{"id":16434,"date":"2025-10-21T10:07:34","date_gmt":"2025-10-21T03:07:34","guid":{"rendered":"https:\/\/nhathuocngocanh.com\/bp\/?p=16434"},"modified":"2025-11-15T16:04:21","modified_gmt":"2025-11-15T09:04:21","slug":"interferon-beta-1a-concentrated-solution","status":"publish","type":"post","link":"https:\/\/nhathuocngocanh.com\/bp\/interferon-beta-1a-concentrated-solution\/","title":{"rendered":"Interferon Beta-1a Concentrated Solution"},"content":{"rendered":"

(Ph. Eur. monograph 1639)<\/em><\/p>\n

C_{908<\/sub>H_{14<\/sub>O_{6<\/sub>N_{246<\/sub>O_{252<\/sub>S_{7<\/sub> Mr approx. 22 500<\/p>\n}}}}}}

Action and use<\/strong><\/p>\n

Cytokine.<\/p>\n

DEFINITION<\/h2>\n
Solution of a glycosylated protein having the same amino acid sequence and disulfide bridge and a similar glycosylation\u00a0 pattern as interferon beta produced by human diploid fibroblasts in response to viral infections and various other inducers. It exerts antiviral, antiproliferative and immunomodulatory activity.<\/p>\n
Content<\/h3>\n
Minimum 0.20 mg of protein per millilitre.<\/p>\n
Potency<\/h3>\n
Minimum 1.5 \u00d7 10 IU per milligram of protein.<\/p>\n
It may contain buffer salts.<\/p>\n
PRODUCTION<\/h2>\n
Interferon beta-1a concentrated solution is produced by a method based on recombinant DNA (rDNA) technology, using mammalian cells in culture.<\/p>\n
Prior to release, the following tests are carried out on each batch of interferon beta-1a concentrated solution, unless exemption has been granted by the competent authority.<\/p>\n
Host-cell-derived proteins<\/h3>\n
The limit is approved by the competent authority.<\/p>\n
Host-cell or vector-derived DNA<\/h3>\n
The limit is approved by the competent authority.<\/p>\n
N-terminal truncated forms<\/h3>\n
Examination for specific N-terminal truncated forms should be performed using a suitable technique such as N-terminal sequence determination. The limits are approved by the competent authority.<\/p>\n
Dimer and related substances of higher molecular mass<\/h3>\n
Not more than the amount approved by the competent authority, using an appropriate validated liquid chromatography method.<\/p>\n
CHARACTERS<\/h2>\n
Appearance<\/h3>\n
Clear or slightly opalescent, colourless or slightly yellowish liquid.<\/p>\n
IDENTIFICATION<\/h2>\n
A. It shows the expected biological activity (see Assay).<\/p>\n
B. Isoform distribution. Mass spectrometry (2.2.43).<\/p>\n
Introduction of the sample: Direct inflow of a desalted preparation to be examined or liquid chromatography-mass spectrometry combination.<\/p>\n
Mode of ionisation: Electrospray.<\/p>\n
Signal acquisition: Complete spectrum mode from 1100 to 2400.<\/p>\n
Calibration: Use myoglobin in the m\/z range of 600-2400; set the instrument within validated instrumental settings and analyse the sample; the deviation of the measured mass does not exceed 0.02 per cent of the reported mass.<\/p>\n
Interpretation of results: A typical spectrum consists of 6 major glycoforms (A to F), which differ in their degree of\u00a0 sialylation and\/or antennarity type as shown in Table 1639.-1.<\/p>\n
Table 1639.-1.<\/p>\n\n\n\n\n\n\n\n\n\n
MS peak\u00a0<\/strong><\/td>\n **Glycoform*\u00a0<\/strong><\/td>\n Expected M_{r\u00a0<\/sub><\/strong><\/td>\n} Sialylation level<\/strong><\/td>\n<\/tr>\n**
A<\/td>\n 2A2S1F<\/td>\n 22 375<\/td>\n Disialylated<\/td>\n<\/tr>\n
B<\/td>\n 2A1S1F<\/td>\n 22 084<\/td>\n Monosialylated<\/td>\n<\/tr>\n
C<\/td>\n 3A2S1F and\/or 2A2S1F + 1 HexNacHex repeat<\/td>\n 22 739<\/td>\n Disialylated<\/td>\n<\/tr>\n
D<\/td>\n 3A3S1F<\/td>\n 23 031<\/td>\n Trisialylated<\/td>\n<\/tr>\n
E<\/td>\n 4A3S1F and\/or 3A3S1F + 1 HexNacHex repeat<\/td>\n 23 400<\/td>\n Trisialylated<\/td>\n<\/tr>\n
F<\/td>\n 2A0S1F<\/td>\n 21 793<\/td>\n Non-sialylated<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
* 2A = biantennary complex type oligosaccharide; 3A = triantennary complex type oligosaccharide; 4A = tetraantennary complex type oligosaccharide; 0S = non-sialylated; 1S = monosialylated; 2S = disialylated; 3S = trisialylated; 1F = fucosylated.<\/p>\n
Results: The mass spectrum obtained with the preparation to be examined corresponds, with respect to the 6 major peaks, to the mass spectrum obtained with interferon beta-1a CRS.<\/p>\n
