HUMAN ALBUMIN-CONTAINING MEDICAL PRODUCT AND PREPARATION METHOD THEREOF
20220313790 · 2022-10-06
Assignee
Inventors
Cpc classification
A61K47/10
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
A61P7/04
HUMAN NECESSITIES
A61P9/02
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
International classification
A61K47/10
HUMAN NECESSITIES
Abstract
A preparation method of a human albumin-containing medical product comprises controlling a content of long-chain fatty acids in the medical product and adding poloxamer to the medical product. The long-chain fatty acid is one or more selected from myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid, and a molar ratio of the long-chain fatty acids and human albumin is 0.2-3.0. A content of the poloxamer is 10-500 μg/g of human albumin. The human albumin-containing medical product prepared by the method has improved stability and does not contain particle of over 30 nm in diameter is formed from aggregation and/or fibrillation of human albumin.
Claims
26. A human albumin-containing medical product, wherein the medical product comprises poloxamer and long-chain fatty acids.
27. The human albumin-containing medical product according to claim 26, wherein a molar ratio of the long-chain fatty acids and human albumin in the medical product is 0.2-0.3:2.0-3.0.
28. (canceled)
29. The human albumin-containing medical product according to claim 26, wherein a content of poloxamer in the medical product is 10-500 μg poloxamer/1 g of human albumin.
30. The human albumin-containing medical product according to claim 26, wherein the poloxamer is one or more selected from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407.
31. The human albumin-containing medical product according to claim 26, wherein the poloxamer is poloxamer 188.
32. The human albumin-containing medical product according to claim 26, wherein the long-chain fatty acid is one or more selected from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4).
33. The human albumin-containing medical product according to claim 26, wherein the long-chain fatty acid is soya fatty acid or fully hydrogenated soya fatty acid.
34. The human albumin-containing medical product according to claim 26, wherein the medical product further comprises ingredients and contents required by local pharmacopoeias, and the ingredients required by local pharmacopoeias comprise sodium caprylate or/and N-acetyltryptophan.
35. (canceled)
36. A preparation method of a human albumin-containing medical product, wherein the method comprises: improving the stability of human albumin in the medical product according to claim 26, by controlling the content of long-chain fatty acids in the medical product and adding poloxamer to the medical product.
37. The method according to claim 36, wherein the method further comprises: improving the stability of human albumin in the medical product by reducing aggregation and fibrillation of human albumin.
38. The method according to claim 36, wherein the method further comprises: improving the stability of human albumin in the medical product so that the stability meets the following conditions: no turbidity or visible particles are formed during incubation at 57° C. for 50 hours, at 30° C. for 14 days and at 20° C. for 28 days.
39. The method according to claim 36, wherein the human albumin is human serum albumin or recombinant human albumin.
40. The method according to claim 36, wherein the long-chain fatty acids are added in the form of salts.
41. The method according to claim 36, wherein the long-chain fatty acids are added in the form of sodium salts.
42. The method according to claim 36, wherein the medical product contains no particle of over 30 nm-200 nm in diameter due to aggregation and/or fibrillation of human albumin.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DESCRIPTION OF THE EMBODIMENTS
[0072] Embodiments of the present invention are illustrated hereinafter through specific examples. Other advantages and effects of the present invention can be readily understood by those of skill in the art via what is disclosed in the specification. The invention can also be implemented or applied in other different embodiments. The details in this specification can also be modified or changed in various ways based on different views and applications without departing from the spirit of the invention. It shall be noted that the following embodiments and the features in the embodiments can be combined with each other where there is no conflict.
[0073] The terms used in this application are defined and explained hereinafter.
[0074] The term “human albumin” means human serum albumin or recombinant human albumin, the recombinant human albumin also being called recombinant human serum albumin or recombinant human albumin mutant. Recombinant human albumin includes at least human albumin derived from microbial genetic recombination, eukaryotic cellular recombinant human albumin, plant transgenic recombinant human albumin and animal transgenic recombinant human albumin and the like.
[0075] The term “medical product” shall be understood to include at least a solution product, a freeze-dried product or a low-temperature spray-dried product, preferably a solution product which is typically filled aseptically in a glass or plastic container of a defined volume.
