic acid is present in an amount of 5mmol/L based on the total volume of the composition;
the phosphate buffer salt is present at a concentration of 10mmol/L based on the total volume of the composition;
the tween 80 content was 0.05wt%, based on the total mass of the composition;
the interferon is present in an amount of 1.0mg/mL based on the total volume of the composition;
the pH of the composition is 6.0-7.0.
2. The composition of claim 1, wherein the composition does not comprise propylene glycol, glycine, alanine, or human serum albumin.
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