C. Peptide mapping (2.2.55) and liquid chromatography (2.2.29).<\/p>\n
Test solution: Add 5 \u03bcL of a 242 g\/L solution of tris(hydroxymethyl)aminomethane R and a volume of the preparation to be examined containing 20 \u03bcg of protein to a polypropylene tube of 0.5 mL capacity. Add 4 \u03bcL of a 1 mg\/mL solution of endoprotease LysC R in 0.05 M tris-hydrochloride buffer solution pH 9.0 R. Mix gently and incubate at 30 \u00b0C for 2 h. Add 10 \u03bcL of a 15.4 g\/L solution of dithiothreitol R. Dilute the solution with the same volume of a 573 g\/L solution of guanidine hydrochloride R. Incubate at 4 \u00b0C for 3-4 h.<\/p>\n
Reference solution: Prepare at the same time and in the same manner as for the test solution but using interferon beta-1a CRS instead of the preparation to be examined.<\/p>\n
Precolumn:<\/p>\n
\u2014 size: l = 0.02 m, \u00d8 = 2.1 mm;<\/p>\n
\u2014 stationary phase: spherical octadecylsilyl silica gel for chromatography R (5 \u03bcm) with a pore size of 30 nm.<\/p>\n
Column:<\/p>\n
\u2014 size: l = 0.25 m, \u00d8 = 2.1 mm;<\/p>\n
\u2014 stationary phase: spherical octadecylsilyl silica gel for chromatography R (5 \u03bcm) with a pore size of 30 nm.<\/p>\n
Mobile phase:<\/p>\n
\u2014 mobile phase A: dilute 1 mL of trifluoroacetic acid R to 1000 mL with water for chromatography R;<\/p>\n
\u2014 mobile phase B: dilute 1 mL of trifluoroacetic acid R in 700 mL of acetonitrile R1, then dilute to 1000 mL with water for chromatography R;<\/p>\n\n\n\n\n\n\n\n\n\n
Time<\/strong><\/p>\n
(min)<\/strong><\/td>\n
Mobile phase A<\/strong><\/p>\n
(per cent V\/V)<\/strong><\/td>\n
Mobile phase B<\/strong><\/p>\n
(per cent V\/V)<\/strong><\/td>\n<\/tr>\n
0 – 30<\/td>\n 100 \u2192 64<\/td>\n 0 \u2192 36<\/td>\n<\/tr>\n
30 – 45<\/td>\n 64 \u2192 55<\/td>\n 36 \u2192 45<\/td>\n<\/tr>\n
45 – 50<\/td>\n 55 \u2192 40<\/td>\n 45 \u2192 60<\/td>\n<\/tr>\n
50 – 70<\/td>\n 40 \u2192 0<\/td>\n 60 \u2192 100<\/td>\n<\/tr>\n
70 – 83<\/td>\n 0<\/td>\n 100<\/td>\n<\/tr>\n
83 – 85<\/td>\n 0 \u2192 100<\/td>\n 100 \u2192 0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
Flow rate: 0.2 mL\/min.<\/p>\n
Detection: Spectrophotometer at 214 nm.<\/p>\n
Injection: Volume that contains 20 \u03bcg of digested protein.<\/p>\n
System suitability: The chromatogram obtained with the reference solution is qualitatively similar to the chromatogram of interferon beta-1a digest supplied with interferon beta-1a CRS.<\/p>\n
Results: The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.<\/p>\n
TESTS<\/h2>\n
Impurities of molecular masses differing from that of interferon beta-1a<\/h3>\n
Polyacrylamide gel electrophoresis (2.2.31) under reducing conditions.<\/p>\n
Resolving gel 12 per cent acrylamide.<\/p>\n
Concentrated sample buffer concentrated SDS-PAGE sample buffer for reducing conditions R containing 2-mercaptoethanol as the reducing agent.<\/p>\n
Sample buffer: Mixture of equal volumes of concentrated SDS-PAGE sample buffer for reducing conditions R and water R.<\/p>\n
Test solution (a): Concentrate the preparation to be examined using a suitable method to obtain a protein concentration of 1.5 mg\/mL.<\/p>\n
Test solution (b): Mixture of equal volumes of test solution (a) and the concentrated sample buffer.<\/p>\n
Test solution (c): Dilute test solution (a) to obtain a protein concentration of 0.6 mg\/mL. Mix equal volumes of this solution and the concentrated sample buffer.<\/p>\n
Test solution (d): Mix 8 \u03bcL of test solution (c) and 40 \u03bcL of the sample buffer.<\/p>\n
Test solution (e): Mix 15 \u03bcL of test solution (d) and 35 \u03bcL of the sample buffer.<\/p>\n
Test solution (f): Mix 18 \u03bcL of test solution (e) and 18 \u03bcL of the sample buffer.<\/p>\n
Test solution (g): Mix 12 \u03bcL of test solution (f) and 12 \u03bcL of the sample buffer.<\/p>\n
Reference solution: Solution of relative molecular mass markers suitable for calibrating SDS-PAGE gels in the range of 15-67 kDa. Dissolve in the sample buffer.<\/p>\n
Sample treatment: Boil for 3 min.<\/p>\n
Application: 20 \u03bcL of test solutions (b) to (g) and the reference solution.<\/p>\n
Detection: Coomassie staining, carried out as follows: immerse the gel in Coomassie staining solution R1 at 33-37 \u00b0C for 90 min with gentle shaking, then remove the staining solution; destain the gel with a large excess of a mixture of 1 volume of glacial acetic acid R, 1 volume of 2-propanol R and 8 volumes of water R.<\/p>\n