[0076] The term “human albumin-containing medical product” shall be understood as medical products for clinical injections, culture media, drug excipients, medical devices, vaccine excipients, cosmetics, analytical diagnostic reagents and many other aspects containing human albumin of varied concentrations, for example, a human albumin concentration of 1%-30%, preferably 10%-25%.
[0077] Poloxamer is a non-ionic surfactant, a block copolymer of ethylene oxide and 1,2-epoxypropane, with the general formula H(C2H4O) a(C3H6O) b(C2H4O) aOH. With a high degree of biosafety, poloxamer 188 is listed as an emulsifier for intravenous fat emulsions and a stabilizer for antibody injections in the Chinese Pharmacopoeia 2015 edition, the US Pharmacopoeia, the European Pharmacopoeia and in approved drugs.
[0078] The term “long-chain fatty acids” refers to fatty acids with more than 12 carbon atoms in the carbon chain and should be understood to mean fatty acids of various origins (synthetic or extracted) that are acceptable to humans, or fatty acids present in various forms (e.g., salts, especially sodium salts), including but not limited to one or more from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4).
[0079] The term “control” with the subject of long-chain fatty acid content in a human albumin-containing medical product, means maintaining the content of long-chain fatty acids in a human albumin-containing medical product within a given range: in a case of long-chain fatty acids inherent in a human albumin-containing medical product prepared by existing methods, such as a human albumin-containing medical product of human blood origin, if the content of long-chain fatty acids is below a given range, “control” means adding long-chain fatty acids during the preparation process of the human albumin-containing medical product such that the amount of long-chain fatty acids in the final human albumin-containing medical product is within a given range; if the content of long-chain fatty acids is already within a given range, “control” means no action is required during the preparation process of the human albumin-containing medical product; if the human albumin-containing medical product prepared by the existing method contains no or a very small amount of long-chain fatty acids, “control” means adding long-chain fatty acids during the preparation process of the human albumin-containing medical product such that the content of long-chain fatty acids in the final human albumin-containing medical product is within a given range.
[0080] The term “long-chain fatty acids” shall be understood as a fixed phrase, wherein there is no logical relationship between the “or” and the context of the phrase. For calculation of the content of “long-chain fatty acids”, “long-chain fatty acids” refers to the sum of amounts of each long-chain fatty acid. In order to have the content of long-chain fatty acids within a given range, either a long chain fatty acid or a mixture of long-chain fatty acids can be added.
[0081] The term “content of long-chain fatty acids” shall be understood as the content of long-chain fatty acids in the final human albumin-containing medical product, including the amount of residual long-chain fatty acids during the preparation process due to the long-chain fatty acids inherent in the source materials, and the amount of the additionally added long-chain fatty acids (if any addition during the “controlling” process).
[0082] The term “add”, when the subject thereof is poloxamer, shall be understood as adding poloxamer during the preparation process of the human albumin-containing medical product such that the poloxamer content in the final human albumin-containing medical product is within a given range.
[0083] The steps of “controlling” and “adding” shall be understood as being carried out during the preparation process of the human albumin-containing medical product such that the content of long-chain fatty acids and the content of poloxamer in the final human albumin-containing medical product are each within a given range. For example, the steps can be performed on the final bulk of the formulation, or performed by ultrafiltration and dialysis processes, or poloxamer and long-chain fatty acids can be added as protein stabilizers before pasteurization, or the steps can be performed in the last one or the last few steps of the purification chromatography process, in order to obtain the content range specified in the present invention for poloxamer and long-chain fatty acids in the final human albumin-containing medical product.
[0084] The term “improve” shall be understood as a higher stability of the human albumin-containing medical product of the present invention than that of existing human albumin, for example, it is achieved in the present invention that no particle of 30-300 nm in diameter is formed during storage, shipping or ambient-temperature cultivation due to aggregation or fibrillation of human albumin.
[0085] The term “particle” shall be understood as a substance of a certain range in diameter and formed during storage, shipping and ambient-temperature cultivation of the human albumin-containing medical product due to aggregation or fibrillation of human albumin, the preferred particle diameter in the present invention being 30-300 nm.