Apparent molecular masses Interferon beta-1a = about 23 000; underglycosylated interferon beta-1a = about 21 000; deglycosylated interferon beta-1a = about 20 000; interferon beta-1a dimer = about 46 000.<\/p>\n
Identification of bands: Use the electropherogram provided with interferon beta-1a CRS.<\/p>\n
System suitability:<\/p>\n
\u2014 the validation criteria are met (2.2.31);<\/p>\n
\u2014 a band is seen in the electropherogram obtained with test solution (g);<\/p>\n
\u2014 a gradation of intensity of staining is seen in the electropherograms obtained with test solutions (b) to (g).<\/p>\n
Limits:<\/p>\n
\u2014 in the electropherogram obtained with test solution (c), the band corresponding to underglycosylated interferon beta-1a is not more intense than the principal band in the electropherogram obtained with test solution (e) (5 per cent);
\n\u2014 in the electropherogram obtained with test solution (b), the band corresponding to deglycosylated interferon beta-1a is not more intense than the principal band in the electropherogram obtained with test solution (e) (2 per cent); any other band corresponding to an impurity of a molecular mass lower than that of interferon beta-1a, apart from the band corresponding to underglycosylated interferon beta-1a is not more intense than the principal band in the electropherogram obtained with test solution (f) (1 per cent).<\/p>\n
Oxidised interferon beta-1a<\/h3>\n
Maximum 6 per cent.<\/p>\n
Use the chromatogram obtained with the test solution in identification C. Locate the peaks due to the peptide fragment comprising amino acids 34-45 and its oxidised form using the chromatogram of oxidised interferon beta-1a digest supplied with interferon beta-1a CRS.<\/p>\n
Calculate the percentage of oxidation of interferon beta-1a using the following expression:<\/p>\n
A_{34-45ox<\/sub>\/ (A_{34-45\u00a0<\/sub>+ A34-45ox)<\/p>\n}}
A_{34-45ox<\/sub> = area of the peak due to the oxidised peptide fragment 34-45;<\/p>\n}
A_{34-45<\/sub> = area of the peak due to the peptide fragment 34-45.<\/p>\n}
Bacterial endotoxins (2.6.14)<\/h3>\n
Less than 0.7 IU in the volume that contains 1 \u00d7 10 IU of interferon beta-1a, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins.<\/p>\n
ASSAY<\/h2>\n
Protein<\/h3>\n
Liquid chromatography (2.2.29). Prepare 3 independent dilutions for each solution.<\/p>\n
Test solution: Dilute the preparation to be examined to obtain a concentration of 100 \u03bcg\/mL.<\/p>\n
Reference solution: Dissolve the contents of a vial of interferon beta-1a CRS to obtain a concentration of 100 \u03bcg\/mL.<\/p>\n
Precolumn:<\/p>\n
\u2014 size: l = 0.02 m, \u00d8 = 2.1 mm;<\/p>\n
\u2014 stationary phase: end-capped butylsilyl silica gel for chromatography R (5 \u03bcm) with a pore size of 30 nm.<\/p>\n
Column:<\/p>\n
\u2014 size: l = 0.25 m, \u00d8 = 2.1 mm;<\/p>\n
\u2014 stationary phase: end-capped butylsilyl silica gel for chromatography R (5 \u03bcm) with a pore size of 30 nm.<\/p>\n
Mobile phase:<\/p>\n
\u2014 mobile phase A: 0.1 per cent V\/V solution of trifluoroacetic acid R;<\/p>\n
\u2014 mobile phase B: to 300 mL of water for chromatography R, add 1 mL of trifluoroacetic acid R and dilute to 1000 mL with acetonitrile R1;<\/p>\n\n\n\n\n\n\n\n
Time<\/strong><\/p>\n
(min)<\/strong><\/td>\n
Mobile phase A<\/strong><\/p>\n
(per cent V\/V)<\/strong><\/td>\n
Mobile phase B<\/strong><\/p>\n
(per cent V\/V)<\/strong><\/td>\n<\/tr>\n
0 – 20<\/td>\n 100 \u2192 0<\/td>\n 0 \u2192 100<\/td>\n<\/tr>\n
20 – 25<\/td>\n 0<\/td>\n 100<\/td>\n<\/tr>\n
25 – 26<\/td>\n 0 \u2192 100<\/td>\n 100 \u2192 0<\/td>\n<\/tr>\n
26 – 40<\/td>\n 100<\/td>\n 0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
Flow rate: 0.2 mL\/min.<\/p>\n
Detection: Spectrophotometer at 214 nm.<\/p>\n
Injection: 50 \u03bcL.<\/p>\n
Retention time: Interferon beta-1a = about 20 min.<\/p>\n
System suitability: Reference solution:<\/p>\n
\u2014 symmetry factor: 0.8 to 2.0 for the peak due to interferon beta-1a;<\/p>\n
\u2014 repeatability: maximum relative standard deviation of 3.0 per cent between the peak areas obtained after injection of the 3 independent dilutions.<\/p>\n