[0086] The human albumin-containing medical product of the present invention can be used to inhibit aggregates, agglomerates and fibrillation formed by freeze and thaw in cold drying, freeze drying, and low-temperature spray drying, and to accelerate reconstitution.
[0087] The human albumin-containing medical product of the present invention includes but not limited to human serum-derived albumin prepared by Cohn method and polishing, and recombinant human albumin purified by varied steps.
[0088] The pH range of the human albumin-containing medical product in the present invention is 6-8, and preferably 6.4-7.4, the pH values in each national pharmacopoeial specification.
[0089] The human albumin-containing medical product of the present invention may further include diluents, buffers, solubilizers, excipients, pH regulators, sulfur-containing reducing agents, antioxidants and the like. The sulfur-containing reducing agents include N-acetyl-DL-tryptophan (N-acetyltryptophan), N-acetyl-L-tryptophan, N-acetylmethionine, N-acetyl-L-methionine, etc. Commonly used buffers and diluents include sodium chloride solution, sodium phosphate buffer solution, sodium acetate solution, and sodium citrate solution, preferably within a range according to each national pharmacopoeial specification.
[0090] The human albumin-containing medical product of the present invention may be further supplemented with medium-chain fatty acids, such as sodium caprylate, sodium heptanoate, sodium decanoate, and preferably sodium caprylate of the pharmacopoeial specification.
[0091] The application of the human albumin-containing medical product of the present invention includes but not limited to aspects like clinical injections, culture media, drug excipients, medical devices, vaccine excipients, cosmetics and analytical diagnostic reagents.
[0092] Embodiments of the present invention are illustrated hereinafter by specific examples.
[0093] Embodiment 1
[0094] Commercial human serum albumin (sample 1, Shanghai RAAS blood products Co., Ltd., batch number: 201708A010) was used, the formulation thereof being: human albumin with excipients sodium caprylate and acetyltryptophan. The formulation thereof is in accordance with pharmacopoeial requirements, i.e., 0.079 mmol of sodium caprylate is added on the basis of 1 g of albumin, and 0.076 mmol of acetyltryptophan is added on the basis of 1 g of albumin, and the pH is 6.8. The content of residual long-chain fatty acids after purification of the serum-derived human albumin was tested by gas chromatography. The detailed method of human albumin analysis by gas chromatography (GC) was as follows:
[0095] Gas chromatography: model: 7890A; manufacturer: Agilent Technologies Co., Ltd.
[0096] GC column: acid-deactivated polyethylene glycol capillary column TG-WAXMS A by Thermo Fisher Scientific Co., Ltd.; specification: 30 m*0.32 mm*0.25 μm;
[0097] Materials and reagents: C17 heptadecanoic acid: grade: USA reference material; batch number: N-17A-JY16X; strength: >100 mg; content: ≥99.0%; palmitic acid: grade: national certified reference material; batch number: 190032-201603; strength: 200 mg/ampoule; stearic acid: grade: national certified reference material; batch number: 190032-201603; strength: 200 mg/ampoule; oleic acid: grade: national certified reference material; batch number: 111621-2015-06; strength: 100 mg/ampoule; content: ≥99.0%; linoleic acid: grade: USA reference material; batch number: U-59A-J6-Y; strength: >100 mg/ampoule; content: ≥99.0%; linolenic acid: grade: USA reference material; batch number: U-62A-S20-B; strength: ≥100 mg/ampoule; content: ≥99.0%; chloroform: grade: AR; batch number: 20170410; strength: 500 ml/bottle; content: ≥99.0%; manufacturer: Sinopharm.
[0098] Experimental method: in accordance with the Pharmacopoeia of the People's Republic of China, 2015 edition: adding the internal standard substance C17 heptadecanoic acid and calculating the absolute peak areas of palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and C17 heptadecanoic acid, plotting a linear regression with the peak area ratio between each fatty acid reference solution and the internal standard against the amount of each fatty acid reference solution, obtaining the linear regression equation, and calculating the absolute peak area of each fatty acid in the test solution, thereby obtaining calculated content of each fatty acid in the test sample.
[0099] Chromatography conditions: temperature: 260° C.; column flow: 2.0 ml/min; purge flow: 20 ml/min; split flow: 2 ml/min; temperature program: initial, hold for 5 min at 80° C., increase to 230° C. at 10° C./min and hold for 25 min; 45 min in total; detector: model: FID; temperature: 280° C.; hydrogen: 40 ml/min; air: 450 ml/min; makeup gas flow rate: 45 ml/min.
[0100] The C17 heptadecanoic acid was weighed accurately to prepare the internal standard solution; the fatty acid standards were weighed accurately and mixed as the reference solution. A standard curve was determined to calculate the content of fatty acids in human albumin.
[0101] Test results of fatty acid content in human albumin injection (Shanghai RAAS, 201703A010):
[0102] Content of C16:0 (palmitic acid) (mol/mol protein): 0.241
[0103] Content of C18:0 (stearic acid) (mol/mol protein): 0.147
[0104] Content of C18:1 (oleic acid) (mol/mol protein): 0.077
[0105] Content of C18:2 (linoleic acid) (mol/mol protein): 0.085
[0106] Content of C18:3 (linolenic acid) (mol/mol protein): not detected
[0107] Content of C20:4 (arachidonic acid) (mol/mol protein): not detected
[0108] Total of the above: proportion of long-chain fatty acids (mol/mol of human albumin): 0.55:1
[0109] Dynamic light scattering was employed for testing:
[0110] dynamic light scattering (DLS) model: Litesizer 500; manufacturer: Anton Paar (Shanghai) Trading Co., Ltd;
[0111] cuvettes: quartz cells;
[0112] sample: bulk sample solution was tested.
[0113] Method: measurement angle: automatic; temperature range: 25° C. -60° C. ; step: 5° C.
[0114] Particles of 30-300 nm due to instability of the commercial human serum albumin needs to be removed by gel filtration; the chromatography conditions thereof are as follows:
[0115] G-25 packing material (General Electric Company), chromatography device: EV 50D chromatography system (Lisure Science (Suzhou) Co., Ltd.), buffer system: 0.1 mol/L phosphoric acid solution, neutral pH and purified water. The sample was concentrated using a 30K ultrafiltration membrane after chromatography and formulated into a product for testing according to the original product formulation.
[0116]
[0117] The particles of 30 nm-300 nm in sample 1 were removed according to the method above, and 75 μg of poloxamer 188 (BASF, batch number: WPAK527B) was added on the basis of 1 g of human albumin to obtain sample 2. The DLS chromatogram of sample 2 is shown in
[0118] As demonstrated by the DLS results in
[0119] It was further found by the inventors of the present application through tests that, as a variation of this specific embodiment, poloxamer 188 can also be replaced with one of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407, and alternatively, a plurality from poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 can be used simultaneously. Tests have demonstrated that the same effects could be achieved with these poloxamers, i.e., after removal of particles, micro-particles were absent between 30nm and 300nm at both 25° C. and 60° C. in human serum albumin with a small amount of additional poloxamer.
[0120] The inventors of this application have also found through tests that the desired effect can be achieved by adding only a small amount of poloxamers. In general, with an additional 10 μg or more of poloxamers/gram of human albumin, micro-particles between 30 nm and 300 nm are basically undetectable or only a small amount of micro-particles between 30 nm and 300 nm are present; and with an additional over 500 μg of poloxamers/gram of human albumin, there is a significant increase in micro-particles between 30 nm and 300 nm.
Embodiment 2
[0121] Commercial human serum albumin (sample 3, Shantou Weilun Bio-pharmaceutical Co.,
[0122] Ltd., batch number: 20170104) was used, the formulation ingredients thereof being human albumin with excipients sodium caprylate and sodium chloride. The formulation is in accordance with pharmacopoeial requirements, i.e., 0.159 mmol of sodium caprylate is added on the basis of 1 gram of albumin, with no additional N-acetyltryptophan. The pH is 6.7, and content of total sodium is not more than 160 mmol/L. The method of Embodiment 1 was adopted to determine the content of residual long-chain fatty acids in sample 3, and the proportion of long-chain fatty acids (mol/mol of human albumin) was 0.34:1.