Calculate the content of interferon beta-1a (C_{908<\/sub>H_{1406<\/sub>N_{246<\/sub>O_{252<\/sub>S_{7<\/sub>) taking into account the assigned content of C_{908<\/sub>H_{1406<\/sub>N_{246<\/sub>O_{252<\/sub>S_{7<\/sub> in interferon beta-1a CRS.<\/p>\n}}}}}}}}}}
Potency<\/h3>\n
The potency of interferon beta-1a is estimated by comparing its ability to protect cells against a viral cytopathic effect with the same ability of the appropriate International Standard of human recombinant interferon beta-1a or of a reference preparation calibrated in International Units.<\/p>\n
The International: Unit is the activity contained in a stated amount of the appropriate International Standard. The equivalence in International Units of the International Standard is stated by the World Health Organization.<\/p>\n
Carry out the assay using a suitable method, based on the following design.<\/p>\n
Use, in standard culture conditions, an established cell line sensitive to the cytopathic effect of a suitable virus and responsive to interferon. The cell cultures and viruses that have been shown to be suitable include thefollowing:<\/p>\n
\u2014 WISH cells (ATCC No. CCL-25) and vesicular stomatitis virus VSV, Indiana strain (ATCC No. VR-158) as infective agent;<\/p>\n
\u2014 A549 cells (ATCC No. CCL-185) and encephalomyocarditis virus EMC (ATCC No. VR-129B) as infective agent.<\/p>\n
Incubate in at least 4 series, cells with 3 or more different concentrations of the preparation to be examined and the reference preparation in a microplate and include in each series appropriate controls of untreated cells.<\/p>\n
Choose the concentrations of the preparations such that the lowest concentration produces some protection and the largest concentration produces less than maximal protection against the viral cytopathic effect. Add at a suitable time the cytopathic virus to all wells with the exception of a sufficient number of wells in all series, which are left with uninfected control cells. Determine the cytopathic effect of the virus quantitatively with a suitable method. Calculate the potency of the preparation to be examined by the usual statistical methods (for example, 5.3).<\/p>\n
The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 64 per cent and not more than 156 per cent of the estimated potency.<\/p>\n
STORAGE<\/h2>\n
In an airtight container, protected from light, at a temperature below -70 \u00b0C. If the substance is sterile, store in a sterile, airtight, tamper-evident container.<\/p>\n
LABELLING<\/h2>\n
The label states:<\/p>\n
\u2014 the interferon beta-1a content, in milligrams per millilitre;<\/p>\n
\u2014 the antiviral activity, in International Units per millilitre;<\/p>\n
\u2014 where applicable, that the substance is suitable for use in the manufacture of parenteral preparations.<\/p>\n","protected":false},"excerpt":{"rendered":"
(Ph. Eur. monograph 1639) C908H14O6N246O252S7 Mr approx. 22 500 Action and use Cytokine. DEFINITION Solution of a glycosylated protein having the same amino acid sequence and disulfide bridge and a similar glycosylation\u00a0 pattern as interferon beta produced by human diploid fibroblasts in response to viral infections and various other inducers. It exerts antiviral, antiproliferative and…<\/p>\n","protected":false},"author":4,"featured_media":16713,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[174],"tags":[],"class_list":["post-16434","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-medicinal-substances"],"acf":[],"_links":{"self":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/16434","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/comments?post=16434"}],"version-history":[{"count":2,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/16434\/revisions"}],"predecessor-version":[{"id":16457,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/16434\/revisions\/16457"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media\/16713"}],"wp:attachment":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media?parent=16434"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/categories?post=16434"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/tags?post=16434"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}