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[0124] The particles of 30-300 nm in sample 3 were removed according to the method in Embodiment 1, and 100 μg of poloxamer 188 (BASF, batch number WPAK527B) was added on the basis of 1 g of human albumin to obtain sample 4. The DLS chromatogram of sample 4 is shown in
[0125] As shown by the DLS results in
[0126] Embodiments 1 and 2 have demonstrated that particles of about 200 nm are present in both standard human serum album formulations of the pharmacopoeia when poloxamer is not added; in a case of a small amount of additional poloxamer, the formation of micro-particles can both be effectively inhibited and prevented.
[0127] It was further found by the inventors of the present application through tests that, as a variation of this specific embodiment, poloxamer 188 can also be replaced with one of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407, and alternatively, a plurality from poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 can be used simultaneously. Tests have demonstrated that the same effects could be achieved with these poloxamers, i.e., after removal of particles, micro-particles were absent between 30nm and 300nm at both 25° C. and 60° C. in human serum albumin with a small amount of additional poloxamer.
[0128] The inventors of this application have also found through tests that the desired effect can be achieved by adding only a small amount of poloxamers. In general, with an additional 10 μg or more of poloxamers/gram of human albumin, micro-particles between 30 nm and 300 nm are basically undetectable or only a small amount of micro-particles between 30 nm and 300 nm are present; and with an additional over 500 μg of poloxamers/gram of human albumin, there is a significant increase in micro-particles between 30 nm and 300 nm.
Embodiment 3
[0129] The recombinant human albumin bulk of high purity obtained after improved purification according to the method in the Chinese patent application (application no. 201010124935.2) was used herein. The tested residual content of long-chain fatty acids of the purified recombinant human albumin as indicated by the proportion of long-chain fatty acids (mol/mol of human albumin) equaled to 0.059:1, which is nearly 10 times lower than the content of long-chain fatty acids in human serum-derived albumin. Without any addition of long-chain fatty acids or poloxamers, sample 5 was prepared according to the method requirements of the Chinese Pharmacopoeia 2015 edition only, i.e., 0.071 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) was added on the basis of 1 g of albumin, and 0.085 mmol of acetyltryptophan (Adamas Reagent Co., Ltd, batch No. P230264) was added on the basis of 1 g of albumin; pH was 6.8; and the total sodium content was controlled to be not more than 160 mmol/L.
[0130]
Embodiment 4
[0131] Preparation of sample 6: taking the recombinant human albumin bulk of high purity obtained by the method of Embodiment 3 above, adding sodium oleate (BBI Life Sciences, batch number: E802BA0026) to the bulk till an 1.0:1.0 molar ratio between long-chain fatty acids and recombinant human albumin is achieved (residual long-chain fatty acids from the bulk were counted), adding 0.081 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) on the basis of 1 g of albumin, and adding 0.067 mmol acetyltryptophan (Adamas Reagent Co., Ltd Batch No. P230264) on the basis of 1 g of albumin, pH being 6.7, and controlling the total sodium to be not more than 160 mmol/L.
[0132]
Embodiment 5
[0133] Preparation of sample 7: taking the recombinant human albumin bulk of high purity obtained by the method of Embodiment 3 above, adding 100 μg of poloxamer 188 (BASF, batch no. WPAK527B) on the basis of 1 g of human albumin, adding 0.079 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) on the basis of 1 g of albumin, and adding 0.066 mmol of acetyltryptophan (Adamas Reagent Co., Ltd Batch No. P230264) on the basis of 1 g of albumin, pH being 6.6, and controlling the total sodium to be not more than 160 mmol/L.
[0134]
Embodiment 6
[0135] Preparation of sample 8: taking the recombinant human albumin bulk of high purity obtained by the method of the Embodiment 3 above, adding 100 μg of poloxamer 188 (BASF, batch no. WPAK527B) on the basis of 1 gram of human albumin, adding sodium oleate (BBI Life Sciences, batch number: E802BA0026) till an 1.0:1.0 molar ratio between long-chain fatty acids and recombinant human albumin was achieved (residual long-chain fatty acids from the bulk were counted), adding 0.083 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) on the basis of 1 g of albumin, and adding 0.084 mmol of acetyltryptophan (Adamas Reagent Co., Ltd Batch No. P230264) on the basis of 1 g of albumin, pH being 6.8, and controlling the total sodium to be not more than 160 mmol/L.
[0136]
Embodiment 7
[0137] Preparation of sample 9: taking the recombinant human albumin bulk of high purity obtained by the method of Embodiment 3 above, adding 100 μg of poloxamer 188 (BASF, batch no. WPAK527B) on the basis of 1 g of human albumin, adding sodium soyate (Qingdao Raynol Chemical Co., Ltd., batch no. 20190121) till a 0.5:1.0 molar ratio between long-chain fatty acids and recombinant human albumin was achieved (residual long-chain fatty acids from the bulk were counted), adding 0.082 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) on the basis of 1 g of albumin, and adding 0.080mmol of acetyltryptophan (Adamas Reagent Co., Ltd, batch No. P230264) on the basis of 1 g of albumin, pH being 6.9, and controlling the total sodium to be not more than 160 mmol/L.
[0138]
Embodiment 8
[0139] Preparation of sample 10: taking the recombinant human albumin bulk of high purity obtained by the method of Embodiment 3 above, adding 50 μg of poloxamer 188 (BASF, batch no. WPAK527B) on the basis of 1 g of human albumin, mixing well sodium stearate (Damas-beta, batch number: P1042477) and sodium oleate (BBI Life Sciences, batch number: E802BA0026) in a molar ratio of 1:2 and adding to the bulk till a 0.6:1.0 molar ratio between mixed long-chain fatty acids and recombinant human albumin was achieved (residual long-chain fatty acids from the bulk were counted), adding 0.086 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) on the basis of 1 g of albumin, and adding 0.076 mmol of acetyltryptophan (Adamas Reagent Co., Ltd Batch No. P230264) on the basis of 1 g of albumin, pH being 7.0, and controlling the total sodium to be not more than 160 mmol/L.
[0140]
Embodiment 9
[0141] Preparation of sample 11: taking the recombinant human albumin bulk of high purity obtained by the method of Embodiment 3 above, adding 50 μg of poloxamer 188 (BASF, batch no. WPAK527B) on the basis of 1 g of human albumin, mixing well sodium stearate (Damas-beta, batch number: P1042477) and sodium oleate (BBI Life Sciences, batch number: E802BA0026) in a molar ratio of 1:2 and adding to the bulk till a 0.6:1.0 molar ratio of long-chain fatty acid mixture to recombinant human albumin was achieved (residual long-chain fatty acids from the bulk were counted), adding 0.153 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, batch No. 20170901) on the basis of 1 g of albumin, pH being 7.0, and controlling the total sodium to be not more than 160 mmol/L.
[0142]
Embodiment 10
[0143] Incubation and thermostability tests of the samples prepared in Embodiments 1-9 (Samples 1-11). The method thereof was in accordance with the Chinese Pharmacopoeia, 2015 edition, Volume III, p. 244-245. The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Observation results for stability of samples 1-11 Incubation Incubation Incubation Sample at 57° C. for at 30° C. for at 20° C. No. 50 hours 14 days for 28 days Sample 1 (−) (−) (−) Sample 2 (−) (−) (−) Sample 3 (−) (−) (−) Sample 4 (−) (−) (−) Sample 5 (+) (+) (+) Sample 6 (+) (+) (+) Sample 7 (+) (+) (+) Sample 8 (−) (−) (−) Sample 9 (−) (−) (−) Sample 10 (−) (−) (−) Sample 11 (−) (−) (−) (Note: − is negative indicating qualified; + is positive indicating unqualified)
[0144] The above embodiments demonstrated the effect of oleic acid, sodium soyate, mixture of sodium stearate and sodium oleate, and mixture of sodium stearate and sodium oleate, in combination with poloxamer in promoting the stability of recombinant human albumin products. It was further found by the inventors of the present application through tests that, as a variation of the embodiments, the long-chain fatty acids can also be one or more selected from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4). Tests have demonstrated that the same effect can be achieved by these long-chain fatty acids, i.e., the combined use thereof with poloxamers can promote the stability of recombinant human albumin products. Negative results were shown in the above stability test.