Content<\/h3>\nMinimum 0.20 mg of protein per millilitre.<\/p>\n

Potency<\/h3>\nMinimum 1.5 \u00d7 10 IU per milligram of protein.<\/p>\nIt may contain buffer salts.<\/p>\n

Host-cell-derived proteins<\/h3>\nThe limit is approved by the competent authority.<\/p>\n

Host-cell or vector-derived DNA<\/h3>\nThe limit is approved by the competent authority.<\/p>\n

N-terminal truncated forms<\/h3>\nExamination for specific N-terminal truncated forms should be performed using a suitable technique such as N-terminal sequence determination. The limits are approved by the competent authority.<\/p>\n

Dimer and related substances of higher molecular mass<\/h3>\nNot more than the amount approved by the competent authority, using an appropriate validated liquid chromatography method.<\/p>\n

CHARACTERS<\/h2>\n

Appearance<\/h3>\nClear or slightly opalescent, colourless or slightly yellowish liquid.<\/p>\n

TESTS<\/h2>\n

Bacterial endotoxins (2.6.14)<\/h3>\nLess than 0.7 IU in the volume that contains 1 \u00d7 10 IU of interferon beta-1a, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins.<\/p>\n

ASSAY<\/h2>\n

STORAGE<\/h2>\nIn an airtight container, protected from light, at a temperature below -70 \u00b0C. If the substance is sterile, store in a sterile, airtight, tamper-evident container.<\/p>\n

Content<\/h3>\n
Minimum 0.20 mg of protein per millilitre.<\/p>\n

Potency<\/h3>\n
Minimum 1.5 \u00d7 10 IU per milligram of protein.<\/p>\n
It may contain buffer salts.<\/p>\n

Host-cell-derived proteins<\/h3>\n
The limit is approved by the competent authority.<\/p>\n

Host-cell or vector-derived DNA<\/h3>\n
The limit is approved by the competent authority.<\/p>\n

N-terminal truncated forms<\/h3>\n
Examination for specific N-terminal truncated forms should be performed using a suitable technique such as N-terminal sequence determination. The limits are approved by the competent authority.<\/p>\n

Dimer and related substances of higher molecular mass<\/h3>\n
Not more than the amount approved by the competent authority, using an appropriate validated liquid chromatography method.<\/p>\n

Appearance<\/h3>\n
Clear or slightly opalescent, colourless or slightly yellowish liquid.<\/p>\n

Bacterial endotoxins (2.6.14)<\/h3>\n
Less than 0.7 IU in the volume that contains 1 \u00d7 10 IU of interferon beta-1a, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins.<\/p>\n

STORAGE<\/h2>\n
In an airtight container, protected from light, at a temperature below -70 \u00b0C. If the substance is sterile, store in a sterile, airtight, tamper-evident container.<\/p>\n