[0145] It was further found by the inventors of the present application through tests that, as a variation of the above embodiments, poloxamer 188 can also be replaced with one of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407, and alternatively, a plurality from poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 can be used simultaneously. Tests have demonstrated that the same effect can be achieved by these poloxamers; the stability of human serum albumin can meet requirements with the addition of a quantity of long-chain fatty acids and a small amount of poloxamer.
[0146] The inventors of this application have also found through tests that the desired effect can be achieved by adding only a small amount of poloxamer. In general, the observation results for stability are substantially negative when 10 μg or more of poloxamer/g of human albumin is added; and when more than 500 μg of poloxamer/g of human albumin is added, positive results start to appear in the observation tests for stability.
[0147] It was further found by the inventors of the present application through tests that, the stability of human albumin medical product is at its best when the molar ratio between long-chain fatty acids and human albumin is 0.2-3.0, and particularly 0.3-2.0. When the molar ratio between long-chain fatty acids and human albumin is lower than 0.3 or greater than 2.0, positive results start to appear in a small amount in the observation tests for stability, whereas when the molar ratio between long-chain fatty acids and human albumin is lower than 0.2 or greater than 3.0, positive results start to appear in a large amount in the observation tests for stability.
Embodiment 11
[0148] Samples 5 to 11 prepared by the method of Embodiments 3 to 9 were subjected to differential scanning calorimetry (DSC) analysis and DSC chromatograms were obtained. DSC model: Nano DSC 602000; manufacturer: TA; sample diluent: 0.01 mol/L phosphoric acid with a neutral pH. Test conditions: the bulk sample was diluted with the diluent to 1-5 mg/ml; temperature range: 45° C.-90° C.; step: 1° C./min. The results are shown in Table 2.
TABLE-US-00002 TABLE 2 DSC results of samples 5-11 Sample no. T.sub.onset T.sub.m1 T.sub.m2 Recombinant human albumin 60.9° C. 65.0° C. 70.1° C. bulk of high purity Sample 5 62.9° C. 66.7° C. 70.5° C. Sample 6 63.2° C. 68.0° C. 73.7° C. Sample 7 62.8° C. 67.0° C. 71.6° C. Sample 8 63.3° C. 68.3° C. 74.2° C. Sample 9 64.0° C. 68.7° C. 72.9° C. Sample 10 63.6° C. 68.2° C. 70.0° C. Sample 11 64.7° C. 68.8° C. 73.1° C.
[0149] The results above of the present invention have demonstrated a synergistic stabilizing effect of poloxamers and long-chain fatty acids. For human blood-derived albumin preparations, the inherent residual long-chain fatty acids and other ligands from circulation in human body can effected inhibit the appearance of particles of 30-300 nm and improve thermostability with only a small amount of additional poloxamers while no further addition of long-chain fatty acids is required.
[0150] For recombinant human albumin of high purity, which is substantially defatted completely, additional poloxamers are required to inhibit the appearance of particles of 30-300 nm. Comparison between the test results demonstrates that, in a case where only poloxamers, conventional pharmacopoeial sodium caprylate (medium-chain fatty acids) and acetyltryptophan are added, requirements for thermostability can not be met due to a lack of mutual support for the various adjacent domain ligands of albumin by long-chain fatty acids, although medium-chain fatty acids can effectively enhance Tonset, Tm1 and Tm2.
[0151] Therefore, controlling the content of long-chain fatty acids can effectively enhance Tonset, Tm1 and Tm2, and while the connective stability of the protein domains are enhanced, a small amount of additional poloxamers is required to inhibit hydrophobic interactions. The human albumin-containing medical product of the present invention meets the requirements of stability for incubation at 57° C. for 50 hours, at 30° C. for 14 days and at 20° C. for 28 days with excellent stability indicators, meeting requirements of stability for long-term storage and shipping at 2° C.-8° C. and room temperature.
[0152] The embodiments above are merely illustrative of the principles of the invention and efficacy thereof, and are not intended to limit the invention. Any person of the skill in the art may modify or change the above embodiments given that it is not against the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the art, without departing from the spirit and technical concept revealed by the present invention, shall still be covered by the claims of the present invention.